mucosa-associated biohydrogenating microbes protect the simulated gut microbiome from stress by a...

5th International Human Microbiome Congress (IHMC) 2015 / Luxembourg

ABSTRACT BOOK

APPLICATION OF TOOLS FOR MICROBIOME RESEARCH

#18 / Short Talk Chemical-based Metaproteomics of the Healthy Human Gut Microbiome Ana Y. Wang (1), Sandip Chatterjee (1), Peter Thuy-Boun (1), John R. Yates III (2,3), Andrew I. Su (1,4), and Dennis W. Wolan (1,2) 1. Department of Molecular and Experimental Medicine 2. Department of Chemical Physiology 3. Department of Molecular and Cellular Neuroscience 4. Department of Integrative Structural and Computational Biology The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. Pioneering metagenomic sequencing methodologies have provided valuable insights into the species diversity and composition of bacteria that colonize the human intestinal tract. We seek to expand on these discoveries with the development and application of new chemical biology tools and mass spectrometry proteomic methods as techniques to interrogate gut flora on the protein level. Here, we present a modified functional proteomics approach that employs activity-based protein probes (ABPPs) to directly measure, characterize, and quantitate gut microbial proteins and their enzymatic functions. Small molecule ABPPs, attached to a biotin enrichment tag, are systematically incorporated to covalently label and isolate target protein families, including hydrolases, proteases, sulfatases, and glycosidases from the complex gut microbial proteome. These ABPP-labeled proteins are then subjected to separation by high performance reverse phase liquid chromatography coupled with tandem mass spectrometry for protein identification. Ultimately, our goal is to elucidate the normal distribution of gut bacterial proteins among individuals (if one exists), identify which protein functionalities are essential for human homeostasis, and determine what proteins and interactions with the host are altered or compromised in dysbiosis. The results from our work will facilitate the discovery of chemical and biological tools to probe the roles that key enteric bacterial enzymes play in human health and microbiome-related diseases. Importantly, our new methods and materials will provide a foundation for further development of proteomic approaches to target a variety of key protein functionalities within microbiomes not only in the gut, but also in other cavities of the human body. App-001#28 Impacts of infection with different toxigenic Clostridium difficile strains on faecal microbiota in children Zongxin Ling (1), Xia Liu (1,2), Xiaoyun Jia (3), Yiwen Cheng (1), Yueqiu Luo (1), Li Yuan (1), Yuezhu Wang (4), Chunna Zhao (3), Shu Guo (3), Lanjuan Li (1), Xiwei Xu (3), Charlie Xiang (1) 1. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310003, China; 2. Intensive Care Unit, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310003, China; 3. Department of Gastroenterology, Affiliated Beijing Children’s Hospital, Capital Medical University, Beijing, 100045, China; 4. Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai 201203, China; Increasing evidence suggests that altered intestinal microbial composition and function result in an increased risk of Clostridium difficile-associated diarrhoea (CDAD); however, the specific changes of intestinal microbiota in children suffering from CDAD and their associations with C. difficile strain toxigenicity are poorly understood. High-throughput pyrosequencing showed that reduced faecal bacterial diversity and dramatic shifts of microbial composition were found in children with CDAD. The Firmicutes/Bacteroidetes ratio was increased significantly in patients with CDAD, which indicated that dysbiosis of faecal microbiota was closely associated with CDAD. C. difficile infection resulted in an increase in lactate-producing phylotypes, with a corresponding decrease in butyrate-producing bacteria. The decrease in butyrate and lactate buildup impaired intestinal colonisation resistance, which increased the susceptibility to C. difficile colonisation. Strains of C. difficile which were positive for both toxin A and toxin B reduced faecal bacterial diversity to a greater degree than strains that were only toxin B-positive, and were associated with unusually abundant Enterococcus, which implies that the C. difficile toxins have different impacts on the faecal microbiota of children. Greater understanding of the relationships between disruption of the normal faecal microbiota and colonisation with C. difficile that produces different toxins might lead to improved treatment. App-002#41 Evaluation of microsphere intergrity in modified fluidized bed bioreator compared to choanoid fluidized bed bioreator Juan Lu, Lanjuan Li State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital,School of Medicine, Zhejiang University, Hangzhou 310003, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, China Bioartificial liver provides a combination of three-dimensional support to create a bio-mimic microenvironment for maintaining cell functions in vitro.Cells in bioartificail bed bioreators could be in the form of microspheres or microencapsules. However, different reatctors have various effects on microspherss or microencapsules. This study is to evaluate the protective capacity of microsphere intergrity in two fluidized bed bioreators. Forty milliliters empty microspheres were placed in modified fluidized bed bioreator (MFBB) and choanoid fluidized bed bioreator(CFBB) and circulated in normal saline(NS) for 24h,48h,72h separately. Residual quantity, mean diameters and viscoelasticity of microspheres in two reactors were determined. The

residual microspheres in CFBB were significantly more than those in MFBB in quantity every 24h.The percentage of the remaining microspheres in two reactors was separately 99.8%,98.1%,96.6% and 94.6%,92.1%,91.2% at the end of every 24h. Secondly, the mean diameter of the microspheres on account of the fluidization effect in reactors was increasing as time extention whereas the mean diameter of the microspheres in CFBB was bigger 10um than those in MFBB. Moreover, the microspheres in CFBB compared to MFBB were more fragile by imposing the same external forces because of the descending viscoelasticity. Taken together, the results above suggested that MFBB could protect the intergrity of microspheres due to its low fluid shear forces, balance of fluidized state and stress evenly supplied. The improvement of the fluidized bed bioreators could be benificial for the application of various cells in reactors, thus for further objective support for clinical advance. App-003#42 In vitro and in vivo analysis of antimicrobial agents alone and in combination against multi-drug resistant Acinetobacter baumannii Songzhe He1,2, Hui He1,2, Yi Chen1,2, Yueming Chen2, Wei Wang2, Daojun Yu1,2 1The First Affiliated Hospital of Hangzhou, Zhejiang Chinese Medical University. 261 Huansha Rd, Hangzhou, 310006, China. 2Department of Clinical Laboratories, Hangzhou First People’s Hospital, 261 Huansha Rd, Hangzhou, 310006, China. Objective To investigate in vitro and in vivo antibacterial activities of tigecycline and other 13 kinds of common antimicrobial agents alone or in combination against multi-drug resistant Acinetobacter baumannii. Methods In vitro susceptibility test was used to detect minimal inhibitory concentration (MIC). A mouse lung infection model established by ultrasonic atomization method was used to detect in vivo antimicrobial activities. Results Multi-drug resistant Acinetobacter baumannii showed a high sensitivity to tigecycline (98% inhibition), polymyxin B (78.2% inhibition), minocycline (74.2% inhibition). When combined with other antimicrobial agents, polymyxin B, tigecycline, minocycline presented synergistic or additive effects. In vivo data showed that white blood cell (WBC) counts in drug combination group C (minocycline+Amikacin) and D (minocycline+Rifampicin) were significantly lower than those in group A (tigecycline) and B (polymyxin B) (P App-004#44 Influence of diet and parasitism on the gut microbiome of African hunter-gatherers, farmers and fishers Elise Morton (1), Joshua Lynch (1), Alain Froment (2), Sophie Lafosse (2), Evelyne Heyer (2), Molly Przeworski (3), Ran Blekhman (1) and Laure Ségurel (2) 1. Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, 55455 2. Eco-anthropology and ethnobiology, UMR 7206, CNRS - MNHN - University Paris 7 Diderot 3. Department of Biological Sciences, Columbia University, New York, NY 10027 The human gut microbiome is influenced by the host’s nutrition and health status. It therefore represents an interesting adaptive phenotype under the influence of metabolic and immune constraints. Previous studies contrasting rural African and industrialized Western populations have shown that geography is an important factor associated with the gut microbiome; however, studies have yet to disentangle the effects of e.g., climate, diet, host genetics, hygiene and parasitism. Here, we focus on fine-scale comparisons of African rural populations in order to (i) contrast the gut microbiomes of populations that inhabit similar environments but have different traditional diets and (ii) evaluate the effect of parasitism on microbiome composition and structure. We sampled rural Pygmy hunter-gatherers as well as Bantu farmers and fishermen in Southwest Cameroon (n=64 individuals). We found that the presence of Entamoeba is the best predictor of microbial composition and diversity, such that an individual’s infection status can be predicted with 81% accuracy based on his/her gut microbiome composition. We identified multiple taxa that differ significantly in frequency between infected and uninfected individuals, notably Elusimicrobiaceae unc., Treponema unc. and Prevotella copri. Furthermore, alpha diversity is significantly higher in infected individuals while beta-diversity is reduced. We found that the second best factor predicting microbial composition is subsistence. Interestingly, Pygmy hunter-gatherers have significantly more Proteobacteria (in particular more Succinivibrio unc. and Ruminobacter unc.) than their farmer and fisher neighbors, two opportunistic pathogens also found enriched in the Hadza as compared to Italians. Furthermore, we found Bifidobacterium to be in higher frequency in fishers, likely reflecting a higher consumption of dairy products in this group. In conclusion, our results stress the importance of taking into account an individual’s parasitism status in studies of the microbiome, and highlight how sensitive the microbial ecosystem is to subtle changes in host’s nutrition. Indeed, we found a higher alpha diversity in fishers as compared to farmers, two groups that share a similar rural unindustrialized environment and the same genetic ancestry but only slightly differ in their diet. Finally, our fine-scale analysis allowed us to identify microbial features that are specific to hunter-gatherers versus ones shared by all rural African populations, increasing our understanding of the influence of subsistence and lifestyle on gut microbiome composition.

App-005#70 The Metabolic Contribution of the Mammalian Gut Microbiota Revealed by a Study of the Urine and Feces of Germ-Free Rats Ping Yi(1), Yong jun Li(1), Qin Xie, Deying Cheng(1), Zhenggang Yang(1), Jiezuan Yang(1), Lanjuan Li(1) State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, Zhejiang University, Hangzhou,China The gut microbiota is generally accepted to play an important role in the maintenance of host health. The gut microbiota operates with the host in some metabolic processes, such as co-metabolism of substrates. However, the detailed mechanism of such interactions is poorly understood. Here, we sought to evaluate the contributions of the mammalian gut microbiota in a metabolic study of urine and feces by ultraperformance liquid chromatography−mass spectrometry (UPLC-MS) in germ-free rats. Data were subjected to reveal characteristics that differed between the metabolisms of GF (germ-free) and CV (conventional) rats. Levels of amino acid and bile acid metabolites were markedly different between the groups. There were higher concentrations of phenylalanine and tryptophan in the GF group, related to the lack of microbiota metabolism. Furthermore, bacterial co-metabolic products of indole-containing metabolite were higher in the conventional group. The GF samples showed higher concentrations of tauro-conjugated bile acids and lower unconjugated bile acids. Raffinose was detected only in the GF group. Collectively, our data suggest that the gut microbiota plays an essential role in human health, affecting the fates of dietary components and many drugs. Understanding the activity of individual members of the gut microbiota will be indispensable for personalized medicine. App-006#76 Large Quantity Cryopreservation of Microencapsulated immortalized human hepatocyte cell line for Application of BAL Jianzhou Li(1),Ying Yang(1),Juan Lu(1),Xiaoping Pan(1),Ermei Chen(1),Ning Zhou(1),Lanjuan Li(1) 1.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou, 310003, China Large quantity cryopreservation of microencapsulated hepatocytes, providing a readily available hepatocytes supply permanently and sufficiently, is required for the application of BAL. Extensive researches on cryopreservation of microencapsulated hepatocytes all based on a 2 ml type of vial, the volume of which is too small for large quantity cryopreservation. In this study, we explored the effect of a large quantity cryopreservation of microencapsulated immortalized human hepatocyte cell line HepLi3 in a 50 ml vial on the subsequent integrity, mechanical stability, viability and functionality comparable to 2 ml vial. Further more, we evaluated the viability and functionality of cryopreserved microencapsulated immortalized human hepatocytes based on a choanoid fluidized bed bioreactor using severe hepatitis plasma..Results showed there is no significant difference between 50 ml vial and 2 ml one in maintenance of the integrity, mechanical stability, viability and functionality of microencapsulated hepatocytes after 30 day’s cryopreservation. And the bioartificial liver system based n a fluidized-bed bioreactor with cryopreserved microencapsulated immortalized human hepatocytes appeared to be effective for improving severe hepatitis plasma parameters. While, the 50 ml vial is easier to handle and of great practical efficiency with a large volume containing more hepatocytes. In conclusion, we present a large quantity cryopreservation of microencapsulated hepatocytes ,which is promising for application of BAL App-008#113 Deciphering the gut microbiome in infants Herbert Pang (1), Nana Jin (2) 1. The University of Hong Kong, Hong Kong SAR, China 2. Harbin Medical University, Harbin, China The gut microbiota plays an important role in human diseases and has been linked to many diseases, such as inflammation, atopic disease, heart disease and obesity. With the advent of high-throughput technologies in the post-genomic era, understanding the human gut microbiome has led to important insights regarding human diseases and potential therapeutic treatments. However, relatively little research has characterized the early microbial communities in infant. As a secondary data analysis, we obtained 10 infants’ gut microbiota measured at 5 and 21 months. SOAPdenovo was used to assemble the short-reads from Illumina HiSeq 2000. We calculated the domain frequency statistic, and identified the top 10 Pfam families and top 10 GO Terms after Bonferroni correction for multiple-testing. To compare the difference between species enriched in the two time points, we used paired t-test. Top 5 species that were significantly different between the two months are Ruminococcus obeum (p <.0001), butyrate-producing bacterium SSC/2 (p=0.0002), Halanaerobium hydrogeniformans (p=0.0015), Coprococcus catus (p=0.0017) and Coprococcus sp. ART55/1 (p=0.0020). Top 5 significant Pfam families were TonB dependent receptor, SusD family, Major Facilitator Superfamily, TonB dependent receptor plug domain, and LysR substrate binding domain. Top 5 significant GO terms were receptor activity, carbohydrate transport, phosphoenolypyruvate-dependent sugar phosphotransferase system, signal transducer activity, and molecular transducer activity. From the domain frequency statistics, we found that the number of sequences with domain hits and the total number of the same infant measured at 5 months were different from month 21. Gastrointestinal microbial composition has an important influence on the functional genomics in human health and disease. More in-depth work and longitudinal data with larger sample size will be needed to deepen our understanding of the complex infant gut microbiome.

#131 Short Talk Pangenome-based, meta'omic analysis highlights association of E. coli accessory gene content with risk of necrotizing enterocolitis in a cohort of pre-term infants. Matthias Scholz (*,1), Doyle V. Ward (*,2), Thomas Tolio (1), Moreno Zolfo (1), Adrian Tett (1), Ardythe Morrow (3), Nicola Segata (^, 1) (1) Centre for Integrative Biology, University of Trento, Italy (2) Center for Microbiome Research, UMass Medical School, Worcester MA (3) Department Pediatrics, Perinatal Institute, Cincinnati Children’s Hospital Medical Center * Equal contribution ^ Presenting/corresponding author This project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272200900018C. Biologically relevant microbial diversity is found at the strain level. However, determining the genomic repertoire of a strain within a community from shotgun metagenomic data is remarkably complex. Read alignment to reference genomes is sensitive to inter-species conservation and gene copy number, while de novo metagenomic assembly is computationally challenging and produces at best fragmented scaffolds with a significant fraction of false negative genes. We developed a novel, assembly-free Pangenome-based Phylogenomic Analysis (PanPhlAn), which comprises multiple mapping and co-abundance normalization steps to capture the strain-specific gene set from metagenomes and metatranscriptomes. We first validated the method on 65 large semi-synthetic metagenomes (Fig. 1A) showing that even at 1X average coverage, more than 87% of gene families are correctly recovered. We then applied PanPhlAn on 1438 publicly available metagenomic samples (Fig. 1B) and integrated them with representative reference genomes. This highlighted how intestinal E. coli strains fall in multiple functionally distinct clades almost all represented by sequenced reference genomes and also proved the PanPhlAn ability to profile the German outbreak strain from metagenomes. In our cohort of pre-term infants, investigated using deep shotgun meta'omic sequencing, we found a significant fraction of infants with high abundances of E. coli. We aimed to understand if functionally-defined clades of E. coli were associated with necrotizing enterocolitis (NEC). Using PanPhlAn we profiled the functional diversity of intestinal E. coli strains from 48 E. coli positive infants (9 term, 38 preterm <30 weeks gestational age, 12 NEC cases). When hierarchically clustered they strongly correlated with the multi-locus sequence types of the strains (Fig. 1C) and revealed a variable risk of NEC within the phylogenetic structure (p=0.051). Moreover, Fisher’s exact test identified genes associated with uro- and extraintestinal pathogenic E. coli as enriched in clades associated with NEC as well as other genes with unclear annotations that should be prioritized for future functional studies. When metagenomic and metatranscriptomic samples from the same specimen are available, PanPhlAn also provides gene-specific transcription rates of individual strains in a sample. In the pre-term infant cohort this resulted in an “in-vivo” transcriptional activity map (Fig. 1D) which is not accessible via culture-dependent approaches. We are currently mining these profiles to understand whether specific transcriptional patterns are associated with NEC. Altogether, our results suggest that understanding the epidemiology of, and rapid identification of E coli strains is critical to understanding NEC pathology. We demonstrate that metagenomic sequencing in combination with PanPhlAn is an effective cultivation-free approach for the epidemiology of intestinal pathogens. App-009#132 Micelle PCR reduces artifact formation in 16S microbiota profiling Stefan A. Boers (1), John P. Hays (1), Ruud Jansen (2) 1. Department of Medical Microbiology and Infectious diseases, Erasmus University Medical Centre, Rotterdam, The Netherlands. 2. Department of Molecular Biology, Regional Laboratory of Public Health, Haarlem, The Netherlands Introduction The cornerstone of microbiota profiling is the sequencing of 16S rRNA PCR amplicons with next generation sequencing (NGS). The main disadvantage of this approach is the formation of PCR amplification artifacts, such as chimeric sequences that can lead to incorrect taxonomic identification and overestimated microbial diversity. Although chimeric sequences can be filtered out with specialized software after the PCR and NGS, the generation of chimeric products can still seriously reduce the amount of useful information obtained in a single sequencing run. Here we introduce a micelle based amplification strategy that greatly reduces artifact production during PCR amplification and subsequent NGS sequencing. Micelle PCR is a single-molecule clonal amplification method in which template DNA molecules are separated into a large number of physically distinct reaction compartments using a water in oil emulsion. Methods Universal 357F and 936R primers were used to amplify the 16S rRNA V3-V5 region from a synthetic microbial community containing equimolar 16S rRNA operon counts derived from 20 different bacterial species. Both micelle PCR and traditional PCR methods were used. Identical protocols were utilized for determining the microbiota for low-concentration DNA (nose swabs), high-concentration DNA (feces) and high-concentration DNA / hyper diverse (soil) samples. Amplicons were sequenced using 454 sequencing (GS Junior, Roche) employing two subsequent PCRs that is known to generate high percentages of chimeric sequences. The degree of chimera formation was determined using UCHIME. Microbiota profiles were determined by clustering operational taxonomic units (OTUs) with 97% similarity (MOTHUR). Results Micelle PCR generated 1.5% chimeric sequences and 20 OTUs in the synthetic community, compared to 56.9% chimeras and 70 OTUs using traditional PCR NGS sequencing. Chimeric products not recognized as actual chimeras were the cause of this overestimation, as most of the 70 OTUs were only found once. In addition, micelle PCR data exhibit an average 0.85-fold difference from the expected percentage in the synthetic community, with a maximum overestimation of 1.83-fold and a maximum underestimation of 0.20-fold. On the other hand, traditional PCR data showed an average 0.63-fold difference from

expected percentage with an overestimated maximum of 2.48-fold and underestimated maximum of 0.03-fold. Similar results were obtained for nose swabs, feces and soil samples. Conclusion Micelle PCR drastically reduces chimera formation without the reliance on complex computational methods, resulting in improved microbial diversity estimates. In addition, micelle PCR prevents PCR competition resulting in highly reliable quantitative microbiota profiles. The compartmentalization of micelle PCR has two major advantages: 1) reduction of chimera formation and 2) the prevention PCR competition due to unequal amplification rates of different 16S template molecules. App-010#147 Type II toxin-antitoxin systems as a functional marker for identification of Bifidobacterium and Lactobacillus strains suitable for metagenomic studies Ksenia Klimina (1), Siarhei Hladyshau (1), Natalia Zakharevich (1), Artem Kasianov (1), Elena Poluektova (1), Vsevolod Makeev (1), Valery Danilenko (1) 1. Vavilov Institute of General Genetics, Moscow, Russia The human gastrointestinal (GI) tract is inhabited with a wide variety of bacteria. Violation of the intestinal microbiota may trigger various diseases: immune, cardiology, oncology, neurodegenerative and others. The gut microbiota (GM) is characterized not just at the level of phyla or species but also at the strain level. Recent research has indicated that each individual may have a unique metagenomic genotype. Essential components of GM are the probiotic bacteria of the genus Bifidobacterium and Lactobacillus. Type II toxin-antitoxin systems are functional biomarkers allowing one to differentiate these groups of bacteria on the strain level [1, 2]. The objective of this work was to study toxin-antitoxin (TA) type II systems (MazEF and RelBE) in Bifidobacterium and Lactobacillus strains, their variability and applicability of these systems for analysis of represented functional genes, known gene variants (SNP), and groups of reference strains in metagenomes. Based on known annotated genes from RelBE and MazEF TA superfamilies we constructed a database of toxin and antitoxin genes, which was used for primary annotation of genes in strains of different Lactobacillus and Bifidobacterium species. We analyzed variability of these genes in different strains after the expanded annotation of genes from TA system in bacteria and we built a diagram showing the presence or absence of the toxin/antitoxin genes in bacterial strains. Distribution of the toxin and antitoxin genes and SNP variants were found to be species and strain specific, the most distant species did not have the same T and A genes. Strains belonging to one species of bacteria had similar but not always identical sets of T and A genes. We developed a pipeline for computational detection of variants of TA genes and selection of subgroups within a species, taking into account uneven and fragmented characteristics of metagenomic sequencing. The pipeline was tested on a number of metagenomic samples (specially sequenced for this purpose or publicly available). The method was compared with existing methods of metagenomic analysis (16S rRNA and MetaPhlAn). 1. Klimina K.M., D.Ch.Kjasova, E.U.Poluektova, H.Krügel, HP Saluz, V.N. Danilenko, Identification and characterization of Toxin-Antitoxin systems in strains of Lactobacillus rhamnosus, isolated from humans; Anaerobe 22 (2013) 82-89 2. Averina OV, Alekseeva MG, Abilev SK, Il'in VK & Danilenko VN (2013) Distribution of genes of toxin-antitoxin systems of mazEF and relBE families in bifidobacteria from human intestinal microbiota. // Genetika 49(3): 315-27. Russian. App-011#151 A method for selectively enriching microbial dna from contaminating vertebrate host dna Erbay Yigit(1), Fiona J. Stewart(1), George R. Feehery(1), Samuel O. Oyola(2), Yan Wei Lim(3), Bradley W. Langhorst(1), Victor T. Schmidt(4,5), Eileen T. Dimalanta(1), Linda A. Amaral-Zettler(4,6), Theodore Davis(1), Michael A. Quail(2), Sriharsa Pradhan( 1. New England Biolabs Inc., Ipswich, MA, USA 2. Wellcome Trust Sanger Institute, Cambridge, UK 3. San Diego State University, San Diego, CA, USA 4. The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA, USA 5. Department of Ecology and Evolutionary Biology, Brown University, Providence, RI, USA 6. Department of Geological Sciences, Brown University, Providence, RI, USA Recent discoveries have implicated the human microbiome as playing a role in certain physical conditions and disease states, and these advances have opened up the potential for development of microbiome-based diagnostic and therapeutic tools. The majority of microbiome DNA studies to date have employed 16S analysis, but these provide very little information regarding function. In contrast, sequencing of the total DNA of a microbiome sample provides a broader range of information including genes, variants, polymorphisms, and putative functional information. However, many samples, including those derived from vertebrate skin, bodily cavities, and body fluids, contain both host and microbial DNA. Since a single human cell contains approximately 1,000 times more DNA than a single bacterial cell, even low-level human cell contamination can substantially complicate the analysis of a sample. In some cases, as low as 1% of sequencing reads may pertain to the microbes of interest and a large percentage of sequencing reads must be discarded, making such experiments impractical. To address this issue, we developed a method to enrich for microbial DNA using methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA. Importantly, microbial diversity and relative abundance is maintained after enrichment. This simple magnetic bead-based method was used to remove human or fish host DNA from bacterial and protistan DNA. We describe the enrichment of DNA samples from human saliva, human blood, a mock malaria-infected blood sample, human cystic fibrosis sputum, and a black molly fish, followed by next generation sequencing on multiple platforms. Sequence reads aligning to host genomes were reduced approximately 50-fold, while the percentage of sequence reads corresponding to microbial sequences increased approximately 10-fold. This new method for microbiome sequence analysis holds promise for use with a variety of sample types, enabling enrichment while accurately reflecting the diversity of the original sample.

App-012#323 Prediction of folate metabolism in gut microbes by whole genome comparison and metabolic modeling Stefania Magnusdottir (1), Ines Thiele (1), Michael Blaut (2), Delphine M. Saulnier (2) 1. Luxembourg Center for System Biology, University of Luxembourg, Luxembourg 2. Department of Gastrointestinal Microbiology, German Institute of Human Nutrition Potsdam-Rehbruecke Folate is one of the key vitamins involved in normal cellular function, growth and development. The colonic microbiota has been suggested to contribute to the folate pool, and affects individual dietary folate requirements. However, the main bacterial contributors to host folate status still have to be defined. We predicted folate metabolic capabilities of 35 main representatives of the human gut microbiota belonging to different phyla (Bacteroidetes, Firmicutes, Proteobacteria, Verrucomicrobia) based on their whole genome sequences. We then assessed in depth folate synthesis and uptake in a sub-selection of 8 microbes using constraint-based genome-scale metabolic models. We predicted previously that these 8 microbes were either de novo folate producers (Bacteroides thetaiotaiomicron, Escherichia coli), folate producers requiring para-aminobenzoic acid as a precursor (Bifidobacterium longum, Lactobacillus plantarum, Lactobacillus reuteri), or folate auxotrophs (Anaerostipes caccae, Clostridium ramosum, Faecalibacterium prausnitzii). In addition, we predicted the different folate vitamers that could be synthesized by these microbes, and the potential yield in a defined medium. We compared these predictions with intra and extracellular folate produced by these strains grown in a folate free medium. We refined the metabolic models when discrepancies between in silico predictions and experimental data were observed. Based on whole genome comparison, folate production was predicted for 19 of 35 bacterial strains. Folate auxotrophy was encountered mostly among Firmicutes, such as Roseburia intestinalis, or Ruminococcus bromii. All 3 strains of Faecalibacteriaum prausnitzii were predicted to require folate for growth. Using metabolic modeling, intracellular folate production was predicted to be the highest among the selected strains in Bacteroides thetaiotaiomicron (~11 mmol h-1 g dry weight-1). Bacteroides thetaiomicron displayed the highest intracellular folate production among the strains tested in vitro. Although in silico and experimental data agreed for most strains, some important discrepancies were observed: for instance, folate polyglutamylation reactions were absent in the E. coli metabolic model, but polyglutamylated folates were measured during in vitro growth. The model was therefore corrected accordingly. In addition, folic acid - a synthetic form of folate which is not produced by bacteria - was initially predicted to be produced in the E. coli metabolic model. This discrepancy was corrected by modifying the metabolic model such as folic acid reductases are uni-directional rather than bi-directional. The results of this study give a better understanding of bacterial folate metabolism. In the future, we aim to combine host-microbe modeling to better define the potential contribution of gut microbes to host folate status, and ultimately to human health. App-013#154 Methodological impact on metagenomic analyses Patrick Robe (1), Cyrille Jarrin (1), Daniel Auriol (1), David Villanova (1), Kuno Schweikert (1,2) 1. Libragen, Toulouse, FR 2. Induchem, Volktswil, CH Metagenomics, defined as the global functional and sequence-based analysis of the collective microbial genomes (microbiome) of a particular environment, has promoted a considerable increase in knowledge of the taxonomic and functional microbial diversity of natural ecosystems. Moreover, the exploration of microbial communities associated with human body sites (gut, skin, mouth, vagina …) enables the deciphering of close relationships linking human health and inhabiting microbiota. Realizing the potential of metagenomics for discovering novel genes from the yet untapped microbial diversity, libragen has been providing for 15 years analyses of microbial communities (Manichanh et al., 2006) as well as new and performant biocatalysts and metabolic pathways that give solutions to industrial issues (Lefevre et al., 2007 and 2008). The apparent practical simplicity of DNA extraction from natural samples using dedicated commercial kits, and the explosion of Next-Generation Sequencing facilities, allowing to define the genetic diversity of bacterial communities and enabling prediction of the associated gene functions, have stimulated metagenomic analyses. However, the procedures applied in the published studies are usually different, sometimes poorly described and results can hardly be compared in particular when considering a defined environment such as human skin. The objective of Libragen was to investigate each step of the metagenomic process with a particular focus on DNA extraction, to be able to provide a reliable and argued answer to a biological question using the most appropriate tools. Many sources of technical biases were identified, which can impact the results: inadequate experimental design, sampling and sample storage, insufficient purity of extracted nucleic acids, inappropriate selection of 16S variable region or poor choice of primers, insufficient control of produced libraries, inappropriate raw data analyses or deficient statistical analyses. These critical technical steps were evaluated through multiple methodological and technical in-house studies, including some achievements on skin microbiota. We have shown that several technical choices, the method of extraction and purification used to produce metagenomic DNA, the targeted DNA biomarker and the primers selected for PCR amplification and sequencing, and the sequencing strategy (amplicon-based sequencing vs WGS direct sequencing) can greatly affect the results of a metagenomic profiling study and may strongly alter the perception of the targeted microbial diversity. The acceptability of the results of any metagenomic study can be seriously hampered by inappropriate experimental conditions, and the extent of the findings is closely linked to the methodological scheme and the technical tools used to produce the results.

App-014#155 Short-term diet drives quick changes in the metabolic activity and composition of human gut microbiota Marisol Aguirre (1,2,3); Anat Eck (4); Paul H.M. Savelkoul (2,4); Andries E. Budding (4); Koen Venema (1,5) 1. TIFN, The Netherlands 2. Human Biology, Maastricht University, The Netherlands 3. MSB, TNO, The Netherlands 4. Microbiology, VU University Medical Center, Amsterdam, The Netherlands 5. Beneficial Microbes Consultancy, Wageningen, The Netherlands Background: Increasing evidence shows that changes in diet influence composition and activity of the gut microbiota. Due to the potential microbiota, host and diet interactions, strategies considering dietary interventions can be used to tackle, prevent or even treat worldwide epidemics such as obesity. However, there is no current consensus on how fast and reproducibly human gut microbiota can respond to short-term changes in the diet and there is scarce available information addressing this question. Studies performed in human individuals are the ideal way to follow this assessment. However, the limitation of such trials are mainly attributed to ethical concerns and high costs. An alternative to this is the performance of in vitro studies. Aim: The aim of this study was to screen how quick changes, in response to diet, are expressed in the human gut microbiota when a 72 h fermentation was performed in a validated dynamic in vitro model of the proximal colon (TIM-2 system). Methods: Two diets with different carbohydrate:protein ratios (high carbohydrate and high protein) were tested in microbiota obtained from healthy volunteers. A control simulating an average western diet was used. Fermentation experiments were performed during 72 h in computer controlled TIM-2 units. SCFA (acetate, propionate, and n-butyrate) and BCFA (iso-butyrate and iso-valerate) were analyzed and the 16S-23S intergenic space of the microbiota was profiled. Results: The activity of the microbiota reflected differences between the diets exhibiting a trade-off between saccharolytic and proteolytic fermentation when compared to the control diet. This was confirmed by the different cumulative total amounts of SCFA and BCFA observed. The increase of luminal pH in the experiment with the high protein diet was higher (6.3) when compared to the other diets. The analysis on the diversity change over time of each phylum group shows different responses of the communities depending on the diet tested. The shifts of specific OTU’s over time also showed the effects of the different diets on the composition of the microbiota. For instance, E. ventriosum, a known butyrate producer, was poorly detected at the starting point of the study in all diets but its growth was stimulated when the microbiota was exposed to the control and high carbohydrate diet and it was inhibited under the high protein diet. Conclusions: The outcome of these set of experiments allows to elucidate how quickly human gut bacteria respond to a change in diet. In addition, it confirms that variations in the concentration of carbohydrates and proteins modify the activity and composition of the microbiota and these changes affect the health status of the host. This study was partly funded by TIFN (GH004) (Wageningen, The Netherlands). App-015#178 Clinical applications of proteomic analysis based on shotgun strategy Wangjie,Lilanjuan State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou, 310003, China Nowadays MS-based proteomics have been an indispensable tool for clinical use, among which shotgun proteomics have played an crutial role . The goal of clinical proteomics is to acquire one or more specific biomarkers using clinical samples, such as plasma and tissues from patients. Identification of useful biomarkers of disease could provide a better chance for early diagnosis, optimization of treatment, and means for monitoring progress during treatment. Shotgun proteomics is the analysis of peptides released from the protein through proteolysis. It avoids some limitations of the respective methods, providing high-throughput, accurate quantification, and reproducible measurements within a single experimental set-up. In this review,we focus on introducing the strategies and clinical application of shotgun proteomics. Additionally , we try to provide a detail guide for different types of isobaric reagents and their reaction chemistry (e.g., amine-, carbonyl-, and sulfhydryl-reactive). App-016#188 A Metagenomics Approach to Studying Blastocystis Lee O'Brien Andersen (1), Ida Bonde (2), Henrik Bjørn Nielsen (2), Christen Rune Stensvold (1) 1. Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, DK 2. Department of Systems Biology, Technical University of Denmark - Center for Biological Sequence Analysis, Copenhagen, DK

Blastocystis is a common single-celled intestinal parasitic protist. Here, we screened data obtained by metagenomic analysis of faecal DNA for Blastocystis by searching for subtype-specific genes in co-abundance gene groups, which are groups of genes that co-vary across a selection of 316 human faecal samples, hence representing genes originating from a single subtype. The 316 faecal samples were from 236 healthy individuals, 13 patients with Crohn’s disease, and 67 patients with ulcerative colitis. We determined the prevalence of Blastocystis to be 20.3% among healthy individuals and 14.9% among patients with ulcerative colitis. Meanwhile, Blastocystis was absent in patients with Crohn’s disease. Individuals with intestinal microbiota dominated by Bacteroides were much less prone to having Blastocystis-positive stool (Matthew’s correlation coefficient = -0.25, P < 0.0001) than individuals with Ruminococcus- and Prevotella-driven enterotypes. This is the first study to investigate the relationship between Blastocystis and communities of gut bacteria using a metagenomics approach. The study serves as an example of how it is possible to investigate microbial eukaryotic communities in the gut using metagenomic datasets targeting the bacterial component of the intestinal microbiome and the interplay between these microbial communities. App-017#194 Saliva and tongue swab based metaproteome analysis Alexander Rabe (1), Manuela Gesell Salazar (1), Stephan Fuchs (2), Helge Senkbeil (3), Thomas Kocher (3), Uwe Völker (1) 1. Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany 2. FG13, Robert Koch Institute, Wernigerode, Germany 3. Unit of Periodontology, Department of Restorative Dentistry, Periodontology and Endodontology, University Medicine Greifswald, Greifswald, Germany Metaproteomic approaches provide information about microbial activities in natural habitats and thus provide complementing information to metagenomic approaches. Several studies revealed the essential role of the human microbiome for human health and disease. Metaproteomic approaches offer great opportunities to gain new functional insights into the behavior of microbes in their natural habitats. The main aim of the study was to establish a community proteomic approach for saliva and the tongue in humans. Therefore, different protocols were tested. We focused on high bacterial protein coverage that then provides information about the active players and their functional role in the human oral cavity. A cohort of 15 healthy subjects was selected. The subjects were divided into two groups. For five subjects saliva was collected via a paraffin gum and Salivette® on two consecutive days (Setup A). For ten subjects we obtained saliva using the paraffin gum and tongue swabs with sterile wooden spatulas (Setup B). Saliva was centrifuged to separate pellet and supernatant. Afterwards, a gel-free approach was performed using shotgun LC-MS/MS (QExactive). The data analyzing pipeline included two steps. First, searching the analyzed spectra against a combined database, which included the “Human Oral Microbiome Database (HOMD) and a human swissprot database. Second, using the Prophane bioinformatics pipeline we performed taxonomical and functional assignments. Peptides with a high confidence level and proteins covered by at least two peptides were considered to be present. Proteins, which were identified in more than 50 % of all biological and the three technical replicates, were assigned as the “core-metaproteome” of saliva and tongue. In the first experimental setup (probands of group A) most bacterial proteins could be identified in the saliva - pellet using the paraffin gum. For the supernatant no significant identification rates could be discerned. Similar results were observed for saliva in setup B. However, many more bacterial proteins could be identified in the tongue swabs in comparison to the saliva - pellet and especially in the supernatant. Energy metabolism and protein synthesis were the most dominant protein functions, which could be assigned. Regarding the taxonomical composition, saliva - pellet and the tongue revealed high similarity. Streptococcus, Rothia, Neisseria, Prevotella, Veillonella and Haemophilus could be demonstrated to be the most prominent genera. We were able to show that for metaproteome analysis of saliva, the sampling procedure via the paraffin gum is the method of choice. Saliva, especially the pellet and tongue swabs offer great opportunities for a community proteomics approach. The samples can be obtained easily and non-invasively. The number of identified proteins enabled us to get an insight into the taxonomical and functional composition of the microbial communities in saliva and of the tongue. App-018#196 Proteome analysis of human sebaceous skin follicles reveals health- and disease-associated proteins of human and microbial origin Hans B. Lomholt (1), Malene Bek-Thomsen (1), Carsten Scavenius (2), Jan J. Enghild (2), Holger Brüggemann (1) 1. Department of Biomedicine, Aarhus University, Denmark 2. Department of Molecular Biology and Genetics, Aarhus University, Denmark Objective: Unlike the gut microbiota, the contribution of the skin microbiota to human health and disease is largely unknown. The pathobiological events leading to acne vulgaris are in the focus of this study. We analyzed sebaceous follicles of the skin and determined human and microbial proteins present in such follicles isolated from healthy individuals and acne patients. Methods:

Human follicles were extracted by cyanoacrylate biopsies and the protein content was determined by mass spectrometry (NanoESI-MS/MS). Results: Health- and acne-associated human proteins were detected. Healthy follicles are enriched in proteins such as prohibitins and peroxiredoxins which are involved in the protection from various stresses, including reactive oxygen species. By contrast, acne-affected follicles specifically contained proteins involved in inflammation, wound healing and tissue remodeling. The most significant biological process among all acne-enriched proteins was ‘response to a bacterium’. Identified bacterial proteins were exclusively from Propionibacterium acnes, supporting the role of P. acnes as an inducer of inflammation. In both diseased and healthy follicles the most abundant P. acnes proteins were surface-exposed dermatan sulphate adhesins, CAMP factors, and a so far uncharacterized lipase. Conclusion: Our study shows that the host inflammatory reaction in acne could be explained as a response to P. acnes. Vimentin was exclusively expressed in acne-affected follicles and a new model is presented for vimentin-mediated invasion of P. acnes into follicle-associated cells that could account for the long-lasting inflammation and difficulties in antibiotic eradication known in acne treatment. App-019#197 Time-varying network construction with CoNet Karoline Faust (1,2,3) & Jeroen Raes (1,2,3) 1. Department of Microbiology and Immunology, Rega Institute KU Leuven, Leuven, Belgium. 2. VIB Center for the Biology of Disease, VIB, Belgium. 3. Laboratory of Microbiology, Vrije Universiteit Brussel (VUB), Brussels, Belgium. Previously, we developed CoNet, a tool to infer microbial association networks from 16S count data. CoNet relies on an ensemble approach, i.e. the results of several network construction methods are merged. The ensemble inference is combined with a stringent assessment of significance. In recent years, the number of metagenomic time series studies that combine long duration with short sampling intervals has increased. Several dynamic network inference tools exploit the extra information present in these time series, but they infer networks that remain constant over time. However, the long time series available to date allow the construction of time-varying networks, i.e. networks that change over time. Here, we present time-varying networks constructed with CoNet from a recent longitudinal study (David 2014) using a sliding-window approach. Time-varying networks allow differentiating between stable edges present in most time windows and unstable edges present only in few time windows or only together with certain events. When analyzing edge stability, we found event- and phylum-specific differences. Time-varying network construction has been applied previously to gene expression time series, but to our knowledge, this is the first time this technique is employed to analyze metagenomic time series. David et al. (2014). Host lifestyle affects human microbiota on daily timescales. Genome Biology 15:R89. App-020#200 Microbiome-Gut-Brain Health Questionnaire and its application in mental health assessment Yunfeng Duan(1, 2),Xiaoli Wu(1), Feng Jin(1) 1.Behavioral biology laboratory, Key Laboratory of mental health, Institute of Psychology, Chinese Academy of Sciences, Beijing, China. 2.Bioinformatics laboratory, Key Laboratory of mental health, Institute of Psychology, Chinese Academy of Sciences, Beijing, China. The purpose of this study is to develop Microbiome-Gut-Brain Health Questionnaire(MGBHQ) and apply it in mental health condition assessment. The gut-brain and the brain are connected through the brain-gut axes. In fact we believe that the gut-brain should be the complex of intestinal tract neurons and human symbiotic microorganisms and it could also be called Microbiome-gut-brain. Some psychiatric disorders and functional gastrointestinal disorders are usually coupled together, such as autism, mood disorders, anxiety disorders. The gut-brain is closely related to the symbiotic microbes in the gut. Germ-free mice showed more anxiety-like behavior ; a Clostridium bacteria was found in the gut of autistic children with bowel disease. However , human microbiome and gut-brain research has just begun. The MGBHQ has not yet appeared both in China and abroad up to now . We developed MGBHQ and tested its reliability and validity . A total of 942 subjects were collected and the subjects' anxiety(State-Trait Anxiety Inventory, STAI), depression(Self-Rating Depression Scale, SDS) and insomnia(Athens Insomnia Scale, AIS) was also assessed. Item analysis and exploratory factor analysis showed that the structure of MGBHQ consists of three dimensions: intestinal status, eating habits and defecation status; reliability analysis shows that each dimension of α coefficient were 0.68-0.80; the gut-brain health scores has a significant negative correlation with STAI, SDS and AIS (P <0.01). We also collected 15 high MGBHQ scores and 15 low MGBHQ scores subjects' fresh fecal and venous blood samples. The fecal genomic DNA was amplified with Real-time fluorescent quantitative PCR by using specific primers of Bacteroidetes,

Firmicute, Clostridium(Clo.), Escherichia coli, Bifidobacterium(Bi.) and Lactobacillus. We found that there was no significant difference except Clo. and Bi. The Clo. was significantly higher in high MGBHQ group (p < 0.05)and the Bi. was significantly higher in high MGBHQ group (p < 0.05). Higher Clo. was also found in autism children's fecal, it was always identified as Bad bacteria. The Bi. was often considered a kind of healthy probiotics. We next tested the plasma serotonin and dopamine levels in both groups, and the plasma serotonin and dopamine levels were significantly higher in high MGBHQ group (p < 0.01). Higher serotonin and dopamine levels was detrimental and could lead to many mental disorders. We concluded that the MGBHQ has good reliability and validity, can quickly and easily assess the individual's gut-brain health status. It’s items are based on the habits and customs and it is more objective than the evaluation of subjective emotion and feeling and it can reflect the objective reality of the individual. Next, we want to test this questionnaire in different population, cultures and nations. Accordingly, we expected it could be used to assess and to help early prevention and identification of mental disorders in the future. App-021#208 A genome-based identification approach for members of the genus Bifidobacterium Chiara Ferrario (1), Christian Milani (1), Leonardo Mancabelli (1), Gabriele Andrea Lugli (1), Francesca Turroni (2), Sabrina Duranti (1), Marta Mangifesta (3), Alice Viappiani (3), Douwe van Sinderen (2), Marco Ventura (1) 1. Laboratory of Probiogenomics, Department of Life Sciences, University of Parma, Italy 2. Alimentary Pharmabiotic Centre and Department of Microbiology, Bioscience Institute, National University of Ireland, Western Road, Cork, Ireland 3. GenProbio ltd, Italy During recent years the significant and increasing interest in novel bifidobacterial strains with health-promoting characteristics has catalysed the development of methods for efficient and reliable identification of Bifidobacterium strains at (sub)species level. We developed an assay based on recently acquired bifidobacterial genomic data and involving 98 primer pairs, called the Bifidobacterium-ampliseq panel. This panel includes multiplex PCR primers that target both core and variable genes of the pan-genome of this genus. Our results demonstrate that the employment of the Bifidobacterium-ampliseq panel allows rapid and specific identification of the so far recognized 48 (sub)species harboring the Bifidobacterium genus, and thus represents a cost- and time-effective bifidobacterial screening methodology. #210 Short Talk Transit time influences gut microbiota species diversity, growth rates and enterotypes Doris Vandeputte (1,2,3 †) , Gwen Falony (1,2 †), Sara Vieira-Silva (1,2), Raul Tito (1,2,3), Marie Joossens (1,2,3), and J. Raes (1,2,3) 1. KU Leuven, Department of Microbiology and Immunology, Rega Institute, Herestraat 49, B-3000 Leuven, Belgium. 2. VIB, Center for the Biology of Disease, Herestraat 49, B-3000 Leuven, Belgium. 3. Microbiology Unit, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. † These authors contributed equally to this work. One of the most important determinants of the human microbiome, transit time, is often neglected in gut microbiome studies because of the invasive nature of its measurement. We assessed the impact of transit time on gut microbiota composition using a proven proxy, the Bristol Stool Scale, and find that it strongly correlates with all major microbiome markers. It is negatively correlated with species richness, positively associated to the widely used Bacteroides-Firmicutes ratio and strongly associated with enterotypes. Fast transit samples often belong to Prevotella individuals while slow transit samples are characterized by a higher abundance of key species as Akkermansia and Methanobrevibacter. Furthermore, we find stool score to be positively correlated to average microbial growth potential, indicating selection of fast growing bacteria in individuals with short transit time. However, in Prevotella individuals, the absence of this trend could hint to host tissue adhesion as an alternate microbial strategy to avoid washout. Together, our results show a profound effect of transit time differences on gut microbiota composition, with implications for gut parameter confounder analysis and personalized gut microbiota modulation strategies. We therefore strongly recommend the inclusion of a measurement of transit time, such as Bristol Stool Scale, in future microbiome studies. App-022#222 Revealing microbial recognition by the immune system Aurea Simon-Soro (1), Giuseppe D’Auria (1), M. Carmen Collado(2), Maria Dzunkova (1), Shauna Culshaw (3), Alex Mira (1) 1. Department of Health and Genomics. FISABIO Foundation, Center for Advanced Research in Public Health, Avda. Cataluña 21, 46020 Valencia, Spain. 2. The Institute of Agrochemistry and Food Technology, Spanish National Research Council (IATA-CSIC), 46980 Valencia, Spain. 3. School of Dentistry, Glasgow, United Kingdom. Glasgow Dental Hospital & School, 378 Sauchiehall Street, Glasgow, G2 3JZ, Scotland, United Kingdom.

There is currently very limited understanding of immune recognition of 50% of the human microbiome which is made up of un-culturable bacteria. We have combined the use of flow cytometry and pyrosequencing to describe the microbial composition of human samples and its interaction with the immune system. We show the power of the technique in faecal, saliva, oral biofilm and breast milk samples by using fluorescent markers which are specific for IgA or IgG. By the use of Fluorescence-Activated Cell Sorting (FACS), bacterial cells can be separated depending on whether they are coated with specific human antibodies. Each bacterial population can then be PCR-amplified and pyrosequenced, characterizing the microorganisms which evade the immune system and those which are recognized by each immunoglobulin. The results show that the average proportion of IgA-opsonized bacteria in the subjects analyzed in the current proof of concept study ranged from 52.3% for faecal samples, 73.6% for saliva, 78.4% for the oral biofilm, and 63.7% for breast milk. Ig-coated microorganisms appear to be individual-specific but some patterns start to emerge. Specific bacteria appear to be able to evade opsonization, including Escherichia and Stenotrophomonas in the gut, or Enterococcus and Prevotella in breast milk. Others, like Veillonella and Fusobacterium, appear mostly opsonized both in saliva and dental plaque. In conclusion, the application of the technique to healthy and diseased individuals may unravel the contribution of the immune response to microbial infections and polymicrobial diseases. Considering immune recognition and opsonization in healthy individuals as the goldstandard, deviations from that balanced microbe-immune interaction can potentially be related to microbial-mediated disorders, and the characterization of individual-specific opsonization profiles can prove fruitful in diagnostic and therapeutic strategies for personalized medicine.

App-023#226 Safety assessment of Lactobacillus gasseri JDM511 based on complete genome sequences Lu Ye, Yan-Xia Wei, Xiao-Kui Guo, Chang Liu Department of Immunity and Medical Microbiology, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China Probiotics are live microorganisms which have beneficial effects on hosts.Lactobacillus gasseri has already been used in commercial product for a long time. There has been an increased focus on the bio-safety of probiotic strains. The whole genome of Lactobacillus gasseri JDM511 was sequenced. Genome-based safety evaluation of JDM511 was conducted in this study. Existence of antibiotic resistance genes, virulence-related genes and the adverse metabolism-associated genes were assessed. Antibiotic susceptibility test and the biogenic amine production of JDM1 were tested to supplement a traditional oral toxicity test. The complete genome of Lactobacillus gasseri JDM511 includes a single, circular chromosome of 1,788,466 bp and 2 plasmids with the size of 93,309bp and 39,184bp respectively.The G+C content of the genome is 35.06% . It also contains 66 tRNA,one 23S RNA,one 16S RNA and one 5S RNA respectively. 43 antibiotic genes were identified by AFDB in JDM511 (E < 0.01, coverage > 70%),152 genes were identified in JDM511 by BLAST with VFDB (E < 0.01, coverage > 70%).The genome of JDM511 has 5 GS genes ,1 GN gene,4 NR genes and 1 DLD

Human Sample

Cell Fixation on Formaldehyde

Fluorescent Labelling

RNA Labelling Anti-‐Ig Labelling(IgA, IgG, IgM)

DNA Labelling

DNA/RNA extraction

Pyrosequencing454 Titanium Roche

Active Cells

PCR 16 S/28S rDNA

Non Active Cells

IgOpsonized

Cell Count(Bacterial /Fungal load)

MicrobialComposition

Ig Non Opsonized

gene. JDM511 could not produce enough amount of tyramine, histamine, tryptamine , , cadaverine, spermine or putrescine to change the color of the plates. There are only 1 questionable CRISPR and 1 predicted prophage fragment in the genome. No phage was discovered. 9 IS were identified in the genome. This approach can be generalized to provide a deep safety investigation of novel probiotic strains and greatly reveal the potential danger determinants and their molecular mechanisms. App-024#230 Enhanced virome sequencing with capture enrichment and its application to the EV-D68 outbreak Kristine M. Wylie (1,2), Todd N. Wylie (1,2), Brandi N. Herter (1), Anthony Orvedahl (1), Richard Buller (1), Vincent Magrini (2), Richard K. Wilson (2), Gregory A. Storch (1) 1. Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA 2. The Genome Institute, Washington University School of Medicine, St. Louis, MO, USA Metagenomic shotgun sequencing (MSS) is an important tool for characterizing viruses in clinical samples. It is culture-independent, requires no a priori knowledge of the viruses in the sample, and may provide extensive genomic information. However, MSS can be less sensitive than targeted molecular tests and in samples with low levels of virus may not yield sufficient sequence data for detailed analysis. We designed a sequence capture reagent, ViroCap, which targets viruses from 38 families that can infect vertebrate or invertebrate hosts. An innovative computational approach condensed 1 billion nucleotides of potential target sequence into 200 million bases of unique sequence suitable for production with Roche NimbleGen SeqCap EZ Developer Library. We tested ViroCap on samples containing 19 viral genera from 10 families, and ViroCap correlated perfectly with molecular assays for virus detection. Depth- and breadth-of-coverage of the genomes were consistently improved, and the percentage of viral sequences in each sequence data set increased 10- to 11,000- fold post-capture. This approach will significantly improve MSS studies while reducing the cost of sequencing. In the fall of 2014, enterovirus D68 (EV-D68) was circulating at an unprecedented level in the United States. There was no specific molecular test for this virus during the outbreak, and specific identification required PCR and sequencing of a region of the VP1 gene for typing. The lack of a rapid molecular assay available in clinical laboratories resulted in vastly under-reported cases of EV-D68 infection. Furthermore, genomic data for EV-D68 was limited, with only 12 complete or nearly complete sequences available in GenBank and no sequences from the 2014 outbreak. We used ViroCap to enrich viral nucleic acid and rapidly generate genome sequence of EV-D68 from clinical samples from St. Louis. These sequences were compared with publicly available EV-D68 sequences and subsequently used to design an EV-D68-specific PCR assay with the ability to detect highly divergent strains of EV-D68, including the original Fermon strain from 1962. This assay can be used for epidemiological studies of the EV-D68 outbreak and for virus monitoring in subsequent seasons. This is the first of many potential applications of the ViroCap library. App-025#233 iCLiKVAL: Community resource for curating the vast wealth of metagenomic-related literature through the power of crowdsourcing Todd D. Taylor (1), Naveen Kumar (1) 1. Laboratory for Integrated Bioinformatics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan Background: There are currently over 24 million citations to various forms of scientific literature in PubMed, including thousands related to metagenomic research. Searching this vast resource does not always give desirable or complete results due to a number of factors such as: missing abstracts, unavailability of full-article text, non-English articles, lack of keywords, etc. Ideally, each and every citation should include a complete set of keywords or terms that describe the original article in enough detail that searches, using natural language, return more relevant results; however, this would require countless hours of manual curation. In the field of metagenomics the terminology can be quite confusing, and there are no comprehensive resources where one can find all related materials – we plan to change that. Objectives: Our objective is to make manual curation 'fun' and social and self-correcting, thus enriching resources such as PubMed so that users are able to extract more valuable and relevant results. We have developed a web-based open-access tool for manual curation of PubMed articles, and other media types, using a crowdsourcing approach which we believe the community will enjoy using. While we encourage the use of ontology terms and support them as auto-suggest keyword terms, we do not restrict users to these terms because we do not want to impose, within reason, any limitations on the types of annotation that one may provide. Non-English annotation is also supported. Through this ‘non-restrictive’ approach, we hope that communities of researchers, no matter where their location or what their language, will take advantage of this tool to work together on the manual curation of the metagenomic and other literature related to their projects. Methods: We have constructed a cross-browser and platform-independent application using the latest web technologies and a NoSQL database. Users perform searches to identify articles of interest, mark articles for later review or review them immediately or add them to a review request queue, load PDFs into the viewer, select annotations (values) within the text, and add appropriate keywords (keys). Article-specific comments can be made, key-value pairs can be edited and rated, live chats between users working on the same article can be held, etc. Users can even add annotations via Twitter.

Conclusion: As more annotations accumulate in the database, the more our semantic search feature will improve and the more relevant the results. Users will then be able to precisely filter the results. Rather than compete with other already-established curation projects, we wish to work with them to incorporate their valuable data and in return, via our REST API, make the iCLiKVAL annotations easily accessible to the entire research community. We hope this will become the default resource for community-based curation of all online metagenomic-related and other scientific literature. App-026#237 Decontaminator: detection and removal of contaminating sequences of unknown source Bettina Halwachs (1,2*), Rene Snajder (1), Gregor Gorkiewicz (2), Gerhard Thallinger (1) 1. Bioinformatics Group, Institute for Knowledge Discovery, Graz University of Technology, Austria 2. Institute of Pathology, Medical University of Graz, Austria (*current address) Targeted amplification and sequencing of specific genetic marker regions using universal primers, is used as the standard technique for microbial community analyses. Major improvements on sequencing technologies and throughput, as well as the drastic decrease of sequencing costs, shifted sequencing from specific genetic marker regions only, towards sequencing of entire genomes. Although, this new approach of whole genome shotgun sequencing allows now also for a functional description of the investigated community, targeted amplicon sequencing benefits of higher sequencing depth, as well as of higher species resolution within the investigated habitat. Therefore, this sequencing approach is still the standard for bacterial, archaeal, or fungal community profiling, although recent investigations revealed, that non-target DNA is also amplified by such marker gene specific primers. These so called contaminating sequences bias the determined community richness and are a reason for OTU inflation. The majority of these problematic fragments originate from genomic DNA of other organisms present in the sample, such as human DNA. Hence, the detection and the removal of any kind of non target sequence fragments are absolutely mandatory for determining the true microbial community profile. Here we present the Decontaminator as an effective tool for the detection and the removal of contaminating sequences in targeted amplicon sequencing datasets. The method is based on a BLAST like homology search using the BLAT algorithm against a specific marker gene reference database. This approach enables fast and effective identification of sequences which do not show any or low similarity to the marker genes such as 16S or ITS, through the characteristic structure and the degree of sequence conservation within these regions. In contrast to other tools for the detection and the removal of contaminating sequences, the Decontaminator does not need information about the source of contaminating sequences, since only reads similar to the reference marker gene sequences are considered as true amplicons. By the analysis of a real targeted amplicon sequencing dataset with and without the removal of contaminating sequences the positive effect of the Decontaminator was confirmed. On the one hand the number of finally determined OTUs was reduced by about 13 % after pre-processing the raw dataset with the Decontaminator. And on the other hand it was shown that the Decontaminator approach already covers the removal of other common error sources such as chimeric, low quality, and noisy sequences. In addition, the introduced method is highly parallelizable, scalable, as well as considerably faster than other tools. Conclusively, the introduced Decontaminator is able to detect and remove any kind of non target amplicon sequences effectively and allows the unbiased determination of the community profile in question. #239 Short Talk Development and validation of a gut microbiota analysis pipeline: from specimen collection to sequencing Vincent Thomas (1), Matthieu Pichaud (1), Nicolas Goffard (1), Florence Levenez (2), Alessandra Cervino (1), James Clark (1), Joël Doré (2) 1. Enterome, Paris, France 2. INRA - MetaGenopolis, Jouy-en-Josas, France Gut microbiota analysis relies on rapidly evolving tools and has the potential to lead to better understanding of major diseases, as well as to the development of new drugs and new biomarkers. The potential applications are countless, giving rise to considerable interest from researchers as well as pharmaceutical and agro-food companies. Despite this, there are still no universally accepted standards to conduct gut microbiota analysis. The vast majority of studies use stool material since it is considered as a good proxy that reflects microbiota composition in the different compartments of the digestive tract. In addition it has the advantage of allowing easy collection and shipping of the samples that can be collected in healthcare settings or directly at home by patients. However many different protocols can be used for specimen collection, DNA extraction, sequencing and biostatistical analysis. Consequently, conducting fecal microbiota studies is not trivial and many important choices face those interested in starting such studies. Enterome together with INRA Metagenopolis develops science-based personalized medical tests and companion diagnostics based on profiling of the human fecal microbiome, with the goal to improve management of health. The method used by Enterome is based on mapping of total fecal microbial gene content and functions, leading to the characterization of a personal metagenome (metagenotype®) for each individual. Each metagenotype® is analyzed using proprietary algorithms (linking individual metagenotype® to associated phenotypes) allowing proper evaluation of personal risk of each subject to develop a wide variety of pathological conditions.

Since its inception in 2012, Enterome has dedicated significant effort to reach a high level of standardisation for its shotgun metagenomics analysis pipeline. Collaborations with several academic and private partners have led to the development and/or validation of dedicated methods for each step of the pipeline, from specimen collection to biostatistical analysis. In this presentation we will give an overview of devices that were developed to answer the specific needs of sample collection at home by patients followed by shipping at room temperature. We will also present some of our validation studies dealing with various aspects of the pipeline, including sample storage, DNA extraction, sequencing platforms comparison and technical variability of the platforms. App-027#242 Assessing V4 and V6-8 16S variable regions for the microbiome analysis of skin samples using Illumina next generation sequencing Christophe Lambert (1), Béatrice Sente (1), Stephanie Van Horn (2), Christopher Traini (2), Lynn Tomsho (2), James Brown (2), Jean-Louis Ruelle (1), Nathalie Devos (1) 1. GSK Vaccines, Rixensart, Belgium 2. GSK Pharma R&D, Collegeville, Pennsylvania, United States Bacterial diversity among biological samples is commonly assessed by PCR-amplification and subsequent sequencing of 16S rRNA genes (16S) or regions within this gene. However, measured diversity and relative abundance can be biased by different factors such as sample preparation, DNA extraction efficiency, PCR primer selection, 16S chosen region, chimeric 16S amplification products or sequencing technology. Until recently, regions V1-V3, V3-V5 and V6-V9 of 16S were typically used to assess bacterial diversity with the Roche 454 GS FLX sequencers that allow to sequence long amplicons of 500-600bp. Illumina introduced sequencers able to dramatically reduce the cost of sequencing with the drawback of providing only short reads (2x150bp). Consequently, Microbiome diversity analyses realised with Illumina sequencers frequently used shorter 16S fragments such as the 16S V4 variable region (~250bp) displaying an overall lower species discriminatory power compared to regions cited above. The latest version of Illumina MiSeq (v3) provides longer reads (2x300bp) that enable good overlap of paired-end reads coming from amplicons with length 400-500bp. In this study we assessed the use of 16S V6-V8 region (~450bp) with the Illumina v3 technology to accurately inform on bacterial diversity and to enhance the identification of species frequently associated with skin disorders or infections. Mock samples were prepared with genomic DNA of species expected to be found in skin samples and some additional species. Primers for amplification of V4 or V6-V8 regions were optimized to allow optimal coverage of all bacteria (RDP database ProbeMatch). The analysis of the samples was performed on an Illumina MiSeq v3 sequencer with paired end reads of length 300 bases (2 x 300). Microbial diversity of samples was evaluated using two different 16S regions, V4 and V6-V8. Different bioinformatics protocols and databases were used to assess the bacterial diversity. The results obtained using the different bioinformatics protocols and the two selected 16S regions to evaluate the bacterial diversity of the mock samples will be presented. Based on these results, the best method will be used to analyse the skin samples. App-028#243 The effect of sampling and storage on the fecal microbiota composition in healthy and diseased subjects. Danyta Tedjo (1,2), Daisy Jonkers (1), Paul Savelkoul (2), Ad Masclee (1), Niels van Best (2), Marieke Pierik (1), John Penders, (2) 1. School for Nutrition and Translational Research in Metabolism (NUTRIM), Division Gastroenterology-Hepatology, Maastricht University Medical Center+, Maastricht, The Netherlands. School for Nutrition and Translational Research in Metabolism (NUTRIM), 2. Department of Medical Microbiology, Maastricht University Medical Center+, Maastricht, The Netherlands. Introduction: Many large-scale cohort studies are currently being designed to study the influence of the human microbiome in health and disease. Adequate sampling strategies are required in such studies to limit bias due to shifts in microbial communities during sampling and storage. The aim of this study is to examine the impact of different sampling and storage conditions on the stability of fecal microbial communities. Methods: Fecal samples from 10 healthy controls, 10 irritable bowel syndrome and 8 inflammatory bowel disease patients were aliquoted immediately after defecation and stored at -80°C, -20°C for 1 week (1wk -20°C), +4°C for 24 hrs (24h +4°C ) or room temperature for 24 hrs (24h RT). Furthermore, a fecal swab (FS) was collected and stored for 48-72 hours at RT. We used pyrosequencing of the 16S gene to investigate the stability of microbial communities. Results: Comparisons between -80°C samples and 1wk -20°C, 24h +4°C or 24h RT samples, showed no significant difference in α-diversity. FS showed a significant higher α-diversity compared to -80°C samples. UPGMA clustering and principal coordinate analyses showed the samples clustered significantly by test subject (p<0.001) for unweighted UniFrac, weighted UniFrac and Bray-Curtis), but not by storage method. Bray-Curtis dissimilarity and (un)weighted Unifrac showed a significant (p<0.05) higher distance between FS and -80°C samples versus the other methods and -80°C samples. The unweighted

UniFrac distance between the reference method and the other sampling and storage methods as well as the between-subject unweighted UniFrac distance of all samples stored at -80°C is depicted in the boxplot (see figure). No significant change in the relative abundance of extreme oxygen species Ruminococcus, Faecalibacterium and Roseburia was observed between -80°C and 1wk -20°C, 24h +4°C or 24h RT. The relative abundance in Ruminococcus (p<0.05, delta median= 1.2•10-2%) and Enterobacteriaceae (p<0.05, delta median= 8.4•10-4%) was significantly higher in the FS compared to the -80°C samples. Conclusions: In this study we demonstrated storage up to 24 hours at room temperature did not significantly alter the fecal microbial community structure as compared to direct freezing of feces from both healthy subjects and patients with gastrointestinal disorders. When using fecal swabs DNA isolation should be optimized to ensure optimal preservation of microbial community structures.

App-029#251 A human infant gut microbial gene catalog by SOLID sequencing Amine Ghozlane* (1), Anne-Sophie Alvarez* (1), Mathieu Almeida (2), Florian Plaza Oñate (1,3), Emmanuelle Le Chatelier (1), Nicolas Pons (1), Edi Prifti (1), Harm Wopereis (4,5), Christophe Lay (4), Rocio Martin (4), Raish Oozeer (4), Jan Knol (4,5), Sean 1.Metagenopolis, INRA, Jouy-en-Josas, France 2.CBCB, University of Maryland, College Park, USA 3.Enterome Bioscience, Paris, France 4.Nutricia Research, Utrecht, Netherlands 5.Wageningen University, Laboratory of Microbiology, Netherlands Background. Development of the gut microbiota in the first years of life is not well understood. Quantitative metagenomic analysis, apt to give new insights in that process, is restricted by the lack of an appropriate gene catalog: infants' gut microbiota is notoriously different from that of adults [Wopereis et al., 2014] and may not be well represented by the currently available adult-based gene catalogs. We describe here a catalog adapted to infant gut microbiota. Methods. Fecal samples from 60 infants at 8 and 26 weeks were sequenced with SOLiD wildfire 5500xl resulting in 58.7 +/- 14.6M sequences of 50-base-long single-end reads. A dedicated pipeline, SOMA was developed to assemble reads in colorspace; it includes aspects from MOCAT [Kultima et al., 2012] augmented with specific features for SOLiD reads metagenomic assembly, gene detection and annotation. SOMA involves: (i) read filtering using k-mer abundance; (ii) assembly using Denovo2 with optimized parameters for metagenomics data; (iii) gene prediction with Metagenemark; (iv) phylogenetic marker genes detection with fetchMG. All unassembled reads from single samples were pooled for an additional round of assembly. Redundancy of the gene set was removed using CD-HIT-EST following the Metahit protocol. Taxonomic and

functional annotation was carried out with BLASTN against NT, WGS, HMDB and BLASTP against EGGNOG, KEGG, CAZY databases. Results. A total of 1,002,952 non-redundant genes were assembled by SOMA. Of these, 67% and 38% were not present in the Metahit 3.9M gene catalog [Nielsen et al., 2014] and the most comprehensive Metahit 9.9M IGC catalog [Li et al., 2014], respectively. A large proportion (56%) of these genes were taxonomically annotated at a species level, with a dominance of Firmicutes (Clostridium 10%, Streptococcus 9%, Enterococcus 8%, Lactobacillus 7%), Bacteroidetes (Bacteroides 6%), Proteobacteria (E. coli 6%) and Actinobacteria (Bifidobacterium 8%), in contrast to adult catalogs (28% species-level annotation). Combination of the Metahit 3.9M gene catalog with the infant gene set led to a 4.6M gene catalog. It will be useful for infants, as it yields a higher mapping rate (60%) than even the 9.9M IGC catalog (58%; only 43% for the 3.9M catalog), while remaining much smaller and thus easier to use. The genes were clustered into Metagenomic Species (MGS) and Units (MGU), enabling comparative studies with adults at species and sub-species levels. App-030#254 Genomic and functional analysis of Romboutsia ilealis CRIB reveals adaptation to the small intestine Jacoline Gerritsen* (1,2), Bastian Hornung* (1,3), Bernadet Renckens (4), Sacha A. F. T. van Hijum (4,5), Vitor A.P. Martins dos Santos (3), Ger T. Rijkers (6,7), Peter J. Schaap (3), Willem M. de Vos (1,8) and Hauke Smidt (1). 1. Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands; 2. Winclove Probiotics, Amsterdam, The Netherlands; 3. Laboratory of Systems and Synthetic Biology, Wageningen University, Wageningen, The Netherlands; 4. Nijmegen Centre for Molecular Life Sciences, CMBI, Radboud UMC, Nijmegen, the Netherlands; 5. NIZO Food Research, Ede, the Netherlands; 6. Laboratory for Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands; 7. Department of Science, University College Roosevelt, Middelburg, The Netherlands; 8. Departments of Microbiology and Immunology and Veterinary Biosciences, University of Helsinki, Helsinki, Finland. *these authors contributed equally to this work. The microbiota in the small intestine relies on its capacity to rapidly import and ferment available carbohydrates to survive in a complex and highly competitive ecosystem. Understanding how these communities function requires elucidating the role of its key players, the interactions among them and with their environment/host. Hence, we present the complete genome of Romboutsia ilealis CRIB, a natural inhabitant and key player of the small intestinal microbiota of rats. R. ilealis CRIB possesses a circular chromosome of 2,581,778 bp and a plasmid of 6145 bp, carrying 2351 and eight predicted protein coding sequences, respectively. Analysis of the genome revealed limited capacities to synthesize amino acids and vitamins. However, multiple and partially redundant pathways for the utilization of different relatively simple carbohydrates could be identified. A transcriptome analysis allowed pinpointing the key components in the degradation of glucose, L-fucose and fructo-oligosaccharides. This revealed that R. ilealis CRIB is adapted to a nutrient-rich environment where carbohydrates, amino acids and vitamins are abundantly available and reveals potential mechanisms for competition with mucus-degrading microbes. Other features of ecological interest include the presence of urease and bile salt hydrolase encoding genes. This work shows how genome mining and functional analyses can provide insights in the functional potential of new intestinal bacteria, and will help us with understanding the microbial communities in us. App-031#257 Mucosa-associated biohydrogenating microbes protect the simulated gut microbiome from stress by a Western-style fat consumption Rosemarie De Weirdt (1), Emma Hernandez-Sanabria (1), Bruno Vlaeminck (2), Eva Mees (1), Ruy Jauregui (3), Dietmar H. Pieper (3), Annelies Geirnaert (1), Florence Van Herreweghen (1), Ramiro Vilchez Vargas (1), Veerle Fievez (2) and Tom Van de Wiele (1) (1) Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Ghent, Belgium. (2) Laboratory for Animal Nutrition and Product Quality (LANUPRO), Ghent University, Melle, Belgium. (3) Microbial Interactions and Processes Research Group. Helmholtz Centre for Infection Research, Braunschweig, Germany. A Western high fat/low fibre diet is increasingly evidenced to disrupt gut microbiome homeostasis and these changes have been associated with the incidence of chronic illnesses such as type-2 diabetes, obesity and IBD. The direct effects of increased levels of dietary fat on the colon microbiome are not well understood. Fat, and in particular poly-unsaturated fatty acids (PUFA), may affect colon microbiome homeostasis in two ways. They may exert (specific) antimicrobial effects, and act as detergents at the gut mucosa disrupting the establishment of a mucosa-associated microbial community. On the other hand, colon microbes may gradually saturate PUFA in a process named biohydrogenation. Here, we investigated how linoleic acid (LA), the main PUFA in the Western diet, and its biohydrogenation to vaccenic acid (VA) and stearic acid (SA) affected the activity and composition of the human colon microbiome in vitro. First, standardized batch incubations of faecal microbiota of a healthy volunteer were supplemented with 1 g/L LA, VA or SA to screen for general metabolic effects (SCFA production) and biohydrogenation activity. Second, the dynamic and validated SHIME-model was used to investigate how daily exposure to 1 g/L LA affects the microbial composition (Illumina-based 16S

rRNA gene profile) and functionality (SCFA, biohydrogenation), either in the absence (L-SHIME) or presence (M-SHIME) of a mucosal environment. In the absence of a mucosal environment, LA specifically inhibited two health-promoting gut microbial parameters: butyrate production and levels of Faecalibacterium prausnitzii. In batch, VA and SA did not or barely inhibit butyrate production. In the presence of a mucosal environment, LA did not affect butyrate production and F. prausnitzii levels. Interestingly, the mucosal environment of the M-SHIME appeared to be a hotspot for LA biohydrogenation, with LA:VA:SA profiles of about 50:10:40 in the mucus compared to 80:10:10 in the lumen. In accordance, 16S rRNA gene profiling showed that the two most important biohydrogenating genera of the human gut – Roseburia and Pseudobutyrivibrio – specifically colonized the mucosal environment of the M-SHIME. Correlation network mapping furthermore revealed that LA supplementation stimulated these genera to shift from a strict mucosal niche (Fig. 1A) towards a more central position in the SHIME microbial network (Fig. 1B), indicating an increased functional interaction between distinct mucosal and luminal microbes. In a final co-culture experiment, we confirmed that, also in the absence of a mucosal environment, biohydrogenating R. hominis could directly protect F. prausnitzii from LA stress. Hence, we concluded that mucosal biohydrogenating species in the M-SHIME were responsible for protection against LA. Overall, these results demonstrate the importance of a healthy mucus layer harbouring biohydrogenating species to provide resilience for the gut microbiome upon exposure to high levels of LA.

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App-032#264 Metabiote®: development and application of an integrated solution for microbiota analysis Nathalie Adele-dit-Renseville (1), Do Ngoc Linh Nguyen (2), Louise-Eva Vandenborght (1), Sébastien Terrat (4), Nausicaa Gantois (2), Laurence Delhaes (2,3), Lionel Ranjard (4), Stéphanie Ferreira (1) 1. GenoScreen, Research, Development and Innovation in Human Health and Environment Team, Lille, France 2. Biology & Diversity of Emerging Eukaryotic Pathogens (BDEEP), Center for Infection and Immunity of Lille (CIIL), INSERM U1019, CNRS UMR8204, IFR142, Lille Pasteur Institute, Lille Nord de France University (EA4547), Lille, France. 3. Department of Parasitology–Mycology, Regional Hospital Center, Faculty of Medicine, Lille, France 4. Plateforme GenoSol, INRA, UMR1347 Agroécologie, Dijon, France The growing need to survey the tremendous microbial diversity in a culture independent manner has led to the development of molecular methods through sequence profiling of phylogenetically conserved genes such as 16S rDNA, in scientific field like ecology, agronomy, biotechnology, plant, animals and of course Human Health. Next-generation sequencing (NGS) technologies providing unprecedented throughput of data, are now used routinely to assess bacterial community composition in complex samples. Consequently, many scientific or clinical studies have been, and are still performed with these NGS technologies. However, several protocols for amplicon library realization exist and are widely used to perform these analyses whereas no study has looked at their respective impact on taxonomical description, relative abundances of taxa, diversity and richness indexes. To address these issues, Genoscreen has performed a comparison of two classical amplicon library preparation (direct PCR and ligation from soil sample) and consequently developed an optimized and standardized (ready to use) solution for the analysis of microbiota called Metabiote®. The data presented here will show that routine protocols induce biases between samples and have impact on taxonomical definition and the observed relative abundances of taxa while our own solution Metabiote® greatly improve these data. Moreover, our internal protocol generates better diversity and richness indexes indicating that routine protocols could underestimate the complexity of a bacterial community. Our solution has successfully been tested on several types of human microbiota (faeces, biopsy, skin, sweat, saliva, and sputum samples…). A concrete application on the human respiratory microbiota in the context of Cystic Fibrosis will be presented. This solution is accessible through our services platform on both 454-Roche (GS Junior, GS FLX) and Illumina (MiSeq2*300pb) and is now available under ready-to-use kits (Metabiote® kits) with the associated fully automated pipeline (Metabiote Online®) for raw data analysis. This bioinformatics pipeline and its web interface allows the users to be autonomous for the analysis and interpretation of their data with no particular IT investment. The first Metabiote® kits have been designed for amplicon library preparation targeting 16S rDNA V3V4 or V4V6 regions that can be sequenced using GS Junior & GS FLX sequencers (Roche Diagnostics). Additional kits targeting fungal communities (mycobime component) and other sequencing platforms are currently under validation. App-033#266 Dynamics of Moraxellaceae in the Nasal Microbiota of Healthy Infants Using Oligotyping Moana Mika (1,2), Daniel Wüthrich (2,3), Insa Korten (2,4), Weihong Qi (5), Urs Frey (6), Philipp Latzin (4, 6), Markus Hilty (1, 7) 1. Institute for Infectious Diseases, University of Bern, Bern, Switzerland 2. Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland 3. Interfaculty Bioinformatics Unit, University of Bern, Switzerland Swiss Institute of Bioinformatics, Lausanne, Switzerland 4. Division of Respiratory Medicine, Department of Pediatrics, Inselspital and University of Bern, Bern, Switzerland 5. Functional Genomics Center, Swiss Federal Institute of Technology Zurich/University of Zurich, Zurich, Switzerland 6. University Children’s Hospital (UKBB), Basel, Switzerland. 7. Department of Infectious Diseases, University Hospital, Bern, Switzerland Objectives: Recently we analyzed the dynamics of the nasal microbiota of infants within the first year of life using 454 Pyrosequencing of the bacterial 16S rRNA gene. However, the conventional definition of operational taxonomic units (OTUs) based on 97% sequence identity limits the identification of potential pathogens, as for example Moraxella catarrhalis of the Moraxellaceae family. Here, we aim an in-depth analysis of the family of Moraxellaceae to identify potential pathogens out of a microbiota dataset using a high-resolution method called oligotyping. Methods: Taxonomic assignment, and the definition of OTUs based on 97% sequence identity, was generated by Pyrotagger using 16S rRNA sequencing data of 872 nasal swabs collected biweekly from 48 unselected infants within the first year of life. Samples were grouped according the data of acquisition, referring either to the age or the season. The 16S sequence reads of all OTUs of the bacterial family of Moraxellaceae were then selected, aligned, and trimmed to the same length using a customized shell script. High-resolution method (oligotyping) was applied using the otu2ot package in R. Results: The family of Moraxellaceae was found at a mean relative abundance of 34.8% (95% CI: 32.5 - 37.1), and thus was the most abundant family in the nasal microbiota of healthy infants within the first year of life. The relative abundance of Moraxellaceae increased exponentially by age (R2=0.59) and was highest at the end of the first year of life (42.95%; 95% CI: 34.01-51.89). However, regarding seasonal changes no clear trend was observed on the taxonomic level of the bacterial family. In total, Moraxellaceae consisted of 247 different OTUs and 391`642 sequence reads, whereas 19`827 out of these were unique sequences. Applying oligotyping onto these 19`827 sequences, revealed 131 distinct `oligotypes`, which were defined based on a single nucleotide variation, indicating a highly diverse Moraxellaceae family in the nasal microbiota.

Conclusion: This study reveals Moraxellaceae as the most abundant bacterial family, and thus as an important key player in the nasal microbiota of healthy infants. The Moraxellaceae family itself is highly diverse and consists of a multitude of different oligotypes. This will now allow the investigation if certain `oligotypes` are associated with infections and/or a disordered microbiota. App-034#275 Fluorescence in situ imaging of placental bacteria in the absence of infection Maxim Seferovic (1) , Eumenia Castro (2,3), Angela Major (3), Michelle Moller (1), Brigid Boggan (1), Kjersti Aagaard (1) 1. Department of Obstetrics & Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX, United States 2. Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, United States 3. Department of Pathology, Texas Children’s Hospital, Houston, TX, United States Background Spontaneous preterm birth (sPTB) confers increased risk of morbidity and mortality, and later deficit in neurological function and metabolic health. Historically, sPTB is associated with ascending intrauterine infection including chorioamnionitis. However we recently established that there is a commensal placental microbiome in all pregnancies, with variation in sPTB. This challenges the paradigm of a ‘sterile’ intrauterine environment, and presents opportunities to investigate the interaction of bacteria in situ. Histological descriptions of chorioamnionic bacteria in the absence of inflammation thus far have been accomplished by gram staining and immunohistochemistry, techniques limited either in their exclusion of taxa or by their lack of specificity. Here we sought to establish a placental microbiome using fluorescence in situ hybridization (FISH). Methods Placentas were collected from four groups including chorioamnionitis, term pregnancies, sPTB, and indicated PTB (n=12), and aseptically dissected. Paraffin embedded sections were stained for hematoxylin and eosin to assess for histological infection, and serial sections probed using Cy3 labeled EUB338 specific to a conserved region of the 16S transcript, or a non-sense control. The chorioamnion was then assessed for inflammation and the presence of bacteria. Results Histological examination confirmed inflammation in all three chorioamnionitis placentas, but not for term or preterm delivery groups. FISH staining revealed observable bacteria in 10 of 12 chorioamnions, averaging 103.3 per section for chorioamnionitis patients, 2.0 for normal term pregnancies, 11.5 for sPTB and 1.7 for indicated PTB. The distribution of bacteria was highly clustered and heterogeneous regardless of group. Localized clusters contain bacteria at a density of 3.9 x 106 bacteria/mm3 of tissue, which was greater than in spontaneous PTB (0.9 x 106) and both normal term pregnancy (0.2 x 106) and indicated PTB (0.2 x 106) (p=0.03). Conclusion: Bacteria were intact and present in greater than half the pregnancies examined in the absence of histological or clinical infection, although much larger densities of bacteria were evident in chorioamnionitis.

App-035#277 Extensive characterisation of the gut resistome yields new insights about the microbiome Amine Ghozlane* (1), Etienne Ruppé* (1), Julien Tap* (1), Nicolas Pons (1), Alexandre G. de Brevern (2), Sean P. Kennedy (1), S. Dusko Ehrlich (1, 3) 1.Metagenopolis, INRA, Jouy-en-Josas, France 2.INSERM UMRS_S1134, Univ Paris Diderot, France 3.Centre of Host-Microbiome Interactions, King’s college, London, United Kingdom Background. The gut microbiota harbours an allegedly vast diversity of antibiotic resistance determinants (ARDs) yet their census (i.e. the resistome) has not been previously determined. Indeed, bioinformatic tools are stymied by the identity gap between known ARDs and those of bacteria from the gut microbiota. Thus, whether subjects can be stratified according to their resistome remains unanswered. Here, we used a new 3-dimensional modeling based approach to accurately identify ARDs. We then stratified MetaHIT subjects with regards to their gut resistome. Methods. We developed a new method of functional annotation named pairwise comparative modelling (PCM). Homology modeling of candidates with templates (PDB) identified as (i) reference on one hand, and (ii) negative on the other hand are compared. Scores generated by the two modeling paths were compared and the candidates classified into the most appropriate category. When tested with an external functional metagenomic dataset, ARD predictions by were 99.1% (1,380/1,391) true. We then queried the 3.9M MetaHIT gene catalogue for ARDs belonging to 20 classes, conferring resistance to nine major antibiotic families. We attempted to stratify 663 subjects from the MetaHIT cohort according to their ARDs class distributions, and assessed the possible connexions between gut resistome, richness and enterotypes. Results. Using the PCM, we identified 6,095 ARDs candidates among which half had an amino-acid identity below 30%. ARDs candidates were assigned to Firmicutes (49%), Bacteroidetes (14%) and Proteobacteria (4%) phyla, while 29% remained unassigned. The distribution of phyla varied according to the ARD family: aminoglycosides-modifying enzymes (AMEs) and class B beta-lactamases (bla) were enriched in Firmicutes while class A bla and Sul were enriched in Bacteroidetes. Of note, we predicted four ARDs in Methanobrevibacter and three in Methanoculleus. A chromosomal localization was suggested for 59.9% of ARDs. Mapping reads frequencies ranged from 0.18% to 0.52% per metagenomes. Six ARD clusters, using distribution patterns of ARDs classes, were detected. We observed that ARDs richness was positively correlated with overall gene richness and that ARDs clusters were associated with enterotypes: Bacteroides driven enterotype was associated with two ARD clusters enriched in class D beta-lactamases and tetracycline resistance conferring Tet(X), while Clostridiales driven enterotype was associated with three ARD clusters enriched in AMEs and Prevotella driven enterotype with a class B1-bla enriched cluster. Conclusions. The human gut resistome was associated with gene richness and enterotypes. Our findings open perspectives in deciphering the variable response of the gut microbiota to antibiotics. Acknowledgement. This research is sponsored by the European Union FP7 projects EvoTAR-282004. App-036#281 A novel sampling method for analysis of the human gut microbiome. Jodie Booth (1), Andrew Llewelyn (1), Cheryl Collins (1) 1. Origin Sciences Limited, Cambridge, UK The gut microbiome is the most complex bacterial community in the human body. Alterations in the composition of intestinal microbiota are associated with various disease states including inflammatory bowel disease, obesity and colon cancer. The majority of studies of the human gut microbiome have analysed stool samples although mucosal biopsy specimens have also been used in numerous studies. In this study, we investigated the utility of OriCol™, a novel sampling device, for profiling the human gut microbiome (Figure 1). The OriCol™ device is a simple, convenient method for sampling the rectal mucosa without the need for prior fasting or bowel preparation. Sampling can be performed by a trained healthcare professional in less than 5 minutes. The device incorporates a nitrile membrane, which after insertion into the rectum via a standard proctoscope, is inflated to make contact with the rectal mucosa. The membrane is then deflated and retracted into the device prior to removal from the patient. Upon retraction the material sampled from the rectal mucosa is retained on the inverted membrane. The device can either be stored frozen or a suitable buffer added to preserve the material for subsequent analysis. Samples from the rectal mucosa (obtained using the OriCol™ device) and stool were obtained from 5 healthy volunteers on three discrete occasions. A single rectal swab sample was taken from each volunteer immediately before the final OriCol™ sample. The microbial community of samples was profiled via 16S V4 sequencing. Preliminary analysis has demonstrated that OriCol™ and stool samples have comparable diversity measures, while the rectal swabs show lower diversity. In the top 9 phyla with highest abundance, OriCol™ and stool samples have comparable abundance levels. The OriCol™ device is able to capture differences between donors, but the microbial community is different to that from stool samples. We hypothesise that this difference is due to the capture of bacteria closer to the mucosa by the OriCol™ device.

App-037#284 Intestinal microbiota composition influences the response to a dietary intervention with resistant starch Fabiana A H Sarda (1), Christian Hoffmann (2), Eliana B Giuntini (2) Carla R Taddei (3), Frederic Bushman (4), Elizabete Wenzel de Menezes (2) (1) Food Science Post-Graduation Program, Department of Food and Experimental Nutrition – Faculty of Pharmaceutical Sciences (FCF), University of São Paulo - USP, São Paulo, Brazil (2) Department of Food and Experimental Nutrition, FCF, USP (3) Microbiology Laboratory, FCF, USP (4) Microbiology Dept of Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA The gut microbiota plays an important role in maintaining host’s overall health. Modulation of these microbial-host interactions could be accomplished by the introduction of nutrients such as non-digestible, fermentable carbohydrates, which in turn modulate the gut microbiota composition to maintain and/or promote health. One such functional ingredient is Unripe banana flour (UBF), which is a good source of unavailable carbohydrates, the resistant starch being its most expressive component. It is unknown whether or not UBF can stimulate or change the intestinal microbial community. The aim of this work was to evaluate the effects of the regular consumption of (UBF) on the intestinal microbiota. Healthy volunteers (n=33) participated in this double blind, parallel, placebo-controlled study, distributed into: Control group (maltodextrin) and UBF group (8 g), over 6 weeks of regular consumption (3 times/week). Stool samples were collected at baseline and at the end of the intervention. DNA was extracted and the bacterial 16S rDNA was sequenced to determine the microbial dynamics in the gut microbiome. Sequences were processed using Qiime and Picrust, and analyzed using the R environment for statistical computing. Two clusters of individuals were detected in the baseline microbiome of Brazilians, one more Prevotella prevalent (Cluster A) and other more Bacteroides prevalent (Cluster B), containing 45% and 55% of subjects, respectively. Host metabolic parameters evaluated at baseline were distinct between these 2 groups of individuals, although all within normality. The metabolic profile inferred using Picrust showed statistically significant differences for cluster A (792 Kegg ortologs were detected using a False Discovery Rate of 0.05) but not cluster B, after the UBF intervention. Metabolic pathways enriched post intervention included glycolysis, pentose phosphate metabolism and propionate metabolism, as well as several phosphotransferase system (PTS) transporter involved in the uptake of carbohydrates, and the B12 biosynthesis pathway. Lipopolysaccharide biosynthesis pathway genes were greatly reduced after the UBF dietary intervention. Biochemical parameters, such as total and HDL cholesterol levels had different outcomes for each cluster of individuals. The results demonstrate the potential of using UBF to promote gut microbiome modulation and the effect of that the individual Enterotype may define the outcome of an intervention. Funding: FAPESP, CNPq and CAPES

App-038#295 Comparative evaluation of metagenomic sequencing and data analysis methods V Manghina (1), C Fraumene (2), M Deligios (1), A Palomba (2), S Uzzau (1,2) 1.Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy. 2.Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio 07041 Alghero, Italy. A very large number of microbiome studies are currently performed, and many different questions are addressed about the relationship between complex microbial communities and their own host. Animals and human microbiomes can be investigated to assess how they might impact on lifelong health, the relationship between the host genetic background and their composition, the lifestyle and diet changes that might affect the taxonomy and metabolism of the hosted microbial community. In addition, microbiome DNA sequencing might be useful to provide a matched database for mass spectrometry, allowing highly successful protein identification and annotation. In this respect, a critical evaluation is needed when selecting the appropriate sequencing pipeline in the context of a microbiome study, depending on the specific goals to which the research is aimed. Currently, most of the research methods are either based on the sequence of 16S rRNA gene hypervariable regions, full length 16S rRNA gene, or shotgun sequencing. In this work, we have compared the impact of different methods for metagenomic sequencing and data analysis, making use of real gut microbiome from human and mouse. Different gut microbiome samples were obtained from intestinal contents and feces of adult mice, from intestinal contents of 14 and 18 days old mice, and from fecal samples of a healthy volunteer. DNA was then amplified with specific primers for the hypervariable region V4 and for the complete 16S rRNA gene. Shotgun sequencing was performed at different depth (28, 40 and 76 million of reads for the same fecal sample). The amplicons and whole metagenomes were sequenced by Illumina NGS technology and the reads analyzed with QIIME. The resulting V4 and 16S datasets were compared in terms of microbial alpha-diversity, beta-diversity, and community structure. In both human and mice samples, Shannon’s diversity index were significantly lower for the V4 metagenomes compared to the 16S ones. Consistently, significantly lower OTUs numbers were obtained from V4 datasets. OTUs relative abundances (community structure) was also dissimilar when defined by either V4 or 16S sequencing. Indeed, a number of OTUs belonging to either Firmicutes or Bacteroidetes were dramatically underrepresented or absent in the V4 metagenome datasets compared to the full length 16S. These V4-dependent variations, in turns, affected the relative prevalence of taxa at different levels. Finally, an artificial gut metagenome dataset was made up with publicly available reads from a selection of genomes, according to the real human fecal sample composition as defined by the 16S sequencing data. A comparison of the relative abundances of KEGG pathways encoded by this artificial dataset and by the shotgun metagenome reads was performed applying the same pipeline of analysis using QIIME, also providing insights in the reliability of the built in model of analysis. App-039#310 Validation of IS-pro for bacterial quantification in complex microbial communities M.L.M. van Doorn-Schepens(1), A. Eck(1) , Martine Bos(1), P.H.M. Savelkoul(1), A.E. Budding(1) Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands Objectives:The advent of culture independent techniques has increased the possibilities in the field of microbiota research. One of these culture independent techniques is the Interspace profiling technique, IS-pro. IS-pro is fast, reproducible and can be performed in a routine laboratory setting. This makes the technique applicable for clinical diagnostics. IS-pro yields bacterial profiles such that the abundance of each specie is reflected by relative fluorescence units (RFU). It was previously shown that IS-pro can accurately quantify bacterial loads in dilution series of bacterial strains. In this study we aimed to validate IS-pro for bacterial quantification in complex microbial communities. Also, we aimed to show proof of principle for applicability of the IS-pro technique in measuring the effect of an intervention on microbiota composition. Methods: Two skin swabs were taken from 47 healthy subjects, one before application and one after application of ethanol 75%-isopropyl alcohol 10% (hand alcohol). Skin microbiota profiles were obtained by means of IS-pro. IS-pro involves bacterial species differentiation by the length of the 16S–23S rDNA interspace region with taxonomic classification by phylum-specific fluorescent labeling of PCR primers. We compared the abundance measured by IS-pro to quantitative-PCR (qPCR). Three qPCR reactions were tested: 16S rDNA Eubacterial qPCR (Nadkarni et. al), Staphylococcus genus qPCR and viridans streptococci (S. viridans) qPCR. For each qPCR tested, spearman correlation was calculated between IS-pro measured intensity and Cp value or copies/10µl DNA, measured by qPCR.Results: In total 94 skin swabs were collected from 47 healthy subjects. IS-profiling showed that skin microbiota composition was dominated by Firmicutes, Actinobacteria and Proteobacteria both before and after application of hand alcohol. Application of hand alcohol mostly decreased the abundance of Firmicutes and Proteobacteria and Shannon diversity decreased significantly for Proteobacteria (P<0.05).The effect of hand alcohol on skin microbiota abundance was positively correlated between IS-pro and qPCR (rho=0.8). With both techniques we measured a decrease in total bacterial load and total abundance of Staphylococcus genus and S. viridans. Cp values of qPCR were negatively correlated to log2 transformed intensity measured by IS-pro (rho= -0.8). Copies/10µl DNA were positively correlated to RFU intensity (rho=0.7). Conclusions: IS-pro and qPCR measured a similar effect of hand alcohol on skin microbiota. Abundance measured with qPCR was positively correlated to abundance measured with IS-pro, both before and after the intervention. We conclude that IS-pro is suitable for bacterial quantification in complex microbial communities. This may allow future applications of IS-pro in disease monitoring and evaluation of the effect of therapy or intervention on microbial communities.

App-040#311 IS-pro: fast and portable clinical microbiota analysis Dries Budding (1), Malieka Degen (2), Suzanne Jeleniewski (2), Linda Poort (2), Paul Savelkoul (1,3) 1. Department of medical microbiology and infection control, VU University medical center, Amsterdam, the Netherlands 2. IS-Diagnostics, VU University medical center, Amsterdam, the Netherlands 3. Department of medical microbiology, Maastricht University medical center, Maastricht, the Netherlands Microbiota analysis has enjoyed enormous interest in recent years and many compositional variations have been described that may be used in clinical diagnostics. Despite the enormous potential, microbiota analysis has not yet found its way into clinical practice. A reason for this is that commonly used sequencing technologies are currently not compatible with a clinical workflow: equipment is not standard in clinical laboratories, techniques are typically slow and large sample batches are needed to make these analyses financially feasible. Furthermore, lack of standardization of sample preparation and reference libraries constitute a big problem for reproducible diagnostics. Finally, important issues like PCR inhibition or contamination are unaccounted for in current protocols. To solve these issues, we have developed IS-pro, a molecular bacterial profiling tool that can be preformed on standard equipment with proven applicability in daily clinical routine. It is fast (from sample to analyzed data in 5 hours), highly standardized and can be run efficiently even with small batch sizes. To monitor potential PCR inhibition and technical errors, we have developed a wholly novel internal amplification control. All data is automatically processed, analyzed and quality controlled by a central server. Finally, bacterial profiles may be classified into clinically relevant categories by comparison to a standardized reference library. Because of the high level of standardization, the IS-pro technique is fully portable between laboratories and may form an important step towards clinical microbiota based diagnostics. App-041#312 Analysis of B-vitamin biosynthesis pathways suggests co-operation among human gut microbes Stefania Magnusdottir (1), Dmitry Ravcheev (1), Valerie de Crecy-Lagard (2), Ines Thiele (1) 1. Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg 2. Department of Microbiology and Cell Science and Genetics Institute, University of Florida, Gainesville, FL, USA The human gut microbiota has been shown to supply its host with B-vitamins. However, it is still unknown which of the hundreds of microbial species in the gut are capable of producing these vitamins and providing them to the host. Using the PubSEED platform, we systematically assessed the presence of genes involved in seven B-vitamin biosynthesis pathways in genomes of 256 common human gut bacteria. On the basis of the presence and absence of genome annotations, we predicted that each of the seven vitamins was produced by 40-65% of the 256 human gut microbes. The phylum Bacteroidetes contained the highest ratio of producers for every vitamin, whereas the Firmicutes phylum contained few vitamin producers. In addition, we identified several pairs of organisms in which every vitamin synthesis pathway that was present or absent in one genome was absent or present, respectively, in the other. Most commonly, the organisms had either all eight vitamin biosynthesis pathways present, or all the pathways absent; suggesting that some bacteria can survive in the gut without producing any B-vitamins. Our analysis suggests that human gut bacteria actively exchange B-vitamins among each other, and the fact that several organisms can survive without synthesizing any of these necessary cofactors indicates their co-operation in the human gut environment. App-042#317 Effects of Long Term Exposure to a Subantimicrobial Dose of Doxyxyxline on the Fecal and Oral Microbiota Bart Keijser(1), Mamun-Ur Rashid (2), Gino Kalkman (2), Guus Roeselers (1), Georgios Panagiotidis (2), Tobias Bäckström (2), Andrej Weintraub (2), Roy Montijn (1), Carl Erik Nord (2) 1. TNO Microbiology and Systems Biology, Zeist, The Netherlands 2. Department of Laboratory Medicine, Karolinska University Hospital, Karolinska Institutet, SE-141 86 Stockholm, Sweden Aim: The purpose of this study was to investigate impact of a subantimicrobial dose doxycycline [40 mg once daily (o.d.)] for 16 weeks on the oral and intestinal microbiota of healthy human volunteers. Study design: 34 healthy volunteers were randomly assigned to receive doxycycline (40 mg) or placebo capsules for 16 weeks. Blood plasma, saliva and fecal samples were collected in the clinical center at baseline and at weeks 4, 8, 16 and 20. Plasma samples were assayed for doxycycline using a validated liquid chromatography tandem mass spectrometry (LC–MS/MS) method. Concentrations of doxycycline in saliva and feces were determined microbiologically using the agar plate diffusion method (1). Saliva and fecal microbiota community structure was analyzed by sequencing the V4 hypervariable region of the small subunit ribosomal gene. Data analysis was performed by ANOVA analysis of Bray Curtis similarity index for inter and intra

individual variation, and changes in abundance at the level of phylum, genus and oligotype level (2) compared to consecutive timepoints as well as the baseline. Results: The 16 week antibiotic exposure had no significant effect on the over-all microbiota community structure, as based on the Bray-Curtis distance measures. A significant decrease in salivary abundance was observed for several Rothia oligotypes throughout the 16 week antibiotic intervention, regaining their original abundance after the 4 week washout period at week 20. A more pronounced effect was observed for the fecal microbiome, showing a temporary impact on the genus Bifidobacterium and Lachnospiraceae and longer impact on several Bacteroides and Peptostreptococcus species. No significant effects compared to the baseline could be detected after the 4 week wash out period at week 20. Conclusions: This work shows that a long term exposure to sub antimicrobial dose of doxycycline has modest transient effect on the salivary and fecal microbiota. References 1) Int J Antimicrob Agents. 2013 Apr;41(4):352-7. 2) Methods Ecol Evol. 2013 Dec 1;4(12) App-043#321 Identification of new MAMPS modulating human intestinal epithelial cells using functional metagenomics. Nicolas Lapaque (1), Véronique Lejard (2), Alexandre Jamet (1), Alhosna Benjdia (1), Moez Rhimi (1), Olivier Berteau (1), Sylvia Guglietta (3), Dario Brunelli (3), Florence Ledue (1), Emmanuelle Maguin (1), Joël Doré (1,2), Maria Rescigno (3), Hervé M. Bl (1) INRA UMR 1319 Micalis, (2) INRA US 1367 MetaGenoPolis, F-78352 Jouy-en-Josas, France; (3) European Institute of Oncology, 20141 Milan, Italy. The human intestinal microbiota has been revealed to control key physiological functions in its host, including mucosal homeostasis and maturation of the immune system. However, the mechanism by which this key organ is contributing to human physiology is still poorly understood. Indeed, it is a complex ecosystem composed of hundreds of different species, and the vast majority of these microbes are not cultured yet. Thus, to decipher the mechanisms of interaction between commensal bacteria and intestinal epithelial cells (IECs), a high throughput cell-based functional metagenomic approach was developed (Lakhdari et al., PLoS one, 2010, de Wouters et al, PLoS one, 2014). Stably transfected human IECs bearing the luciferase reporter gene under the control of promoter of key genes or binding element of key signaling pathways (NF-kappaB, PPARgamma, AP1) were used to screen metagenomic libraries bearing large DNA fragments (~40 kb) derived from human fecal microbiota, as well as cultured commensal strains. High throughput screening showed that short chain fatty acids are key regulators of gene expression and led to the identification of bioactive metagenomic clones modulating key pathways in human IECs. Sequencing, annotation and transposon mutagenesis allowed the identification of putative genes implicated in such processes. Up to now, about 30 metagenomic clones were identified. For one stimulatory clone derived from a Bacteroides vulgatus, we identified 2 loci involved in the NF-κB stimulatory effect. Additional clone, derived from a Firmicutes, was selected for the stimulation of NF-kappaB, AP1 and TSLP reporter systems as well as IL-8 secretion. Biochemical characterization indicated that a small heat resistant compound was secreted and transposon mutagenesis in an ABC transporter system abolished this effect. In a co-culture system, this clone indirectly activated dendritic cells through IEC stimulation and further modulated T cell activity. More important, on its own, it also protected gut mucosa from pathogen- or chemical-induced injury. Thus, our Functional Metagenomic approach allowed the identification of new bacterial genes involved in the cross-talk with gut epithelium with functional consequences for the host. App-044#327 Identification of factors impacting reproducibility and quality of microbiome profile analysis Evgueni Doukhanine (1), Anne Bouevitch (1), Lindsay Pozza (1), Carlos Merino (1), Rafal M. Iwasiow (1) 1. DNA Genotek, Ottawa, Canada Recent discoveries relating the gut microbiome to health and disease have stimulated interest in biomarker discovery and therapeutic development. The study of the host-microbiome interaction relies on stabilization of the microbial community at the point of collection, cost-effective, scalable processes and reproducible analysis. We assessed the impact of pre-analytical (i.e. biological and environmental) and analytical (i.e. DNA extraction, sequencing and data analysis) factors on the variability in microbial profiles. We observed that these factors can impact the assessment of the donors’ microbiome as measured by Bray-Curtis distance and Shannon Diversity Index. Pre-analytical: Exposure of fecal samples to environmental conditions can have a major impact on the microbiome profile. Using normalized sequencing read counts, we identified taxonomic units that were significantly changed following exposure to conditions encountered during sample transport. These changes represented the major source of variability and were not consistent across donors; thus, they could not be filtered using algorithms. We observed that OMNIgene•GUT stabilization technology significantly reduced microbiome changes of samples under these conditions.

DNA Extraction: Methods of extraction can vary largely between labs. We evaluated discrepancies between bead-beating and bead-vortexing methods. We found a significant difference in the resulting microbiome profiles of identical samples that underwent two different extraction methods. In addition, we identified that the stabilization/homogenization capabilities of OMNIgene•GUT significantly reduced the difference seen between extraction methods as compared to unstabilized samples. Sequencing and data analysis: It is known that experimental procedures, including PCR and sequencing contribute to microbiome profile variability. We observed significant variability after sequencing multiple aliquots of the same sample extraction; suggesting the need for more robust methods of amplification. In addition, choice of data analysis package can have a significant impact on the taxonomic resolution that can be assigned to sequencing reads. We compared the data analysis using GreenGenes and a curated metagenomic database. We identified that the latter increased confidence in taxanomic calls and the richness of profile due to reduced ambiguity. Accurate measurement of a host microbiome clearly requires standardization in methods of sample collection, handling, extraction, sequencing and data analysis. In conclusion, our data illustrates that of the above factors which contribute to microbiome variability, unbiased biospecimen stabilization is by far the most critical. Furthermore, OMNIgene•GUT collection, stabilization and homogenization technology effectively mitigates the negative effect of several pre-analytical and analytical conditions, enabling accurate representation of the in vivo biology. App-045#328 Gist – an ensemble approach to the taxonomic classification of short read data Samantha Halliday (1), John Parkinson (2) 1. Department of Computer Science, University of Toronto, Toronto, Canada 2. Hospital for Sick Children, Toronto, Canada & Departments of Biochemistry and Molecular Genetics, University of Toronto, Toronto, Canada Metatranscriptomics, unbiased mRNA shotgun sequencing, is emerging as a powerful technology to functionally interrogate microbiomes. Through characterising gene expression across a large diversity of species simultaneously, metatranscriptomics offers the potential to identify specific functional contributions associated with each taxon within a microbiome. A significant challenge in these studies however, is assigning accurate functional and taxonomic information to each read. High-quality identifications are important not only for community profiling, but to also ensure that metabolic reconstructions and functional assignments properly compartmentalize unrelated reaction pathways from different strains; thereby providing a deeper understanding of how the loss or gain of key taxa alters microbiome functionality. Further, the ability to bin reads on the basis of taxonomic assignment has the potential to significantly streamline sequence assembly. However, since current approaches typically rely on sequence-similarity searches, accuracy is compromised by the high degree of bacterial diversity associated with environmental samples. Here we introduce Gist (Generative inference of sequence taxonomy), an ensemble method that integrates several statistical and machine learning methods for compositional analysis of both nucleotide and amino acid content with the output from the Burroughs-Wheeler Aligner to produce high quality taxonomic assignments for metatranscriptomic RNA read datasets. Key to the success of this approach is the assignment of genome-specific weights that optimize the balance of methods for each genome. Applied to real as well as synthetic datasets, generated using Genepuddle, a synthetic metatranscriptome generator based on Flux Simulator, the Gist pipeline is found to significantly outperform existing taxonomic assignment methods. We are currently developing Gist as a standalone, open source package that may be readily integrated into new and existing analytical pipelines.

length

25 nt 50 nt 100 nt 250 nt 0% 1% 2% 5% 10%

subclass

phylum

no guesssuperkingdom

ordersuborderfamilygenusspecies groupspeciessubspeciesstrain

% reads classified at this level or better

MetaCV

read length corrupted nucleotides

Performance of taxonomic assignment of short read data for Gist and the current leading taxonomic classifier, MetaCV.Genepuddle, a synthetic metatranscriptome gen-

erator based on Flux Simulator, was used to

generate training and test datasets of ~300,000

short reads based on geneome sequence data

from 434 genomes and and plasmids from 359

bacterial strains. Here we compare the ability of

Gist and MetaCV to accurately infer taxonomic

reads at specific levels of phylogenetic resolution

for (a) different read lengths;; and (b) different

levels of introduced noise. Compared to MetaCV,

Gist is able to capture a greater proportion of

accurate strain and species level assignments.

App-046#340 Influence of nisin intake on gut microbiome and physiological parameters in healthy volunteers Fabiana A Hoffmann Sarda (1,2), Eliana B Giuntini (1,2), Christian Hoffmann (1), Bernadette DBG Franco (1,2), Elizabete Wenzel de Menezes (1,2) 1. Department of Food and Experimental Nutrition – School of Pharmaceutical Sciences, University of São Paulo, Brazil. 2. Food Research Center - University of São Paulo, Brazil. Technological alternatives in the food industry that present less adverse effects to human health are gradually replacing the use of antibiotics for improvement of animal health, food productivity, and microbial food safety, in line with the novel concept of biopreservation. Nisin is a bacteriocin used as an alternative to chemical preservatives in several food products, especially cheeses. Although recognized as safe and approved for use in foods, only few studies evaluated the effects of nisin on the human organism. The present work investigated the influence of nisin intake on the gut microbiome and physiological parameters in healthy volunteers. Volunteers of both genders were oriented to consume a soup supplemented with nisin (5.625 mg/person/day) (Nisin group, n=14), or not (Control group, n=16), three times a week during six weeks. Stool samples were collected at baseline and at the end of the intervention. DNA was extracted and the microbial dynamics in the gut microbiome was determined by sequencing the bacterial 16S rDNA. Sequences were processed using Qiime, and analyzed using LEFSE and the R environment for statistical computing. Biochemical parameters (total, HDL and LDL cholesterol, triglycerides, creatinine, fasting glucose, ALT and AST hepatic enzymes) did not change significantly at the end of the intervention. There were only minor alterations in the intestinal microbiota in the Nisin group, when compared to the Control group. LEFSE showed possible changes in the Bacteriodales order, with decreasing relative abundance of the Barnesiellaceae family. Barnesiella has been implicated in the clearance of intestinal vancomycin-resistant Enterococcus (VRE) and may prevent colonization and spread of highly antibiotic-resistant bacteria. A logistic regression was able to discriminate changes in several genera due to nisin intervention, such as Catenibacterium, Bulleidia and Enterococcus. The effect of nisin on the healthy intestinal microbiome was minor, even after gastrointestinal digestion. It is still unclear if this effect would compromise defenses against pathogenic bacteria in this environment. Funding: CAPES, CNPq, FAPESP. #341 Short Talk Lactobacillus spp. genotypes may drive vaginal community stability Bing Ma (1), Mike Humphrys (1), Pawel Gajer (1), Hongqiu Yang (1), Doug Fadrosh (1), Larry Forney (2) and Jacques Ravel (1) 1The Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21209 2 Institute for Bioinformatics and Evolutionary Studies, University of Idaho, Moscow, ID 83844 Four Lactobacillus species, including L. iners, L. crispatus, L. gasseri, and L. jensenii, are often found as members of healthy vaginal microbiota, and are noted for their ability to produce copious amount of lactic acid resulting in a low and protective pH (3.5-4.5). Recent studies have shown that certain types of vaginal communities remain stable over time while others exhibit frequent transition in and out of a dysbiotic state lacking significant numbers of Lactobacillus spp and associated with increased risk to STIs, including HIV. However, little is known about the factors that determine vaginal microbial community membership and the frequency of vaginal dysbiosis. To determine if genomics factors associated with different species or strains of Lactobacillus play a role in this processes, we designed a de novo assembly-based bioinformatics pipeline to reconstruct whole genome sequences of bacterial species using culture-independent metagenome sequencing data. Our approach surpassed commonly used reference-based methods and recruit an additional ~5-50% reads per genome. This results in nearly complete genomes with 91-99% coverage compared to published reference genomes, even for species making up less than 10% of the community. We applied this pipeline on a data set obtained from the analysis of 18 swab samples from 9 subjects collected two-years apart. From these we were able to reconstruct 20 genomes that included all 4 major vaginal Lactobacillus species with >95% genome completeness. We further performed comparative genomics analyses on these reconstructed genomes, and evaluated whether the same or different strains of a species exist in the communities of subjects 2 years after the initial sample was collected. We also determined the number of SNPs and average nucleotide identity in the reconstructed Lactobacillus species genomes. By correlating genome sequences and 10-week patterns of community composition and structure, we observed that unstable temporal profile are associated with changes in the Lactobacillus strains present, while highly stable communities retained the same strains over time. These findings suggest that genetic properties of vaginal Lactobacillus strains may influence the frequency of vaginal dysbiosis. This work led to the development of bioinformatics tools that can be effectively used to study the genomic properties of individual microbial community members and further advance our understanding of the genomic determinants associated with vaginal community stability and how they increase risk to vaginal dysbiosis.

App-047#344 Microbiome analysis of early childhood caries from Chinese She and Han race Wen Jiang(1), Zongxin Ling(2), Xiaolong Lin(1), Yadong Chen(1), Jie Zhang(1), Jinjin Yu(1), Hui Chen(1) 1.Department of Conservative Dentistry and Periodontics, Affiliated Hospital of Stomatology, College of Medicine, Zhejiang University, China; 2.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, College of Medicine, Zhejiang University, China Early children caries remains one of the most common chronic childhood disease in the worldwide. It is a multi-risk disease resulting from a susceptible host, cariogenic bacteria and cariogenic diets. Despite strong evidence of biogeography of oral bacterial communities, there have been few researches focused on the microbiota of early children caries from different races. This study aimed to explore the bacterial microbiota in dental plaque from Chinese Han and She children by pyrosequencing analysis and to identify behavioural risk factors related to oral health condition from different ethnic minority. The study population of children from She and Han people were ranging from 3-6 years. The “dmft” index was used to assess dental caries. Supragingival plaque was collected from children with or without dental caries. The V1–V3 hypervariable regions of bacterial 16S rDNA genes were amplified by multiply primers and the high-throughput pyrosequencing was performed. Questionnaire were completed by children's caregivers. A total of 609,735 reads passed the quality control, belonging to eighteen phyla and 101 genera. Fifteen genera including Actinomyces, Corynebacterium, Derxia, Leptotrichia, Neisseria, Streptococcus, Veillonella, Capnocytophaga, Prevotella, Granulicatella, Fusobacterium, Johnsonella, Kingella, Porphyromonas, Abiotrophia were shared by 97% She and Han children with or without dental caries. These genera constituted 86.2-88.9% of the total microbiota of dental plaque in all subjects. However the quantity of dental plaque microbiota composition of She and Han subjects were various. Thirty genera such as Actinomyces, Campylobacter, Neisseria, Leptotrichia, Prevotella, Porphyromonas and Capnocytophaga showed significantly different between the Han and She subjects. Three genera including Gemella, Bergeyella, Granulicatella were associated with dental caries in She group, whereas other nine genera including Streptococcus, Actinomyces were associated with dental caries in Han group. Moreover the caries experience was associated with snacking habits, dental visit habits and oral hygiene. Our study first explored the framework of normal plaque microbiota from She and Han race and found that dental caries were associated with potential cariogenic microbes. Our findings could be extended to correlating oral microbiomic changes after caries treatment. App-048#346 Disposable For Faster And More Convenient Stool Sample Sreparation : Metagenomic Application. Hervé Rostaing (1), Agnes Dupond Filliard (1), Elise Simonazzi (1), Mollon Patrick (3), Emmanuelle Santiago-Allexant (3), Sandrine Gicquel (2), Bertrand Bonnaud (3), Magali Jaillard (3), Ghislaine Guigon (3), Patrick Broyer (1), Jerôme Blaze (1), Frederic 1. Innovation and system, bioMerieux, Grenoble, France 2. Molecular Unit, bioMerieux, Grenoble, France 3. Innovation and system, bioMerieux, Marcy l’Etoile, France Feces sample is one of the main sample used in diagnostic after blood and urine. But if blood and urine can be processed in fully automated platforms and have standardized protocols, feces sample preparation is still requiring very manual steps (vortex, weight sample, centrifugation, pipetting…) and protocols varies a lot from laboratories to laboratories and are complex enough to be hardly repeatable between laboratories. Need for simplification and standardization is not limited to human clinical application but also extended to veterinary or industry application. Innovation & System department of bioMerieux designed a prototype disposable ‘’easyStool’’ that strongly simplify the sample preparation of feces samples. It is composed of a screw cap attached to a rigid spoon that enable precise and easy calibration of the sample. Sample is then released by vortexing and mixed with an already enclosed buffer. After homogenization the technician squeeze the bottle and dispense the filtrate in vials trough a set of filters that let viruses and bacteria go through and retain PCR inhibitors. This device has been tested for some human diagnosis applications. In the case of AdenoVirus diagnostic the hospital that performed the study in comparison to their own reference protocol claim to decrease the processing time from 2h00 to 5 minutes with a significant gain of viral quantities detected. We also performed an internal study for metagenomic application of the device. For this particular application the protocol has been modified and include a mechanical lysis step to ensure full lysis of all kind of microorganisms. The aim is to evaluate our system in comparison to two other commercial protocols. Comparison is first of all based on PCR sensitivity. One stool sample have been spiked with 3 different microorganisms: Gram negative, Gram positive and Yeast. The three protocols were performed according to the notice for commercial kits and to our internal protocol for the easyStool device. Results (see figure) show that our protocol is simpler but enable extraction of lower quantities of DNA. Nevertheless level of PCR detection (Cq) is significantly better for the internal protocol. Surprisingly only internal protocol can detect the spiked yeast in the sample. Then the same samples have been sequenced on PGM system (IonTorrent). Data shows no differences between protocols in terms of repeatability and diversity. At the phylum level of taxonomy all protocols are in good agreements (70% of agreements for the three protocols and 90% for the closest protocols). Deeper analysis shows significant differences at lower level of taxonomy.

Finally we show that our protocol is in line with other protocols while strongly simplifying the process and enable larger studies while limiting the number of required technician for the sample preparation. The ease of use will increase reproducibility between analysis centers in studies involving multiple centers.

App-049#347 Exploring the functional capacity of the Faecalibacterium prausnitzii pan-genome Páraic Ó Cuív (1), Nur Sabrina Mohd Najman (1), Sriti Burman (1), Harry Sokol (2, 3), Philippe Langella (3) & Mark Morrison (1) 1. The University of Queensland Diamantina Institute, The University of Queensland, QLD 4101, Australia 2. Department of Gastroenterology, Saint Antoine Hospital, Paris, France 3. Commensal and Probiotics-Host Interactions Laboratory, Institut National de la Recherche Agronomique, Jouy-en-Josas, France Faecalibacterium prausnitzii is a Gram positive bacterium that is known to possess potent anti-inflammatory properties and to enhance intestinal barrier function, and consistent with this F. prausnitzii is an abundant constituent of the healthy human core gut microbiota. In contrast it is regularly found to be depleted in patients suffering from Crohn’s disease (CD) and longitudinal studies of CD patients suggest that the poor restoration of F. prausnitzii populations is predictive of recurrent disease. The specific factors underpinning the ability of F. prausnitzii to colonise and persist in the gut remain poorly defined but their identification could support new strategies to increase its abundance in the CD gut, and attenuate the inflammatory response. To address this issue we undertook a comprehensive comparative genomic analysis of the five publically available genome sequences: F. prausnitzii SL3/3 and M21/1 (assigned to F. prausnitzii phylogroup I) and F. prausnitzii A2-165, L2-6 and KLE1255 (assigned to F. prausnitzii phylogroup II). We first examined the genomic architecture of F. prausnitzii and determined that there was a greater degree of genome synteny between strains assigned to phylogroup I (F. prausnitzii SL3/3 and M21/2) than for strains assigned to phylogroup II (F. prausnitzii A2-165, L2-6 and KLE1255), suggesting that the latter group may be more susceptible to genetic flux. Next, we determined that F. prausnitzii is characterised by significant intraspecies variations that are underpinned by the presence of an extensive pan-genome. In particular the 5 strains possess a pan-genome consisting of almost 9,000 individual gene clusters and mathematical modelling suggests that the addition of each additional genome will add 400 new gene clusters to the pan-genome. In contrast the core genome consists of 834 gene clusters and could be largely identified from as few as three strains. All five F. prausnitzii genomes appear to lack the common pathway for tryptophan biosynthesis, and a candidate tryptophan uptake system was assigned to the core genome, suggesting that tryptophan acquisition may be rate-limiting to the growth of F. prausnitzii and its persistence in the human gut.

Protocol DNA (µg) Practicability MRSA KPC S. pombe

bioMerieux 2,5-‐3 ++ 26,6 Cq 28,1 Cq 35,2 Cq

Com. kit 1 7-‐10 + 28,4 Cq 29,2 Cq /

Com. kit 2 0,8-‐1 +++ 31 Cq 30,2 Cq /

bioMerieux

Com. Kit 1

Com. Kit 2

Comparison of quantities of DNA extracted and PCR detection of spiked microorganisms in stool.

Log radargraph representing the number of sequences of each phylum for each protocol.

App-050#349 Profiling Vaginal Microbiota Using Real-Time PCR with OpenArray® Technology Kelly Li, Boli Huang, Sunali Patel, Ioanna Pagani, Pius Brzoska, Evan Diamond, and Kamini Varma Genetic analysis R&D, Life Science Division, Life Technologies, part of Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080 Highly diverse and dynamic human microbiome plays important roles in maintaining health and is implicated in various diseases. For example, vaginal microbiota is complicated as they vary among individuals and ethnic groups but also affected by age, and physiological or pathological conditions. Vaginal microbiota disequilibrium may result in bacterial vaginosis (BV) and Candida vaginitis and trichomonas vaginitis. These infections are the most common vaginal diseases and associated in pre-term birth, infertility, and increased risk of pelvic inflammatory disease (PID) and sexually transmitted diseases (STD). Therefore, profiling vaginal microbiota may help to define vaginal healthy and disease status, study the susceptibility and risk factors for vaginal diseases, or potentially benefit diagnosis and clinical intervention. Next generation sequencing (NGS) has advanced human vaginal microbiome understanding tremendously, especially using 16s rRNA gene sequencing. However, it cannot detect other non-bacterial species that are important in vagina health like Candida, protozoa and virus. The lengthy NGS workflow and complicated data analysis could be intimidating and more challenging to some researchers. In addition, it is desirable to have orthogonal platforms to verify the discovery from NGS or to screen large sample size or to focus on subset of targets. Although widely used PCR-based molecular detection is sensitive and straightforward, currently most of them lack of target throughput – only detecting single species and a very small panel. To fulfill these unmet needs, we have developed a new application to profile vaginal microbiota by leveraging our existing high throughput OpenArray® technology and the real-time PCR assay design expertise. We designed a panel of assays that target 33 unique species that are important for either healthy or disease-associated vaginal microbial community, including bacteria, fungi, protozoa and even virus. We evaluated these assay performance on OpenArray®, a microscope slide–sized plate with 3,072 through-holes (48 subarrays/plate and 64 through-hole/subarray). For sensitivity, we demonstrate 7 log linear dynamic range (with R2 >0.99) with limit of detection (LOD) down to ~50 copies with spike-in of plasmid templates. Furthermore, we tested each assay with all 33 targets and did not observe any cross-reactivity using both ATCC controls and plasmids. We also ran some vaginal swaps that were tested previously and found a high concordance. For the detected targets that were not tested previously, we did DNA sequencing to verify qPCR results. In summary, our studies demonstrate high sensitivity, specificity, accuracy and reproducibility of the panel on OpenArray®. Complementary to NGS, the application provides researchers a powerful and cost-effective tool with simple workflow, fast turnaround time, and high throughput yet flexible sample/target combinations. #351 Short Talk Standards for human intestinal samples identification, collection and DNA preparation Joel Dore (1), S.Dusko Ehrlich (1), Florence Levenez (1), Eric Pelletier (2), Adriana Alberti (2), Laurie Bertrand (2), Peer Bork (3), Paul I. Costea (3), Sinishi Sunagawa (3), Francisco Guarner (4), Chaysavanh Manichanh (4), Alba Santiago (4), Liping Zha 1. INRA, France 2. CEA Genoscope, France 3. EMBL, Germany 4. HUVH, Spain 5. SJTU, China 6. BCM, USA 7. BGI, China 8. WESTERN, Canada A detailed understanding of human-microbe symbiosis requires a precise characterization of human-associated microorganisms, the human microbiome. To progress towards this ambitious goal it is of utmost importance that the data generated in each of many large 6projects involved in human metagenome research be optimally comparable. The International Human Microbiome Standards (IHMS) project coordinated the development of standard operating procedures designed to optimize data quality and comparability in the human microbiome field. Overall, IHMS focused on all key aspects of metagenomics from human sample identification, collection and processing to DNA sequence generation and analysis. We herein outline IHMS contribution concerning sample identification, collection and processing. Consideration was given to the requirement for high throughput treatment of large sample sets. A suite of sample collection procedures were compared allowing the management of different conditions and delivery-time for appropriately identified samples. The 8 partners of IHMS and 15 contributors across 12 different countries further participated in a study designed to identify the critical features and optimal protocols for DNA preparation. Contributors were offered to treat aliquots of two fecal samples using their own lab procedures. Stringent selection criteria were applied for DNA yield and quality. Full metagenomic sequencing and analysis allowed assessing the recovery of diversity and specific bacterial taxa. An initial subset of 3 satisfactory protocols was selected and analysed with respect to sequencing and data analysis standards. A final set of 14 SOPs, covering all stages of the process, have been produced. It must be kept in mind that the specific area of nucleic acids preparation does see constant evolutions and improvements, such that IHMS protocols may not be regarded as optimal in the long run. Yet they will highlight critically key feature, may serve as benchmark and help move towards automation. Supported by the European Commission under the 7th Framework Programme, IHMS Project organizes public access to downloadable SOPs and enables exchanges between users and providers of the standards (www.microbiome-standards.org).

App-051#352 Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation Michael Reck (1), Jürgen Tomasch (1), Michael Jarek (2), Peter Husemann (2), Irene-Wagner Döbler (1) (1) Research Group Microbial Communication, Helmholtz-Centre for Infection Research, Braunschweig, Germany. (2) Research Group Genome Analysis, Helmholtz Centre for Infection Research, Braunschweig, Germany. The complex microbiome of the gut has an enormous impact on human health. Analysis of the transcriptional activity of microorganisms through mRNA sequencing (metatranscriptomics) opens a completely new window into their activity in vivo, but it is highly challenging due to numerous technical and bioinformatical obstacles. Here we present an optimized pipeline for extraction of high quality mRNA from stool samples. Comparison of three commercially available RNA extraction kits with the method of Zoetendal revealed that the Powermicrobiome Kit (MoBio) performed best with respect to RNA yield and purity. Next, the influence of the stabilization reagent during sample storage for up to 15 days was studied. RIN analysis and qRT-PCR of spiked-in and indigenous genes revealed that RNA Later preserved mRNA integrity most efficiently, while samples conserved in RNA Protect showed substantial mRNA decay. Using the optimized pipeline developed here, recovery rates for spiked-in E.coli cells expressing fluorescing proteins were 8.7-9.7% for SuperfolderGFP and 14.7-17.8% for mCherry. The mRNA of stabilized stool samples as well as of snap-frozen controls was sequenced with Illumina Hiseq, yielding on average 74 million reads per sample. PCoA analysis, taxonomic classification using Kraken and functional classification using bwa showed that the transcriptomes of samples conserved in RNA Later were unchanged for up to 6 days even at room temperature, while RNA Protect was inefficient for storage durations exceeding 24 h. However, our data indicate that RNA Later introduces a bias which is then maintained throughout storage, while RNA Protect conserved samples are initially more similar to the snap frozen controls. RNA Later conserved samples had a reduced abundance of e.g. Prevotellaceae transcripts and were depleted for e.g. COG category “Carbohydrate transport and metabolism”. Since the overall similarity between all stool transcriptional profiles studied here was >0.92, these differences are unlikely to affect global comparisons, but should be taken into account when rare but critically important members of the stool microbiome are being studied. App-052#354 MetaFun : a functional metagenomic platform allowing to decipher gut microbiota-host cell cross-talk Véronique Lejard (1), Aline Letur (1), Camille Bruneau (1), Adeline Dubreuil (1), Stanislas Dusko Ehrlich (1), Joël Doré (1,2), Hervé M. Blottière (1, 2) 1. US 1367 MetaGenoPolis, INRA, Jouy-en-Josas, France 2. UMR 1319 Micalis, INRA, Jouy-en-Josas, France Exploration of microbes-hosts interactions have led to extended recognition of the role of commensal intestinal microbes in several physiological mechanisms, from epithelial barrier development to immune development and metabolism as well as neurological aspects. However, because of the complexity of the intestinal ecosystem and of our inability to cultivate most of its microorganisms (80%), the cross-talk mechanisms between the intestinal cells and the gut commensal microbiota are poorly understood. To study these interactions and explore the functionality of the human intestinal microbial communities, the MetaFun platform has developed an innovative functional metagenomic strategy allowing such explorations independently from phylogenetic identity or cultivability of the microorganisms producing the cross-talk molecules. Our approach is based on the High Throughput Screening (HTS) of metagenomic or large fragment genomic libraries on various reporter human intestinal cells targeting different intestinal functions (metabolism, immunity, proliferation …). It enables the identification of bioactive clones modulating crucial pathways. Sequencing, annotation and transposon mutagenesis performed on these bioactive clones allow the identification of the bacterial genes involved in host-microbiota dialog. The potential of our approach has been demonstrated in several publications and so far, 30 bioactive clones have been identified and are under further investigation. To cover this whole functional metagenomic screening process, MetaFun has developed four activities : 1) the construction of metagenomic and large fragment genomic libraries, 2) the production of new HTS screens, 3) the high throughput screening using SEAP and luciferase reporter systems, and 4) the High Content Screening (HCS) for fluorescent screens. With a screening capacity which can reach 200,000 clones per year, MetaFun can provide expert advice to academic and industrial partners to design the study and to achieve functional metagenomics projects. Our strategy opens perspective of discovery of novel signaling molecules relevant for physiological situations and preventive recommendations as well as pathological contexts and therapeutic applications.

App-053#363 Flexible Method for Targeted Transcript Depletion from RNA-Seq Libraries Svenja Debey-Pascher, Steve Kain, Lin Pham, I-Ching Wang, Marie Eide, and Doug Amorese This poster describes a novel method, Insert Dependent Adapter Cleavage (InDA-C), for effective removal of specific transcripts from RNA-Seq libraries without impacting non-targeted transcripts. InDA-C employs specific and robust enzymatic steps to eliminate undesirable transcripts such as rRNA during library construction without perturbing the original total RNA population as with hybridization capture methods. The specificity of transcript depletion relies on InDA-C primers which can be designed to target virtually any class of unwanted transcripts from any species. The library construction workflow uses as little as 10 ng of input total RNA, produces a strand-specific library, and is highly adaptable for depletion of any unwanted transcript(s). Here we report the unbiased removal of rRNA from RNA-Seq libraries across a variety of prokaryotic organisms and mixed species samples. Use of InDA-C primers designed against rRNAs from both bacterial and host species resulted in >98% reduction in rRNA transcripts compared to samples prepared without the InDA-C approach. As a result, through the use of InDA-C technology a greater percentage of RNA-Seq sequencing reads can be directed towards desired coding and non-coding transcripts. App-054#364 Overcoming the Challenges of Fecal Sample Handling – Use of Frozen Aliquotting Technology to Improve Efficiency and Performance of Fecal Microbiome Analyses Long, G.S., Fraone, J.M. CryoXtract Instruments, LLC, 5 Constitution Way, Woburn, MA 01801, USA The gut Microbiome has become an increasingly important system for scientific study with a potentially broad application base in drug development, nutritional research, and preventative and diagnostic medicine. However, the logistics of working with fecal samples pose significant operational and health and safety challenges, and physical handling of these samples is often undesirable. Due to the semi-solid nature of the samples, they are not readily amenable to manual or automated liquid handling methodologies making it difficult to scale from small pilot studies to larger studies requiring high throughput capabilities. Frozen sample aliquotting offers a novel and efficient solution to such challenges. By maintaining fecal samples in a frozen state at -80oC or below, and eliminating freeze-thaw cycling, the in vivo profile of the Microbiome is preserved. Additionally, frozen sample aliquotting provides a safe and uniform processing method, while suppressing unpleasant odors, helping to facilitate increased usage of such samples in laboratory environments. There are currently only two instruments capable of frozen sample aliquotting available for laboratory use. One is a bench top, semi-automated instrument that can accommodate a broad range of sample tube types, suitable for smaller pilot studies and method development, and the other is a fully automated platform that is best suited for more standardized workflows and sample formats for larger cohort studies. Both these instruments can process frozen raw, or frozen suspended fecal samples and are currently actively deployed in Microbiome research focused laboratories. App-055#365 Gut Microbiota analytical platform Françoise LE VACON BIOFORTIS, a Mérieux NutriSciences Company 3 route de la Chatterie, 44800 Saint-Herblain, FRANCE Tel : +33(0)2 40 20 57 99 Fax: +33(02) 40 35 46 95 Mail: francoise.le.vacon@mxns.com Abstract: From simple discomfort in developed countries or malnutrition in developing countries to severe pathologies like cancer, the gastrointestinal tract is the key point of Human Health. Gut health depends on a balance between three components: Host Physiology, Environmental Factors and Gut Microbiota. Disequilibrium leads to dysbiosis which is involved in numerous diseases. It’s the reason why taking into account the gut microbiota balance will help to define its impact and interactions with a disease, a product, a therapy, a patient and their nutrition needs.

With more than 1 thousand scientific papers published worldwide last year, gut microbiota study is one of the most promising field of investigation of this century, especially because of the technological improvement of the investigation tools. The establishment and the use of an analytical platform is the central point in the study of the intestinal microbiota. However beyond the need for technical solutions, several dimensions should be considered in structuring the project: biological question, target population (preclinical, healthy/diseased volunteers, screening approach…), biological sample required (size, number, sampling strategy…), biobanking (short to long term), pre-processing and logistics (mono or multicenter study…), analytical target within the gut microbiota (taxonomical levels, methods…), data processing and output format results (study report, scientific paper…). As an Contract Research Organization, specializing in the design and running of experimental, preclinical and clinical research projects for gut health, Biofortis can help you on all these aspects, independently or integrated into a major project. #361 Short Talk A gut-scale computational model reveals the potential of precise dietary interventions and rational microbiota design Hsuan-Chao Chiu (1), Elhanan Borenstein (1,2,3) 1) Department of Genome Sciences, University of Washington, Seattle, USA 2) Department of Computer Science and Engineering, University of Washington, Seattle, USA 3) Santa Fe Institute, Santa Fe, USA Hsuan-Chao Chiu (1), Elhanan Borenstein (1,2,3) 1) Department of Genome Sciences, University of Washington, Seattle, USA 2) Department of Computer Science and Engineering, University of Washington, Seattle, USA 3) Santa Fe Institute, Santa Fe, USA A complex microbial community resides in the human gut and contributes greatly to human health. Observed associations between abnormal composition of the gut microbiota and multiple diseases have raised considerable attention to the potential of microbiome-based therapies and interventions. Yet, the complexity of the gut microbiota, the lack of a predictive understanding of this system, and the limited availability of well-characterized intervention mechanisms forced preliminary therapeutic efforts to rely mostly on complete microbiome transplantation rather than on more targeted approaches or on the modulation of specific microbial species. One promising, non-invasive, and cost-effective approach for obtaining such targeted manipulations is precise dietary interventions, tailored specifically toward desired microbiota alterations. A comprehensive understanding of the impact of diet on the gut microbiota is therefore essential. Here, we accordingly set out to provide a predictive, systems-level model of the impact of diet on a representative gut ecosystem. Specifically, we extend a previously developed framework for modeling multi-species metabolic systems, constructing a gut-scale model of a representative gut community and its interaction with the gut environment. Our framework incorporates a metabolic representation of various diets and supports multiple initial compositions. We demonstrate that the impact of diet predicted by our framework based solely on metabolic modeling agrees with experimental observations of community composition under various dietary regimes. We additionally show that the impact of diet effectively and consistently overwhelms any initial variation in the gut microbiota. We finally use our modeling framework to explore the potential of customized precise dietary interventions, demonstrating that carefully designed dietary perturbations can lead to desired and tailored microbiota modulation. These findings suggest a promising therapeutic route for rational microbiome design with multiple environmental and clinical applications.

BIOBANKING AND THE MICROBIOME: USE, APPLICATION, ETHICS

Bio-001#19 Draft genome sequence of Staphylococcus gallinarum DSM20610 Ding Shi (1,2) , Xinjun Hu (1,2) , Ang Li (1,2), Lanjuan Li (1,2*) 1.State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, Zhejiang University, Hangzhou, PR China. 2.Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China * Corresponding author: To whom correspondence may be addressed Abstract Members of the Staphylococcus gallinarum group are widespread in nature. The coagulase-negative staphylococcus Staphylococcus gallinarum, originally isolated from the skin of a chicken, is a rare pathogen in humans. Staphylococcus gallinarum DSM20610 belongs to family Staphylococcaceae in the order Bacillales, class Bacilli and phylum Firmicutes. It has been reported that S. gallinarum isolates can cause traumatic endophthalmitis and bacteremia in patients. So we determine the genomic sequence of Staphylococcus gallinarum DSM20610 because of the increasing clinical relevance of this group. Here we describe the features of S. gallinarum DSM20610, together with the genome sequence and its annotation. And this is the first genome sequence of the species Staphylococcus gallinarum. Bio-002#21 Saccharomyces boulardii ameliorates carbon tetrachloride-induced liver fibrosis in rats Ming Li, Lin Zhu, Ao Xie, and Jieli Yuan Department of Microecology, School of Basic Medical Science, Dalian Medical University, Dalian, China To investigate the effects of orally administrated Saccharomyces boulardii (S. boulardii) on the progress of carbon tetrachloride (CCl4)-induced liver fibrosis, 34 male Wistar rats were randomly divided into four experimental groups including the control group (n=8), the cirrhotic group (n=10), the preventive group (n=8), and the treatment group (n=8). Results showed that the liver expression levels of collagen, type I, alpha 1 (Col1A1), alpha smooth muscle actin (αSMA), transforming growth factor beta (TGF-β) and the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA) increased significantly in cirrhotic rats compared with control and decreased by S. boulardii administration. Treatment of S. boulardii also attenuated the increased endotoxin levels and pro-inflammatory cytokines in CCl4-treated rats. And, these were associated with the changes of intestinal permeability and fecal microbial composition. Our study suggested that oral administration of S. boulardii can promote the liver function of CCl4-treated rats, and the preventive treatment of this probiotic yeast may decelerate the progress of liver fibrosis.

Bio-003#23 Screening and characterization of purine nucleoside degrading lactic acid bacteria isolated from Chinese sauerkraut and evaluation of the serum uric acid lowering effect in hyperuricemic rats Ming Li (1), Dianbin Yang (1), Lu Mei (2), Lin Yuan (3), Ao Xie (1), Jieli Yuan(1) 1, Department of Microecology, School of Basic Medical Science, Dalian Medical University, Dalian, China 2, Department of Gastroenterology, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China, 3, Faculty of Agricultural, Life and Environmental Sciences, University of Alberta, Edmonton, Alberta, Cannada Hyperuricemia is well known as the cause of gout. In recent years, it has also been recognized as a risk factor for arteriosclerosis, cerebrovascular and cardiovascular diseases, and nephropathy in diabetic patients. Foods high in purine compounds are more potent in exacerbating hyperuricemia. Therefore, the development of probiotics that efficiently degrade purine compounds is a promising potential therapy for the prevention of hyperuricemia. In this study, fifty-five lactic acid bacteria isolated from Chinese sauerkraut were evaluated for the ability to degrade inosine and guanosine, the two key intermediates in purine metabolism. After a preliminary screening based on HPLC, three candidate strains with the highest nucleoside degrading rates were selected for further characterization. The tested biological characteristics of candidate strains included acid tolerance, bile tolerance, anti-pathogenic bacteria activity, cell adhesion ability, resistance to antibiotics and the ability to produce hydrogen peroxide. Among the selected strains, DM9218 showed the best probiotic potential compared with other strains despite its poor bile resistance. Analysis of 16S rRNA sequences showed that DM9218 has the highest similarity (99%) to Lactobacillus plantarum WCFS1. The acclimated strain DM9218-A showed better resistance to 0.3% bile salt, and its survival in gastrointestinal tract of rats was proven by PCR-DGGE. Furthermore, the effects of DM9218-A in a hyperuricemia rat model were evaluated. The level of serum uric acid in hyperuricemic rat can be efficiently reduced by the intragastric administration of DM9218-A (P

#47 Short Talk Method optimization for fecal sample collection and fecal DNA extraction Conny Mathay (1), Gael Hamot (1), Estelle Henry (1), Laura Georges (1), Camille Bellora (1), Laura Lebrun (2), Brian de Witt (1), Wim Ammerlaan (1), Anna Buschart (2), Paul Wilmes (2), Fay Betsou (1) 1. Integrated BioBank of Luxemburg (IBBL), Luxembourg 2. Luxembourg Centre for Systems Biomedicine (LCSB), Luxembourg Optimization and method validation of a fecal sample collection procedure and fecal DNA extraction protocol was performed in this study. The complete stool processing workflow was validated for Biobanking fitness-for-purpose in terms of downstream DNA metagenomic analyses. Methods: Stool collection was initially optimized in terms of sample input quantity and supernatant volume using canine stool. Three DNA extraction methods (PerkinElmer MSM I®, Norgen Biotek All-In-One®, MoBio PowerMag®) and six collection container types (Sarstedt feces plain tube, Sarstedt 25 ml container plain tube, DNA Genotek P-084 for DNA extraction, DNA Genotek P-085 for DNA and RNA extraction, Stratec PSP Spin stool DNA plus kit, Sarstedt brown tube with RNAlater stabilizer) were evaluated with human stool in terms of DNA quantity and quality: DNA yield and its reproducibility were evaluated by spectrophotometry, spectrofluorometry, and quantitative PCR; DNA purity, SPUD assay and 16S rRNA gene sequence-based taxonomic signature assays were performed. Results: The optimal MSM I protocol involves a 0.2 g stool sample and 1000 µl supernatant. The MSM I extraction was superior in terms of DNA yield and quality than the other two methods tested. Optimal results were obtained with plain Sarstedt tubes (without stabilizer, requiring immediate freezing and storage at -20°C or -80°C) and DNA Genotek ® tubes (with stabilizer and RT storage) in terms of DNA yields (total, human, bacterial, and double-stranded) according to spectrophotometry and spectrofluorometry, with low yield variability and good DNA purity. No inhibitors were identified at DNA concentration 25 ng/µl. The protocol was reproducible in terms of DNA yield among different stool aliquots, and compatible with automated processing on Perkin-Elmer MSM I platform. Conclusions: We validated a stool collection and processing method suitable for downstream DNA metagenomic analysis. DNA extraction with the MSM I® method using DNA Genotek® tubes is considered optimal, promotes simple logistics in terms of collection and shipment and offers the possibility of future automation. Laboratories and biobanks should ensure preanalytical conditions are systematically recorded. Bio-004#51 Identification of a toxin-antitoxin module implicating in osmotic stress from Bifidobacterium longum Yanxia Wei(1,2), Lu Ye(2), Dianbin Liu(3), Xiaokui Guo(2), Chang Liu(2) 1.Department of Pathogenic Biology and Immunology, Laboratory of Infection and Immunity, Xuzhou Medical College, Xuzhou, Jiangsu 221004, China; 2.Department of Microbiology and Immunity, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. 3.School of Stomatology, Xuzhou Medical College, Xuzhou, Jiangsu 221004, China. AIM: To identify a toxin-antitoxin (TA) module-relBE in Bifidobacterium longum JDM301 (relBEBif) and evaluate its function in stress response. METHODS: Bioinformatic analysis of genome sequences discovered a pair of linked genes encoding a RelBE-like TA system (RelBEBif) in JDM301. The identification of RelBEBif was carried out by expressing the single relEBif gene and co-expressing the relBEBif genes in Escherichia coli (E.coli). To confirm the genetic organization and transcriptional coupling between relEBif and relBBif, reverse transcription-PCR was performed using primers spanning the 3’ end and 5’ end of relBBif and relEBif, respectively. The activity of RelBEBif under osmotic stress was detected by assessing the expression of relBBif with quantitative real-time PCR. RESULTS: Our results discovered a bicistronic operon formed by relBEBif in JDM301. Over-expression of RelEBif had a toxic effect on E.coli which can be neutralized by coexpression of its cognate antitoxin, RelBBif. We found that the expression level of RelBEBif increase during osmotic stress, which show that RelBEBif is activated under this adverse condition. CONCLUSION: Our results suggested that the RelBEBif TA module might represent a cell growth modulator helping B. longum to deal with the harsh conditions. Key words: Bifidobacterium longum; toxin-antitoxin module; osmotic stress

Bio-005#50 Activation of the Chromosomally Encoded mazEFBif Locus of Bifidobacterium longum under Acid Stress Yanxia Wei(1,2), Lu Ye(2), Dianbin Liu(1), Zhuoyang Zhang(2), Chang Liu(2), Xiaokui Guo(2) 1.Department of Pathogenic Biology and Immunology, laboratory of Infection and Immunity/School of Stomatology, Xuzhou Medical College, Xuzhou, Jiangsu 221004, China 2.Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. Toxin-antitoxin (TA) systems are distributed within the genomes of almost all free-living bacteria. Although the roles of chromosomally encoded TA systems are still under debate, they are suspected to be involved in various stress responses. Here, we provide the first report of a type II TA system in the probiotic bacterium Bifidobacterium longum. Bioinformatic analysis of the B. longum JDM301 genome identified a pair of linked genes encoding a MazEF-like TA system at the locus BLJ_811- BLJ_812. Our results showed that B. longum mazEFBif genes form a bicistronic operon. The over-expression of MazFBif was toxic to Escherichia. coli and could be neutralized by the co-expression of its cognate antitoxin MazEBif. We demonstrated that MazEFBif was activated during acid stress, which would most likely be encountered in the gastrointestinal tract. In addition, we found that the protease ClpPXBif, in addition to MazEFBif, was induced under acid stress. Furthermore, we examined antitoxin levels over time for MazEFBif and observed that the antitoxin MazEBif was degraded by ClpPXBif, which suggested that MazEFBif was activated through the hydrolysis of MazEBif by ClpP1XBif and ClpP2XBif under acid stress. Our results suggest that the MazEFBif TA module may play an important role in cell physiology and may represent a cell growth modulator that helps bacteria to cope with acid stress in the gastrointestinal tract and environment. Keywords: Bifidobacterium longum; Toxin-antitoxin system; Acid stress; ClpPX Bio-006#53 Fecal Microbiota Transplant for Treatment of Food Allergic Proctocolitis in Pediatric Patients: a Clinical Observation Yan Liu(1), Chen Dong(1) ,Zhi-hua Huang(1) Department of Pediatrics, Tongji Hospital, Tongji Medical College Affiliated to Huazhong University of Science and Technology, Wuhan , China OBJECTIVES: Fecal microbiota transplantation (FMT) appears effective for the treatment of Clostridium difficile infections (CDI). For other diseases, evidence is still limited. The aim of this study was to assess the efficiency of FMT for treatment of food allergic proctocolitis in infants following the failure of other standard therapies. METHODS: We have prospectively enrolled 11 exclusively breast or formular--fed infants diagnosed food allergic proctocolitis with refractory to allergen avoidance or failure to other standard therapies. Then fecal microbiota replacement was used alone. Following-up ranged from 1 day to 4 months after the transplantation. All the patients were diagnosed with endoscopic examinations and clinical history of food allergy, and at the same time, intestinal infection or inflammation was excluded. RESULTS: We compared the clinical symptoms and quality of life in these patients before and after FMT treatment and estimated the adverse events of FMT transplantation. Before FMT, the clinical manifestation of bloody stool or/and mucous mixed with blood in the stool were reported in all patients, while flatus was reported in only 18.2%(2/11) of cases. The quality of life was affected in all patients, including bad sleep in 81.8% (9/11)of patients and poor appetite in 63.6%(7/11) of cases. However, the symptom of flatus ceased after FMT in all patients. 90.9% (10/11)of patients had resolution in symptoms of blood or mucous mixed with blood in the stool after FMT. Improvement of sleep and appetite were observed in all patients. None suffered infections definitely related to FMT, but two patients developed unrelated infections. One patient had a Rotavirus enteritis after 2 times of FMT. Another patient suffered an acute bronchitis after5 times of FMT treatment. CONCLUSIONS: This clinical study demonstrates the effective use of FMT for food allergic proctocolitis in pediatric patients with refractory to allergen avoidance or failure to other standard therapies. Importantly, there were no related infectious complications in these infants. Bio-007#85 Draft Genome Sequence Of Staphylococcus Gallinarum Ding Shi (1,2), Xinjun Hu (1,2), Ang Li (1,2), Lanjuan Li*（1,2） 1.State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, Zhejiang University, Hangzhou, PR China. 2.Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China Members of the Staphylococcus gallinarum group are widespread in nature. The coagulase-negative staphylococcus Staphylococcus gallinarum is a rare pathogen in humans. It has been reported that S. gallinarum isolates can cause traumatic endophthalmitis and bacteremia in patients. Staphylococcus gallinarum strain L18 was isolated from human gut. It belongs to family Staphylococcaceae in the order Bacillales, class Bacilli and phylum Firmicutes. We determine the genomic sequence of

S. gallinarum strain L18 because of the increasing clinical relevance of this group. Here we describe the features of S. gallinarum strain L18, together with the genome sequence and its annotation. The draft genome of S. gallinarum strain L18 was sequenced by using next-generation sequencing technologies. Sequence data assembly revealed a genome size of 2,691,655 bp with a G + C content of 33.73%. This genome comprises one chromosome and no plasmids. To the best of our knowledge, this is the first genome sequence of the species S. gallinarum. Availability of S. gallinarum strain L18 genome could prompt the development of post-genomic tools for its rapid discrimination from S. gallinarum. Bio-008#135 Circulating long non-coding RNA NEAT1 is a novel potential biomarker for HIV-1 infection Changzhong Jin (1, 2), Xiaorong Peng (1, 2), Tiansheng Xie (1, 2), Xiangyun Lu (1, 2), Fumin Liu (1, 2), Haibo Wu (1, 2), Zongxing Yang (1, 2), Juan Wang (1, 2), Linfang Cheng (1, 2), Nanping Wu (1, 2) 1 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases，The First Affiliated Hospital，School of Medicine，Zhejiang University, Hangzhou 310003, China. 2 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, China. Objectives The Long non-coding RNAs (lncRNAs) in HIV-1 infection are not well studied. Here we detected two lncRNAs, NEAT1 and MALAT1, in peripheral blood mononuclear cells (PBMC) and plasma of HIV-1 infected patients. Methods Fifty nine HIV-1 infected patients and 21 healthy controls were recruited, of whom 31 patients were HAART-naïve and 28 patients received HAART for more than one year. Total RNA was extracted from PBMC and plasma, and levels of NEAT1 and MALAT1 were detected by quantitative real time polymerase chain reaction. Results We found levels of NAET1 and MALAT1 in PBMC were up-regulated in HAART naïve patients, and were reduced in HAART treated patients. NEAT1 was down-regulated in plasma of infected patients and expression was correlated with CD4+ T-cell counts. Conclusions This suggests that NEAT1 and MALAT1 may interact with HIV-1 in vivo and that the presence of NEAT1 in plasma is a potential biomarker of HIV-1 infection. Bio-009#149 Transmission of neonatal intensive care unit microbes to the gut of premature infants Brandon Brooks (1), Seema Bhangar (2), Robyn Baker (3), Brian A. Firek (4), Xiaochen Tang (2), Michael J. Morowitz (4), William W Nazaroff (2), Jillian F. Banfield (1) 1. Department of Earth and Planetary Sciences, University of California, Berkeley, CA 94720 USA 2. Department of Civil and Environmental Engineering, University of California, Berkeley, CA 94720 USA 3. Division of Newborn Medicine, Magee-Womens Hospital of UPMC, Pittsburgh, PA 15224 USA 4. Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 USA The objective of this study is to characterize and quantify occupant-room microbial interactions, using the preterm, low birth weight infant as a model system. Many low birth weight infants receive broad-spectrum antibiotic treatment within the first week of life and often spend several months in a neonatal intensive care unit (NICU). These circumstances largely decouple infants from source inocula acquired during the birthing process, resetting the infant’s gut microbiome, and leaving a period of risk for atypical colonization. Such abnormal colonization is characterized by low bacterial diversity, abrupt shifts in community composition, and an abundance of opportunistic pathogens. Recent studies have shown that microbes in the NICU resemble those found in the gut of premature infants; however, strain-level resolution is needed to better understand occupant-room interactions. Here, we prospectively collected fecal samples concordantly with NICU room samples for 16 infants during their first month of life. Room samples included 13 surface types sampled via swabbing and 4 via wipes for small and large surface areas, respectively. Room samples also included size-resolved airborne particle number concentrations, settled particles collected passively with suspended petri dishes, size-resolved bioaerosols collected using a NIOSH two-stage cyclone sampler, and other environmental measurements such as carbon dioxide, relative humidity and occupancy levels. Microbial community DNA from 288 fecal samples were sequenced on an Illumina platform, reads assembled, contigs binned, and high quality genomes recovered. In all, 3445 biological room samples underwent a 16S rRNA gene survey analysis. To quantify biomass, room and fecal samples were subjected to droplet digital PCR (ddPCR) using universal bacterial primers. To further define reservoirs of gut colonizing microbes in the NICU, primers for unique genes mined from gut metagenomes were generated and ddPCR performed on room samples guided by the 16S rRNA gene survey. Ongoing analyses are revealing a multitude of strain-specific reservoirs distributed throughout the NICU, suggesting that both dispersal and host selection contribute as major filters to gut colonization in the NICU. These results provide unprecedented characterization of occupant-room interactions, highlighting the need for prospective surveillance of microbial communities in hospitals, and furthering our understanding of preterm infant gut colonization.

Bio-010#238 Microbiomic Mapping reveals Structural Shift of Fecal Microbiota as a Predictor of the Progression of Nonalcoholic Fatty Liver Disease Baohong Wang (1), Xiangyang Jiang (2), Qiongling Bao (3), Jianping Ge (4), Zhenya Lu (5), Lingling Tang (6), Yu Chen (7), Lanjuan Li (8). Affiliations: Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China. Email: ljli@zju.edu.cn Background and Aims: Evidence is increasingly suggesting that the role of the intestinal microbiota in the pathogenesis of obesity and the development of NAFLD, which is associated with high morbidity and mortality. Rapid, economic, and non-invasive methods to monitor this condition may improve the prevention and clinic care substantially. This prospective, cross-sectional study was aimed at identifying the structural shift of the fecal microbiota between adult patients with NAFLD and healthy subjects and investigated its potential correlation between biochemical indicators of NAFLD progression. Materials and Methods: Totally 166 subjects were recruited: 93 patients with NAFLD and 73 healthy controls (HCs). A stool sample was collected from each subject to extract genomic DNA and a blood sample was taken for metabolic, inflammatory parameters and endotoxin (Lipopolysaccharide, LPS). The microbial community was profiled by denaturing gradient gel electrophoresis. The correlation between key microbes and biochemical indices was assessed. Results: Multivariate data analysis revealed a significant division-wide change in the fecal microbiomic profiles in patients with NAFLD compared with HCs. The Gram-negative (G-) bacteria were found to be the key microbes contributing to the difference between these two groups (p = 0.002). And the serum G- bacteria derived LPS concentrations in patients with NAFLD were higher than those of HCs (108.69 pg/ ml vs. 96.23 pg/ ml). Among the patients, the LPS level in the patients differentiates between those with or without normal liver enzymes (107.17 pg/ ml vs. 110.66 pg/ ml). Interestingly, the serum expression level of LPS significantly correlated with the uric acid level (p = 0.04, r = 0.16), which independently predicts the increase risk for incident NAFLD. The area under the curve (AUC) of the receiver operator characteristics curves based on LPS level was significant (AUC: 0.6; 95% CI: 0.5~0.69, p = 0.04) in predicting patients with or without normal liver enzymes. Conclusion: There was an association between the structural shift of intestinal microbiota and the NAFLD progression. And our study firstly indicated that G- bacteria derived circulating LPS could be a surrogate marker for the progression of NAFLD, which may be useful in the detection of NAFLD progression and facilitate the microbial research for the therapeutic approach. Bio-011#209 Creating a US registry of fecal microbiota for FMT Gary D. Wu Perelman School of Medicine, University of Pennsylvania Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection (CDI) and a growing number of clinical practitioners now offer this treatment to their patients. Despite its therapeutic utility, the human-to-human transfer of feces may be associated with long-term health risks to the recipient because the gut microbiota is composed of many components that have not yet been characterized and can change over time in ways that cannot be currently predicted. In addition to its therapeutic utility, FMT provides an important opportunity to characterize the role of the gut microbiota in the pathogenesis of, or protection against, several diseases in humans. There is growing evidence and interest for interventions that alter the gut microbiota as novel strategies to promote health and treat disease. Compelling evidence for a functional effect of the gut microbiota has been demonstrated in animal models and intriguing associations with disease states have been reported in humans. Based on the scientific foundation established by the NIH-sponsored Human Microbiome Project and other related efforts, the entire field is now at an important inflection point where translational human studies are needed to develop meaningful gut-microbiota-based therapeutic interventions. The overall goal is to develop a national FMT registry that will collect clinical data from both the donor and recipient for the following purposes: 1) To assess short- and long-term safety; 2) To gather information on practice in the U.S. and assess effectiveness of the intervention; 3) To promote scientific investigation; and 4) To aid practitioners and sponsors in satisfying regulatory requirements. The leadership of the American Gastroenterological Association (including the Scientific Advisory Board of the AGA Center for Gut Microbiome Research and Education), IDSA, NASPGHAN, and CCFA, with support from the ACG and together with the FDA and CDC, have worked together to develop the plans for this registry based on the consensus opinion on the importance of a registry by participants at an FDA/NIH-sponsored public workshop on FMT held at the NIH in May 2013. The AGA is coordinating efforts to develop this national resource using its expertise and experience in registry development. A national FMT registry will greatly enhance our ability to identify potential short-term adverse outcomes, to search for long-term safety concerns, and provide the scientific community a rich resource of information about manipulation of the gut microbiota in humans. Acknowledgments: This study was supported by the Natural Science Foundation of China (30901190, 81172702), National Program on Key Basic Research Project (2013CB531401) and the Health Bureau of Zhejiang Province Foundation (2008QN010).

COMPUTATIONAL AND INTEGRATIVE 'OMICS ANALYSES FOR MICROBIOME RESEARCH

Comp-002#26 Characterization of skin microbiome of Chinese individuals of Hong Kong Marcus H. Y. Leung (1), David Wilkins (1), Patrick K. H. Lee (1) 1. School of Energy and Environment, City University of Hong Kong, Hong Kong The advent of high-throughput sequencing technologies is met with the increasing focus of analyzing the human microbiome, which rests on the premise that microbial life is ubiquitous on the human body. One of the most studied areas of human microbiome research is the human skin, where microbial abundance may rise over 10^7 cells/cm2. The Human Microbiome Project (HMP) has extensive collection of data, and generated some of the most informative reports pertaining to the skin microbiome. However, the majority of these studies involve subjects of western descents, and effects of racial differences in skin microbiome remain elusive. In particular, only a single piece of work focuses on skin microbiomes of Chinese individuals, and none thus far for the city of Hong Kong, a metropolis where the majority of its residents are of Chinese descent, but leads very different lifestyles to that of residents of China. In this study, skin samples from 40 individuals living in Hong Kong were collected. The Illuimna MiSeq sequencing technology was employed to target the V4 region of the 16S rRNA gene. Following conventional sequence quality filtering, we revealed that the bacterial phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes, which are commonly detected on other skin microbiome analyses, constituted over 94% of all high-quality reads. Extensive inter-personal variations were detected for both α (within-sample) and β (between-sample) diversities, with majority of the operational taxonomic units and genera present were non-core. Household location of the selected occupants was the strongest factor explaining variation in microbial communities (ANOSIM Global R = 0.363, p = 0.001). Distance-based redundancy analysis revealed that demographic and biological variables, such as gender, age group, and skin sites also explained community differences, some of which may be driven by specific skin-associated genera including Propionibacterium and Staphylococcus. The high abundance of Enhydrobacter (> 5%) in our study is consistent with a previous Chinese study, suggesting that this genus is common in Chinese individuals. This prompt a comparison of our Hong Kong dataset with skin microbiomes of subjects from USA, Tanzania, and China, revealing strong geography-based clustering of skin microbiomes (ANOSIM Global R = 0.750, p = 0.001). In addition, SparCC-derived co-abundance network analysis indicates co-exclusion between Enhydrobacter and other genera across all sites, suggesting that this previously overlooked genus may be ecologically relevant in this cohort. We strongly believe that the results from this study will set stage for studies analyzing additional parts of the human microbiome in Chinese individuals (such as the gut, oral, nasal microbiomes) to increase the current representation of human microbiome works featuring non-Western cohorts, which is desperately needed. Comp-003#29 The Expression of SIgA in the Intestinal Mucosa of IBD Mice and the Effect of Probiotics Guina Wang (1), Zhiqin Mao (1) 1. Department of Pediatrics, Shengjing Hospital of China Medical University , Shenyang, China. Objective Rat model of inflammatory bowel disease (IBD) was established by administration of trinitrobenzene sulphonic acid (TNBS). Investigate the correlation between the IBD and SIgA level. Investigate the effect of different probiotics (Saccharomyces Boulardii SB, Clostridium Butyricum CB, Bifidobacterium BD, CB+BD) on SIgA expression. Methods 60 BABL/c mice were randomly divided into 6 groups: WT group, TNBS group, TNBS+SB group, TNBS+CB group, TNBS+BD group, TNBS+CB+BD group, with 10 mice respectively; The general situation and weight of mice were observed ,the disease activity index（DAI）was evaluated. Colon tissue was collected to be general observed and tissue injure evaluated. Alterations of colon inflammation were observed by means of haematoxylin-eosin (HE). The level of SIgA in tissue was evaluated by enzyme linked immunosorbent assay ( ELISA ). The expression of SIgA were respectively located and measured by Immunohistochemistry and Western blotting. Result Compared with WT group, the mice symptoms of diarrhea and weight loss were increased in TNBS group. After treatment of probiotics, the symptoms were reduced. In TNBS group, the disease activity index（DAI）showed more than probiotics groups (p Comp-004#55 Identification of new potential CAZymes in the gut microbiome from Hadza hunter gatherers. Simone Rampelli (1) , Matteo Soverini (1), Stephanie L. Schnorr (2) , Silvia Turroni (1) , Elena Biagi (1), Sara Quercia (1), Clarissa Consolandi (3) , Alyssa N. Crittenden (4), Amanda G. Henry (2), Patrizia Brigidi (1) and Marco Candela (1). 1.Department of Pharmacy and Biotechnology, University of Bologna, Italy. 2.Plant Foods in Hominin Dietary Ecology Research Group, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany. 3.Institute of Biomedical Technologies, Italian National Research Council, Segrate, Milan, Italy.

4.Metabolism, Anthropometry, and Nutrition Laboratory, Department of Anthropology, University of Nevada, Las Vegas, USA. Gut microbiota (GM) co-evolves with the human host and provides physiological flexibility for facilitating nutritional acquisition from food intake. By whole genome shotgun sequencing, we can directly observe the metabolic specializations of this commensal biome and better understand how host evolutionary and ontogenetic history is reflected in the GM function. However, there has been no information on the metabolic GM configuration in hunter-gatherer populations, posing a substantial gap in our knowledge of the configuration arising from a lifestyle that describes over 90% of human evolutionary history. To better understand how GM adapted to diet and lifestyle changes occurring along human evolution, we have detected novel microbial enzymes by sequencing fecal microbial metagenomic libraries of Hadza hunter-gatherers of Tanzania and Italian controls. The Hadza are one of the last remaining hunter-gatherer communities in the world. They live around the shores of Lake Eyasi in northwestern Tanzania and maintain a subsistence strategy that relies on wild foods and natural water sources. Here, we focus on GM enzymes involved in complex carbohydrate degradation. We assembled the GM metagenomic reads into contigs and screened the obtained sequences for carbohydrate-active enzymes (CAZymes), i.e. enzymes able to act on a vast range of glycosidic monomers, oligomers or polymers. We found a peculiar enrichment of CAZymes in the Hadza GM, which aligns with the dietary and environmental factors characteristic of their foraging subsistence. Furthermore we find a unique set of alpha-amylases that have never before been identified in the human GM. These new alpha-amylases were taxonomically assigned not only to commensal bacteria, but also to the genus Treponema, which is not typically present in the human GM. Indeed, Treponema is generally considered an opportunistic pathogen in industrialized populations because of T. pallidum, the bacterium responsible for syphilis. However, Treponema is increasingly recognized as a mutualistic member of the human GM in nonwestern populations, including the Hadza, in which it possibly facilitates degradation of refractory or resistant polysaccharides. Our results depict nuanced adaptations in the Hadza GM, responding to a broad spectrum of complex polysaccharides in the diet, and further illustrating that the metabolic specificity of the GM is directly correlated to environmental and lifestyle factors. These findings improve our understanding of the essential, and perhaps unique, functional role of the GM in a foraging lifestyle, similar to that which was practiced by our ancestors. Comp-005#57 The gut microbiome in response to temporal and technical variability Anita Voigt (1,2,3), Paul Costea (1), Shinichi Sunagawa (1), Jens Roat Kultima (1), Georg Zeller (1), Simone Li (1), Peer Bork (1,3) 1. Structural and Computational Biology Unit, European Molecular Biology Laboratory Heidelberg, Heidelberg, Germany. 2. Dept. of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany. 3. Molecular Medicine Partnership Unit, University of Heidelberg and European Molecular Biology Laboratory Heidelberg, Heidelberg, Germany. The role of the human gut microbiota in health and disease is increasingly being recognized. However, the temporal variability of the healthy gut microbiome has not yet been studied in depth using metagenomics and the knowledge about how sampling and preservation conditions can introduce variability into the readout of the fecal microbial composition is limited. To address this limitation, we collected fecal samples from seven subjects over up to two years. Preservation-induced technical variability was assessed in this context comparing fresh frozen versus RNALater-preserved samples. RNALater was chosen as alternative to freezing in order to make sample collection more robust. Short-term disturbances caused by antibiotics treatment for example were also monitored. We performed metagenomic sequencing to investigate the biological temporal and the technical variability. We find that the human gut microbiome exhibits high temporal stability and individuality. Over multiple time-points, samples cluster per individual, even in the context of a large dataset of European and US American fecal metagenomes. One exception was the antibiotic intervention case, where samples up to twelve months after the antibiotics treatment did not resemble the pre-treatment state and harbored many resistance genes. The preservation in RNALater did not affect the individuality or time point specificity of the respective samples and showed high similarity to frozen samples, indicating RNALater-preservation does not constitute a major confounder. In conclusion, the technical variability (within-sample and RNALater-induced variability) is small compared to the biological temporal within-subject variability of the unperturbed gut microbiome, which in turn is much smaller than the observed between-subject variability. Thus, short-term storage of fecal samples in RNALater is an appropriate and cost-effective alternative to freezing of fecal samples and for their use in metagenomic studies. Antibiotics treatment however lastingly affects the gut microbiome composition and it is unclear whether the composition returns to its initial state. Comp-006/#66 Analysis of the genome of Bifidobacterium longum GT15: focus on the unique genes and genes potentially involved in the microbiota-gut-brain communication. Natalia Zakharevich (1), Olga Averina (1), Artem Kasianov (1), Ksenia Klimina (1,2), Vsevolod Makeev (1), Valery Danilenko (1,2) 1. RAS, Vavilov Institute of General Genetics, Moscow, Russia 2. Nonprofit Organization "Scientific Research Center for biotechnology of antibiotics BIOAN", Moscow, Russia Bifidobacteria represent an important group of the human intestinal microbiota(1).There is increasing attention of research to the microbiota–gut–brain axis.Much attention is paid to the bifidobacteria in these studies and their role in the interaction with central nervous system via neural, neuroendocrine, neuroimmune and humoral mechanisms(2).We present the analysis of the

genome sequence for Russia origin strain Bifidobacterium longum subsp. longum GT15.This strain was isolated from the feces of a healthy adult inhabiting Central Russia.This complete genome project has been deposited in GenBank under the accession no. CP006741. Basic characteristics of this strain are published as a brief genome announcement(3).We have analyzed the genes - candidates for neuromodulators: genes encoding small proteins(4) and neurotransmitters.In the GT15 genome and in available complete genomes of species B.longum, we found small proteins (less than 50 amino acids long).In the GT15 genome, we have identified 46 such small proteins (their functions is unknown) and 9 of them are present only in GT15 genome.Some of these small proteins may be secreted proteins.We search genes encoding enzymes involved in chemical messenger metabolism of bacteria in the GT15 genome: histidine decarboxylase, glutamate decarboxylase, monoamine oxidase, spermidine synthase, acetylcholine esterase and amino-acid decarboxylase.Only genes encoding monoamine oxidase and spermidine synthase were found.Also, we detected unique genes (UGs) that present only in GT15 genome, and not found in any genome of B.longum species (not of Russian origin).The GT15 genome contained 35 ORFs of such genes.Some of the UGs are adjacent to each other, forming clusters.In the GT15 genome, we found seven such gene clusters.Some of the clusters are flanked by different mobile elements and display significant divergence from the average G+C genome content, suggesting acquisition through horizontal gene transfer.A large proportion of UGs encode proteins with unknown function; thus, they may be genes - candidates for neurotransmitters.The strain GT15 has been used as a component of probiotic drug.This work was supported by the Ministry of Education and Science of the Russian Federation under state grants 14.N08.12.0021 1. Turroni F, Ventura M, Buttó LF, Duranti S, O'Toole PW, Motherway MO, van Sinderen D. Molecular dialogue between the human gut microbiota and the host: a Lactobacillus and Bifidobacterium perspective. Cell Mol Life Sci. 2014. 71(2):183-203. 2. Lyte M. Microbial endocrinology and the microbiota-gut-brain axis. Adv Exp Med Biol. 2014. 817:3-24. 3. Zakharevich NV, Averina OV, Klimina KM, Kudryavtseva AV, Kasianov AS, Makeev VJ, Danilenko VN. Complete Genome Sequence of Bifidobacterium longum GT15: Unique Genes for Russian Strains. Genome Announc. 2014. 18;2(6). 4. Hobbs EC, Fontaine F, Yin X, Storz G. An expanding universe of small proteins. Curr Opin Microbiol. 2011. 14(2):167-173. Comp-007#65 Seqlib: an integrated website to process amplicon reads Li Ang(1) Li Lanjuan(1) 1.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University. No. 79, Qingchun Road, Hangzhou, Zhejiang Province, P.R.China We developed a free, fast and friendly website for researchers who have no experience in bioinformatics to analyze and manage amplicon reads from microbial communities. Seqlib will automatically parse datasets and describe user&039;s microbial community in detail via an online report after users submit unprocessed sequences and required metadata. Users can publish their dataset, share dataset with collaborators and easily combine datasets from different studies archived in Seqlib&039;s public database. (The website is not released yet and you could find a mirror-site here: http://paranoia88.w118.mc-test.com/seqing/, it is just a premature version and we are still working on it, especially the layout and interface.)

Comp-008#75 A preliminary study of gut microbiomes in early diagnosis of liver cirrhosis Li Shao (1,2), Ang Li (2), Nan Qin (1,2), Lanjuan Li (1,2) 1. State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, College of Zhejiang University, 310003 Hangzhou China. 2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, Zhejiang University, 310003, Hangzhou China. Liver cirrhosis is the final stage attained by various chronic liver diseases, being the 14th most common cause of death worldwide. Increasingly, cirrhosis has been seen to be not a single disease entity, but a dynamic process that can be classified into distinct clinical prognostic stages with varying 1-year mortality. Thus it is proposed that management of patients with cirrhosis should be prevention and early intervention to stabilize disease progression and to avoid or delay clinical decompensation and the need for liver transplantation. However, most chronic liver disease is notoriously asymptomatic until cirrhosis with clinical decompensation occurs, and conventional imaging can lead to false-negative diagnosis in early diagnosis. Therefore, developing early diagnosis strategy has been the key issue in improving liver cirrhosis treatment. We found in our previous study that gut microbiomes is significantly perturbed in liver cirrhosis patients, and can provide markers for cirrhosis diagnosis. Here we continue to evaluate the perturbation of gut microbiomes in compensated stage, and the potential of it in early diagnosis using next-generation sequencing. It is found that 23,946 genes are significantly regulated in compensated stage, and most of them (91.7%) persist in disease progression. The SVM (support vector machine) model based on 35 markers could diagnose compensated patients in the validation cohort with an AUC of 0.945. In conclusion, the results provided here confirmed the potential of gut microbioes in early diagnosis of liver cirrhosis, which will be of great value in disease management and also in improving the life quality of cirrhotic patients. #92 Short Talk Disentangling complex diseases with strain level resolution time-series metagenomes Chengwei Luo (1,2), Rob Knight (3,4), Heli Siljander (5,6), Mikael Knip (5,6,7,8), Ramnik J. Xavier (1,2), Dirk Gevers (1) 1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA 2. Gastrointestinal Unit, Center for the Study of Inflammatory Bowel Disease, and Center for Computational and Integrative Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA 3. Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado, USA 4. Howard Hughes Medical Institute, Boulder, Colorado, USA 5. Children’s Hospital, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland 6. Research Program Unit, Diabetes and Obesity, University of Helsinki, Helsinki, Finland 7. Folkhälsan Research Center, Helsinki, Finland 8. Department of Pediatrics, Tampere University Hospital, Tampere Finland Complex autoimmune diseases, such as Crohn’s disease, ulcerative colitis, and Type 1 Diabetes, have shown by mounting evidence that they are results from inappropriate immunological responses to microbes in a genetically susceptible host. A dysbiosis is found to be associated with these diseases, and hence monitoring of the microbiome over time, particularly the transition from healthy to disease state, or from disease throughout treatment, will be very valuable. Time-series whole genome shotgun metagenomics is thought to be well suited for such goals. The current state-of-the-art metagenomics approaches provide insight at the genus or species level, and have already offered important insights in these diseases. However, numerous studies have indicated that a microbial strain is the basic operational unit; yet no good approach to study a community at that resolution currently exists despite its potential importance. Hence, it is urgent to develop novel methods to fully utilize the longitudinal aspect to advance the investigation on these diseases at fine strain level. We therefore developed ConStrains, a novel algorithm that can rapidly and accurately profile and genotype microbial communities of large cohorts. Our results using both in silico and host-derived data show that ConStrains recovers intra-specific strain profiles and phylogeny with high accuracy, and captures critical signals including dominant strain switches and rare strains. The simulated data sets address performance in the context of different intra-population diversities, different numbers of strains, the interference from other species within the same community, as well as the scalability of the method using a large in silico cohort with 322 samples. Applying this method to a large metagenomic infant gut development data set reveals new insights of strain dynamics with underlying functional importance. ConStrains is implemented in Python, and the source code and documentation are freely available at https://bitbucket.org/luo- chengwei/constrains. Comp-009#98 Omics of Acne: Microbiome and Metabolic Profile in Human Skin – A Preliminary Report Alessandro Afornali (1), Rodrigo Makowiecky Stuart (1), Ariane Caroline Campos Paschoal (1), Andressa Katiski da Costa (1), Sarah da Costa Amaral (1), Aline Raquell Leck (1), Carla Abdo Brohem (1), Márcio Lorencini (1), Marcelo Távora Mira (2)

1. Research and Development Department, Biomolecular Research Management, Center for Biological Studies and Alternative Methods, Cencoderma - Boticario Group, Curitiba, Brazil. 2. School of Medicine, Pontifícia Universidade Católica do Paraná (PUCPR), Curitiba, Brazil. Human skin is a complex ecosystem that hosts a variety of bacteria, fungi, viruses, archaea and small arthropods, collectively comprising the skin microbiome. Under specific conditions, the sebum naturally produced by the skin accumulates in a clogged pore originating a comedo, a structure that favours the aggregation of bacterial pathogenic species contributing to the development of skin inflammation and acne. The aim of the present study is to describe the bacterial populations and the metabolite profile of skin comedones obtained from the different groups of volunteers stratified according to the severity of acne. A total of 76 volunteers of both sexes were phenotypically classified as presenting degree I (45), II (26) or III (5) of acne (Habif, 2005). Bacterial DNA and metabolites were isolated from alar crease comedones of all individuals and submitted to next generation sequencing of 16S small-subunit ribosomal RNA genes and molecular mass spectrometry (ESI-Q-Tof), respectively. A preliminary metagenomic analysis of 11 volunteers revealed 219 operational taxonomic units (OTUs) at the similarity level of 98%, covering 6 bacterial phylotypes: Actinobacteria (35.2%), Firmicutes (26.9%), Proteobacteria (21.5%), Bacteroidetes (12.3%), Fusobacteria (2.3%) and Deinococcus-Thermus (1.8%). Despite an observed interindividual variation, Actinobacteria dominated the microbiota of comedo in all analyzed subjects, with Propionibacteriaceae and Dietziaceae being the most abundant within this specific phylotype. The metabolomic analysis of the entire sample set identified 453 putative metabolites, as identified by the mass-to-charge ratio (m/z); partial least squares discriminant analysis (PLS-DA) using the entire sample set revealed two groups, highly correlated with gender (31 males and 45 females, P≤0.01). When males and females were compared, eighty-eight metabolites were found differently expressed (fold change ≥ 2, P < 0.05). The same PLS-DA strategy of analysis, applied only for males, revealed three separate groups in high correlation with degrees I, II and III of acne. The 15 most important compounds and their relative abundance sorted by the Variable Importance in the Projection (VIP) for the first component showed an interesting correlation with the severity of the disease. As a future perspective, the analysis will be extended to additional comparisons, and the top fifteen candidates from each of the comparisons will be selected for fragmentation by MS/MS, aiming to obtain higher accuracy in the identification and validation of metabolites. Finally, further analysis will be performed to correlate metabolomics and metagenomics data and its relationship to severity of acne. This study can contribute to a better understanding of the balance between host and the skin microbiome, directing future research addressing the importance of microbiome and metabolome composition in acne.

Figure 01. Interpersonal profile of the skincomedobiome. Characterization of the skinmicrobiota, as determined by 16S ribosomal RNA(rRNA) sequencing, on eleven healthy volunteers(V:01 - V:11) is depicted. The main bacterialfamilies are represented.

Figure 02. The partial least square-discriminateanalysis (PLS-DA) score scatter plot of the first 2components to the data obtained frommetabolomic analysis. Dashed circles indicate the95% confidence interval for each class. In (A),PLS-DA analysis represents the differentiation of76 samples (male, n= 31 [in green]; female, n = 45[in red]) showing the formation of two distinctgroups. In (B), PLS-DA analysis represents thedifferentiation of 31 samples (degree I, n = 11 [inred]; degree II, n = 16 [in green]; degree III, n = 4[in blue]). The PLS-DA model revealed threeseparate groups in high correlation with degrees I,II and III of acne.

Figure 03. Contribution of individual compounds to PLS-DA component 1. The 15 most important compounds and their relative abundance in theirrespective compositions are shown, sorted by the Variable Importance in the Projection (VIP) for the first component. In (A), comparisons between genders(F = female; M = male). In (B), comparison between the degrees of acne in females (F_G1 = degree I; F_G2 = degree II; F_G3 = degree III). In (C),comparison between the degrees of acne in the male group (M_G1 = degree I; M_G2 = degree II; M_G3 = degree III).

Comp-010#101 Viral and microbial communities in the oral cavity are part of a complex ecosystem Rodrigo García-López(1,2,3), Andrés Moya(1,2,3), José V. Bagan(4), and Vicente Pérez-Brocal(1,2,3) 1Área de Genómica y Salud de la Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO)-Salud Pública, Valencia, Spain. 2Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, Valencia, Spain. 3CIBER Epidemiología y Salud Pública (CIBERESP), Madrid, Spain. 4Oral Medicine. Department of Stomatology, Hospital General Universitari, Universitat de València, Valencia, Spain. Proliferative verrucous leukoplakia (PVL) is a rare high-risk variant of Oral leukoplakia (OL) affecting multiple zones in the oral cavity characterized by the formation of white asymptomatic hyperkeratotic plaques that may change into exophytic wart-like forms on gingiva and other oral mucosal tissues. Long-term studies revealed ~70% of PVL lesions develop oral carcinoma, especially Oral squamous cell carcinoma (OSCC). The high malignant transformation rate is aggravated by the inefficacy of clinical procedures in the long term and a particularly complicated diagnosis. No etiological agent has been identified. Current high-throughput DNA sequencing technologies allow for the study of whole microbial and bacterial populations found in the human oral cavity. Previous metagenomic studies have demonstrated the correlation between microbial dysbioses and the onset or development of diseases from chronic gut inflammation to diabetes. Although causality has not been established in most cases, it is undeniable that the microbial communities play an important role in human health. Viruses, on the other hand, have recently become an interesting group to study as microbial communities are influenced and even limited by viral populations. In this study we present the exploration of viral and bacterial communities in biopsies from a cohort of 40 individuals comprised of 4 groups: 10 subjects with PVL, 10 with OL, 10 with OSCC and 10 healthy controls. We extracted DNA and RNA in order to study a broad spectrum of the viral population whereas the bacterial community has been assayed by the amplification of the 16S rRNA gene and later pyrosequencing in a 454 Titanium FLX platform. All viral samples were sequenced using Illumina MiSeq genmome sequencer for paired end reads. Sequences were quality trimmed and filtered, demultiplexed and cleansed from low complexity or quality sequences. Alpha and beta diversity analyses were carried for intra and inter individual diversity. Linear Discriminant Analyses were used to identify significant biomarkers that may be predominant in each group in the study. Relations between biomarkers were used to construct Bayesian networks. These just slightly reflect the different groups of biopsies. However, relevant links can be traced within and between both viral and bacterial communities, which provide an insight into the complexity of the systems. Bacterial and viral communities influence and limit each other as the analyses demonstrate. This exposes the need to further develop new strategies in order to understand the systems as complex viral and bacterial ecosystems. Comp-011#111 Global metabolic interaction network of the human gut microbiota Jaeyun Sung (1), Seunghyeon Kim (1,2), SungHo Jang (3), Yong-Su Jin (4,5), Gyoo Yeol Jung (3), Nicholas Chia (6), Pan-Jun Kim (1,2) 1. Asia-Pacific Center for Theoretical Physics, Pohang, South Korea 2. Department of Physics, Pohang University of Science and Technology, Pohang, South Korea 3. Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, South Korea 4. Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, USA 5. Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA 6. Department of Surgery, Mayo Clinic, Rochester, MN, USA Despite recent advances in knowledge of the microbial diversity inside the human gastrointestinal tract, the global interaction dynamics between the myriad microbial species, and its influence on host health and disease remains poorly understood. To better understand the emergent properties arising from the interactions between human gut microbes, we constructed a global network model based on interspecies cross-feeding relationships. We used transport reaction information from public databases (e.g. KEGG, TransportDB), from published genome-scale metabolic models, and from literature annotations to identify small-molecule metabolic compounds that are imported and/or exported by microbes found to reside in the human gut. Next, we defined an interaction between two microbes when one class of species can uptake a metabolic compound that is secreted by another (i.e. interspecies cross-feeding). Following this approach, we linked all interacting microbes into a global Microbe-Microbe Network. Using microbiome samples collected from patients across healthy and Type-2 Diabetes phenotypes (Qin et al. Nature, 2012), we superimposed each sample’s microbial abundance information upon our Microbe-Microbe Network, thereby removing low-quantity species (i.e. nodes), along with their interactions (i.e. edges). This led to interaction networks specific to individuals, and, in turn, networks specific to phenotype. Interestingly, we identified network-based topological features, as well as enrichment of biologically meaningful interspecies interactions, unique to Type-2 Diabetes. We present the first microbial community structure within the human gastrointestinal tract based on interspecies cross-feeding of small-molecule metabolites. Network analysis of the global microbial symbiosis provides novel insight into the molecular basis of pathophysiology. Furthermore, our results can be utilized for clinical applications in the form of network-based disease classifiers.

Comp-012#108 Temporal dynamics of gut microbiome in people sharing a confined environment, the ground space-simulation MARS500 Silvia Turroni (1), Simone Rampelli (1), Clarissa Consolandi (2), Marco Severgnini (2), Clelia Peano (2), Elena Biagi (1), Sara Quercia (1), Matteo Soverini (1), Franck Carbonero (3), Giovanna Bianconi (4), Petra Rettberg (5), Francesco Canganella (4), Pa 1. Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy 2. Institute of Biomedical Technologies, Italian National Research Council, Segrate, Milan, Italy 3. Department of Food Science, University of Arkansas, Fayetteville, United States 4. Department for Innovation in Biological, Agrofood, and Forest Systems, University of Tuscia, Viterbo, Italy 5. Radiation Biology Department, Institute of Aerospace Medicine, German Aerospace Center (DLR), Köln, Germany Each human being possesses a specific intestinal microbial fingerprint, whose main contributing factors include genetic relatedness, age, diet, and lifestyle. Also the environments around us and people with whom we come in contact likely shape our gut microbiome. To investigate more in depth the impact of sharing the same confined environment on the human intestinal microbiome, we sampled the crew of MARS500 over the entire duration of the experiment, analyzing the temporal dynamics of their fecal microbial communities. The MARS500 project was conducted between 2007 and 2011 by the Institute for Bio-Medical Problems of the Russian Academy of Sciences, with extensive participation by ESA. It involved more than hundred experiments simulating some of the aspects of an interplanetary manned flight. A crew consisting of three Russians, a Frenchman, an Italian and a Chinese citizen was shut into an isolated five-module infrastructure for 520 days in order to explore the influence of the conditions of a simulated manned mission to MARS on their physiological and psychological state. Fecal samples were collected prior to entering the isolation modules, every 15-30 days during the 520 days of stay in the modules and up to six months after exiting. The fecal microbiota composition was characterized by 16S rRNA sequencing. According to our preliminary findings, each participant of MARS500 maintained a strong individuality in his own gut microbiota structure during the whole course of the study, describing a unique trajectory over time. However, a partially convergent change in the composition of their microbiome was evident during the stay, suggesting a common adaptive gut microbiota response to life in the MARS500 modules. Comp-013#122 Association of Preterm Birth with Alterations in the Microbiome and Linkage to Host mtDNA Variants Kjersti Aagaard (1), Jun Ma (1), Amanda Prince (1), Derrick M. Chu (1), Lori Showalter (1), Kathleen M. Antony (1), James Versalovic (1) 1. Baylor College of Medicine, Houston, TX, United States Objective: Although we and our microbial community and genomes (the human microbiome) have co-evolved over millions of years, to what extent the human host maternally inherited ancestral genome (mitochondrial genome) informs both microbial composition and risk of human disease is unclear. We have previously employed metagenomics to demonstrate a significant association between the placental microbiome and risk of preterm birth, as well as (in nonpregnant subjects) linkage of mtDNA variants with the vaginal and gut microbiome. In this study, we aimed to explore complex interactions between human host mtDNA SNPs and the microbiome with risk of preterm birth. Study Design: mtDNA samples from 105 gravid subjects and their offspring were deep sequenced (65X average coverage), and haplogroup and mtDNA variants were called with high confidence. The microbiome taxonomy abundance of the vagina (introitus and post fornix), placenta (maternal and fetal side) and meconium were obtained via 16S and WGS-based metagenomics. Taxa with significant differential abundance among preterm births were identified (using Boruta feature selection and LEfSe) and examined for association with mtDNA haplogroups and SNPs variants employing multiple linear regressions modeling for both genotype and clinical covariates. Results: Consistent with ours and others recent published observations, we observed an association between preterm birth and composition of the microbiome community (vaginal, placental & meconium) by virtue of preterm delivery. Significant association between mtSNPs with the preterm microbiome were observed (PLINK quantitative trait association) in the vagina and placenta (Table). Of note, several functional mtSNPs (within coding regions for electron transport genes) demonstrated significant and robust associations (Figure). Conclusion: We have demonstrated for the first time that maternally inherited human mtDNA SNPs are associated with variations in the placental and vaginal microbiome, and consequentially are linked to preterm birth.

Comp-014#127 Application of metagenomics in human gut microbiome Shaoyan Xu(1,2,3), Zhigang Ren(1,2,3), Weilin Wang(1,2,3), Jianwen Jiang(1,2,3), Shusen Zheng(1,2,3) 1Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003, Hangzhou, China. 2Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, College of Medicine, Zhejiang University,310003, Hangzhou, China. 3Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003, Hangzhou, China. Purposes: We aim to review and discuss the application of metagenomics in human gut microbiome. Methods：The studies in English about the application of metagenomics in human gut microbiome are searched and read, trying to obtain insightful conclusions. Results：There are more than one thousand microbial species living in the complex human intestine. The gut microbial community plays an important role in protecting the host against pathogenic microbes, modulating immunity, regulating metabolic processes and is even regarded as an endocrine organ. However, the traditional culture methods are very limited in identifying microbes. With the application of molecular biology technology in the field of the intestinal microbiome, especially metagenomic sequencing of the next-generation sequencing technology, progress has been made in the study of the human intestinal microbiome. Metagenomics can be used to study intestinal microbiome diversity and dysbiosis, as well as its relationship to health and disease. Moreover, functional metagenomics can identify novel functional genes, microbial pathways, antibiotic resistance genes, functional dysbiosis of the intestinal microbiome and determine interactions and co-evolution between microbiota and host, although there are still some limitations. For example，metagenomics is not possible to identify microbial expression. Metatranscriptomics, metaproteomics and metabolomics are huge complement to understand human gut microbiome. Conclusions：Metagenomics can be a powerful tool in studying human gut microbiome and it has bright prospects. The limitations of metagenomics are urgently needed to be overcome. Metatranscriptomics, metaproteomics and metabolomics in relation to the study of human gut microbiome are needed to applied more widely. Comp-015#128 Identifying biomarkers of vaginal diversity and dysbiosis: a multiplatform untargeted metabolomics approach. Amy McMillan (1,2), Jean M. Macklaim (1,3), Justin Renaud (4), Stephen Rulisa (5), Mark Sumarah (4), Gregory B. Gloor (3), and Gregor Reid (1,2,6). 1. Human Microbiology and Probiotics, Lawson Health Research Institute, Western University, London, Ontario, Canada 2 . Department of Microbiology and Immunology, Western University, London, Ontario, Canada 3. Department of Biochemistry, Western University, London, Ontario, Canada 4. Agriculture and Agri-food Canada, London, Ontario, Canada 5. University Teaching Hospital of Kigali, National University of Rwanda, Kigali, Rwanda 6. Department of Surgery, Western University, London, Ontario, Canada A low diversity, Lactobacillus-dominated vaginal community is characteristic of most women, but it can rapidly shift to a diverse biota and a condition termed bacterial vaginosis (BV) that afflicts 30% of women in Canada at any given time. High throughput sequencing studies by our group and others have uncovered increased bacterial diversity in BV, and bacterial transcriptome studies have demonstrated that gene expression differs significantly. To date, little is known about the metabolome (the complete set of small molecules in a given environment). We hypothesize that the vaginal metabolome of women with a diverse, BV-like microbiota will be distinct from women with low diversity. Using untargeted gas chromatography-mass spectrometry (GC-MS) techniques we identified over 100 compounds in vaginal samples from 64 healthy non-pregnant Rwandan women. We also recently developed an alternative metabolomics method using LC-MS in order to expand the coverage of metabolites detected, and confirm trends identified by GC-MS. Partial Least Squares (PLS) regression analysis of these metabolites indicates that the vaginal metabolome is driven by bacterial diversity, and women with a more diverse, BV-like microbiota have a distinct metabolic profile. Compounds associated with increased diversity include the novel biomarkers gamma-hydroxybutyrate (GHB) and 2-hydroxyisovalerate, and amines tyramine, cadaverine, and putrescine, which are known to cause malodour. The same markers associated with diversity were identified in a replicate cohort of 67 pregnant women, indicating these metabolic changes are independent of pregnancy status. Furthermore, by combining microbiota profiles with metabolic data we have pinpointed the organism responsible for producing one of these markers, GHB, and confirmed production in vitro. Finally we demonstrate that many of these biomarkers are likely universal across different populations by replicating the findings of the Rwandan study in a groupsof Canadian women (n=35). This work may lead to improved rapid diagnostic tools for BV, and will vastly improve our understanding of the dynamics of the vaginal microbiota

Comp-016#146 Functional analysis of type II toxin-antitoxin systems of the MazEF, RelBE, VapBC families of Bifidobacterium longum subsp. infantis ATCC 15697 strain Olga Averina (1), Maria Alekseeva (1), Andrei Shkoporov (2), Valery Danilenko (1) 1. Vavilov Institute of General Genetic, Moscow, Russia 2. Pirogov Russian National Research Medical University, Moscow, Russia The bacteria of Bifidobacterium genus are the significant representatives of the human intestine microbiota according to the relative density in microbiocenosis. The interrelation between the bifidobacteria and psychophysiological, neurohumoral factors of host has been identified. In order to generate healthy activities the bifidobacteria have to survive the challenging conditions of the gastrointestinal tract. The bifidobacteria have to possess specific adaptation traits to the environment in which they live. Among mechanisms of adaptation, toxin-antitoxin systems (TAS) play an important regulatory role by adjusting RNA levels. Using bioinformatic analysis of genome sequences we identified in B.infantis ATCC 15697 strain genes of type II TAS of MazEF, RelBE, VapBC families. It were revealed a pairs of linked genes encoding of a RelBE-like proteins (two pairs), RelB-MazF-like proteins (three pars), pair of RelB-VapC-like proteins, one gene, encoding MazF toxin and one gene, encoding RelB antitoxin [1]. Functional activities of all these TAS genes were tested in E.coli strain. It was showed that cloned single relE and mazE genes encoding toxins had a toxic effect on E. coli, which was neutralized by coexpression of its cognate antitoxins. Only two proteins RelE (Blon0012) and VapC(Blon_0934) were not toxic to E.coli. These proteins were purified for the analysis of

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Figure 1. The vaginal metabolome is most correlated with bacterial diversity. All analyses were carried out independantly for non-pregnant (left) and pregnant (right) cohorts. Row (a) PLS regression scoreplot built from 128 metabolites detected by GC-MS using bacterial diversity as a continuous latent variable. Each point represents a single woman (n=131). The position of points display similarities in the metabolome, with samples closest to one another being most similar. Circles are colored by diversity of the microbiota measured using the Shannon Index, where darker circles indicate higher diversity. Row (b) PLS regression loadings. Each point represents a single metabolite. Shaded circles indicate metabolites robustly associated with diversity in either cohort (Jackknifing, 95% CI < 0 > ). Shading of circles correspond to the size of the CI for each metabolite, where darker circles indicate narrower CIs. Venn diagram depicts overlap between metabolites associated with diversity in either cohort. Cad:Cadaverine, Tya:Tyramine, Put:Putrescine, MPh:Methlphosphate, 5AV:5-aminovalerate, HIC:2-hydroxyisocaproate, HMV:2-hydroxy-3-methylvalerate, HV:2-hydroxyisovalerate, GHB:γ-hydroxybutyrate. Ser:serine, Asp:aspartate, Glu:glutamate, Gly:glycine, Tyr:tyrosine. NAcLys:n-acetyl-lysine, Phe:phenylalanine, Orn:ornithine.

5828 16

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Non-Pregnant Pregnant

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91% of Comp 1

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91% of Comp 1

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phosphate

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tyramine

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91% of Comp 1

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tyramine

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91% of Comp 1

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95% of Comp 1

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tyramine

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phosphateglucose

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91% of Comp 1

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tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2−0

−0.1

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown!

−10 −5 0 5

−6−4

95% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

5828 16

Diversity

Confidence Interval

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2−0

−0.1

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2−0

−0.1

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown!

−10 −5 0 5

−6−4

95% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

5828 16

Diversity

Confidence Interval

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

1595% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 142

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 142

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 142

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown!

−10 −5 0 5

−6−4

95% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

< 0.510.51-0.830.84-1.83> 1.83

95% Confidence Interval

−10 −5 0 5

−6−4

91% of Comp 1

5828 16

Diversity

Confidence Interval

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

1595% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

1595% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 142

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 142

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 142

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown!

−10 −5 0 5

−6−4

95% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

5828 16

Diversity

Confidence Interval

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 142

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 142

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 142

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown!

−10 −5 0 5

−6−4

95% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

5828 16

Diversity

Confidence Interval

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2−0

−0.1

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2−0

−0.1

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

1595% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

1595% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

1595% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown!

−10 −5 0 5

−6−4

95% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−10 −5 0 5

−6−4

91% of Comp 1

5828 16

Diversity

Confidence Interval

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2−0

−0.1

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2−0

−0.1

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown!

−10 −5 0 5

−6−4

95% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−10 −5 0 5

−6−4

91% of Comp 1

5828 16

Diversity

Confidence Interval

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine!

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown!

−10 −5 0 5

−6−4

95% of Comp 1

−10 −5 0 5

−6−4

91% of Comp 1

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

CI crosses 0> 0.0880.087-0.0750.076-0.064<0.064

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1 0.2

−0.2

−0.1

91% of Comp 1

5A_valerate

Cad2HI_valerate

PutNAc_Put2H3MV

phosphate

2HI_caproate

tyramine

glucaratethreose

aspartate

ornithine

glycineserine

NAc_lysine

glutamate

phenylalanine

tyrosine

unknown

−0.2 −0.1 0.0 0.1

91% of Comp 1

HMVHIC

AspCad

GluGly

NAcLysOrn

−10 −5 0 5

−6−4

95% of Comp 1

−10 −5 0 5

−6−4

95% of Comp 1

mp 2 !

Non-Pregnant

−0.2 −0.1 0.0 0.1

−0.20

−0.15

−0.10

−0.05

91% of Comp 1

mp 2 !

HMVHIC

AspCad

GluGly

NAcLysOrn

−0.2 −0.1 0.0 0.1

−0.2

−0.1

91% of Comp 1

GHB Tya

MPhHMV

5AVPut Tyr

AspPheGlu

OrnNAcLys

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

NAc_Put

tyramine

5A_valerate

thymine2HI_caproate

2HI_valerate

phosphateglucose

glycine

serine

lysine

glutamate

gluconate

xylulose

aspartate

NAc_lysine

ornithinefructose

phenylalanine

tyrosine

−0.2 −0.1 0.0 0.1 0.2

95% of Comp 1

AspCad

GluGlyNAcLys

OrnPhe

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

AspCad

GluGlyNAcLys

OrnPhe

−5 0 5

95% of Comp 1

−5 0 5

−4−2

95% of Comp 1

Pregnant

−0.2 −0.1 0.0 0.1 0.2

−0.1

−0.0

95% of Comp 1

TyrGluGly NAcLys

PheOrn

5AVHIC

−5 0 5

−4−2

95% of Comp 1

their activities in vitro and electrophoretic assays showed the endoribonuclease activity. The quantitative real-time PCR analysis revealed transcription of all genes encoding toxin and antitoxin in B. infantis ATCC 15697 strain. The expression level of genes encoding toxins increased during the nutrient starvation and entry into late stationary phase. Functionality of two relBE bicistronic operons: Blon_0013-Blon_0012 and Blon_1398-Blon_1399 from B. infantis ATCC 15697 genome were studied in recipient B. longum NCC2705 strain. The obtained results showed that RelE2 (Blon_0012) toxin may increase the rate of persisters, resulting in multidrug tolerance and RelE1 (Blon_1399) toxin can participate in growth control. Identified gene, genomic polymorphism and functional activity of TAS II type allow used them as functional biomarkers in the metagenomic analysis of human microbiome for determination of biodiversity the bifidobacteria on species and strain level. 1. Averina OV, Alekseeva MG, Abilev SK, Il'in VK & Danilenko VN (2013) Distribution of genes of toxin-antitoxin systems of mazEF and relBE families in bifidobacteria from human intestinal microbiota. // Genetika49(3): 315-27. Russian. Comp-017#148 A Fungal Signature in the Gut Microbiota of Pediatric Patients with Inflammatory Bowel Disease Christel Chehoud (1), Lindsey G. Albenberg (2), Colleen Judge (2), Christian Hoffmann (1) Stephanie Grunberg (1), Kyle Bittinger (1), Robert N. Baldassano (3), James D. Lewis (2), Frederic D. Bushman (1), and Gary D. Wu (2) 1. Department of Microbiology, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA. 2. Division of Gastroenterology, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA. 3. Division of Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, PA. Inflammatory bowel disease (IBD) involves dysregulation of mucosal immunity in response to environmental factors such as the gut microbiota. The bacterial microbiota is altered in IBD, but the connection to disease is not fully clarified. Evidence suggests that gut fungi may play a role in the pathogenesis of IBD. In this study, we compared microbes from all three domains of life, bacteria, archaea, and eukaryota, in patients with IBD to healthy human subjects. A stool sample was collected from pediatric patients with IBD (n=34) or health control subjects (n=90), and bacterial, archaeal, and fungal communities were characterized by deep sequencing of rRNA gene segments specific to each domain. IBD patients (Crohn’s disease or ulcerative colitis) had lower bacterial diversity and distinctive fungal communities. Two sequences annotating as Candida (OTUs GU370744 and EF197997) were significantly more abundant in IBD patients (p = 0.0034 and p=0.00038, respectively) while a different Candida taxon (OTU EU490138) was more abundant in healthy subjects (p=0.0025). There were no statistically significant differences in archaea, which were rare in pediatric samples compared to those from adults. Pediatric IBD is associated with reduced diversity in both fungal and bacterial gut microbiota. Specific Candida taxa were increased in abundance in the IBD samples. These data emphasize the potential importance of fungal microbiota signatures as a useful biomarker of pediatric IBD, supporting their possible role in disease pathogenesis. Comp-018#192 Building a disease specific gut microbiome catalogue and early evaluation Emna Achouri (1), Alban Mathieu (1), Nicolas Goffard (1), Alessandra Cervino (1) and Matthieu Pichaud (1) 1. Enterome Bioscience, Paris, France In 2010, the MetaHIT Consortium published the first catalogue of microbial genes identified in the human intestinal tract [Qin, 2010] and several other catalogues have been constructed since [The Human Microbiome Jumpstart Reference Strains Consortium, 2010; Li, 2014; Karlsson, 2014]. These catalogues are extensively used in many studies of the gut microbiota [Arumugam, 2011; Le Chatelier, 2013; Nielsen, 2014] for the interpretation of NGS data obtained from stool samples. We assume that the study of a disease of interest will be improved when using a specific catalogue of genes, produced using samples from patients showing this particular disease phenotype as done by others [Qin, 2012; Karlsson, 2013; Qin, 2014; Zeller, 2014]. To the best of our knowledge, no such catalogue was developed for Crohn’s disease. In order to establish a Crohn’s disease specific catalogue, we selected a set of 161 sequenced metagenomic samples from Enterome CrohnOmeter study and other public studies [Qin, 2010; Qin, 2012; Karlsson, 2013] and performed metagenomic assembly of reads along with prokaryotic genes prediction. We obtained a non-redundant set of 1.8M complete ORFs that we compared to the two MetaHIT catalogues of 3.3M [Qin, 2010] and 10M [Li, 2014] genes. Our Crohn’s disease Catalogue contains almost 600k complete genes unshared with the two public catalogues. We enriched the MetaHIT catalogue of 3.3M with our own Crohn’s disease catalogue, which led to a new set of ~4M non-redundant genes. The number of genes seen in each samples have significantly increased when using the enriched catalogue, especially when they were low on the initial catalogue of 3.3M. This work shall shed a new light on the analysis of Crohn’s patient microbiota, providing biomarker candidates that have been identified in patients with Crohn’s disease and not identified in previous published studies.

Comp-019#158 Analysis of antibiotic resistance genes in ascites and feces of SBP-complicated hepatocirrhosis patients Lin Liu, Yonghong Xiao State Key Lab for Diagnosis and Treatment of Infectious Diseases, Zhejiang First Hospital, Hangzhou, Zhejiang, P.R.China 310003 China has a highest incidence of hepatocirrhosis, usually resulting from chronic infection of Hepatitis B Virus(HBV). The cirrhosis is often complicated by bacterial infection leading to a high percentage of death. Bacterial antibiotic resistance (AR) is one of the most important threats in bacterial infection therapy. The human gut microbiota is a great reservoir of antibiotic resistance genes, and intestinal bacterial translocation (BT) probably play an important role in the progress of bacterial infection in cirrhosis, but little is known about AR genes diversity and richness within the gut，and none has done in the comparison in spontaneous bacteria peritonitis (SBP) ascites bacteria and their AR genes with gut microbial AR genes. Taking advantages in quick advances in the next generation sequencing (NGS) and metagenomic technologies, our lab has successfully found gut microbial markers for cirrhosis development (N Qin, et al. 2014, Nature), and it also provided basic database for this study; additionally, we collected 20 ascites and feces samples from SBP-complicated liver cirrhosis patients, and performed the following analysis: 1,Genomic analysis of bacteria community structure and AR genes in ascites of SBP-complicated cirrhosis patients, and compare with clinical examination results; 2,Analyze AR genes in the gut microbiota of SBP-complicated and non-complication hepatocirrhosis patients; 3, Compare AR genes between ascites and feces samples of SBP-complicated cirrhosis patients. The preliminary results show that: 1. Through 16s rRNA amplicon analysis, we found multiple bacteria DNA in ascites, which is not always and fully reflected in clinical culturing results. 2. Through metagenomic analysis, we constructed the reference gene catalogues from ascites and feces in SBP-complicated hepatocirrhosis patients using the methodology developed by MetaHIT (J Qin, et al. 2010, Nature). We built a reference gene set of gut microbiome in SBP-complicated liver cirrhosis for the cohort containing 0.98 million genes. When compared it with the gut microbial catalogues in liver cirrhosis, 0.25 million unique genes were found in SBP-complicated liver cirrhosis gut catalogue. 3. According to the Antibiotic Resistance Database (ARDB), AR genes in the gut of cirrhosis patients are much more enriched than healthy people, and SBP-complicated cirrhosis patients adopted most abundant AR genes. 4. In most cases, AR genes in ascites can also be found (>95% identity) in gut microbiota, suggesting bacteria translocation. 5. To compare the microbial origin of antibiotic resistance genes with other genes in the ascites and feces of SBP-complicated liver cirrhosis, different assignments at the phylum level were observed, that is, antibiotic resistance genes are less prone to occur in Bacteroidetes but more prone to exist in Proteobacteria. Comp-020#160 Metatranscriptomic sequence analysis pipeline for human gut microbiome Xavier Martinez (1), Suchita Panda (1), Marta Pozuelo (1), Ivo Gut (2), Marta Gut (2), Fernando Azpiroz (1,3), Francisco Guarner (1,3), Chaysavanh Manichanh (1,3) 1. Digestive Research Unit, Vall d’Hebron Research Institute, Barcelona, Spain 2. Centro Nacional de Análisis Genómico, Barcelona, Spain 3. CIBERehd, Instituto de Salud Carlos III, Madrid, Spain Background: To date, meta-omics approaches use high-throughput sequencing technologies to produce massive data that challenge the modern computers to effectively and efficiently process them and recover reliable results. The aim of this project is the development of a reliable and efficient pipeline to perform metatranscriptomic analysis using the power of multi-threading computers. Methods: The pipeline was tested on 300 million pair-end reads generated by Hi-Seq 2000 Illumina sequencing of total RNA extracted from eight fecal samples. The feces were collected from four individuals subjected to a flatulogenic diet. The pipeline performed quality-control assessment, rRNA removal process, mapping of reads against different databases such as COG, EggNOG and MetaHIT database. Finally, the pipeline also performed metabolic pathways analysis. Results: We developed our pipeline with a modular design that allows the easy interchange or improvement of any of the stages involved in the analyses. The pipeline conducted a reliable functional analysis on our dataset, as validated with data obtained from previous studies. Furthermore, the pipeline revealed different microbial composition when comparing 16S rDNA with 16S rRNA, thus indicating that metatranscriptomic analysis should also be performed to studying active microbial communities. Finally, as an effect of the flatulogenic diet, the pipeline also showed a tendency of decrease in the functions involved in translation, carbohydrate metabolisms and energy production. Conclusions: We developed an open-source, effective and efficient metatranscriptomic pipeline for paired-end RNA-seq, easily adaptable to different analysis scenarios.

Comp-021#168 Deep learning of human microbiome in health and disease Sultan Imangaliyev (1,2,3), Bart Keijser (1,2), Wim Crielaard (1,3), and Evgeni Tsivtsivadze (1,2) 1. Top Institute Food and Nutrition, Wageningen, The Netherlands 2. Research Group Microbiology and Systems Biology, TNO Earth, Environmental and Life Sciences, Zeist, The Netherlands 3. Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, Amsterdam, The Netherlands Research on human microbiome has seen dramatic growth over the past decade in terms of new available data as well as new computational approaches for studying microbial compositions and their associations with health or disease status. Frequently, we need to apply and develop novel methods to analyze diverse and high-dimensional metagenomic datasets because standard techniques do not lead to satisfactory results. In this work, we turn to modern statistical machine learning algorithms, namely deep neural networks, to build state-of-the-art predictive models of microbial composition and its association with health or environmental factors. Deep learning, which involves training artificial neural networks with many layers, became one of the most significant recent developments in machine learning. Deep learning has been recently demonstrated to work particularly well on complex, high dimensional dataset in variety of domains such as natural language processing, computer vision, etc. To demonstrate efficacy of our deep learning algorithm on metagenome data, we use several biomedical datasets, including the one from the National Institutes of Health Human Microbiome Project (NIH HMP) study, which is publicly available. We show that our method is well suited for learning on complex metagenome datasets and it notably outperforms standard statistical methods in modelling microbial composition profiles. Comp-022#170 Metagenomic sequencing analysis of microbial communities in the oropharynx of individuals with schizophrenia and controls Eduardo Castro-Nallar (1), Matthew L. Bendall (1), Sarven Sabuncyan (2), Emily Severance, (2), Faith Dickerson (3), Jennifer R. Schroeder (4), Robert Yolken, (2), and Keith A. Crandall, (1) 1. Computational Biology Institute, George Washington University, Ashburn, VA 20147 2. Stanley Neurovirology Laboratory, Johns Hopkins School of Medicine, Baltimore, MD 3. Sheppard Pratt Hospital, Baltimore, MD4Schroeder Statistical Consulting LLC, Ellicott City MD The role of the human microbiome in schizophrenia remains largely unexplored. The microbiome has been shown to alter brain development and modulate behavior and cognition in animals through gut-brain connections, and research in humans suggests that it may be a modulating factor in many disorders. This study reports findings from a shotgun metagenomic analysis of the oropharyngeal microbiome in 16 individuals with schizophrenia and 16 controls. We further validate our findings on an exapanded 50+ individual dataset. High-level differences were evident at both the phylum and genus levels, with Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria dominating both schizophrenia patients and controls, and Ascomycota being more abundant in schizophrenia patients than controls. The three most abundant microbial genera found in schizophrenia samples were Pseudomonas, Candida, and Lactobacillus. In particular, Pseudomonas fluorescens, was found more abundant in schizophrenia samples along with 13 species of Lactobacilli, both of which have been associated to chronic inflammation. Functionally, the microbiome of schizophrenia patients was characterized by an increased number of metabolic pathways related to metabolite transport systems including siderophores and vitamin B12. In contrast, carbohydrate and lipid pathways and energy metabolism were abundant in controls. These findings suggest that the oropharyngeal microbiome in individuals with schizophrenia is significantly different compared to controls, and that particular microbial species and metabolic pathways differentiate both groups. We also compare our results with preliminary findings from schizophrenia gut metagenomes and metatranscriptomes.

case control

Samples

OTUsActinomyces odontolyticusAtopobium parvulumRothia dentocariosaRothia mucilaginosaActinobacteria (other)Prevotella melaninogenicaBacteroidetes (other)Streptococcus gordoniiStreptococcus mitisStreptococcus oralisStreptococcus pneumoniaeStreptococcus salivariusStreptococcus sp. oral taxon 071Veillonella parvulaVeillonella sp. 3_1_44Veillonella sp. 6_1_27Firmicutes (other)Fusobacterium periodonticumFusobacteria (other)Campylobacter concisusDelftia acidovoransNeisseria flavescensNeisseria subflavaProteobacteria (other)rare (other)

Comp-023#175 Mining of the incidental virome in the whole-exome sequences from patients at different stages of liver diseases Kunkai Su (1), Xin Huang (2), Dasong Hua (1), Jingjing Tao (1), Xianzhong Jiang (1), Mingding Li (1), Lanjuan Li (1) 1. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, 1st Affiliated Hospital, Zhejiang University 2. Cardiovascular Key Laboratory of Zhejiang Province, 2nd Affiliated Hospital, Zhejiang University Exome analyses powered by next-generation sequencing is an invaluable way to investigate the mutations related to certain phenotype or disease. Current technologies used to capture the exome are still of low efficiency and specificity. However these disadvantage offers a look into the virome of the participants in a current project designed to find causal mutations responsible for susceptibility to HBV (Supported by the Fundamental Research Funds for the Central Universities (2014XZZX008). In the present pilot study, five typical patients from a cohort of 300 pairs of siblings were selected to investigate the possibility to detect their plasma virome. These five selected patients are at different stages of liver diseases: acute hepatitis B, chronic hepatitis B (first visit), chronic hepatitis B (treated for 1 year), liver cirrhosis and liver cancer. Blood was sampled according to a signed agreement with patients, and then DNA was extracted followed by a standard library construct step for Illumina Hiseq2000, during which an exome capture using TruSeq Exome Enrichment Kit. The exome-seq was performed at a depth of 20. Exome-seq data of the five selected patients were cleaned and filtered. The fastq files were then mapped to the human genome and bacterial genomes to remove the human and bacterial sequences. Left sequences were mapped against a set of virus genomes to get the classification of possible existing virus in the patients’ plasma. We found that an average of 4.1% of the whole exome data was not mapped to human or bacterial genomes. And out of that, only 0.4% can be mapped to the set of known virus genomes. The results show that chronic hepatitis B patients, either been treated or not, have more virus sequences both in total amount and virus types. Not surprisingly, the treated patient showed a slightly less virus sequences (17,813 reads mapped to 732 viruses) than the first visiting patient (23,655 reads mapped to 813 viruses), which may be an outcome of the anti-virus therapy. The acute hepatitis B patient (6,723 reads) and cirrhosis patient (4,362 reads) have a lower level of virus sequences. Very interestingly, the patient with liver cancer have the least virus load and diversity of virus distribution (2,396 reads mapped to 41 viruses), and this phenomena need a further validation by analyzing more subjects in the future. The study showed the possibility to detect virome from the data which usually considered useless part in exome-seq data. And this may be a way to illustrate not only the abundance of virus in plasma but also the possible mutations to some extent. In the extremes, certain new virus genome can be assembled out of the lost data of the exome-seq. The present work is supported by Young Researcher Foundation of Health Bureau in Zhejiang Province (2012RCA023) and Postdoctoral Foundation of Zhejiang Province (BSH1202076).

Fig1. Pipeline of detection incidental virome from whole-exome sequences

Comp-024#184 The infant airway microbiome in health and disease impacts later asthma development Shu Mei Teo (1,2), Danny Mok (3), Kym Pham (4), Merci Kusel (3), Michael Serralha (3), Niamh Troy (3), Barbara J. Holt (3), Belinda J. Hales (3), Michael L. Walker (1), Elysia Hollams (3), Yury A Bochkov (6), Kristine Grindle (6), Sebastian L. Johnston (7 1. Medical Systems Biology, Department of Pathology and Department of Microbiology & Immunology, The University of Melbourne, Parkville, Victoria 3010, Australia. 2. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria, Australia. 3. Telethon Kids Institute, The University of Western Australia, West Perth, WA, Australia. 4. Melbourne Translational Genomics Platform, Department of Pathology, The University of Melbourne, Parkville, Victoria 3010, Australia. 5. Queensland Children's Medical Research Institute, The University of Queensland, Brisbane, Australia. 6. University of Wisconsin School of Medicine and Public Health, Madison, USA. 7. Airway Disease Infection Section and MRC & Asthma UK Centre in Allergic Mechanisms of Asthma, National Heart and Lung Institute, Imperial College London, Norfolk Place, London W2 1PG, United Kingdom. Respiratory infections are common in young infants and are a major cause of morbidity and mortality. In the past decade, respiratory infections during infancy have been recognized as an important factor driving the development of asthma during childhood. In addition, bacterial colonisation of the airways has been shown to influence viral infection and asthma development. We set out to characterize the bacterial composition of the nasopharyngeal microbiome in a cohort of 234 infants at high risk of allergy, which was previously established to investigate the role of respiratory infection in asthma development. The infant nasopharyngeal microbiome had a simple structure with six major types and was subject to dynamic changes during the first year of life, influenced by childcare, siblings, season, and incident respiratory infections. Importantly, antibiotic usage disrupted asymptomatic colonization patterns, resulting in increased frequency of subsequent respiratory illness associated bacteria. Early colonization with Streptococcus was associated with the development of allergic asthma by age five. Streptococcus, Haemophilus and Moraxella were associated with the presence and severity of symptoms of respiratory infection, regardless of the presence of virus. Moraxella was also a remarkably stable colonizer of the infant airway, whose presence was associated with increased incidence and severity of infections with respiratory syncitial virus (RSV). Preprint available at: http://biorxiv.org/content/early/2014/12/02/012070 Comp-025#186 Metagenomic Deconvolution Improves the Draft Genomes of Metagenomic Species Youwen Qin (1), Henry Chi Ming Leung (2), Pak Chung Sham (1) 1. Department of Psychiatry, The University of Hong Kong, Pokfulam, Hong Kong, China 2. Department of Computer Science, The University of Hong Kong, Pokfulam, Hong Kong, China Introduction: Binning millions of genes into genetic groups, particularly metagenomic species (MGS), is both meaningful and challenging in metagenomic studies. Due to the complexity of the metagenomic data and the interactions among microbial species, it is difficult to group all the genes of one genome into one MGS. Furthermore, species living in the same niche and phylogenetically close species (strains) are expected to share a proportion of genes because of convergent adaption or recent divergence. Here we proposed a new way to improve the draft genome of MGS by metagenomic deconvolution. Methods: The 3.9 million genes from 396 published human gut microbiome samples (Nielsen et al., Nature Biotechnology, 2014) were binned into co-abundance gene groups (CAGs) according to their pair-wise correlations in abundance. The CAGs with more than 700 genes were defined as MGS. The MGSs correlated with each CAG were recovered by metagenomic deconvolution (Carr et al., Plos Computational Biology, 2013), which decomposes metagenomic community-level gene content into taxa-specific gene profile. Least Absolute Shrinkage and Selection Operator (LASSO) was used for variable selection, and the optimal linear regression model was selected by 10-fold validation. Each MGS was augmented by reassembling all CAGs that include the MGS in their optimal linear predictors. The augmented MGS assembly results were compared to known reference genomes and evaluated for completeness in functional composition. Results: 29 CAGs abundances were predicted by the weighted sum of related MGSs abundances with correlation coefficient greater than 0.9. Eleven of them were consistent with their related MGSs in terms of taxonomic results, and 9 of them were reported by Nielsen et al. Intriguingly, 10 MGSs which represented different species of genus Faecalibacterium or different strains of species F. prausnitzii jointly predicted (R2 = 0.97) CAG:578 assigned to genus Faecalibacterium. CAG:578 included 30 ribosomal protein genes found in the F. prausnitzii reference genome, 28 of which were not found in any of the 10 un-augmented MGSs. The ribosomal proteins are essential to bacteria and CAG:578 elegantly bridged the gaps in these 10 related MGSs. Moreover, many of the other genes in CAG:578 which involved in carbohydrate transport and metabolism, are essential in the F. prausnitzii reference genome. Augmentation of the 10 MGSs by CAG:578 improved assembly results, with the length of longest contig increasing from 63K to 252K and N50 increasing from 19K to 53K, while reserving the similarity with the F. prausnitzii reference genome. In addition, we also found 6 other CAGs that contained ribosomal protein genes and essential genes which were not found in the related MGSs. Conclusions: Our results suggest that metagenomic deconvolution of co-abundance gene groups can augment multiple related MGSs, and result in more complete draft genomes of metagenomic species.

Comp-026#189 BioMaS: a friendly web service for an accurate taxonomic assessment of microbiomes through target-oriented metagenomics. Fosso B(1), Santamaria M(1), Marzano M(1), Donvito G(2), Monaco A(2), Notarangelo P(2), Maggi GP(2)(3), Pesole G(1)(4) (1) Institute of Biomembranes and Bioenergetics, CNR, Bari, Italy. (2) National Institute of Nuclear Physics, Bari, Italy. (3) Department of Physics, Politecnico di Bari, Bari, Italy (4) Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari “Aldo Moro”, Bari, Italy The unprecedented opportunity provided in recent years by Metagenomics for completely disclosing the huge landscape of microbial communities living in any environmental niche, plant or animal host can be fully exploited only with the support of the most advanced high-throughput sequencing (HTS) and specifically intended computational tools. In particular, the bioinformatic analysis platforms should be powerful and sophisticated enough to faithfully reveal the entire microbial biodiversity from HTS data and, at the same time, easily accessible to the vast number of researchers, also without any specific computer skills, who study this fascinating subject from many different perspectives. By developing BioMaS (Bioinformatic analysis of Metagenomic AmpliconS) web service we aimed to provide an easy-to-use, versatile and completely automatic workflow for the analysis of target-oriented Metagenomics (also referred as Meta-barcoding) data. The pipeline integrates selected state-of-the-art and new bioinformatic tools in an automated modular framework ranging from raw HTS data upload and quality assessment to final qualitative and quantitative characterization, up to deeper taxonomic levels, of the investigated microbiome. An optional module was also included in order to remove the inevitable background noise generated by unspecific amplification of host sequences when a symbiotic microbiome (e.g the human microbiome) is analysed. BioMaS currently allows the analysis of both bacterial and fungal communities and, starting directly from data produced by Roche 454 or Illumina platforms, provides a report including a detailed tree representation of the microbiome composition and interactive pie-charts summarizing the inferred taxonomy for each rank level (from species to phylum). Moreover, a tabular file is supplied in order to support the comparative analysis between different samples by means of the BioMaS Post-Processing Tools and METAGENassist (http://www.metagenassist.ca/). In order to cope with the computational requirements of this analysis both in terms of computational power and storage required, the workflow has been embedded in the Cloud infrastructure available at INFN-Bari by using the JST (Job Submission Tool) and a framework based on web services technologies (REST-SOAP interface). The Illumina version of BioMaS has been exposed in the Biodiversity Catalogue as a REST service and it is also available within a Galaxy-based portal at http://galaxy.cloud.ba.infn.it:8080. Moreover, both the 454 and Illumina BioMaS versions are freely available in a web-service hosted by the ReCaS portal (https://recasgateway.ba.infn.it). A comprehensive documentation about how to register and use both the Galaxy and the ReCaS hosted web-services of BioMaS is available at https://wiki.biovel.eu/display/doc/BioVeL+Service+-+BioMaS. Comp-027#190 MetaShot: a complete workflow for the characterization of the human associated microbiome from shotgun metagenomic data. Fosso B(1), Santamaria M(1), Lovero D (1), Corrado G (2), Vizza E (2), Passaro N (3), Crescenzi M (3), and Pesole G(1,4). (1) Institute of Biomembranes and Bioenergetics, CNR, Bari, Italy. (2) Department of Oncological Surgery, Gynecologic Oncology Unit, "Regina Elena" National Cancer Institute, Roma, Italy (3) Dept. of Cell Biology and Neurosciences, Italian National Institute of Health, Rome, Italy (4) Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari “Aldo Moro”, Bari, Italy The analysis of human microbiomes, through a shotgun metagenomics approach, is opening new and fascinating avenues for understanding microbes-host interactions and related pathologies. However, the accurate microbiome characterization through computational analysis of the large amounts of HTS shotgun data produced in a typical DNA-Seq or RNA-Seq experiment is still a largely unsolved issue. Robust and computationally effective workflows for data management and analysis are highly needed for the accurate identification of the total microbiome composition, including bacteria, microbial eukaryotes and viruses, in a context dominated by human sequences. Moreover, since the comparison of a significantly high number of cases is generally critical in clinical studies, it is essential that the analysis is rapid and fully automated. In order to address these requirements, we developed the MetaShot (Metagenomic Shotgun) pipeline designed for supporting the study of human microbiome complexity by means of shotgun HTS approaches. Third party tools and ad hoc written Python and Bash scripts are integrated to analyse paired-end Illumina sequences, offering an automated procedure covering all the required steps from the raw data management to taxonomic profiling. The MetaShot analysis procedure can be distinguished in four processes: Raw data quality check: metagenomic sequences are treated to remove low-quality and low-complexity regions; Comparison with reference databases and taxonomic annotation: cleaned metagenomic sequences are compared against Prokaryotes, Virus, Fungi and Protista reference collections from GenBank as well as against the human genome (hg19) and trascriptome (UCSC RefSeq). The results are then intersected to remove the sequences that map ambiguously on multiple divisions. Finally, the sequences are taxonomically annotated by using the TANGO (Taxonomic assignment in metagenomics) tool. Assembly of unassigned sequences and their taxonomical characterization: all the sequences that were not taxonomically annotated in the previous step are extracted and then assembled. The obtained contigs are then taxonomically annotated as at point 2 relaxing previous similarity threshold.

Report generation: a CSV file, an HTML interactive table summarizing the taxonomic assignment and a high-resolution tree representation of the obtained taxonomy are created for each division. MetaShot will be released as a standalone package including an automatic installation procedure. We report a case study application of MetaShot using DNA-Seq (530 millions PE reads) and RNA-Seq (61 millions strand oriented PE reads) shotgun data from a uterine cervix carcinoma. MetaShot was able to identify about 27,000 and 21,000 microbial sequences in the DNA-Seq and RNA-Seq data, respectively, including a remarkable number of reads assigned HPV serotype 31, a finding truly validated by PCR. Comp-028#198 Towards population-level microbiome analysis: the Flemish Gut Flora Project Jeroen Raes (1,2,3) 1. Department of Microbiology and Immunology, Rega institute, KU Leuven 2. VIB Center for the Biology of Disease 3. Department of Microbiology, VUB Alterations in the gut microbiota have been linked to various pathologies, ranging from inflammatory bowel disease and diabetes to cancer. Although large numbers of clinical studies aiming at microbiome-based disease markers are currently being performed, our basic knowledge about the normal variability of the human intestinal microbiota and the factors that determine this still remain limited. Here, I will present a large-scale study of the gut microbiome variation in a geographically confined region (Flanders, Belgium). A cohort of >5000 individuals from the normal population is sampled for microbiome analysis and extensive metadata covering demographic, health- and lifestyle-related parameters is collected. Based on this cohort, a large-scale cross-sectional study of microbiome variability in relation to health as well as parameters associated to microbiome composition is being performed. In this presentation, I will discuss our experiences in large-scale microbiome monitoring and show how the development of dedicated computational approaches can assist in microbiome analysis and interpretation. Comp-029#205 PathoScope: a multi-omic approach to characterize human microbiomes Marcos Pérez-Losada (1,2), Eduardo Castro-Nallar (1), Evan Johnson (3), Keith A. Crandall (1), Robert J. Freishtat (2) 1. Computational Biology Institute, George Washington University, Ashburn, USA 2. Division of Emergency Medicine, Children’s National Medical Center, Washington, USA 3. Division of Computational Biomedicine, Boston University School of Medicine, Boston, USA BACKGROUND Emerging next-generation sequencing (NGS) technologies have revolutionized the collection of genomic data for applications in bioforensics, biosurveillance, and for use in clinical settings. However, to make the most of these new data, new methodologies that can accommodate large volumes of genomic data from different sources (DNA-seq and RNA-seq) in a computationally efficient manner need to be applied. Here we present a new statistical framework to accurately and quickly analyze host and microbial NGS reads for microbiome characterization and transcript differential expression, and apply it to the study of pediatric asthma. METHODS PathoScope is a newly developed bioinformatic platform that can map host and microbial NGS reads to databases of known target genomes, transcriptomes and amplicons. PathoScope capitalizes on a Bayesian statistical framework that accommodates information on sequence and mapping quality, and provides posterior probabilities of matches. Importantly, our approach also incorporates the possibility that multiple species can be present in the sample and considers cases where the sample species/strain is not in the reference database. PathoScope can also discriminate between closely related strains of microbes (pathogens and commensals) with very low genome coverage, while assessing differential expression of genes – including those involved in the host immune response to specific pathogens. We applied PathoScope to the analysis of RNA-Seq data from nasal epithelial cells in 14 children and adolescents (8 asthmatic and 6 healthy controls) enrolled in the AsthMaP (Asthma Severity Modifying Polymorphisms) Project. We used an Illumina HiSeq 2500 platform to generate an average of 41.4 million single-end 100bp reads per sample (mix of host and microbial RNA) after rRNA depletion. RESULTS PathoScope revealed significant differences in the metagenomic composition of the nasal microbiomes of asthmatic and healthy patients. Moraxella catarrhalis was identified as the predominant microbe in 5 of the 8 asthmatic patients and was 5.6 times more abundant in cases than controls. Subsequent transcriptomic RNA-seq analysis showed a strong host immune response to M. catarrhalis in 4 of the 5 asthmatic patients identified in our metagenomic analysis, but not response was detected in the healthy controls. CONCLUSIONS Our approach not only characterizes microbiomes from genomic data, but can also distinguish pathogens from commensals by determining if the host is mounting an immune response against specific infectious agents. Our results support and expand previous 16S rRNA metagenomic studies suggesting that M. catarrhalis is an airway pathogen and one of the driving factors of pediatric asthma.

Comp-030#206 Changes in the human gastrointestinal microbiome during cancer treatments Anne Kaysen (1), Anna Heintz-Buschart (1), Patrick May (1), Cédric C. Laczny (1), Shaman Narayanasamy (1), Norbert Graf (2), Arne Simon (2), Jörg Bittenbring (2), Stephanie Kreis (3), Jochen G. Schneider (1,2), Paul Wilmes (1) 1. Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Belval, Luxembourg 2. Saarland University Medical Center, Homburg, Saar, Germany 3. Life Sciences Research Unit, University of Luxembourg, Luxembourg, Luxembourg Chemotherapeutic treatments for malignant diseases are known to greatly impact patients’ gastrointestinal (GI) microbiomes. In particular, immune ablative chemotherapy prior to allogeneic stem cell transplantation (Tx) represents a major perturbation of host-microbiome interactions. Resulting imbalances at the mucosal interface may culminate in mucositis, which is considered the major complication associated with chemotherapy and radiotherapy. Another frequent adverse effect of allogeneic stem cell Tx potentially linked to the microbiome is graft-versus-host disease (GvHD). To identify quantitative and qualitative changes in the GI microbiomes of patients undergoing chemotherapy and allogeneic stem cell Tx associated with treatment side effects, in particular mucositis and GvHD, we are characterizing the GI microbiomes of patients undergoing anticancer therapies using integrated omic analyses. We are specifically aiming at providing important first insights into the complex interactions between the host and the intestinal microbiota during and after chemotherapy and link these to clinical parameters and patient outcome. Modulation of the microbiota may potentially help to increase tolerance and/or improve the overall efficacy of the therapy. First results from 16S rRNA gene amplicon sequencing reveal major shifts in the GI microbiome during the course of anticancer treatment, and observed changes are more pronounced in patients undergoing an intensive treatment regime. More specifically, following the initiation of a treatment, a reduction in overall microbial diversity is accompanied by a decrease in abundance of members belonging to the order Clostridiales whereas the abundance of Lactobacillales increases. In several patients receiving an intensive treatment, an overgrowth by individual genera directly after the allogeneic stem cell Tx, as for example by Enterococcus spp. and Shigella spp., is also observed.

Figure 1. Heatmap of Moraxella catarrhalis signature genes distinguishes the asthma samples from the controls. The Moraxella catarrhalis signature strengths are highly concordant with the PathoScope read proportions in control and asthma samples.

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Our initial findings confirm that cancer treatment has an important impact on the composition of the GI microbiome. In a next step, we aim to provide knowledge on how the host's immune system influences the GI microbiome and on the role and involvement of the GI microbiota in development of mucositis and GvHD. Importantly, this could help in the formulation of measures to prevent mucositis and GvHD development in patients undergoing cancer treatment. Comp-031/#212 Human gut virome epigenetics and how DNA modifications can block CRISPR attack Alexandra Bryson (1), Young Hwang (1), Sam Minot (1), Scott Sherrill-Mix (1), Anatoly Dryga (1),Tyson Clark (2), Lindsay Black(3), Frederic D. Bushman (1) 1. University of Pennsylvania School of Medicine, Department of Microbiology, 3610 Hamilton Walk, Philadelphia, PA 2. Pacific Biosciences, 1380, Willow Rd., Menlo Park, CA 94025 3. Biochemistry and Molecular Biology University of Maryland Medical School, 108 N. Greene St., Baltimore, Md 21201-1503 Phages are the most abundant biological entities on Earth and play a dynamic role in regulating bacterial populations. We are investigating how the complex interactions between bacteria and phage in the gut influence human health and disease. In a longitudinal study of the human gut virome spanning 2.5 years, we used deep sequencing to identify phage contigs targeted by both bacterial and viral CRISPR arrays from the same individual, confirming the presence of CRISPR pressure in the human gut microbiome. In separate studies, we found extensive covalent DNA modification in gut bacteriophage using single molecule sequencing, leading us to wonder whether covalent DNA modifications allow phage to evade CRISPR attack. We thus analyzed the efficiency of CRISPR/Cas9 against T4 phage strains with glucosyl-hydroxymethylcytosine (Glc-HMC), hydroxymethylcytosine (HMC), and unmodified cytosines. We found that wild-type T4 containing Glc-HMC was insensitive to attack by CRISPR/Cas9, but mutants with unmodified cytosines were sensitive. Phage with HMC showed intermediate sensitivity. While this work was in progress, another lab published a synthetically engineered CRISPR/Cas9 system that was able to restrict wild-type T4. We verified their results and confirmed that their particularly potent spacers, fused artificially to a single-guide RNA, were indeed able to cleave modified DNA. However most spacers in a biologically relevant CRISPR/Cas9 system were unable to cleave modified DNA. Thus it seems probable that many of the bulkier forms of DNA modification seen in DNA phage have evolved, at least in part, to reduce sensitivity to cleavage by CRISPR/Cas systems. Further preliminary data from single molecule sequencing indicate that over 80% of DNA phages in the human gut contain some form of covalent DNA modifications. We have found new sequence motifs that signal nucleotide modification, and a possible novel covalent guanine modification. To our knowledge, this is the first comprehensive study of the epigenetics of the human gut virome. Elucidating the complex relationships between bacterial and viral species of the human gut mediated by covalent DNA modification can provide new windows into the functions of the gut microbiome. Comp-032#218 Metagenomics of a Population-Based Prospective Cohort Reveals No Discernable Impact of Mode of Delivery (MOD) on the Infant Microbiome by 6 Weeks of Age Derrick M. Chu (1), Jun Ma (1), Amanda Prince (1), Kathleen M. Antony (1), Michelle Moller (1), Brigid Boggan (1), Kjersti Aagaard (1) 1. Baylor College of Medicine, Houston, TX, United States. The infant microbiome is relatively sparse at birth, and populated early in life. The reported relative impact of MOD on the neonatal microbiome has garnered attention due to Cesarean (CD) prevalence and its association with later in life disease. Although previous small studies suggested that CD adversely impacted microbiome diversity, they were limited by potential confounding and lack of longitudinal sampling. We thus sought to interrogate whether MOD impacts the infant microbiome over time in a robust population-based cohort. An at-large representative cohort (n=277 gravidae) was prospectively enrolled, and a subset (n=81) consented to longitudinal sampling (3rd trimester, delivery & 4-6 wks postpartum). Non-contaminated samples were uniformly collected at 5 infant sites (meconium/stool, oral supragingival plaque, respiratory anterior nares, and skin auricular & antecubital fossa) and 7 maternal sites (aforementioned plus vaginal introitus and posterior fornix). DNA was extracted (MolBio), NextGen sequenced, and subjected to robust comprehensive 16S and WGS-based metagenomic analysis. High quality sequence data (>3Tb) was obtained, with complete longitudinal maternal and infant sample sets in a nested cohort (22 CD & 52 VB). At delivery, the infant microbiome was significantly different between CD and VB infants along the first principal component (PC1) axis for all body sites (panel A; Mann-Whitney *p<0.05,**p<0.01). However, by 6 weeks the infant microbiome clusters by body site (panel B; PERMANOVA p=0.001) but not MOD (PERMANOVA p=0.073 & PC1 Mann-Whitney, all p>0.20). Although MOD marginally influenced the infants microbiome at delivery, there was no lasting discernable impact by 6 weeks of age. Based on our prior findings, we speculate that other factors (i.e., the placental microbiome, gestational age & breastfeeding) may bear greater effect on establishment and early development of the infant microbiome.

Comp-033#219 Alterations in the Intestinal Microbiome of Infants are Associated with Breastfeeding Practice Derrick M. Chu (1), Jun Ma (1), Amanda Prince (1), Kathleen M. Antony (1), Diana Racusin (1), Michelle Moller (1), Brigid Boggan (1), Kjersti Aagaard (1) 1. Baylor College of Medicine, Houston, TX, United States.

Cesarean delivery (CD) has been implicated in dysbiosis of the neonatal microbiome (totality of microbes and their genomes). However, it is possible that colinearity of CD with breastfeeding practice may confound these findings. Formula does not mimic the complexity of human breastmilk, which retains both macromolecules as well as a unique microbiome. We thus sought to employ a population-based cohort of mothers and their infants to determine if breastfeeding impacts the neonatal microbiome, potentially negating early cesarean-mediated dysbiosis. A representative cohort (n=277 gravidae) was prospectively enrolled, and a subset (n=81) consented to longitudinal sampling (3rd trimester, delivery & postpartum). Stool swabs were collected from neonates at delivery and by 6 weeks of age. Primary BF practices were determined in multiple interviews (breastfed-only (BF; n=23), formula fed-only (FF; n=2), or both (BF&FF; n=34). DNA was extracted (MolBio) and subjected to 16S and WGS metagenomics. Quality sequences were analyzed with QIIME and causal inference (heirarchical clustering by Manhattan distance & metagenomic biomarker discovery by LEfSe). We detected a distinct intestinal microbiome profile in infants with primary BF practice (panel A). Moreover, the infant gut microbiome did not cluster by virtue of Cesarean delivery (A). LEfSe analysis (panel B) identified significantly distinct taxa in unique association with both exclusive BF and BF&FF (LDA score >3.0). Notably, the phylum Firmicutes was enriched in BF neonates (panel C; p=0.0155; Mann-Whitney) while Enterococcus was enriched in BF&FF neonates (p=0.0179; Mann-Whitney). Casual inference analysis of a robust population cohort enabled us to detect a distinct intestinal microbiome associated with breastfeeding practice, rather than mode of delivery. Our findings underscore the importance of incorporating breastfeeding practices when examining the dynamics of the neonatal microbiome.

Comp-034#225 Healthy human microbiota as reservoir of antibiotic resistance genes: a multi- body habitat analysis Yanjiao Zhou(1), John Martin (2), Richard Wilson(3), Makedonka Mitreva (4) 1. Department of Pediatrics 2. The Genome Institute 3. Department of Medicine Washington University School of Medicine, St. Louis, MO, USA Recent metagenomic studies revealed the human gut microbiota is a reservoir of antibiotic resistant genes. However, little is known about the diversity and abundances of antibiotic resistance genes in other body habitats. By leveraging the most comprehensive human microbiome dataset of healthy adults generated by the human microbiome project, we characterize the human microbiome resistome from four body habitats including stool, oral, anterior nares and vagina from ~1500 samples. The resistance genes were identified using a metagenomic shotgun sequencing-based approach. We specifically attempt to address (1) the profiling of antibiotic resistance genes per body site (2) the phylogenetic similarity of antibiotic resistance genes within individuals across different body sites (3) the spatial and temporal distribution of antibiotic resistance genes over 2 years period within specific individuals and (4) the effect of age, gender, geography and other demographic factors on the profile of antibiotic resistance genes. These findings illustrate that the healthy human microbiota in general, beyond the gut microbiota, is a reservoir for antibiotic resistance genes. This reservoir may serve as a mobile resistance gene pool that facilitates the transmission of antibiotic resistance genes. Comp-035#227 A Robust Approach For Identifying Differentially Abundant Features In Metagenomic Samples Michael B. Sohn (1), Ruofei Du (2), Lingling An (1,2) 1. Interdisciplinary Program in Statistics, University of Arizona, Tucson, USA 2. Department of Agricultural & Biosystems Engineering, University of Arizona, Tucson, USA Metagenomics has a great potential to discover previously unattainable information about microbial communities. Detecting differentially abundant features plays a critical role in revealing the contributors (i.e., features) to the status (e.g., disease) of microbial samples. However, currently available methods lack power in detecting differentially abundant features across different conditions. We have proposed a robust procedure to meet with the challenges in detecting differentially abundant features (e.g., species or genes) from metagenomic samples under different biological/medical conditions. The new approach, Ratio Approach for Identifying Differential Abundance (RAIDA), utilizes the ratio between features in a modified zero-inflated lognormal model. Thus it avoids possible issues/concerns that all other existing methods encounter, as these issues are associated with counts or proportions that these methods rely on. In addition, the use of modified zero-inflated model in RAIDA takes care of the undersampling issue that microbial samples undergo. Compared with other existing methods the new approach shows either best or one of the tops in performance in the comprehensive simulation studies. The new method is also applied to a real metagenomic dataset relating to human health. Our findings are consistent with those in previous reports. Comp-036#236 Investigating Microbial Co-Occurrence Patterns Based On Metagenomic Compositional Data Yuguang Ban (1), Lingling An (2), Hongmei Jiang (1) 1. Department of Statistics, Northwestern University, Evanston, USA 2. Department of Agricultural & Biosystems Engineering, University of Arizona, Tucson, USA The high-throughput sequencing technologies have provided a powerful tool to study the microbial organisms living in an environment as a community. Characterizing the interactions among the organisms can give us insights into how they work and live together as a community. Metagonomic data are usually summarized in a compositional fashion due to varying sampling/sequencing depths from sample to sample. We study the co-occurrence patterns of microbial species across multiple subjects using their relative abundance information. Analyzing compositional data using conventional correlation methods has been shown prone to bias that produces artifactual correlations. We propose a novel method to identify significant co-occurrence patterns by finding sparse solutions to a system with deficient rank. To be specific, we construct the system using log ratios of counts data and solve the system using the L1-norm shrinkage method. Our comprehensive simulation studies show that the proposed method outperforms conventional correlation methods in all cases, and achieves higher accuracy than other existing methods when large microbiome samples are provided while controlling the false positives at a pre-specified level. The proposed method is also applied to several real datasets.

Comp-038#248 Integrated omics analyses of the human gut microbiome in a multiplex family study of type 1 diabetes mellitus Anna Heintz-Buschart (1), Patrick May (1), Cédric Laczny (1), Laura Lebrun (1), Linda Wampach (1), Angela Hogan (2), Jochen Schneider (1), Carine de Beaufort (1,3), Paul Wilmes (1) 1. Luxembourg Centre for Systems Biomedicine, Luxembourg 2. Integrated BioBank of Luxembourg, Luxembourg 3. Centre Hospitalier de Luxembourg, Luxembourg Recent phylogenetic and metagenomic studies of human microbiota have provided evidence for links between the composition of the gastrointestinal (GI) microbiome and the development of chronic, inflammatory and autoimmune diseases. In this context, integrated omic analyses are expected to further our understanding of the role of individual microbial community members in health and disease. We have recently developed wet-lab and computational workflows to investigate the GI microbiota by integrated analyses of metagenomic, metatranscriptomic and metaproteomic data. We have applied this workflow within the framework of a multiplex family study into type 1 diabetes mellitus (T1DM), considering also anthropometric and clinical data, as well as records of dietary habits of the study participants. T1DM is a disease associated with genetically triggered auto-immunity, and links to the GI microbiome are currently being pursued. Here, we discuss the results of the analyses of the first four families of the study. To facilitate per-sample integration of biomolecular data, we established a protocol for the sequential isolation of biomolecular fractions (DNA, RNA and proteins) from single faecal samples. By applying this protocol to samples collected over several time points from patients with diabetes and healthy family members, we retrieved high-quality molecular fractions that were subsequently subjected to high-throughput metagenome and metatranscriptome sequencing, as well as metaproteome analysis by liquid chromatography coupled to mass spectrometry. Metagenomic and metatranscriptomic information were combined in a novel assembly workflow, in addition to analysing community structure and activity by assembly-independent techniques. Functional annotation of genes within the assemblies was carried out using HMMs, which we found to be able to assign a 30 % wider taxonomic range with function thanthe commonly used BLAST. While no significant differences in terms of community structures of patients with T1DM and healthy individuals were readily identifiable, differential transcript abundance of functional units were found. These functions with differential enrichments primarily relate to hexose and vitamin metabolism, as well as the interaction of bacteria with the human host and bacteriophages. In a further step, our workflow allows the identification of microbial populations expressing genes for specific functions of interest, e.g. thiamine (vitamin B1) metabolism, which was more active in the microbiota of the healthy sub-cohort. Identified populations included Prevotella spp. and Akkermansia spp. with family-specific patterns in abundance. In conclusion, the presented workflow allows for the in-depth comparative analysis of the functional potential and the activity of whole microbiota, as well as detailed description of functionally important microbial populations. Comp-039#250 Integrative metagenomic analyses of gut microbiota development during infancy Yvonne Vallès (1), Alejandro Artacho (1), Alberto Pascual-García (2), María José Gosalbes (1, 3), M. Pilar Francino (1) 1. Unidad Mixta de Investigación en Genómica y Salud, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (FISABIO)-Salud Pública/Institut Cavanilles de Biodiversitat i Biologia Evolutiva (Universitat de València), València, Spain 2. Centro de Biología Molecular ‘‘Severo Ochoa’’ (CSIC-Universidad Autónoma de Madrid), Madrid, Spain 3. CIBER en Epidemiología y Salud Pública (CIBERESP), Spain Development of the human gut microbiota occurs through a complex process of microbial succession during infancy and early childhood, and has a major impact on life-long health. We are jointly analyzing the patterns of taxonomic and functional change in the gut microbiota during the first year of life for a birth cohort of 13 infants. Metagenomic sequencing shows that individual instances of gut colonization vary in the temporal dynamics of microbiota richness, diversity, and composition at both functional and taxonomic levels, and that the taxonomic composition of the microbiota shapes its functional capacities. Nevertheless, trends discernible in a majority of infants in the cohort indicate that gut colonization occurs in two distinct phases of succession, separated by the introduction of solid foods to the diet. This change in resource availability causes a sharp decrease in the taxonomic richness of the microbiota due to the loss of rare taxa, but the number of core genera shared by all infants increases substantially. Moreover, although the gut microbial succession is not strictly deterministic, we detect an overarching directionality of change through time towards the taxonomic and functional composition of the maternal microbiota. Succession is however not complete by the one year mark, as significant differences remain between one-year-olds and their mothers in terms of taxonomic and functional microbiota composition, and in taxonomic richness and diversity. Different network analyses aimed at reconstructing potential taxa interactions during infant gut microbiota assembly indicate that positive interactions among core genera contribute to ensure their permanence within the community, and to progressively establish an interaction network similar to that of the adult. We detect a substantial expansion of network complexity at the one-year time mark, although the network still remains less extensive than that of the mothers. Interestingly, the network complexity increase in the infant occurs in a delayed manner with respect to the appearance of taxa in the community, as many new interactions are apparent at one year that involve core taxa already present right after the introduction of solid foods. Also, a substantial fraction of interactions are established between genera belonging to the same phylum, especially during the milk-feeding period. In particular, a direct positive link between the Bacteroidales genera Bacteroides and Prevotella is present in the infant networks up to the seven-months timepoint. This positive association is in contraposition to the negative correlation between these two

genera in the adult gut microbiome, and to the fact that their relative abundance is one of the main distinctions among gut enterotypes. This indicates that any competitive interactions between Bacteroides and Prevotella do not initiate during the first year of life and that differentiation into enterotypes would likely also start at a later date. Comp-040#252 Enterotype based determination of statistical power for microbiome case-control studies Bie Verbist (1), Luc Bijnens (1), Federico Matiello (2), Olivier Thas (2), Karoline Faust (3) and Jeroen Raes (3). 1. Quantitative Sciences, Janssen pharmaceutical Companies of Johnson and Johnson, Turnhoutseweg 30 Beerse, Belgium. 2. Laboratory of Molecular Bacteriology (Rega Institute) and Department of Microbiology and Immunology KULeuven, Campus Gasthuisberg, Herestraat 49, Leuven, Belgium. 3. Ghent University, Mathematical Modeling, Statistics and Bioinformatics, Coupure Links 653, 9000 Gent, Belgium. This project focusses on the design of a case control trial to investigate the differences in microbial composition in stool samples of patients with potential precursors of colon cancer versus control subjects. As a starting point of the study design historical real data were used. Statistical power was compared between two different approaches based on a Dirichlet-multinomial distribution according to La Rosa and Brooks et al. (PloS One, 7, 12: e52078, 2012). The Dirichlet-multinomial distribution is described by 2 parameters, the first, π, is a vector of the expected units of genera and the second parameter, θ, indicates the over-dispersion giving an idea about the variation between the subjects within the population. The first procedure considered the definition of the parameters at the study level and the second looked at the inclusion of the enterotype data (Nature, 473, 174-80, 2011) in the power calculations. The results of the comparison suggest a considerable gain in power when stratified tests are applied. Comp-041#255 Unravelling the fate of dietary fibers in the murine cecum. Floor Hugenholtz (123), Katja Lange (134), Guido Hooiveld (134), Michiel Kleerebezem (1356), Hauke Smidt (123) 1. Top Institute Food and Nutrition, Wageningen, The Netherlands 2. Laboratory of Microbiology, Agrotechnology and Food Sciences, Wageningen University, The Netherlands 3. Netherlands Consortium for Systems Biology, Amsterdam, The Netherlands 4. Nutrition, metabolism, and Genomics Group, Division of Human nutrition, Wageningen University, the Netherlands 5. Host-Microbe Interactomics, Wageningen University, the Netherlands The microbiota of the gastrointestinal tract plays a key role in the digestion of our food. Complex metabolic networks of interacting microbes in the gastrointestinal tract of humans and other mammals yield a wide range of metabolites of which the short chain fatty acids, in particular butyrate, acetate, and propionate are the most abundant products of carbohydrate fermentation. So far metabolic networks were documented in in vitro models. In this project interactions between diet, microbiota and host will be quantitatively studied and subsequently modeled using a Systems Biology approach. In initial experiments the cecum of conventionally raised mice on different fiber diets are analyzed. Determinations of the microbiota composition using phylogenetic microarray technology were complemented with metatranscriptome, metabolome and host derived transcriptome data. This revealed distinct activities of bacterial families in the fermentation of fibres into short chain fatty acids. The Bifidobacteriaceae, Lachnospiraceae and Clostridiaceae families are active in glycoside hydrolysis and saccharide transport, while Bacteroidetes and the Erysipelotrichaceae families express mainly glycosidases and the Ruminococcaceae family is mainly active in the sugar transport. All families express in different ratios enzymes involved in the production of short chain fatty acids. Moreover the butyrate producing bacteria correlate with a set of host genes involved in processes such as energy metabolism, transcriptional regulation and immune system. The metadata obtained is expected to result in refinement of our understanding of the interactions between diet, microbiota and host. Comp-043#265 Seeking differentially abundant taxa: Parkinson’s gut microbiome as an example Velma T. E. Aho (1), Pedro A. B. Pereira (1), Lars Paulin (1), Eero Pekkonen (2), Petri Auvinen (1) and Filip Scheperjans (2) 1. Institute of Biotechnology, DNA Sequencing and Genomics Laboratory, University of Helsinki,Helsinki, Finland, 2. Department of Neurology, Helsinki University Central Hospital, Helsinki, Finland A common task encountered in human microbiome studies is looking for specific taxa whose abundance is associated with a variable of interest. There are many different statistical methods that can be used to do this, and a multitude of existing tools that make their implementation easy. In a recently published study we showed that there are differences between the gut

microbiota of patients with Parkinson’s disease and control subjects without parkinsonism, based on pyrosequenced 16S rRNA gene amplicon data. Particularly, bacteria of the family Prevotellaceae appear to be underrepresented in Parkinson’s patients’ feces. The study relied mainly on one method, metastats, for discovering this difference. To further confirm the finding, in this study we re-examine the original data, which consists of bacterial V1-V3 16S rRNA sequences from fecal samples of 72 Parkinson’s disease patients and 72 control subjects, using three additional tools. A secondary goal is to explore how varied the results from these different tools are. The tools used in this comparison are LEfSe, metagenomeSeq, and the implementation of DESeq2 via Phyloseq. The results of each test suggest a lower amount of Prevotellaceae in Parkinson’s patients’ samples, supporting the earlier finding. However, they otherwise produce quite varying lists of taxa, underlining the importance of selecting one’s tools carefully. Comp-044#268 The comparative genomic analysis of Lactobacillus rhamnosus strains, isolated from human gut, saliva and vagina. Ksenia Klimina, Kirill Emelyanov, Natalia Zakharevich, Artem Kasianov, Elena Poluektova, Vsevolod Makeev, Valery Danilenko Vavilov Institute of General Genetics, Moscow, Russia; Essential components of gut microbiota (GM) are the probiotic bacteria of the genus Lactobacillus. L.rhamnosus is a widespread species inhabiting various ecological niches: plants, food, gastrointestinal and urogenital human tracts. L.rhamnosus strains in a human body inhibit the propagation of pathogenic bacteria, contribute to the digestive process, participate in the formation of the innate and acquired immunity; they also participate in communication in gut-brain area. For this reason they constitute a part of many probiotic drugs. Properties of various L.rhamnosus strains significantly differ. The main goal of our work was to study comparative genomics of five L. rhamnosus strains isolated from different part of human body and to determine genes associated with specific body niches. The strains studied were 51В (vagina), К32 and 116 (gut); 24 and 308 (saliva). All strain was originally isolated from healthy adults inhabiting Central Russia. The strain 116 (gut), 308 (saliva) were isolated from one person. The nucleotide sequences of the chromosomes were determined and deposited in GenBank under the accession no.: 51B (JMSI00000000), K32 (JNNV00000000), 24 (JPZB00000000), 116 (JTDC00000000), 308 (JWHC00000000). For the annotation of identified protein sequences we used the Cluster of Orthologous Groups database (COG). The groups are clustered into super-groups called functional groups. The comparative genomic analysis was estimated by the gene and protein composition. All five strains had a large set of shared genes (1536). Difference in genes composition between strains isolated from different niches and genes specific for every niche were determined. We also performed phylogenetic analysis of strains and phylogenetic trees were built. In all five strains 5 genes involved in the metabolism of glutathione were identified. These data indicate that strains may have antioxidant properties. For strain B51 this fact was demonstrated experimentally. The strain L. rhamnosus К32 has been used as a component of probiotic drug. This work was supported by the Ministry of Education and Science of the Russian Federation under state grants 14.N08.12.002 Comp-045#274 Country specificity of the human gut microbiome revealed by comparative metagenomics Suguru Nishijima (1), Wataru suda (1), Hidetoshi Morita (2), Masahira Hattori (1) 1. University of Tokyo, Center for Omics and Bioinformatics, Chiba, 277-8561, Japan 2. Azabu University, School of Veterinary Medicine, Kanagawa, 252-5201, Japan Gut microbiome has profound influences on host’s various physiologies. Recent development of next-generation sequencing technologies (NGS) has enabled us to comprehensively elucidate the overall structure of gut microbiomes. However, little is known about variation and similarity in human gut microbiome among different countries/populations. In this study, we compared metagenomic data of Japanese gut microbiomes with those from other countries including United States, Spain, Denmark, China, Sweden and Russia to explore differences in their ecological and functional features. We generated about 350 Gb filter-passed metagenomic data from fecal DNA samples of 106 Japanese individuals by using Roche 454, Ion PGM/Proton, and Illumina MiSeq sequencers. We obtained about 10.8 Gb assembled sequences, from which about 4.8 million non-redundant unique genes were identified. We then evaluated the microbial composition by mapping of the metagenomic reads on the reference genomes. Comparative analysis showed significantly high inter-country variability in the microbial composition. Randomforest classifier could predict host’s country with 87% accuracy using the composition. Particularly, Japanese gut microbiomes were discriminated from the other countries by the enrichment of Bifidobacterium and depletion of Methanobrevibacter. We also performed correlation analysis between the microbial composition and various environmental factors publically available including diet information (FAOSTAT) and antibiotics consumption, and found significant associations between several species and the factors.

Comp-046#280 Tools for fast and comparative metagenome analysis Kathrin Petra Aßhauer (1), Heiner Klingenberg (1), Robin Martinjak (1), Thomas Lingner (2), and Peter Meinicke (1) 1. Department of Bioinformatics, Institute for Microbiology and Genetics, Georg-August University of Göttingen, Germany 2. Department of Developmental Biochemistry, University Medical Center Göttingen, Georg-August University of Göttingen, Germany Metagenomics has become a standard approach to analyze microbial communities from environmental and clinical samples. In particular, numerous studies of the human microbiome emphasize the increasing importance for biomedical research. However, the vast amount of data demands new bioinformatics tools which can efficiently deal with metagenomic data sets on a large-scale. We developed UProC [1], Taxy-Pro [2], Mixture-of-Pathways (MoP) model [3], and CoMet-Universe to quickly determine and compare the functional and phylogenetic composition of large metagenomic datasets. Taxy-Pro implements a mixture model for inferring the taxonomic composition. The mixture model is based on Pfam protein domain frequencies and allows to assess a metagenome's taxonomic coverage by known organisms in terms of a model quality index. Taxy-Pro is currently among the computationally most efficient taxonomic profiling approaches and is freely available at www.gobics.de/TaxyPro. The MoP model extends the taxonomic mixture model to a statistically adequate modeling of the metabolic potential of metagenomes. To overcome computationally intense homology searches, we implemented a shortcut to estimate the metabolic profile of a metagenome. Here, we link the taxonomic profile of the metagenome to a set of pre-computed metabolic reference profiles. Our results on a large-scale analysis of human microbiome data show the utility of our methods for fast model-based estimation of taxonomic and pathway abundances. Further, the results indicate that the pathway abundances provide a good summary of the functional capacity of a microbial community, well-suitable for the identification of relevant metabolic differences. The CoMet-Universe web-server (http://comet2.gobics.de/) provides a unique platform for comparative analysis of metagenomic sequence data based on protein domain frequencies. CoMet-Universe provides a comprehensive suite for taxonomic, functional and metabolic profiling of metagenomes. The basis for all analyses is a computationally efficient identification of protein domains that allows to process large amounts of unassembled short read data by orders of magnitude faster than with a conventional BLAST-based approach [1]. Beyond the analysis of uploaded metagenome data, in CoMet-Universe the user has the possibility to compare a particular metagenome with more than thousand precomputed profiles. For offline computation, our Pfam domain detection approach (UProC) is available as an open source C library. [1] P. Meinicke. UProC: tools for ultra-fast protein domain classification. Bioinformatics, 2014. [2] H. Klingenberg, K. P. Aßhauer, T. Lingner, and P. Meinicke. Protein signature-based estimation of metagenomic abundances including all domains of life and viruses. Bioinformatics, 2013. [3] K. P. Aßhauer and P. Meinicke. On the estimation of metabolic profiles in metagenomics. German Conference on Bioinformatics 2013, OpenAccess Series in Informatics (OASIcs), 2013. Comp-048#285 SM3: statistical modeling & marker identification from metagenomic data Georg Zeller(1), Shinichi Sunagawa(1), Julien Tap(1,2), and Peer Bork(1) 1. Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany 2. Institut National de la Recherche Agronomique, Metagenopolis, Jouy en Josas, France Studies of microbial communities have been revolutionized by the application of high-throughput sequencing to DNA directly extracted from the environment. A primary goal of analyzing such data is to determine changes in microbial community composition that are associated with environmental factors. For example, linking human microbiome composition to host phenotypes such as diseases has become an area of intense research. Robust statistical modeling and biomarker extraction from microbial community data, however, is a challenging task. Large variation in the abundance range between different community members and in particular large variation (beta-diversity) between different human hosts – even healthy individuals [e.g. Arumugam et al. Nature 2011] – poses challenges for any analysis approach. Ideally, statistical models of host-microbiome associations should allow for quantification of the overall association strength and prediction of host status from microbiome composition to be useful as diagnostic tools. Moreover, for the identification of microbial biomarkers, parsimonious (sparse) models relying on only the most specific associations are desirable. Currently, however, tools with these properties are lacking, despite a number of software packages published for similar purposes [Segata et al., Genome Biol. 2011; Paulson et al., Nat. Methods 2013]. Here we present SM3, a statistical computing framework that relies on sparse linear models for the identification of robust microbial associations and biomarkers. It is able to efficiently handle abundance data from thousands of microbial taxa (e.g. species, genera, or OTUs) or functional community descriptors (gene families or metabolic pathways) estimated in hundreds of samples (using tools such as MOCAT, MetaPhlAn or HUMAnN [Kultima et al., PLoS One 2012; Segata et al., Nat. Methods 2012; Abubacker et al., PLoS Comput. Biol. 2012]). The key component of SM3 consists of linear models that can be optimized by several machine learning techniques, such as LASSO logistic regression [Tibshirani, J. R. Statist. Soc. B 1996] or support vector machines [Fan et al., JMLR 2008], both of which are L1-regularized to ensure that only a small (parsimonious) selection out of the large number of input features is included in the model. This is a distinctive advantage over existing methods [e.g. Segata et al., Genome Biol. 2011] for the interpretation of the model and the selection of microbial biomarkers. Importantly this workflow also avoids a frequently encountered overfitting issue arising in naive two-step procedures that consist of feature selection and subsequent cross-validation of statistical models [see e.g. Chapter 7 of Hastie et al., The Elements of Statistical Learning 2009]. Recently, we have applied the SM3 framework to demonstrate the potential of fecal metagenomics as a novel approach to non-invasive screening for colorectal cancer [Zeller et al., Mol. Syst. Biol. 2014].

Comp-049#286 Genomics analysis of the respiration the microbiota of human intestine Dmitry A. Ravcheev (1), Ines Thiele (1) 1. Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg Due to the features of anatomy and physiology, the human intestine creates the oxygen gradient, which influences the intestinal microbiota. Because of this gradient, human gut provides a variety of ecological niches for both aerobic and anaerobic microorganisms. The intestinal microbiota has been intensively studied during last years; however, respiratory capacities of the gut microbiota have been investigated for only a limited number of model organisms. Here, we present a systematic analysis of respiration genes encoded by the genomes of human gut habitants. Our study included an analysis of genes for ATP synthases, respiratory reductases for electron acceptors, and quinone biosynthesis. We applied our genomic analysis to 254 microorganisms commonly found in the human gut. The investigated genomes belonged mostly to Firmicutes, Bacteroides, Proteobacteria, Actinobacteria, and Fusobacteria phyla of Bacteria. We found ATP synthases of F- and/or V-type in all analyzed genomes. Of the investigated genome, 111 had both F- and V-type ATP synthases, while 132 had only F-type ATP synthases, and 9 had only V-type ATP synthases. Additionally, the reference genomes demonstrated perceptible variations in the distribution of respiratory reductases. The analysis of studied genomes revealed aerobic and anaerobic reductases for tetrathionate, thiosulfate, polysulfide, sulfite, adenylyl sulfate, heterodisulfides, fumarate, trimethylamine N-oxide, dimethyl sulfoxide, nitrate, nitrate, nitrogen oxide, nitrous oxide, selenate, and arsenate. We did not find any genes for the respiration of chlorate, perchlorate, or metals. In addition to previously known terminal reductases, the two novel reductases were predicted. One of these reductases was a microaerobic extracellular one, dependent on flavins and thiols. This enzyme was found in 14 studied genomes. The second predicted reductase was an anaerobic one, reducing thiosulfate. Among the analyzed genomes, this enzyme was found only in the one genome. Based on the distribution of the respiratory reductases for the nitrogen oxides, we found that certain studied genomes did not contain the full set of respiratory enzymes for the reduction of nitrate to ammonia or molecular nitrogen. For the genomes with an incomplete reductive pathway, we predicted a set of exchange interactions. For example, Lactobacillus spp. can only reduce nitrate to nitrite, which in turn could be reduced to ammonia by the other bacteria, such as Bacteroides spp. and Parabacteroides spp. Finally, the analysis of the pathway distribution for ubiquinone biosynthesis and the two alternative pathways for menaquinone biosynthesis revealed four alternative forms for the known enzymes involved in these pathways. Additionally, we predicted three previously enzymes for the menaquinone biosynthesis through futalosine. Taken together, this work substantially expands our knowledge on physiology of the human gut microbiome. Comp-050#293 Associations between intake of vitamin D and other micronutrients and maternal gut microbiota composition at delivery SiddharthaMandal(1), Keith M Godfrey(2), Daniel McDonald(3,4), Tore Midvedt(5), Shyamal D. Peddada(6), Merete Eggesbø(1) 1. Department of Genes and Environment, Norwegian Institute of Public Health, Oslo, Norway 2. MRC Lifecourse Epidemiology Unit and NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust 3. BioFrontiers Institute, University of Colorado at Boulder, USA 4. Computer Science Department, University of Colorado at Boulder, USA 5. Karolinska Institute, Stockholm, Sweden 6. Biostatistics and Computational Biology Branch, National Institute for Environmental Health Sciences, NC, USA While diet is known to have a major modulating influence on gut microbiota, knowledge is limited regarding the specific roles of particular vitamins, minerals and other nutrients in the diet. The composition of the microbiome in pregnant women at delivery has a direct impact on the gut microbiome of the infant as maternal microbes are transferred during passage through the birth canal and initiate the colonization process in the infant. Understanding whether intake of particular nutrients can modulate gut microbiota in pregnant women is thus of importance as it could enable manipulation of infant gut microbiota. In a subset of the NoMic cohort, we examined the relations between intakes of 34 dietary macro- and micronutrients (derived from food frequency questionnaires administered to 60 women in the second trimester) and observed variations in their gut microbiota four days post-partum (assessed through Illumina 16S rRNA microbial analysis). Specifically, we analyzed microbial diversity and quantified shifts in microbial phyla composition in relation to nutrient intakes. Higher estimated intakes of Vitamin D, Retinol and Vitamin E showed the strongest associations with maternal gut microbiota and were associated with significantly decreased microbial alpha diversity. Further, intakes of the same vitamins, as well as variations in saturated, mono-unsaturated, poly-unsaturated and trans- fat intakes were associated with relative increases or decreases in four major phyla, Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Among the strongest findings was the significant shift towards Proteobacteria associated with higher intake of Vitamin D. Trans-fat was also positively associated with a shift towards Proteobacteria. Higher intake of saturated fat was associated with a decrease in all four major phyla, while higher mono-unsaturated fat intake was associated with the exact opposite shifts. The results suggest that the four main phyla in pregnant women have differential sensitivity to dietary intakes vitamins and fat, with Proteobacteria as the most sensitive phylum. At a finer level of classification, using genus-specific data, we also detected modulation of the genera Sutterella and Methanobrevibacter, previously implicated in autism and methane production, respectively. Our results suggest that fat soluble vitamins and different types of fat may be important mediators of gut microbiota composition during pregnancy; randomized interventions studies are needed to confirm effects of particular micronutrients and/or micronutrient-rich foods.

Comp-051#297 A Longitudinal Study of the Crohn's Disease Microbiome During Treatment with Exclusive Enteral Nutrition Christopher Quince (1) Nick Loman (2) Konstantinos Gerasimidis (3) 1 Warwick Medical School, University of Warwick, Warwick, CV4 7AL, UK 2 Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK 3 School of Engineering, University of Glasgow, Glasgow, G12 8LT, UK Background & Aims: Exploring associations between the gut microbiota and colonic inflammation and assessing sequential changes during treatment may offer clues about its role in Crohn’s disease (CD). We characterised the fecal microbiota in CD children, explored correlations with calprotectin and studied changes during exclusive enteral nutrition (EEN). Methods: A total of 117 fecal samples were collected from 23 CD children and 21 healthy controls. Sequencing of the 16S rRNA gene and shotgun metagenomics were performed to characterise the bacterial community structure and genetic functional capacity. Results: Taxonomic profiles distinguished CD patients before EEN from controls (p=0.006). Thirty six genera, 141 operational taxonomic units (OTUs) and 44 oligotypes differed between the two groups. During EEN, microbial diversity decreased and the community structure became even more dissimilar from that of controls. Thirty four genera significantly decreased and one (Lactococcus) increased during EEN. Fecal calprotectin correlated with 35 OTUs, 14 of which explained 78% of calprotectin variation. OTUs which correlated positively or negatively with calprotectin, decreased during EEN. CD microbiota presented a broader functional capacity than controls but this diversity also decreased during EEN. Genes involved in membrane transport, sulfur reduction, biosynthesis of fatty acids, lipids and carbohydrates differed between the two groups. The relative abundance of genes encoding for biotin (p=0.005) and thiamine (p=0.0166) biosynthesis decreased whereas spermidine/putrescine (p=0.0307) biosynthesis and shikimate pathway (p=0.058) increased during EEN. Conclusions: EEN, a highly efficacious treatment for CD, probably acts by modulating the entire microbiota, suppressing inflammation-associated bacteria rather than stimulating abundance of presumably beneficial commensals.

Comp-053#309 In-situ profiling of microbial growth in the human gut Damian R. Plichta (1), Martin F. Laursen (2), Tine Licht (2), H. Bjørn Nielsen (1) 1. Center for Biological Sequence Analysis, Technical University of Denmark, Kgs. Lyngby, Denmark 2. Division of Food Microbiology, National Food Institute, Technical University of Denmark, Søborg, Denmark Growth rate is a key measure used extensively in microbiology. In cultivation experiments it is straightforward to calculate it from optical density. The growth rate is often used to monitor microorganisms’ response to various substrates and conditions and reflect the dynamics of the population. In microbiome, growth rates have largely not been measured as it poses several challenges. 1) It is difficult and costly to re-sample the same microbial population and 2) indirect measures like rRNA or ribosomal abundance in-situ are rarely available and they reflect both activity and growth. Here, we present an alternative method that uses the widely employed shotgun-sequencing data to measure the replication rate from a single snapshot sample. In most bacteria and some archaea initiation of DNA replication occurs at a single region in their circular genome. While the replication fork propagates in both directions along the circular chromosome, the abundance of the origin of replication in a cell will be twice compared to the terminus of replication. For a fast growing species that ratio could be even higher, since more replication forks can be started at the same time. By comparing the ratio of abundance between origin and terminus of replication we calculate the DNA replication rate, which implicitly represents the growth rate of the population. This parameter can be used to (A) distinguish alive (dividing) from dormant or dead populations and (B) profile microbial response to different conditions. In this way we can estimate the replication rate for the species in the human gut and derive sample specific replication rates across 396 human stool samples. Comp-054#314 Gene expression of Bifidobacterium pseudocatenulatum CECT 7765 in response to lactulose-derived galacto-oligosachharides Alfonso Benítez-Páez (1), F. Javier Moreno (2), M. Luz Sanz (3), Yolanda Sanz (1) 1. Microbial Ecology, Nutrition and Health Laboratory. Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), C/ Catedràtic Agustín Escardino 7, 46980 Paterna-Valencia, Spain. 2. Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), CEI (UAM+CSIC). C/ Nicolás Cabrera 9, 28049 Madrid, Spain. 3. Instituto de Química Orgánica General (IQOG-CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain. Genome information from Bifidobacterium strains has become widely available thanks to the next generation of sequencing technologies, but much less is known about gene expression and function associated with diet and host-microbe interactions. Bifidobacterium species are common inhabitants of the human intestine and show a highly specialized ecological adaptation to specific dietary components, including oligo- and poly-saccharides. Indeed, galacto-oligosaccharides exert a bifidogenic effect, enriching the gut microbiota with bifidobacteria. Here, we have investigated the molecular response of B. pseudocatenulatum CECT 7765, a potentially probiotic strain, to lactulose-derived GOS (GOS-Lu) as alternative to glucose as a source of carbohydrates. Using massive sequencing based on Illumina MiSeq technology and DNA sequence analysis we have assembled the draft genome of B. pseudocatenulatum CECT 7765 and, throughout comparative genomics, we have detected certain strain-specific genome regions in this strain, indicating gain-of-functions associated with genome stability and protection against phage infections as well as the presence of a great diversity of genes involved in sugar transport and metabolism. Genome-wide transcriptome analysis revealed over-expression of several genes associated with GOS-Lu fermentation. Of these genes we detected an up-regulation of a wide variety of sugar transporters and permeases as well as five of out seven beta-galactosidases encoded by the B. pseudocatenulatum CECT 7765 genome. Particularly, we found a specific gene cluster where higher fold-change values were observed in response to GOS-Lu as carbon source. Taking into account all these data together with the pattern of GOS-Lu species consumed, analyzed by Gas Chromatography and Mass Spectrometry (GC-MS), we hypothesize such a gene cluster could be specialized in the uptake and hydrolysis of di- and tri-saccharides taken-up by B. pseudocatenulatum CECT 7765. To identify particular genes associated with GOS-Lu fermentation, we have evaluated the global metabolic output of this process. We found that GOS-Lu fermentation activates the Galactose Degradation, Saccharide, Polyol, and Lipid Transport, Antioxidant Response, and DNA Repair processes as well as Purine/Pyrimidine and Branched-chain Amino Acid biosynthesis. The analysis of non-carbohydrate related metabolites resulting from GOS-Lu fermentation is underway to progress in the understanding of the possible generation of bioactive metabolites, such as leucine that could act as nutrient sensor and inducer of satiety and, thereby, play a role in diet-related diseases. Comp-055#330 Mechanistic understanding of polysaccharide-degrading enzymes involved in xylan hydrolysis and utilization in human colonic Bacteroides

Meiling Zhang (1), Jonathan R. Chekan (3) Dylan Dodd (1,4), Pei-Ying Hong (2), Satish K. Nair (3), Roderick I. Mackie (1,2) and Isaac Cann (1,2,4) (1) Institute for Genomic Biology, Departments of (2) Animal Sciences, (3) Biochemistry and (4) Microbiology, University of Illinois, Urbana, IL 61801, USA Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of the majority of the glycan-degrading enzymes are understudied and poorly understood. Initially, to identify the hemicellulose targeting genes utilized by Prevotella bryantii B14, a rumen bacterium that efficiently degrades hemicellulose, the transcriptomes of P. bryantii cultured on either wheat arabinoxylan or a mixture of its monosaccharide components were compared by DNA microarray and RNA sequencing approaches. The most highly induced genes formed a cluster that contained putative outer membrane proteins analogous to the starch utilization system identified in the prominent human gut bacterium Bacteroides thetaiotaomicron. The arrangement of genes in the cluster was highly conserved in other xylanolytic Bacteroidetes, suggesting that the mechanism employed by xylan utilizers in this phylum is conserved (Dodd et al. 2010, J. Biol. Chem. 285:30261). To extend our findings in the human colonic Bacteroides, we selected B. ovatus ATCC 8483 and B. intestinalis DSM 17393 for further study (Zhang et al. 2014, PNAS 111:E3708). Each strain was cultured on either wheat arabinoxylan or xylose. Cells were harvested at mid-log growth phase, total RNA extracted and submitted for RNA-Seq analysis using the Illumina HiSeq 2000 platform. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (Bixyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system, and also previously identified in P. bryantii B14 (PbXyn10C). The BiXyn10A had a modular architecture consisting of a GH 10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBM’s) with affinity for xylose- configured oligosaccharide ligands, the substrates of the BiXyn10A catalytic activity. Mutational analyses confirmed the importance of these modules for xylan degradation by the GH10 enzyme. Crystal structures of CBM1 from BiXyn10A (1.8 Å), a co-complex of BiXyn10A CBM1 and xylohexaose (1.13 Å) and the CBM from its homolog in P. bryantii B14 Xyn10C (1.68 Å) revealed an unanticipated mode for ligand binding that pinches the xylan substrate inducing a kink in the backbone for subsequent enzymatic hydrolysis. More importantly, a minimal enzyme mix comprised of the four most highly up-regulated genes during growth on wheat arabinoxylan depolymerized the polysaccharide to its component monosaccharides. These studies provide a mechanistic understanding of energy capture from fiber by human colonic Bacteroides that are important in human gut health, as well as the breakdown of hemicellulose, a major structural polysaccharide constituent of plant biomass. Comp-056#333 Human intestinal microbiota dynamics and stability in large population cohorts Leo Lahti (1,2), Anne Salonen (3), Jarkko Salojärvi (1), Marten Scheffer (4), Willem M de Vos (1,2,3) 1. Department of Veterinary Biosciences, University of Helsinki, Finland 2. Laboratory of Microbiology, Wageningen University, The Netherlands 3. Department of Bacteriology and Immunology, Immunobiology Research Program, Haartman Institute, University of Helsinki, Finland 4. Department of Aquatic Ecology, Wageningen University, The Netherlands The diverse microbial communities living in the human gut have a profound impact on our physiology and health. Although the composition and function of these microbial communities have been studied extensively, we have only a limited understanding of the temporal dynamics governing this complex ecosystem. The available longitudinal time series remain limited to relatively small numbers of individuals or time points. Together with the remarkable individual variation in microbiota composition and dynamics this sets challenges for statistical analysis. We demonstrate how combining information across individuals and the accumulating background data collections helps to uncover population-level dynamics that extend beyond individual variation. We combine multiple short time series from a number of individuals with deep phylogenetic profiling of the intestinal microbiota in a thousand healthy western individuals from the phylogenetic HITChip microarray database to assess the dynamics and stability in specific taxonomic groups. A systematic analysis of a thousand species-like bacterial phylotypes that represent majority of the known intestinal microbial diversity indicates that in parallel to the dominating gradual variation in bacterial abundances, specific subpopulations within the intestinal microbiota exhibit contrasting, stable configurations of low and high abundance that are associated to host physiology and health. The bi-stable variation in these sub-communities is often masked by continuous variation in more abundant taxa and hence easily overlooked in ecosystem-level analyses (Fig. 1). The bi-stable sub-communities vary quite independently and are frequently observed in a host in various combinations. Moreover, they appear robust to dietary interventions and exhibit notable differences in their stability and contributions to the overall community composition. We report that host factors such as age can affect the resilience of the alternative states and move the system towards a tipping point of an abrupt switch between the contrasting states. Establishing links between community composition, stability and host health presents a fundamental challenge for microbiome studies. We discuss the emerging approaches based on stochastic nonlinear models and propose how combining information across multiple sources and targeting specific sub-populations can simplify the characterization and possible manipulation of the intestinal microbiota.

Comp-057#337 Metatranscriptomics of colonic lesions in inflammatory bowel disease Feargal Ryan (1,2), Emilo Laserna (1,2), John O’callaghan (1), Ian B Jeffery (1,2), Aldert Zomer (3), Aine Fanning (2), Fergus Shanahan (2), Marcus J Claesson (1,2) 1. School of Microbiology, University College Cork, Ireland 2. Alimentary Pharmabiotic Centre, University College Cork, Ireland 3. Radboud University Nijmegen Medical Centre, The Netherlands Crohn’s disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) characterized by chronic and relapsing inflammation of the gastro-intestinal tract (GIT). They cause lifelong suffering, as well as considerable drainage of health care resources. Although their aetiology is still unclear there is a growing body of evidence for a significant microbial factor. Previous research in this area has focused on examining the microbiota composition in stool and in the GIT, producing conflicting and inconclusive results. Here, we adapt a new approach and focus on the metatranscriptome through RNA sequencing of colonic biopsies. Biopsies were collected from inflamed and non-inflamed colonic mucosa from 6 CD and 12 CD patients and sequenced using RNA-Seq using Illumina HiSeq at 15Gb/sample. Raw reads were quality filtered and trimmed using Trimmomatic before aligning to the human genome (hg20) with STAR. The SILVA database along with Bowtie2 was used for identifying and removing rRNA sequences. Remaining reads were aligned using bowtie2 against a non-redundant gene catalogue constructed from multiple previously published metagenomic studies of the human GIT. DESeq2 was subsequently used to analyse the count data and identify differentially expressed genes. This led to the finding of microbial genes which are significantly differentially expressed between inflamed and non-inflamed mucosa in bacterial species. Furthermore the count data from these samples show a clear distinction between bacterial gene expression of UC and CD. Thus, our analysis has revealed a clear difference in the gene expression of bacteria in the colon of IBD patients, and demonstrated that novel approaches are required in order to understand complex multi-factorial diseases. Comp-058#353 Metatranscriptomics unravels the social life of periodontal microbes Szymon P. Szafrański (1), Zhi-Luo Deng (1), Jürgen Tomasch (1), Michael Jarek (1), Sabin Bhuju (1), Jan Kühnisch (2), Helena Sztajer (1), Irene Wagner-Döbler (1) 1. Helmholtz Centre for Infection Research, Braunschweig, Germany 2. Ludwig-Maximilians-University München, München, Germany Periodontal disease is the most prevalent inflammatory disease worldwide and is associated with a number of systemic conditions. It is caused by a subgingival microbial community that interferes with the host immune system, resulting in tissue destruction and bone loss. Here we studied the activity of the subgingival community in periodontal health and disease in vivo using a metatranscriptomic approach. We identified a shift in the active community from Bacilli and Actinobacteria highly active in health, to Bacteroidia, Deltaproteobacteria, Spirochaetes, and Synergistetes dominating gene expression in disease. The linear discriminant analysis (LDA) effect size algorithm (LEfSe) identified “red complex bacteria” – i.e., Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia – but also numerous other species as key players in disease. Streptococcus species were associated with health. We observed a shift from COGs (clusters of orthologous groups) related to carbohydrate transport and catabolism in health to those related to protein degradation and amino acid catabolism in disease. The expression of iron acquisition systems, chaperones, the cobalamin synthesis pathway, and cell motility related genes was also increased in disease, and at least five species expressed genes coding for enzymes involved in pathways leading to butyrate production, in contrast to Fusobacterium nucleatum alone in health. We discovered and evaluated four gene biomarkers – coding for: succinate-semialdehyde dehydrogenase, heme binding protein HmuY (both from P. gingivalis),

flagellar filament core protein FlaB3 from T. denticola and repeat protein of unknown function from F. alocis – that can be applied to diagnose periodontal disease. The commensal Prevotella nigrescens up-regulated the expression of putative virulence factors in periodontal disease, e.g. genes encoding metalloproteases and the heme transporter. This observation supports the polymicrobial synergy and dysbiosis model where the key-stone pathogens are able to transform commensals into accessory pathogens during dysbiosis. Comp-059#358 Microbiome-based biomarker discovery for periodontitis diagnosis Zhi-Luo Deng (1), Szymon P. Szafrański (1), Jürgen Tomasch (1), Michael Jarek (1), Helena Sztajer (1), Irene Wagner-Döbler (1) 1. Helmholtz Centre for Infection Research, Braunschweig, Germany Periodontitis is an extremely widespread oral disease in the population which causes progressive loss of the alveolar bone around the teeth and can even lead to tooth loss. Since there are no apparent symptoms in the early stage of periodontitis, timely diagnosis remains a big challenge. The oral microbiome might be a promising source of clinical biomarkers for the early stage diagnosis of periodontal disease. Here, the metatranscriptome data of the periodontal pocket communities from 10 healthy individuals and 4 patients with periodontitis were used to discover biomarkers. After quality control and rRNA removal, 25.1 million reads in 14 samples were able to be mapped to 366,055 ORFs from HOMD database. Besides, a taxonomy assignment was applied to investigate the overall transcriptional shift of each taxon in the oral microbial community across health and disease using Kraken. In order to identify disease relevant genes, a differential expression analysis and a feature selection process were performed on these 14 samples. The top 100 ranked genes were selected from the 2000 most highly expressed genes by using LEfSe in accordance with the LDA score and p value at the 0.01 level. Finally, from these 100 genes 4 potential biomarker genes were discovered by a recursive feature elimination (RFE) process and a random forest (RF) feature importance evaluation step in terms of the distinction of gene profiles across healthy and sick individuals. PCA analysis for these 4 genes showed a significant difference between healthy individuals and patients. To further evaluate the discriminatory ability of these 4 biomarker candidates, a support vector machine (SVM) classification model with linear kernel built on the basis of the 4 potential biomarkers was tested on 20 samples consisting of 14 training samples and an external dataset with 6 samples from another recent study (Jorth et al. 2014). Nineteen samples (95% accuracy) were correctly classified including all training samples and 5 external samples except the second external test sample which was considered as an outlier according to the PCA analysis. Hopefully, these discovered 4 biomarker candidates will enable the diagnosis of the disease in an early stage and help us gain more insights into the underlying mechanisms of periodontitis. Comp-060#360 An integrative computational framework for identifying taxonomic drivers of functional shifts in the human microbiome Ohad Manor (1) and Elhanan Borenstein (1,2,3) 1) Department of Genome Sciences, University of Washington, Seattle WA 98102 2) Department of Computer Science and Engineering, University of Washington, Seattle WA 98102 3) Santa Fe Institute, Santa Fe NM 87501 The human microbiome is tightly linked to our health and alterations to its composition have been implicated in numerous diseases, calling for the identification of specific intervention targets and therapeutic routes. To detect such targets, comparative surveys of the microbiome have characterized both taxonomic and functional shifts that may be associated with disease. To date, however, taxonomic shifts and functional shifts are often studied in isolation, and a comprehensive framework for linking disease-associated shifts in the functional profile of the microbiome to shifts in the taxonomic profile is lacking. This gap hinders our ability to fully understand taxonomic determinants of functional shifts. It is not clear, for example, whether functional shifts in the microbiome can be explained solely by shifts in the abundances of specific taxa, and whether the same set of taxa drive shifts in multiple functions? Most importantly, without a formal framework for linking taxonomic and functional shift, our ability to rigorously quantify the contribution of each taxon to specific shifts in functional capacity and ultimately to detect taxa that could be targeted in order to restore desired microbiome-level functional capacity, is limited. To address this challenge, here we introduced a novel statistical and computational framework, termed FiShTaCo, which aims to link taxonomic and functional profiles in a comparative analysis scenario, and to decompose functional shifts observed in a specific disease into individual taxa-level contributions. Importantly, our framework relies on a permutation-based approach to preserve overall community taxonomic characteristics, thereby accounting not only for variation induced by each taxon but also for the way this variation affects community-wide context. Our framework additionally employs carefully designed normalization and scaling schemes to maintain the compositional aspect of the data. Finally, to account for inter-species statistical dependencies, our framework can linearize the obtained taxa contribution profile, utilizing a sophisticated game-theory mathematical technique for optimally estimating the contribution of each player in a multiplayer game. Applying this framework to a set of samples from the Human Microbiome Project, we first demonstrate that it can successfully identify the taxonomic drivers that underlie functional shifts across different body sites. We then apply this framework to a previously characterized type 2 diabetes cohort, demonstrating that it can shed light on disease-associated functional shifts by

pinpointing specific microbial clades that drive these shifts. This analysis confirms, for examples, that seemingly similar functional shifts are in fact driven by a completely different set of microbial taxa. We finally incorporate statistical inference of taxon-specific genomic content into our framework, facilitating the discovery of novel drivers of microbiome functional shifts.

EVIDENCE FOR CAUSE AND EFFECT IN MICROBIOME DISEASES

Evi-001#362 Gut microbiota and lipid metabolism in humans Jingyuan Fu1,2 , Marc Jan Bonder1, Maria Carmen Cenit1, Ettje Tighchelaar1, Floris Imhann3, Rinse K. Weersma3, Lude Franke2, Tiffany W. Poon4, Ramnik J. Xavier4,5,6, Dirk Gevers4, Marten H. Hofker1,*, Cisca Wijmenga2,, Alexandra Zhernakova1 1. University Groningen, University Medical Center Groningen, Department of Pediatrics, Groningen, the Netherlands 2. University Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands 3. University Groningen, University Medical Center Groningen, Department of Gastroenterology and Herpetology, Groningen, the Netherlands 4. Broad Institute of MIT and Harvard, Cambridge, MA02142, USA 5. Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, USA 6. Center for Computational and Integrative Biology, Massachusetts General Hospital and Harvard Medical School, Boston, USA Introduction: Lipids are risk factors for many diseases, including obesity, diabetes and cardiovascular disease. Previous studies have shown that lipid levels can be affected by an individual’s genetic make-up, and that intestinal microbiota has an effect on metabolic state. In this work we aimed to investigate the combined effect of gut microbiome and host genetics on human lipid metabolites. Methods: After filtering the subjects with antibiotic or lipid-lowering medication, this study included 893 individuals from a population cohort LifeLines Deep. In all samples the blood lipid metabolites (LDL, HDL, triglycerides (TG) and total cholesterol) were measured. 157 SNPs that modify lipid levels were directly genotyped or imputed from genome-wide SNP platforms, and combined in the risk score. Microbiome composition was accessed by 16S rRNA gene sequencing method. We clustered the reads using QIIME and GreenGenes May 2013 as reference. These reads were referred as bacterial operational taxonomic units (OTUs) and the total number of OTU reads per sample was further rarefied to 15,000. We developed the 2-part analysis model to account for both binary (presence/absence) and qualitative (different abundance level) features of OTUs. The variation of lipids explained by genetics risk and gut microbes were estimated using machine-learning technique with 80% random samples as discovery and 20% as validation set. Results: After adjusting for age and gender, we identified 114 OTUs associated with TG; 34 OTU for HDL and 66 for body mass index at false discovery rate < 0.05. Gut microbiota can explain 5.5% triglycerides and 2.9% for HDL. Interestingly, gut microbiota don’t seem to contribute the variation in low-density lipoprotein cholesterol (LDL) and total cholesterol level. We also did not observe strong interaction between genetics and gut microbiome in the respect to human lipids. Conclusion: Gut microbiome can have similar strong effect on human lipid metabolism as human genome (2.9% TG variation explained by genetics but 5.5% explained by gut microbiome). We did not observe strong association between host genome and gut microbiomial composition. Host genome and gut microbiome, together with age and gender, collectively explain 18% variation in TG and 26.4% variation in HDL. These results provided rational for developing microbiome-targeting therapy. Evi-002#46 Active and IgA-coated fractions of gut microbiota of patients with Clostridium difficile infection Mária Džunková* (1,2), Giuseppe D'Auria* (1,2), Alejandro Artacho (1,2), Jorge Vázquez-Castellanos (1,2), Xinhua Chen (3), Ciaran Kelly (3), Andrés Moya (1,2) 1. Área de Genómica y Salud, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO-Salud Pública), Avenida de Cataluña 21, 46020 Valencia, Spain - Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universitat de València, C/ Catedrático José Beltrán 2, 46980 Paterna – Valencia, Spain 2. CIBER en Epidemiología y Salud Pública (CIBEResp), C/ Melchor Fernandez Almagro 3-5, Madrid, Spain 3. Department of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston (MA), USA The human immune system recognizes pathogens as well as commensal bacteria. Independently on health or disease, there are always 25-75% of bacteria coated by human IgA. Gut bacteria can be also divided into groups according to the different levels of their RNA activity; the species composition of the most active bacterial group is influenced by antibiotics. One of the gastrointestinal complications associated to antibiotics is nosocomial Clostridium difficile infection (CDI). We aimed to investigate how medical treatment influences bacterial composition of active or IgA-coated bacterial fractions and how microbial disbiosis in these fractions influence onset of CDI. We analyzed fecal samples of 24 patients, 12 of them were positive to C. difficile toxin. The influence of antibiotic types taken by the patients, other intestinal complications (as ulcerative colitis or celiac disease) as well as chemotherapy treatment was studied, too. Bacteria (1) coated/not coated with human IgA and (2) active/inactive bacteria were sorted by FACS and their 16S amplicons were sequenced by Illumina MiSeq. The association of the bacterial composition of the four sorted fractions with medical data, including C.difficile counts from the original non-sorted samples, has been analyzed by Bayesian networks. The bacterial composition of the four fractions significantly differed (p-value less than 0.05). Faecalibacterium and Lactobacillus were significantly associated with active fraction of CDI negative patients, suggesting that they possibly protect against CDI. Clostridium XI (cluster where C. difficile belongs) was associated with recurrent CDI in both active and inactive fractions, however its co-occurrence in the two fractions can be explained by the administration of different types of antibiotics. For example, Clostridium XI was found to be significantly active in patients who took antibiotics inhibiting protein synthesis (previously associated with CDI), however, it was found in the inactive (dying) fraction in patients that apart from antibiotics

associated with CDI took also vancomycin and metronidazol which are antibiotics used to combat CDI. If fractionation of the gut microbiota had not been used, the exact influence of antibiotics on bacterial composition would have remained undiscovered. Clostridium XI was found to have significantly increased presence in IgA coated fraction in CDI positive patients, while in contrast, in CDI negative patients, Clostridium XI was significantly increased in fraction of bacteria not coated by human IgA. In conclusion, fractionation of gut microbiota detects species which are active and being recognized by human immune system under influence of antibiotics and C. difficile toxins. The information about the bacterial activity and about opsonization of bacteria by the human IgA can explain discrepancies which are usually observed in bacterial composition studies where no fractionation of bacteria is used. Evi-004#27 Metagenomic analysis of human gut microbiome in acute on chronic liver failure Jing Guo(1),Nan Qin(1), Lihua Guo(1), Yanfei Chen(1), Guirong Qian(1), Daiqiong Fang(1),Ding Shi(1), Xinjun Hu(1), Fengling Yang(1), Lanjuan Li(1) 1, The First Affiliated Hospital, School of Medicine,Zhejiang University Object: There is closely relationship between liver and gut. The variation of gut microbes in acute on chronic liver failure is unknown. The aim of this study was to compare the intestinal flora between acute on chronic liver failure patients and healthy individuals, to investigate the relationship between acute on chronic liver failure and the intestinal flora. Methods：28 acute on chronic liver failure patients and 28 controls were selected. Their gut microorganism were extracted. High-throughput sequencing method with bioinformatics analysis were used to reveal the variation of phylogenesis and functional genomics in acute on chronic liver failure patients. Result : We got an average of 4.49 GB data per samples, 252 GB in total. Through the enterotype analysis, we found that there are three kinds of enterotype, driven by the genera Bacteroides, Prevotella and Veillonella respectively. 67303 gene markers specific to ACLF were find. 35562 were enriched in ACLF group, and 31741 were in control group. Then gene markers were clustered, and 27 clusters were found. 12 patient-enriched clusters were closely related with CTP. Most of the clusters enriched in the patients group were from Veillonella. We also identified 1,791 KEGG orthologues and 22,200 eggNOG orthologues associated with the disease. The most enriched orthologues in patients group were transcription and translation, replication and repair. Drug resistance genes and infectious disease genes were also enriched in ACLF group. Conclusion: Significant differences between patients with ACLF and healthy individuals in fecal microbial community and functions were detected. Veillonella were prevail in patients with ACLF, and may affect the prognosis of ACLF, and may be able to predict the risk of ACLF. Evi-006#31 Alterations of Bacteroides sp., Neisseria sp., Actinomyces sp. and Streptococcus sp. in oropharyngeal microbiome are associated with liver cirrhosis and pneumonia Zhang-Hua(1),Lu-Hai Feng(2), Qian-Gui Rong(3), Zhang-Chun Xia(4), Ren-Zhi Gang(5) State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, P.R. China. Objectives: Human microbiomes are associated with liver and lung inflammation. We identified and verified alterations of oropharyngeal microbiome, and assessed their potential associations with cirrhosis and pneumonia. Methods: The study included three parts: (1) confirmation of temporal stability of oropharyngeal microbiome in a follow-up study; (2) identification of oropharyngeal microbial variation in 90 subjects; (3) quantitative verification of disease-associated bacteria. Whole genome amplification (WGA) enriched DNA from low-biomass oropharyngeal swabs for DGGE analysis. Results: Combination of WGA and DGGE successfully monitored oropharyngeal microbial variations, and oropharyngeal microbiome in each subject kept relatively stable during follow-up periods. Microbial composition in cirrhotic patients with pneumonia differed from others and respectively clustered together by the subgroup through cluster analysis. Meanwhile, Species richness, Shannon’s diversity and evenness index increased obviously in cirrhotic patients with pneumonia versus others, respectively (all p

Evi-007#34 Altered Fecal Microbiota Composition in Patients with Major Depressive Disorder Haiyin Jiang (1), Zongxin Ling (1), Yonghua Zhang (2), Hongjin Mao (2), Yan Yin (2), Wenxin Tang (2), Zhonglin Tan (2), Jianfei Shi (2), Lanjuan Li (1), Bing Ruan(1) 1. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China 2. Department of Psychiatry, The Seventh People’s Hospital of Hangzhou, Hangzhou, Zhejiang, China Increasing preclinical evidence suggests that exposure to a stressor can influence the diversity of the gut microbiota, but its actual structure and composition in patients with major depressive disorder (MDD) remain unclear. Microbial diversity and composition were analyzed by parallel barcoded 454 pyrosequencing targeting the 16S rRNA gene hypervariable V1-V3 regions in the feces of 46 patients with depression (29 active-MDD and 17 responded-MDD) and 30 healthy controls (HCs). A significant increase in bacterial α-diversity was noted in patients with active-MDD (A-MDD) vs. controls by the Shannon index (P < 0.05) but not in responded-MDD (R-MDD) vs. controls. We also showed that several key MDD-associated bacterial phylotypes changed significantly in the fecal microbiota of patients with MDD. The Bacteroidetes and Proteobacteria phyla were significantly more abundant, whereas the Firmicutes phylum was significantly less abundant in the A-MDD group compared to those in HCs (P < 0.05). MDD-reduced phylotypes, Faecalibacterium, were negatively correlated with the severity of depressive symptoms. Fecal microbial communities differ between A-MDD patients and healthy individuals; improvement in depressive symptoms could partially restore normal bacterial composition. The presence of potentially pathogenic bacteria such as Enterobacteriaceae, and reduced prevalence of beneficial populations such as Faecalibacterium might contribute to the interaction between gut microbiota and depressive symptoms.

Figure 1: (A) Comparison of bacterial diversity by Shannon index among three groups. (B) Comparison of relative aboupled

with effect size measurements identifies the most differentially abundant taxons between A-MDD and healthy controls.

Healthy control-enriched taxa are indicted with a positive LDA score (green), and taxa enriched in A-MDD have a negative

score (red). Only taxa meeting an LDA significant threshold of > 2 are shown. (D) A cladogram representation of data

shown in panel A. red, A-MDD-enriced taxa; green, taxa enriched in healthy controls. The brightness of each dot is

proportional to its fect size. (E) Linear discriminant analysis (LDA) coupled with effect size measurements identifies the

most differentially abundant taxons between R-MDD and healthy controls. Healthy control-enriched taxa are indicted with a

positive LDA score (green), and taxa enriched in R-MDD have a negative score (red). Only taxa meeting an LDA

significant threshold of > 2 are shown. (D) A cladogram representation of data shown in panel A. red, R-MDD-enriced taxa;

green, taxa enriched in healthy controls. The brightness of each dot is proportional to its effect size.

Evi-008#35 NKT cells play a role in modulating ConA-induced hepatitis by gut microbiota Jianing Chen(1, 2), Yingfeng Wei(1, 2), Guangying Cui(1, 2), Yunan Zhu(1, 2), Chong Lu(1, 2), Yulong Ding(1, 2), Toshimitsu Uede(3), Lanjuan Li(1, 2), Hongyan Diao(1, 2) 1. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China 2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China 3. Molecular Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Background: Gut microbiota are implicated in many liver diseases. Concanavalin A (Con A)-induced hepatitis is a well-characterized murine model of fulminant immunological hepatic injury. The correlation between intestinal microbial alteration and immunological hepatic injury, particularly the influence of intestinal microbial alteration on immune cell activation and migration in the intestine and liver, remains obscure. Methods: Oral administration of pathogenic bacteria or gentamycin given to the mice before ConA injection, liver injury, lymphocyte distribution and activation in liver and intestine were assessed. Results: Our data show that administration of pathogenic bacteria exacerbated the liver damage. There was more downregulation of activation-induced natural killer T (NKT) cells in the liver of pathogenic bacteria-treated ConA groups. Also, there was a negative correlation between the numbers of hepatic NKT cells and liver injury in our experiments. Moreover, intestinal dendritic cells (DCs) were increased in pathogenic bacteria–treated ConA groups. The activation of DCs in Peyer’s patches and the liver was similar to the intestine. However, depletion of gut gram-negative bacteria alleviated ConA-induced liver injury, through suppressed hepatic NKT cells activation and DCs homing in liver and intestine. In vitro experiments revealed that DCs promoted NKT cell cytotoxicity against hepatocyte following stimulation with pathogenic bacteria. Conclusion: Our study suggests that increased intestinal pathogenic bacteria facilitate immune-mediated liver injury, which may be due to the activation of NKT cells that mediated by intestinal bacterial antigens activated DCs. Evi-009#36 Intestinal bacterial translocation in rats with cirrhosis is related to intestinal immune dysfunction Haiyan Shi, Longxian Lv, Lanjuan Li 1.State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, Zhejiang University, Hangzhou, PR China. 2.Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China Background The development of spontaneous bacterial infections in liver cirrhosis is associated with a high rate of bacterial translocation (BT) from the gut to mesenteric lymph nodes (MLNs) and/or extraintestinal organs. Most translocating bacteria belong to the common gut flora, suggesting a breakdown of intestinal defense function. Attempts to explain the high rate of BT in cirrhosis include intestinal barrier function damage, which leads to increased intestinal permeability and alterations in

Figure 2: (A) Correlation between BDNF and the relative abundance of the genera Clostridium XIVb. (B) Correlation

between IL-6 and the relative abundance of the genera Lachnospiraceai ncertae sedis. (C) and (D) Correlation between

severity of depressive symptoms and the relative abundance of the genera Faecalibacterium. The spearman rank correlation

(R) and probability (P) were used to evaluate statistical importance. !

intestinal flora. However, the potential contribution of another component of the intestinal barrier, specifically the immune system, to BT has not been addressed so far. Aim To delineate immunological defense mechanisms involved in the process of BT in Cirrhosis. Methods In this prospective study, cirrhosis was induced in male pathogen-free Sprague Dawley rats (200-220 g initial weight) by subcutaneous injections in the dorsal region twice a week CCl4 administration. Phenobarbital (0.35 g/L) was given in drinking water. BT was defined as the growth of bacteria in MLNs culture. T cells were isolated from the intestinal lamina propria and analyzed by flow cytometry. Results In the cirrhotic rat with BT, there is marked submucosal edema , organization structure disorders and inflammation of the intestines. BT to MLNs did not occur in any of the normal control rats. BT was detectable in 46% of cirrhotic rats with ascites. In addition, in the cirrhotic rat with BT, CD3+T and CD4+T cells were significant reduced in the small intestine and colon, CD8+T cells were increased. Conclusions Compromised gut immunity seems to predispose to BT in experimental rat cirrhosis. Understanding this gut associated lympatic tissue immunity including the underlying mechanisms could help us to find new treatment avenues. Evi-010#39 Implication of commensal microbiota in anxiety and alzheimer's disease Mei-Ling Liu (1,2,3), Ben-Hua Zeng (4), Hong Wei (4) & Peng Xie (1,2,3) 1. Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China 2. Chongqing Key Laboratory of Neurobiology, Chongqing, China 3. Institute of Neuroscience, Chongqing Medical University, Chongqing, China 4. Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China Recent evidences suggest that commensal microbiota could influence brain function and behaviors. However, there is a paucity of data pertaining to the influence of commensal microbiota on hippocampal redox homeostasis. Here, we measured the expressions of 84 oxidative stress and antioxidant defense-related genes in the hippocampus of germ-free (GF) mice and specific pathogen free (SPF) mice (n=9 for each group). Totally, 16 genes were differentially expressed between the two groups. Compared to SPF mice, the GF mice were associated with upregulation of RecQ protein-like 4, stearoyl-coenzyme A desaturase 1, glutathione peroxidase 6, glutathione S-transferase pi 1, EH-domain containing 2, peroxiredoxin 6, adenomatosis polyposis coli, apolipoprotein E, superoxide dismutase 1, thyroid peroxidase, uncoupling protein 3 and neuroglobin, and downregulation of flavin containing monooxygenase 2, nitric oxide synthase 2, NADPH oxidase 1 and glutathione peroxidase 2. Majority of upregulated genes were involved in antioxidant defense, while downregulated genes mainly exerted prooxidant activities. These altered genes were highly enriched for gene sets related to anxiety and Alzheimer’s disease. Moreover, behavioral analyses confirmed that GF mice displayed reduced anxiety and Alzheimer-like behaviors compared with SPF mice. Interestingly, constitution of microbiota to adolescent GF mice enabled normalizing 62.5% of aberrant gene expression and reducing enrichment of anxiety and Alzheimer’s disease genes, but was insufficient to reverse the abnormal behavioral phenotypes. These results demonstrated that hippocampal redox homeostasis could be profoundly influenced by commensal microbiota, which may be associated with onset of anxiety and Alzheimer’s disease. Evi-011#40 Gut microbiota metabolites in term infants at 3-4 months of age according to breastfeeding status Sarah L Bridgman (1), Petya Koleva (1), Meghan B Azad (2), Catherine J Field (3), Andrea M Haqq (1), Allan B Becker (2), Stuart E Turvey (4), Piush J Mandhane (1), Padmaja Subbarao (5), Malcolm R Sears (6), David S Wishart (7), Anita L Kozyrskyj (1), and 1. Department of Pediatrics, University of Alberta, Canada 2. Department of Pediatrics & Child Health, University of Manitoba, Canada 3. Department of Agricultural, Food and Nutritional Science, University of Alberta, Canada 4. Department of Pediatrics, University of British Columbia, Canada 5. Department of Pediatrics, University of Toronto, Canada 6. Firestone Institute for Respiratory Health, St Joseph's Healthcare and McMaster University, Canada 7. Department of Biological Sciences, University of Alberta, Canada 8. Canadian Healthy Infant Longitudinal Development Study The combined genomes of the gut microbiota contain more than 5 million genes and have a metabolic capacity equivalent to the liver. The metabolic function of the gut microbiota is closely associated with diet and has also been linked to disease states such as obesity. As part of a larger study to determine early fecal biomarkers of childhood obesity, this pilot study examines differences in fecal short-chain fatty acids (SCFA) and lactate in infants at 3-4 months of age according to breastfeeding status. Metabolic profiling (using nuclear magnetic resonance (NMR)) of fecal samples taken at mean age 14.8 weeks (±4.38) from 17 infants enrolled in the Canadian Healthy Infant Longitudinal Study (CHILD) general population cohort was conducted. Infant and maternal characteristics, including breastfeeding status at 3 months of age (none, partial breastfeeding, exclusive breastfeeding), were collected using standardized questionnaires. Differences in absolute concentrations of SCFA (acetate, butyrate, propionate, isobutyrate, valerate, isovalerate) and lactate in fecal samples of infants’ (breastfed or not breastfed) at 3 months of age were tested using non-parametric Mann Whitney test. Differences in total concentration and relative proportions

of SCFA were also analyzed. Correlation between extent of breastfeeding exposure (none, partial and exclusive) and SCFA and lactate concentration was also examined using Spearman’s rho. All infants were vaginally delivered with mean gestational age of 39.1 weeks (±1.1) and birth weight of 3547g (±452). No infants were exposed to antibiotics at birth or during the first 3 months of life. At 3 months of age, ten infants (58.8%) were breastfed (4 exclusively, 6 partially) and seven infants (41.2%) were not breastfed. Median concentration of total SCFA was significantly higher in infants who were breastfed at 3 months compared to those that were not breastfed (p=0.01). Median concentration of acetate and lactate were higher (p=0.007 and p=0.003 respectively) and isovalerate lower in breastfed infants (p=0.019). Median relative proportions of isovalerate were significantly lower in breastfed infants (p Evi-012#45 Mycobacterium avium-intracellulare Complex infection mistreated as Mycobacterium tuberculosis infection in immunocomplete person: Two cases Yu haiying(1), Sheng jifang(1) Collaborative innovation center of diagnosis and treatment of infectious diseases Although Mycobacterium tuberculosis (MTB) is still the most important mycobacterium species from a public health perspective in China, the prevalence of human disease caused by nontuberculous mycobacteria (NTM) has increased over the past several years. However, routine detections such as acid-fast staining or mycobacteria culture are still not able to differentiate between M. tuberculosis and NTM, which lead to high misdiagnosis rate and unsatisfactory therapeutic efficacy of NTM diseases. The most common NTM pathogens are Mycobacterium avium complex (MAC), and about 90% of cases involve the pulmonary system; the rest involve lymph nodes, skin, soft tissues, and bones. We reported 2 immunocompetent cases of MAC infections in pulmonary and bone respectively mistreated as MTB infections. Evi-013#52 Analysis of infant with auditory nerve injury and their breast milk infection human cytomegalovirus Yu Luo(1),Kaixian Du(2),Wei Guo(1),Hongmei Zhang(1), Guoxin Zhao(1),Linmin Meng(1) 1.Department of Immunology Research，Academy of Medical and Pharmaceutical Sciences，Zhengzhou University，Zhengzhou 450052，China 2.Department of Pediatrics，the Third Affiliated Hospital,Zhengzhou University，Zhengzhou 450052，China Corresponding author: Yu Luo，Email: 1289299548@qq.com Background The infant is the people infected by human cytomegalovirus (HCMV) easily. HCMV is a kind of weak pathogenic factor. It do not have obvious toxicity for the immune normal healthy individuals, but in the body's immune suppression and the virus proliferation activity,it can cause disease or aggravate the disease easily. The aim of this article is to know and analysis the HCMV infections of the infant with auditory nerve injury quantitatively, and to guide the clinical treatment. Methods The HCMV DNA was detected by FQ-PCR in the 120 cases urine of infants and their breast milk,the infants with auditory nerve injury were admitted in our hospital from January 2013 to April 2014 . Rusults The positive rate (≥5×102 gene copies /ml) of urine was 68.33% (82/120 cases), the cases with 5×102 ,103,104,105,106,107 and 108 gene copies/ml were 5, 11, 15, 28, 16, 5, and 2 cases, respectively.The posities rate (≥5×102 gene copies /ml) of breast milk was 50.33%(64/120cases),the cases with 5×102 ,103,104,105,106 and 107 gene copies/ml were 9,13,16,14,11 and 1 cases, respectively.In 82 infants and 64 breast milk with HCMV, The 54 cases of infants urinary and their breast milk had HCMV at the same time,the percentage was 65.85%(54/82 cases) and 84.38%(54/64 cases)，respectively.In 38 infants urinary with not HCMV（＜5×102 gene copies /ml), the 28 cases of their breast milk had not HCMV ,the percentage was 73.68% . Conclusions The positive rate of HCMV infection was higher than 68.00% at the infant with auditory nerve injury. The amount of virus was mainly in 104, 105 and 106 gene copies/ml in the urine. Breastfeeding with HCMV may be the main way of HCMV to infecte infant.

Evi-014#56 Modulation of gut microbiota dysbioses in type 2 diabetic patients by macrobiotic Ma-Pi 2 diet Elena Biagi (1), Marco Candela (1), Matteo Soverini (1), Sara Quercia (1), Clarissa Consolandi (2), Marco Severgnini (2), Francesco Fallucca (3), Mario Pianesi (4), Paolo Pozzilli (5), Simone Rampelli (1), Silvia Turroni (1), Patrizia Brigidi (1) 1. Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy; 2. Institute of Biomedical Technologies, Italian National Research Council, Segrate, Milan, Italy; 3. Department of Clinical Sciences, La Sapienza University II Faculty, Rome, Italy; 4. International Study Center for Environment, Agriculture, Food, Health and Economics, Rome, Italy; 5. Deparment of Endocrinology and Diabetes, University Campus Bio-Medico, Rome, Italy Type 2 diabetes (T2D) is dramatically increasing in prevalence worldwide and represents a challenging problem for national healthcare systems. Gut microbiota (GM) has been recognized as a key environmental determinant for the onset and progression of T2D due to its capability of modulating host metabolism and immune function. Indeed, T2D subjects show peculiar dysbioses of the gut ecosystem that can favor T2D. Thus, GM has been recognized as a new potential therapeutic target for T2D, and robust dietary interventions strategies to deal with GM deregulation in T2D needs to be defined. In this scenario, the Ma-Pi 2 macrobiotic diet – enriched in whole grains, vegetables and legumes – shows a great potential to modulate GM dysbioses in T2D subject, favoring the recovery of a mutualistic layout. Our study is specifically aimed at exploring the efficacy of Ma-Pi 2 diet in redressing GM dysbioses in T2D subject. Forty T2D patients were enrolled and their GM was compared to that of 16 healthy controls by NGS of the V3-V4 region of the 16S rDNA gene. T2D patients showed strong alterations of the gut microbial ecosystem, being increased in pro-inflammatory Enterobacteriaceae, Eubacterium, Collinsella and Streptococcus and depleted in health-promoting short chain fatty acids (SCFA)-producers, such as Bacteroidetes, Lachnospira, Faecalibacterium, and Roseburia. The overall pro-inflammatory layout of the gut microbial ecosystem detected in T2D patients can contribute to the systemic inflammation, favoring insulin resistance. In a randomized, controlled, open-label 21-day trial, half of the enrolled T2D patients were treated with Ma-Pi 2 diet and half with a standard (control) diet recommended by professional societies for T2D management. At the end of the dietary interventions, the GM of all T2D patients was characterized. Even if both diets were successful in the modulation of the GM layout of T2D patients, Ma-Pi 2 was more effective for the recovery of mutualistic GM components. Indeed, Ma-Pi 2 diet was efficient in boosting SCFA-producers like Faecalibacterium and Bacteroidetes, which were largely depleted in T2D subjects before the intervention, as well as the health promoting Akkermansia. On the other hand, control diet resulted in a further reduction of SCFA-producing Roseburia and Prevotella in T2D patients, favoring an extra increase of pro-inflammatory Enterobacteriaceae and Streptococcus. Supporting the importance the Mi-Pi diet induced GM changes in the recovery of metabolic homeostasis in T2D patients, the intervention with Mi-Pi diet resulted in significantly greater improvements in metabolic control in T2D patients respect to control diet. In conclusion, according to our findings, the Ma-Pi 2 favor a mutualistic GM configuration that can support the recovery of metabolic homeostasis, being a good candidate diet to manage GM dysbioses in T2D patients. Evi-015#169 Dynamic Patterns of Serum Metabolites in Fulminant Hepatic Failure Pigs Analysis by GC-MS Ermei Chen (1) ,Ning Zhou (1), Jianzhou Li (1), Danhua Zhu (1), Lanjuan Li (1) (1)State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases，The First Affiliated Hospital, College of Medicine, Zhejiang University Backgroud: Fulminant hepatic failure (FHF) is still an intractable disease associated with serious metabolic disorder. Investigating the dynamic changes of serum metabolites during the development of FHF will reveal the cooperation between the microbiome and the metabolism of mammalian hosts.Therefore,thisstudycharacterized the dynamic metabonome of serum extracted from FHF pigs with gas chromatography–massspectrometry. Methods: 8 Bama experimental miniature pigs were housed for 7 days for acclimatization, and their food intake were controlled. FHF was induced via intravenous administration of D-galactosamine (1.3 g/kg body weight). Serum samples were collected at four different given time points: before the administration of D-gal (healthy group), at 12 h36hand 48h after its administration, and at death time(death group). Before GC-MS analysis，90uL of MSTFA with 1%TMCS as catalyst was added for derivatization.Serum metabolites were studied with gas chromatography mass spectrometry. Metabolic features were then statistically analyzed using commercial mass spectral library for the peak identification .After peak deconvolution, identification, and matching, the acquired GC-MS data were normalized and processed by principal component analysis (PCA). Results: The result of multivariate analysis based on metabonomic data showed that the whole metabolism pattern of the FHF pigs was distinctive among different groups in the process of FHF pigs. From the result of canonical discriminant analysis, we found that metabonomic data was able to predict the severity of liver failure. We also found that specific biomarker in the metabolic composition of serum samples including aromatic amino acids and glucose were shown in GC-MS total ion current (TIC) chromatograms. This study indicated that the GC-MS technique is an alternative tool for the metabonomic study, and the metabonomics approach promises to provide an integrative criterion to evaluate the severity degree of liver failure. disposal these aromatic amino acids . Since gut microflora imbalance in liver disease has been described previously,it is possible that metabolism disorders had a potential link to the changes in altered gut microflora.

Evi-016#58 Germline HIF2A mutation in a patient with clear-cell variant hepatocellular carcinoma and polycythemia Jiong Yu1*,Hongcui Cao1*, Chunzhang Yang2*, Cody L. Nesvick2, Xiaoru Su1, Pauline Dmitriev2, Zhengping Zhuang2, Lanjuan Li1 1. The State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University；Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,79 Qingchun Rd., Hangzhou City 310003, China 2. Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke (NINDS), National Institute of Health (NIH), 9000 Rockville Pike, Bethesda, MD, 20892, USA * equal contribution. Hypoxia-inducible factors (HIFs) play a major role in regulating oxygen sensing and expression of genes that involved in angiogenesis, erythropoiesis, cell metabolism, proliferation, migration and survival. Mutations in HIF2A have been reported in clinical syndromes consisting of neuroendocrine tumors associated with polycythemia. Here we report a novel germline HIF2A mutation in a patient with hepatocellular carcinoma (HCC) and polycythemia. We further delineated that this mutation resulted in a HIF-2a protein that retained transcriptional activity but was resistant to degradation. HIF-2a target genes EDN1, EPO, GNA14, and VEGFA were significantly upregulated in the tumor bed but not the surrounding liver tissue, indicating that hyperactivation of the hypoxia signaling pathway was confined to the hepatocellular carcinoma. Evi-017#60 Early Bacterial coinfection in H7N9 influenza Meifang Yang，Hainv Gao，Jiajia Chen，Xiaowei Xu，Lingling Tang，Yida Yang，Weifeng Liang，Liang Yu，Jifang Sheng，Lanjuan Li. 1.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University. 2.Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China. No. 79 Qingchun Road, Hangzhou 310003, China. Background: Patients contracting influenza A (H7N9) often develop severe disease. However, information on the contribution of bacterial coinfection to the severity of avian influenza A (H7N9) is limited. Methods: A retrospective study was conducted in 83 patients with confirmed avian influenza A (H7N9) infection from April 2013 to February 2014. Data on bacterial coinfection within the first 72 hours of admission were collected. The severity of patients with bacterial coinfection and those without was compared. Meanwhile we analyzed markers for early diagnosis of bacterial coinfection in influenza A (H7N9) infection patients. Results: Bacterial coinfection was confirmed in 16 of 83 patients (19.3%), and Staphylococcus aureus (4/16, 25%) was the most prevalent pathogen. Higher Acute Physiology and Chronic Health Evaluation II score (25.63 ± 5.30 vs 18.57 ± 8.27, p = 0.000), shock (62.5% vs 28.4%, p = 0.010), renal replacement treatment (81.2% vs 23.9%, p = 0.000), mechanical ventilation (93.8% vs 43.3%, p = 0.000), and extracorporal membrane oxygenation treatment (50.0% vs 20.9%, p = 0.018) were more frequently observed in patients with bacterial coinfection. Elevated procalcitonin was an independent marker for bacterial coinfection. Procalcitonin at a cut-off of 0.81 µg/l had an area under the receiver operating characteristic curve of 0.958 (91.7% sensitivity, 90.2% specificity) for diagnosis of bacterial coinfection. Conclusions: Influenza A (H7N9) infection patients with bacterial coinfection had a more severe condition. Elevated procalcitonin is an accurate marker for diagnosing bacterial coinfection in influenza A (H7N9) patients, thus enabling earlier antibiotic therapy. Evi-018#73 Biode gradable antimicrobial polycarbonates with In vitro and In vivo efficacy against methicillin-resistant staphylococcus aureus(MRSA) infection Junchi Cheng(1), Willy Chin(2), Huihui Dong(1), Liang Xu(1), Yuan Huang(1),Lanjuan Li(1), YiYan Yang (2)and Weimin Fan(1) 1.Department of the State Key Laboratory for Infection Diseases Diagnosis and Treatment, Zhejiang University, Hangzhou, China 2.Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore High prevalence and mortality combined with the threatof multi-drug resistance requires development of new medicines to treat methicillin-resistant Staphylococcus aureus(MRSA). We recently reported a series of biodegradable polycarbonate polymers designed and synthesized via organocatalytic ring-opening polymerization of benzyl chloride-functionalized cyclic carbonate monomer (MTC−OCH2BnCl) followed by quaternization using different quaternizing agents. Among them, two polymerspButyl_20 and pButyl0.5Benzyl0.5_20having degree of polymerization (DP)of 20 synthesized using the quaternization agent N,N-dimethylbutylamineor a mixture of N,N-dimethylbutylamine and N,N-dimethylbenzylamine (1:1 molar ratio), respectively, showed highest selectivity towardS.aureus. In this study, the antibacterial properties of these polymers against clinically isolated MRSA as well as their toxicities were studied bothin vitro andin vivo.Minimuminhibitory concentrations(MICs)of these polymers were demonstrated to be much lower than those of cefoxitinagainst all 31 isolates, but slightly higher than those

of vancomycin. Both polymers showed low hemolytic activity toward human red blood cells (hRBC), making them highlyselective toward MRSA in vitro. A subsequent time-kill study revealed that these polymers had high bactericidal efficiency. Results from a resistance development study also attested to the polymers’ low tendency toward resistance. Furthermore, the in vivo study showed pButyl0.5Benzyl0.5_20 was highly efficacious in a mouse systemic infection model. The administration ofpButyl0.5Benzyl0.5_20to mice further indicated that it did not cause any dysfunctions of liver and kidney as well as blood electrolytes. Taken together, these biodegradable antimicrobial polycarbonates may be potentially useful for future clinical application in combating MRSA. Evi-019#79 Bioartificial liver system ameliorated the enteric dysbiosis in fulminant hepatic failure pigs Liang Yu(1,2), Qiongling Bao(1,2), Lanjuan Li(1,2) 1.State key laboratory for diagnosis and treatment of infectious diseases, the first affiliated hospital, college of medicine, Zhejiang University. 2.Collaborative innovation center for diagnosis and treatment of infectious diseases. Background & Aim: Enteric dysbiosis is frequent in the patients with liver failure and is a clinically adverse event for the progression of liver failure. Bioartificial Liver (BAL) System has been proposed as a promising treatment for advanced liver disease. However the efficiency of BALsystem on adjusting the imbalance of intestinal flora is poorly documented. Methods:Fulminant Hepatic Failure (FHP) was induced by the administration of D-galactosamine, 1.5g/kg body weight, in the male Chineseexperimental miniaturepigs. The FHP pigs were divided into two groups: an FHP group which only received the intensive care (n = 8) and an BAL group which was treated with BAL system (n = 7). Samples were collected at three timings: (1). Before FHP induction; (2). 24h after FHP induction; (3). 24h after BAL system treatment. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rDNA Realtime-PCR were applied to analyze the fecal microbial community. Results:Community-wide changes of fecal microfloraafter FHP induction were observed compared with pre-induction through DEEG profile analysis. The 16S rDNA gene copies of Bacteroides(p = 0.049), Bifidobacterium spp.(p< 0.001), Lactobacillus(p< 0.001) and C. coccoides(p = 0.013) were significantly decreased, while E.coli (p< 0.001) and Enterobacteriaceae (p< 0.001) were highly enriched in FHP pigs. The numbers of Clostridium leptum, Bacteroides fragilis, Prevotella and Faecalibacterium prausnitziishowed no differences after D-galactosamineadministration. After the BAL system treatment, the proportion of E.coli was remarkably increased (p = 0.014) and the number of Lactobacillusand Bifidobacterium spp.had a restored trend (p = 0.04 and p = 0.052, respectively). Conclusions:BAL system can promote the restoration of intestinal microbiota after FHP occurred. Evi-020#78 Metabolomic and Microbiological Analysis of the Dynamic Changes of Faeces in the Acute Liver Failure Pigs Ning Zhou (1), Jianzhou Li (1), Yimin Zhang (1), Ermei Cheng (1), Danhua Zhu (1), Lanjuan Li (1) 1. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases，The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. Backgroud: Acute liver failure (ALF) is an intractable disease associated with serious metabolic disorder. Investigating the dynamic changes of faecal metabolites during the development of ALF will reveal the cooperation between the microbiome and the metabolism of mammalian hosts. Therefore, this study characterized the dynamic metabonome of faecal extracted from ALF pigs with ultra performance liquid chromatography–mass spectrometry. Methods: 12 Bama experimental miniature pigs were housed for 14 days for acclimatization, and their food intake were controlled. ALF was induced via intravenous administration of D-galactosamine (1.3 g/kg body weight). Faeces samples were collected at four different given time points: before the administration of D-gal (healthy group), at 12 h (acute liver injury group) and 24 h (acute liver failure group) after its administration, and at death time(death group). Faecal water was extracted by mixed with methanol at a ratio of 5 mL/g. Faecal metabolites were studied with ultra performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF MS). Metabolic features were then statistically analyzed using partial least squares to latent structure-discriminant analysis (PLS-DA) models to discriminate between healthy, liver injury, liver failure and death states. PLS-DA was also used to identify potential biomarkers whichexpressed at significantly different amounts. Results: Score plots of pattern recognition analysis distinguished acute liver injury group, acute liver failure group and death group from healthy group. Based on the variable of importance in the project (VIP) values and S-plots, we identified four major groups of metabolites, which were considered as potential biomarkers: lysophosphatidylcholines, aromatic amino acids, fatty acids amides and bile acids. The results demonstrate that the potential of UPLC-MS is an efficient and convenient method that can be applied to screen fecal samples and aid in the early diagnosis of acute liver injury and acute liver failure. During the progress of liver injury, gut microbiota changed significantly. The increase of species diversity was observed in the liver failure group, which was proved by the increased number of DNA bands appearing on the DGGE. Metabolism disorders had a potential link to the changes in gut microflora.

Evi-021#81 Imbalace of Treg cells to Th17 cells Ratio in an Animal Model of Acute Liver Failure induced by LPS/D-galactosamine Qiongling Bao (1,2), Jianping Ge (1,2),Liang Yu (1,2) 1.the First Affliated Hospital, Zhejiang University,Hangzhou,China; 2.Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, P.R. China; Background & Aim: The appropriate balance between intestinal Th17 and Treg cells is crucial for the maintenance of gut immune barrier. This study aimed to investigate the change of circulating and intestinal Th17, Treg phenotype under conditions of acute liver failure. Methods: Female Sprague-Dawley rats received LPS/D-galactosamine (LPS, 40µg/kg body weight, D-GalN, 500mg/kg body weight) intraperitoneal injection to induce acute liver failure (ALF) (n = 10). Serum and intestine were collected on 48h after the animal models was established. PBMC and intestinal lamina propria lymphocyte were isolated and peripheral and intestinal Th17/Treg cells were analyzed by flow cytometry. Serum concentrations of corresponding cytokines and endotoxin were examined by enzyme-linked immunosorbent assay (ELISA). Results: Higher level of serum IL-17 (34.3 ± 12.1 pg/ml) and IL-23 (95.3 ± 24.5 pg/ml) were observed in ALF rats compared with normal control (p < 0.001 and p = 0.034). In agree with the cytokine profile, ALF rats had a significant increase of Th17 cells frequency in peripheral blood (3.5 ± 1.2%) and intestine (1.2 ± 1.1%). In contrast, the percentages of peripheral and intestinal Treg cell was lower in ALF groups than those in control group (both p < 0.001). Moreover, the elevated prevalence of Th17 cells was positively correlated with the high level of serum LPS (r = 0.65, p < 0.001). Conclusions: The balance between Th17 and Treg cells was destroyed in acute liver failure. Evi-022#86 Dynamic pattern of frequency and skewed TCRBV of peripheral CD4+CD25+ Tregs in HBeAg-positive chronic hepatitis B patients during antiviral treatment Jiezuan Yang, Guoping Sheng, Li Wei, Ping Ye, Yixin Zhu, Hongcui Cao, Lanjuan Li State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, College of Medicine, Zhejiang University, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Changes in the quantity and TCRBV pattern of peripheral CD4+CD25+ regulatory T cells (Tregs) for chronic hepatitis B (CHB) patients undergoing or not HBeAg seroconversion (SC) following tenofovir disoproxil fumarate (TDF) treatment are ambiguous. The percentage of circulation Tregs in HBeAg-positive (HBeAg+) CHB patients was determined by flow cytometry, and the molecular profiles of TCRBV for Tregs were analyzed using gene melting spectral pattern. The relationship between HBV DNA or ALT levels and Treg frequency or the number of skewed TCRBVs was analyzed in SC or non-SC CHB patients following TDF treatment. The positive correlation between HBV DNA and Treg level was significant in both SC and non-SC patients. Between HBV DNA and skewed TCRBV amount, however, the relationship was different significance. Three TCRBVs (BV11, BV15, and BV22) were prevalently in the non-SC group during the treatment period. SC was associated with a decline in the Treg frequency, which may be associated with declining levels of HBV DNA and HBeAg in HBeAg+ patients with TDF treatment. Pattern analysis of peripheral Treg and TCRBV could be used to predict SC in CHB patients. Furthermore, the preferential TCRBVs may be associated with the immune response related to seroconversion in CHB patients. Evi-023#87 Immunological Characteristics Of The Patients With Liver Cirrhosis Daiqiong Fang(1), Jing Guo(1), Yanfei Chen(1), Ding Shi(1), Lanjuan Li(1) 1.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases，The First Affiliated Hospital, College of Medicine, Zhejiang University Objective Bacterial translocation (BT) from the enteric cavity to mesenteric lymph nodes followed by circulatory system plays a significant role in the pathogenesis leading to spontaneous bacterial peritonitis in patients with cirrhosis and ascites. The aim of the current study is to clarify the immunological characteristics of the patients with advanced cirrhosis and ascites which has been or will develop into spontaneous bacterial peritonitis (SBP). Methods 59 patients with liver cirrhosis and ascites and 3 healthy controls were recruited. The levels of 48 kinds of cytokines in peripheral venous blood and ascites which were collected within 48 hours of hospital admission were measured by suspension array. Results Compared with healthy controls，18.75% of plasma cytokines were elevated(P

Evi-024#88 Correlation between liver enzyme levels and human cytomegalovirus antigenemia after hematopoietic stem cell transplantation Hong Zhao, Huihui Dong, Jianhua Hu, Hainv Gao, Meifang Yang, Xuan Zhang, Lichen Xu, Jun Fan, Weihang Ma the State Key Laboratory of Infectious Diseases, Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, zhejiang China ABSTRACT Human cytomegalovirus (HCMV) infection is a common viral infection, which causes morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HSCT). HCMV infection may cause hepatitis and elevate aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The present research aimed to analyze the correlation between liver enzyme levels and infection with HCMV antigenemia after HSCT. 270 blood samples were collected from 30 transplant recipients for six months after HSCT at different time points (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, and 6.0 months after HSCT). The patients were divided into two groups based on the results of a peripheral blood pp65 antigen assay. HCMV antigen was detected by immunohistochemical methods assay, and liver enzyme levels were detected by conventional methods. 12 (40%) and 18 (60%) patients formed HCMV antigenemia positive and negative groups, respectively. 10 of 12 patients were positive for HCMV antigenemia during the first 3 months posttransplant (83.3%). On the time of HMCV antigenemia positive, ALT levels, but not AST levels, were significantly elevated (P=0.0015). However, both ALT and AST were significantly elevated in the HCMV antigenemia positive group than the negative group (P=0.034 and P=0.018, respectively). The study results have confirmed that HCMV antigenemia could occur in the early stage after HSCT, and patients whose liver function increasing could be easily infected with HCMV after HSCT. Evi-025#89 Correlation between liver enzyme levels and human cytomegalovirus antigenemia after hematopoietic stem cell transplantation Hong Zhao, Huihui Dong, Jianhua Hu, Hainv Gao, Meifang Yang, Xuan Zhang, Lichen Xu, Jun Fan, Weihang Ma the State Key Laboratory of Infectious Diseases, Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, zhejiang China Human cytomegalovirus (HCMV) infection is a common viral infection, which causes morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HSCT). HCMV infection may cause hepatitis and elevate aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The present research aimed to analyze the correlation between liver enzyme levels and infection with HCMV antigenemia after HSCT. 270 blood samples were collected from 30 transplant recipients for six months after HSCT at different time points (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, and 6.0 months after HSCT). The patients were divided into two groups based on the results of a peripheral blood pp65 antigen assay. HCMV antigen was detected by immunohistochemical methods assay, and liver enzyme levels were detected by conventional methods. 12 (40%) and 18 (60%) patients formed HCMV antigenemia positive and negative groups, respectively. 10 of 12 patients were positive for HCMV antigenemia during the first 3 months posttransplant (83.3%). On the time of HMCV antigenemia positive, ALT levels, but not AST levels, were significantly elevated (P=0.0015). However, both ALT and AST were significantly elevated in the HCMV antigenemia positive group than the negative group (P=0.034 and P=0.018, respectively). The study results have confirmed that HCMV antigenemia could occur in the early stage after HSCT, and patients whose liver function increasing could be easily infected with HCMV after HSCT. Evi-026#94 Virus opportunistic infections after hematopoietic stem cell transplantation Jianhua Hu(1), Hanying Liang(1), Anbing Liu(1), Hainv Gao(1), Meifang Yang(1), Xuan Zhang(1), Hong Zhao(1), Yaping Huang(1), Jun Fan(1) State key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, Zhejiang University. 【Abstract】Objectives To investigate opportunistic virus, especially herpesvirus, including human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), human herpes virus type 6 (HHV6) infections after hematopoietic stem cell transplantation (HSCT) , their risk factors of infection and their relationship. Methods 44 hematopoietic stem cell transplant recipients (HSCTR) were enrolled in our study, their clinical data were collected. Peripheral blood specimens of HSCTR were collected and each virus DNA was detected using quantified polymerase chain reaction assays. The suspicious risk factors were analyzed by binary logistic regression. And the relationship of the virus were analysed by chi-squared test. Results EBV, HCMV, HHV6 were detected in 50% (22 patients), 45.45% (20 patients), 25% (11 patients) of hematopoietic stem cell transplant recipients (HSCTR). Male donor (P﹤0.05) was more susceptible to EBV infection than female donor, conditioning regimen including ATG (P﹤0.05) and GVHD prophylaxis including prednisone(P﹤0.05) extremely increase the risk of EBV infection. GVHD prophylaxis including prednisone (P﹤0.05) would also significantly increase the risk of HCMV infection. EBV infection(P﹤0.05).

was found to be an independently risk factor for HHV6 infection. HHV6 pre-infection have lower rate of HCMV infection(P﹤0.05); HCMV pre-infected also an protective factor for HHV6 infection, extremely decreased HHV6 further infection (P﹤0.05). Conclusion EBV, HCMV, HHV-6 were frequently detected in HSCT, EBV is the most susceptible. GVHD prophylaxis including prednisone, ATG, male donor were the risk factor of EBV infection. GVHD prophylaxis including prednisone was the risk factor for HCMV infection. Either HCMV or HHV6 pre-infected would be a protective factor, preventing another further infection. EBV when infected firstly would precipitate HHV6 infection. Thus, it is necessary to extensively monitor EBV, HCMV, and HHV6 infection in HSCT recipients, especially, HHV6 infection when EBV infected. 【Key words】 Opportunistic virus infection, Hematopoietic stem cell transplantation(HSCT), risk factors, EB Virus(EBV), Human Cytomegalovirus (HCMV), Human Herpes Virus type 6 (HHV6). Evi-027#97 Enterotypes of the human gut microbiome varied between diseases and ethnicities Fengling Yang(1,2), Nan Qin(1,2), Ang Li(1,2), Edi Prifti(3), Yanfei Chen(1,2), Li Shao(1,2), Jing Guo(1,2), S. Dusko Ehrlich(3), Lanjuan Li(1,2) 1.State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003 Hangzhou, China. 2.Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, 310003 Hangzhou, China. 3.Metagenopolis, Institut National de la Recherche Agronomique, 78350 Jouy en Josas, France. To investigate the phylogenetic and functional composition of the cohorts, enterotypes were indicated to demonstrate the stability of the whole human population rather than only between individuals. In previous study, the human gut microbiota could be partitioned into three enterotypes. We therefore investigated whether the participants of different cohorts could also be classified into enterotypes. First we performed enterotype analysis with multiple clustering methods in genus level in a Chinese cohort associated with liver cirrhosis. All methods recommended two or three clusters as the optimum clusters number. We finally chose three clusters, which has been reported in other cohorts, to display the result. Two of the clusters were driven by the genera Bacteroides and Prevotella, whereas the third cluster was mostly driven by the genus Veillonella. To investigate whether the enterotyping was unaffected by addition of further samples, we combined our data with the sequencing data from the Chinese T2D (Type 2 Diabetes) study to create the “LT group”. Two enterotypes of the three enterotypes were driven by the genera Bacteroides and Prevotella, which was consistent with LC cohort. However, the third enterotype was mostly driven by the genera Bifidobacterium and Escherichia. While in the Chinese T2D study, the third cluster was mostly driven by the genera Bifidobacterium and Ruminococcus. The difference in the main contributors to the third cluster was most likely caused by increasing the sample pool so that Veillonella was no longer the dominant genus. To understand whether the constituents of the enterotypes were influenced by patients’ ethnicity, we combined the LT group with the data from the MetaHIT study, which selected healthy control and IBD (Inflammatory Bowel Disease) samples from European patients, to create the LTI group. The main contributors of the two clusters were the genera Bacteroides and Prevotella, while the genera Faecolibacterium and Eubacterium were the main contributors to the third cluster. Conclusion: In each cohort, the main contributor of the third cluster differed between the different cohorts while the first two clusters were stable. We concluded that the main contributor of the third cluster varied depending on the disease and the ethnicity of the cohorts. Evi-028#100 Gut microbiota composition and associations with obesity and impaired glucose metabolism in seniors Kathrin Lippert (1), Livio Antonielli (1), Ludmilla Kedenko (2), Igor Kedenko (2), Miriam Leitner (3), Carolin Gemeier (2), Alexandra Kautzky-Willer (3), Bernhard Paulweber (2), Evelyn Hackl (1) 1. AIT Austrian Institute of Technology GmbH, Bioresources Unit, 3430 Tulln a.d. Donau, Austria 2. Universitätsklinik für Innere Medizin I, SALK, 5020 Salzburg, Austria 3. Universitätsklinik für Innere Medizin III, Medizinische Universität Wien, 1090 Wien, Austria The prevalence of obesity and associated metabolic disorders has increased dramatically within the last twenty years. Besides environmental, behavioral, and genetic factors, the human intestinal microbiota has been implicated an important role in the host energy metabolism. We studied the composition of the intestinal bacterial communities in two study groups including participants aged 58 to 79 years and explored whether specific signatures of the microbiota could be found under conditions of impaired glucose metabolism and obesity, indicating potential cause-and-effect relationships of the gut microbiome and metabolic disorders. In a pilot study, the gut microbiota composition of 20 individuals aged 58 to 71 years with normal glucose metabolism, prediabetes, or type 2 diabetes mellitus was analyzed through barcoded 454 sequencing of 16S rRNA (V1-V3) genes amplified from microbial DNA extracted from stool samples. Among the clinical data assessed, proinsulin, leptin, and glucagon-like-peptide 1, which are involved in the glucose and lipid metabolism, were found linked to the intestinal bacterial community composition in the study cohort. In addition, differences in the abundances of members of certain bacterial families (e.g. Prevotellaceae, Christensenellaceae) were apparent in individuals with impaired versus healthy glucose metabolism. Successively, the gut microbiota of 40 obese and 40 normal weight individuals aged 60 to 79 years was analyzed through barcoded Illumina Miseq sequencing of 16S rRNA amplicons (V3-V4), besides assessing clinical parameters related to energy

metabolism. Preliminary analysis of the sequencing dataset suggested that the alpha-diversity of the gut bacterial communities (assessed via the Chao1, ACE, Shannon, and Simpson indices) was not different in obese versus normal weight individuals. Ongoing analysis of the comprehensive microbial and clinical dataset centers on exploring relationships between specific microbiota community members, parameters of the glucose and lipid metabolism, and dietary behaviors in the study group. Increasing our understanding of host-microbiota relationships in various population groups shall finally contribute to the development of novel, microbiota-based pharmacological and dietary approaches to controlling obesity and associated metabolic disorders. Evi-029#109 The association of cervical microbiota with an increased risk for cervical intraepithelial neoplasia in Korea Mi Kyung Kim (1), Hea Young Oh (1), Bong-Soo Kim (2, 3), Sang-Soo Seo (4), Ji-Sook Kong (1) 1. Division of Cancer Epidemiology and Management, National Cancer Center, Goyang, Korea 2. Chunlab Inc., Seoul National University, Seoul, Korea 3. Department of Life Science, Hallym University, Chuncheon, Korea 4. Center for Uterine Cancer, National Cancer Center, Goyang, Korea Recent studies have suggested potential roles of microbiome in cervicovaginal diseases. However, there has been no report on cervical microbiome in cervical intraepithelial neoplasia (CIN), cervical precancerous lesion. We aimed to identify the cervical microbiota of Korean women and assess the association between cervical microbiota and CIN, and further determined the combined effect of the microbiota and HPV on the risk of CIN. The cervical microbiota of 70 CINs and 50 controls was analyzed using a pyrosequencing based on 16S rRNA gene. The association between specific microbial pattern and CIN risk was assessed using multivariate logistic regression, and relative excess risk due to interaction (RERI) and synergy index (S) were calculated. The phyla Firmicutes, Actinobacteria, Bacteroidetes, Preoteobacteria, Tenericutes, Fusobacteria, and TM7 were predominant microbiota and four distinct community types were observed in all subjects. A high score of the pattern characterized by predominance of A. vaginae, G. vaginalis, and L. iners with minority of L. crispatus (A in Figure and Table attached) had a higher CIN risk (OR, 95% CI 5.80, 1.73‒19.4, B) and abundance of A. vaginae had a higher CIN risk (6.63, 1.61‒27.2, C). Synergistic effect of a high score of this microbial pattern and oncogenic HPV was observed (34.1, 4.95‒284.5; RERI/S, 15.9/1.93, Table 2). A predominance of A. vaginae, G. vaginalis, and L. iners with a concomitant paucity of L. crispatus in cervical microbiota was associated with CIN risk, suggesting that bacterial dysbiosis and its combination with oncogenic HPV may be a risk factor for cervical neoplasia.

A. Distribution of main species

of risky microbial pattern

according to the pattern score

B. Odd ratio for CIN according to the scores of risky microbial pattern and relative abundance of species

OR and 95% CI for CIN 1) Low Medium High p trend 2)

Risky microbial pattern score 1 (ref.) 1.85 (0.65 5.31) 5.80 (1.73 19.4) 0.004 Relative abundance of A. vaginae 1 (ref.) 3.85 (1.22 12.2) 6.63 (1.61 27.2) 0.003 1) Odds ratio was estimated after adjustment for age, marital status, menopausal status, oral contraceptive use and smoking status as categorical variables. 2) P is for trend of odd ratio of the groups.

C. Synergistic effect of risky microbial pattern with high risk (HR)-HPV on an increase of CIN

Joints Symbol 1) OR (95% CI) 2) RERI / S 3) p for interaction 4) Risky microbial pattern 0, 0 1 (ref.) 15.9 / 1.93 0.018 & HR-HPV 0, 1 8.32 (2.18 31.7)

1, 0 10.8 (1.71 68.8)

1, 1 34.1 (4.95 234.5) 1) S stands for a low score of risky microbial pattern or non-HPV infection. Sscore of risky microbial pattern or HPV infection. 2) Odds ratio (OR) was estimated after adjustment for age, marital status, menopausal status, oral contraceptive use and smoking status as categorical variables. 3) The interaction of joint based on additive model was assessed using relative excess risk due to interaction (RERI) and synergy index (S). RERI > 0 and S > 1 indicate a synergistic effect. 4) P is for the interaction of multiplicative term.

The association of cervical microbial pattern with an increase of CIN risk

Evi-030#115 Disease specific differences in intestinal microbiota between pediatric inflammatory bowel disease and healthy control Evelien F.J. de Groot(1), Nanne K.H. de Boer(1), Marc A. Benninga(2), Andries E. Budding(3), Ad A. van Bodegraven(4), Paul H.M. Savelkoul(3), Tim G.J. de Meij(5) 1. Gastroenterology and Hepatology, VU University Medical Center, Amsterdam, Netherlands. 2. Pediatric Gastroenterology, Academic Medical Center, Amsterdam, Netherlands. 3. Medical Microbiology and infection control, VU University Medical Centre, Amsterdam, Netherlands. 4. Gastroenterology and Hepatology, ORBIS Medical Center, Sittard, Netherlands. 5. Pediatric Gastroenterology, VU University Medical Center, Amsterdam, Netherlands. Introduction: In the etiology of inflammatory bowel disease (IBD), comprising Crohn’s disease (CD) en ulcerative colitis (UC), intestinal microbiota seems to play a crucial role. However, data is limited and conflicting. Here we studied intestinal microbiota in a large cohort of children during onset of IBD and followed-up until achieving remission. We compared results to healthy controls. Methods: Children suspected for IBD were included. All patients were recruited from 2 tertiary centers in Amsterdam, The Netherlands. Fecal samples were collected, prior to bowel cleansing (t0) and at week 1, 3, 6 and 18 after initiation of therapy. All patients were treated according to standard care guidelines. CD patients received thiopurines and were offered 6 weeks exclusive enteral nutrition (EEN) and in case of reluctance, unresponsiveness or intolerance, corticosteroids were prescribed instead. UC patients received aminosalycilates, dependent on disease severity, combined with corticosteroids, and also as maintenance (mono)therapy. Healthy controls collected samples at similar intervals. Disease activity was assessed by Global-Physician-Assessment (GPA) score, fecal calprotectin and CRP. Fecal samples were analyzed by IS-pro, a clinically applicable PCR-based microbiome profiling technique. Results: Fecal samples of 101 newly diagnosed IBD-patients (median 14 years) and healthy controls (median 8 years) were collected. All patients were in clinical remission at t6. Preliminary results showed distinct differences in microbiota for CD (n=60), UC (n=41) and controls (n=61). For the phylum Firmicutes diversity and total abundance were significantly higher in UC compared to controls at t0 (resp. p0.003, p0.001). Total abundance in CU increased even further at t6 (p0.039). For the phylum Bacteroidetes total abundance was lower in both UC and CD compared to controls at t0 (resp. p0.003, p0.07). Furthermore one of the core microbiota in controls, Alistipes putredinis, had a lower abundance or was totally absent in almost all IBD patients. For the phylum Proteobacteria diversity was higher in CD compared to controls at t0 (p0.041) with a higher total abundance (p0.001). In CD, microbiota changed towards normal when patients went into remission, while this effect was not seen in UC. Conclusion: Microbiota-analysis demonstrated clear disease specific differences in composition between pediatric-IBD and controls. Furthermore one of the core microbiota in controls, Alistipes putredinis, had a lower abundance or was totally absent in almost all IBD patients. Shifting towards normal control microbiota was only seen for CD patients. Evi-031#116 Distinct Alterations in the Placental Microbiome Among Spontaneous Preterm Births Amanda L. Prince (1), Jun Ma (1), Kathleen M. Antony (1), Derrick M. Chu (1), Renata Benjamin (1), Claire Cook (1), Lori Showalter (1), Michelle Moller (1), Brigid Boogan (1), James Versalovic (1), and Kjersti M. Aagaard (1) 1. Baylor College of Medicine, Houston, TX Objective: Previously, we and others have shown that the vaginal and gut microbiome are altered during pregnancy and vary further by gestational age. Similarly, we have recently demonstrated that the placenta harbors a low biomass microbiome which varies in association with spontaneous preterm birth (sPTB). Given inherent limitations to longitudinal placenta collection in any given pregnancy, delineating causation from association is problematic. We reasoned that robust causal inference analysis across multiple body sites in a prospective longitudinal cohort inclusive of both spontaneous and indicated PTB could potentially overcome such obstacles. Study Design: Gravid subjects were enrolled (n=277) in the early third trimester or at delivery (170 term, 107 preterm). Extensive clinical metadata (such as comorbidities and indications for inductions) enabled covariate analytics and gestational age comparison. Oral, vaginal, stool, and placental swabs and tissue were uniformly clean collected from subjects and their infants. DNA was extracted (MO BIO) and subjected to 16S and whole genome shotgun (WGS) metagenomics. Quality filtered sequences were analyzed with custom tools (QIIME and MG-RAST) and causal inference approaches (hierarchical clustering by Manhattan distance and regression modeling). Results: Consistent with recent findings, we did not detect likely causal differences in the posterior fornix of preterm subjects (p=0.254) but did observe significant variation in the placental microbiome (p=0.012; A left) which were attributable to cases of sPTB (Fig. A right, p=0.0323 PC2 axis). Moreover, significantly altered taxa abundance (including Mycoplasma and Fusobacterium; Fig. B) persisted amongst sPTB comparative of gestational age and covariates. Conclusions: Causal inference analysis in a longitudinal cohort enabled us to delineate a persistent and significant association of the placental microbiome with sPTB, which appears distinct form indicated births at akin gestational ages.

p = 0.0323

Spontaneous PTB Indicated PTB 260 days 260 days 166 days 214 days

Vagina

Placenta

Spontaneous P

Indicated PTB

Preterm

Evi-032#117 Chorioamnionitis Induced by Intramniotic Injection of IL-1, LPS, or Ureaplasma parvum is Associated with an Altered Microbiome in a Primate Model of Inflammatory Preterm Birth (PTB) Amanda L. Prince (1), Jun Ma (1), Lisa Miller (2), Min Hu (1), Alan Jobe (3), Claire Chougnet (2), Suhas Kallapur (3), and Kjersti Aagaard (1) 1. Baylor College of Medicine, Obstetrics & Gynecology, Division of Maternal-Fetal Medicine, Houston, TX 2. California National Primate Research Center, Davis, CA 3. Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH Objective: Chorioamnionitis has been historically attributed to ascending vaginal microbes. Recently, we have demonstrated that the placenta harbors a unique microbiome markedly dissimilar to the vaginal flora. Therefore, it’s plausible that inflammation or co-infection may alter the resident placental microbiome, tipping the commensal balance and resulting in inflammatory-mediated preterm birth (PTB). Here, we aim to examine the changes in the maternal and fetal microbiome that occur in a primate model of inflammatory PTB. Study Design: Under ultrasound guidance, sterile intramniotic injections (IA) of control (saline) and treatment (LPS, IL-1, or Ureaplasma parvum) were given to preterm (126-132 days) Rhesus macaques (n=32). Fetuses were delivered within one week post-injection via cesarean. Oral, stool, placental, vaginal, meconium and amniotic fluid swabs and tissue were collected for DNA extraction (MO BIO) and subjected to NextGen metagenomic sequencing. V1V3 and V3V5 amplicons and whole genome shotgun (WGS) sequences were analyzed (QIIME, HUMAnN, and MG-RAST). Integrative analysis in each cohort integrated relative abundance of each microbe to bacterial metabolic pathways Results: Sterile IA injections of treatment cohorts, but not control, induced chorioamnionitis as detected by histology, neutrophil infiltration, and increases in inflammation (p<0.01). Further, alterations in the microbiome of the maternal endometrium, placenta and fetal meconium were detected while the microbiome of the maternal oral, rectum and stool remained unaltered by treatment. Detected alterations in abundant taxa were associated with changes in the bacterial metabolic pathways (Figure). Conclusions: Employing a clinically relevant primate model, we demonstrate that sterile IA infection of inflammatory agents and mediators in the preterm interval results in significant alterations in the placental, endometrial and fetal gut microbiomes and their metabolic pathways.

Endometrium

Meconium

Placenta

Evi-033#118 The Microbiome of the Placenta is Altered Among Subjects with Severe Chorioamnionitis and Spontaneous Preterm Birth Amanda Prince (1), Jun Ma (1), Paranthaman Kannan (2), Manuel Alvarez (2), Tate Gisslen (2), Kathleen M. Antony (1), Christine Knox (3), Alan Jobe (2), Claire Chougnet (2), Suhas Kallapur (2), and Kjersti Aagaard (1) 1. Baylor College of Medicine, Obstetrics & Gynecology, Division of Maternal-Fetal Medicine, Houston, TX 2. Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States 3. Queensland University of Technology, Brisbane, QLD, Australia Objective: Chorioamnionitis (IAI) is frequently associated with preterm birth and has long been associated with the presence of bacteria in the placenta. We have recently demonstrated that the placenta harbors a unique microbiome, which can be robustly characterized employing metagenomics. Here, we aimed to examine the differences in the placental microbiome in association with preterm birth and IAI. Study Design: This was a cross-sectional analysis with six nested spontaneous birth cohorts (n=9-15 subjects/cohort, Fig. A): Term without IAI (Cohort 1), term with IAI (Cohort 2), preterm without IAI (Cohort 3), preterm with mild IAI (Cohort 4), preterm with severe IAI (Cohort 5), and preterm with IAI and funisitis (Cohort 6). Clean samples were obtained with sterile placental swabs immediately at delivery, and DNA was extracted (MO BIO) and whole genome shotgun (WGS) sequencing was performed on the Illumina HiSeq platform. Filtered microbial DNA sequences were annotated and analyzed using MG-RAST and R. Results: The mean gestational age (GA) for spontaneous preterm infants was 35.1, while term was 39.6 weeks (p<0.05). We observed distinct clustering of placental microbiome communities among preterm subjects with and without severe IAI (Fig. B, p=0.07) and associated bacterial metabolic pathways were significantly altered between preterm subjects with and without severe IAI (Kendall’s rank correlation, Fig. C). Surprisingly, these alterations in metabolism were not associated with detectable Ureaplasma parvum and Mycoplasma hominis (bacterial species previously reported as associated with preterm birth). Conclusions: Consistent with ours and others prior findings, women who experience spontaneous preterm labor harbor placental microbiota which differed by virtue of severity of IAI. Integrative metagenomic analysis revealed significant variation in distinct bacterial metabolic pathways, which we speculate may contribute to risk of preterm birth with severe IAI.

+ Correlation

- Correlation

Chorioamnionitis

Spontaneous

Corynebacterium accolens Parvibaculum lavamentivorans

Burkholderia ambifaria Burkholder vietnamiensis

Cupriavidus pinatubonensis Acidovorax avenae

Albidiferax ferrireducens Acinetobacter haemolyticus

Mycoplasma hominis Mycoplasma genitalium

Ureaplasma parvum

Corynebacterium accolens Parvibaculum lavamentivorans

Burkholderia ambifaria Burkholder vietnamiensis

Cupriavidus pinatubonensis Acidovorax avenae

Albidiferax ferrireducens Acinetobacter haemolyticus

Mycoplasma hominis Mycoplasma genitalium

Ureaplasma parvum

No Chorioamnionitis

Mild Chorioamnionitis

Severe Chorioamnionitis (+/- funisitis)

p = 0.07

Preterm

Cohort 1 Cohort 2

Cohort 3 Cohort 4 Cohort 5 Cohort 6

Chorioamnionitis

Funisitis

Severe

Evi-034#126 The role of gut microbiome in the initiation and progression of cancer Shaoyan Xu（1,2,3）, Zhigang Ren（1,2,3）, Jianwen Jiang（1,2,3）, Weilin Wang（1,2,3）, Shusen Zheng（1,2,3） 1Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003, Hangzhou, China. 2Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, College of Medicine, Zhejiang University,310003, Hangzhou, China. 3Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003, Hangzhou, China. Purposes: we aim to review and discuss the relationship between gut microbiome and cancer initiation and progression. Methods：the studies in English about the relationship between gut microbiome and cancer initiation and progression are searched and read, trying to obtain insightful conclusions . Results: the latest metagenomic sequencing technology has revealed that there are more than 1000 microbial species in the human gut. They are not only important to keep human healthy, but also closely related to the initiation and progression of some disorders. Recent years, more and more studies focus on the relationship between gut microbiome and cancer and there may be several mechanisms supporting it. Nearly all of human body cells harbor TLRs. Gut microbiome and its products, especially LPS, can combine with TLRs such as TLR-4, leading to the activation of nuclear factor kB (NF- kB), which resulting in the expression of the inflammatory cytokines such as tumour necrosis factor-alpha, interleukin (IL)-1, IL-6 and IL-8. Consquently, the survival advantages of precancerous or cancer cells will be enhanced. Gut microbiome can also lead to the initiation and progression of cancers such as colorectal cancer（CRC）through the biosynthesis of genotoxins interfering with the cell cycle regulation or directly damaging DNA,the production of toxic metabolites, and the activation of dietary heterocyclic amines to pro-carcinogenic compounds. So far, studies researching the association between gut microbiome and cancer mainly focus on CRC and hepatocellular carcinoma（HCC）. Recently, animal experiment found that using of antibiotics to reduce the level of endotoxin or removing TLR- 4 of hepatic cells could effectively slow down the growth of HCC. Researches on other cances such as breast cancer, esophageal adenocarcinoma and prostate cancer are still very limited. Conclusions：current studies have shown that gut microbiome are closely associated with the initiation and progression of certain types of cancers, mainly on CRC and HCC. The relationship is urgently needed to be studied in depth and breadth. Making clear the mechanisms how gut microbiome contribute to occurrence and development of cancer has the potential of providing a novel direction for cancer diagnosis, prevention and treatment. Evi-035#129 Selective Sweeps Drive Pseudomonas aeruginosa Evolution in the Cystic Fibrosis Lung Julio Diaz Caballero (1), Shawn T. Clark (2,3), Bryan Coburn (1), Yu Zhang (2), Pauline W. Wang (4), Sylva L. Donaldson (4), D. Elizabeth Tullis (5), Yvonne C. Yau (6), Valerie J. Waters (7), David M. Hwang (2,3,8), David S. Guttman (1,4) 1. Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada 2. Latner Thoracic Surgery Laboratories, University Health Network, University of Toronto, Toronto, Ontario, Canada 3. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada 4. Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Ontario, Canada 5. Adult Cystic Fibrosis Clinic, St. Michael's Hospital, Toronto, Ontario, Canada 6. Department of Pediatric Laboratory Medicine, Division of Microbiology, The Hospital for Sick Children, Toronto, Ontario, Canada 7. Department of Pediatrics, Division of Infectious Diseases, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada 8. Department of Pathology, University Health Network, Toronto, Ontario, Canada. The opportunistic human pathogen Pseudomonas aeruginosa is highly resistant to eradication once established in the lungs of cystic fibrosis (CF) patients. While the clinical implications of these infections are well studied, we know little about the evolutionary processes that enable this pathogen to persist in this environment. Here, we describe the longitudinal analysis of a single CF patient chronically infected with P. aeruginosa. A total of 235 P. aeruginosa isolates were collected from twelve sputum specimens obtained over a one-year period. Each isolate was subjected to whole-genome sequencing and antibiotic resistance profiling. Our genome analysis finds that P. aeruginosa population dynamics can be driven by recurrent selective sweeps. One selective sweep resulted in replacement of the entire population with a clade that coalesces within three years of the last sample, while a second selective sweep, which originated less than a year before the last sample, has only gone to partial completion. These sweeps result in two distinct populations: the ancestral population characterized by higher diversity and recombination, and the sweep population characterized by a stronger signal of positive selection. These dynamics are associated with mutations in the gene encoding penicillin-binding protein 3 (PBP3) and correlate with resistance to anti-pseudomonal antibiotics. The segregating PBP3 alleles show latent potential for selection under different treatment regimes, which may be responsible for the maintenance of two sub-populations. These intraspecies dynamics are not apparent with lower resolution analyses, and demonstrate a mechanism allowing P. aeruginosa to persist in the CF lung during therapy.

Evi-036#133 Effect of metronidazole on the growth of common strains of vaginal Lactobacillus in vitro Rui Zhang(1), Qinping Liao(2) 1. Department of Obstetrics & Gynecology, Peking University First Hospital, Peking University 2.Department of Obstetrics & Gynecology, Beijing Tsinghua Changgung Hospital Medical Center, Tsinghua University Background Vaginal lactobacilli were very important in keeping the balance of vaginal microbiota. Metronidazole was a very commonly used drug in vaginal infectious diseases, while very few study has investigated if metronidazole would prohibit the growth of vaginal lactobacillus and destruct the balance of vaginal microbiota. Methods MRS broth including different concentration of metronidazole was used to culture six different strains of vaginal lactobacillus recovered from vagina of healthy Chinese women. The turbidities of the lactobacilli were detected every three hours, and the growth curves were dipicted to assess the effect of metronidazole on growth. Results The effect of different concentration of metronidazole was not the same. When the concentration of metronidazole was ≤128 µg/ml, the growth of the six lactobacillus strains were not affected; when the concentration was ≥ 512 µg/ml, the growth of the six lactobacillus strains were apparently prohibited; when the concentration was between 128 µg/ml and 512 µg/ml, L. delbrueckii, L. jensenii and L. vaginalis were prohibited to some extent, while L. crispatus, L. gasseri and L. fermentum were not affected. In addition, the growth of the six strains of vaginal lactobacillus were not the same, and they responded differently to the metronidazole. Table. Lactobacillus turbidity for different metronidazole concentrations (after 24 h)

Metronidazole concentration (µg/ml)

Lactobacillus turbidity （Mean±S.D） L.crispatus L.gasseri L.jensenii L.vaginalis L.fermentum L.delbrueckii

0 21.5±0.86 41.4±1.6 18.5±0.74 22.2±0.80 28.0±1.10 34.2±1.3 128 20.0±0.63 32.4±1.1 12.0±0.42 23.6±0.67 25.0±0.87 31.2±1.0 256 18.0±0.74 28.8±1.0 4.0±0.14 8.8±0.32 3.0±0.11 27.4±1.0 512 6.4±0.29 9.1±0.41 3.0±0.13 4.0±0.18 3.1±0.14 11.4±0.52 1024 2.9±0.17 2.6±0.16 1.9±0.10 3.4±0.21 2.6±0.26 3.5±0.21 2000 1.3±0.06 0.6±0.03 2.1±0.02 1.9±0.10 1.6±0.08 1.5±0.07

Conclusions The effects of metronidazole on the growth of vaginal lactobacillus strains were closely related to the concentrations of metronidazole. The recommended regimens (0.4g, oral or vaginal administration twice a day) might not affect the growth of vaginal lactobacillus. While the vaginal administration of 37.5 mg (a 5 g applicator dose) of 0.75% metronidazole gel might destruct the vaginal microbiota to some extent. Evi-037#139 HIV-1 infection is affected by has-miR-191-5p in 293T and Tzmbl Zongxing Yang (1,2), Jin Yang (3), Juan Wang (1,2), Xiangyun Lu (1,2), Nanping Wu (1,2) 1. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Institute of Infectious Diseases, The First Affiliated Hospital of Zhejiang University, School of Medicine, Zhejiang University, Hangzhou, China 2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China 3. Department of Medicine, Blood Center of Zhejiang Province, Hangzhou, China Abstract Background: microRNAs are are critical regulators of mRNA translation and turnover. The important role that miRNAs play in HIV-1 pathogenesis is only now emerging. Accumulating evidence demonstrates the interaction between miRNAs and HIV. Methods: miRNA expression profile in peripheral blood mononuclear cells from HIV-infected patients on HAART or without HAART and normal people were analyzed by microarray. Differentially expressed miRNAs were tested by qPCR. Influence of the differentially expressed miRNAs on HIV infection was further tested in vitro in 293T cells with HIV pseudotype virus and HIV-infected Tzmbl cells. Then HIV-related targets of differentially expressed miRNAs were test with qPCR and report gene system containing 3’ UTR of the targets. Results: Eleven miRNAs were identified by microarray analysis, and one of them (has-miR-191-5p) were tested by qPCR. Experiments in vitro revealed the anti-HIV effect of has-miR-191-5p. Thirteen potential targets of miR-191-5p related to HIV from MAGIA were test by qPCR and report gene system, and five of them were tested. Conclusions: has-miR-191-5p might affect HIV infection by ragulating its targets.

Evi-038#141 Gut colonization with Methanobrevibacter smithii is associated with childhood overweight Catherine A. Mbakwa (1,2), John Penders (2,3), Paul H. Savelkoul (3), Carel Thijs (2), Pieter C. Dagnelie (2,4), Monique Mommers (2), and Ilja C.W. Arts (2,4) 1. Top Institute Food and Nutrition 2. Department of Epidemiology, Maastricht University, CAPHRI School for Public Health and Primary Care, Maastricht, The Netherlands. 3. Department of Medical Microbiology, Maastricht University Medical Centre, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht, The Netherlands. 4. Department of Epidemiology, Maastricht University, CARIM School for Cardiovascular Diseases, Maastricht, The Netherlands. Background: Recent studies have indicated that, next to the many bacterial species, indigenous archaea play a crucial role in the metabolic capacity of the gut microbiota. However, few of these studies have been conducted in humans. Moreover, to date no study has investigated intestinal archaea with respect to weight gain over time in children. Aim: Prospectively investigate whether the presence and abundance of archaea in faeces of 472 children at school age are associated with weight development from 6-10 years of age. Methods: The present study was conducted within the prospective KOALA Birth Cohort study. Faecal samples of 472 children were analysed by qPCR to quantify archaea (M. smithii and M. stadtmanae). Parents were sent repeated questionnaires to collect anthropometric data and information on potential confounders. Main outcomes were overweight (BMI≥85th percentile), and age- and sex-standardized BMI z-scores, weight z-scores and height z-scores at ages (mean±sd) of 6.2±0.5, 6.8±0.5, 7.8±0.5, and 8.8±0.5 years. Generalised Estimating Equations (GEE) for repeated measures was used for the statistical analysis while controlling for the following confounders: maternal educational level, weight gained during pregnancy, place and mode of delivery, birth weight, household size, antibiotic use, physical activity, sex, and dietary intake of fibre, fat, carbohydrates and total energy. Results: The presence and high abundance of M. smithii were associated with an increased risk of overweight (adjusted odds ratio (OR) = 2.69; 95% CI 0.96-7.54; OR=3.27; 95% CI 1.0-9.83, respectively) from 6-10 years of age. Moreover, the presence of M. smithii was also associated with higher weight z-scores (adjusted beta 0.18; 95% CI 0.01-0.36), but not with height z-scores. We found a statistically significant interaction (P=0.008) between M. smithii and age for BMI z-scores, implying that the strength of this association increased over time. Conclusion: The presence/higher abundance of M. smithii in the gut of children is associated with higher weight z-scores, higher BMI z-scores and overweight.

Table 1. Participant characteristics of the study populationStudy Population

(N=472)a Mean ± SD ‘Present’(n=369) Mean ± SD

‘Absent’ (n=103) Mean ± SD

‘Present’ (n=39) Mean ± SD

‘Absent’ (n=433) Mean ± SD

Dietary factorsTotal energy intake (KJ) 6143.2 ± 1255.0 6131.6 ± 1223.3 6185.7 ± 1370.4 6318.9 ± 1402.9 6127.2 ± 1241.8% energy intake from fats 29.7 ± 4.2 29.6 ± 4.2 29.8 ± 4.0 29.5 ± 4.6 29.7 ± 4.1% energy intake from carbohydrates 56.7 ± 5.9 55.7 ± 4.9 55.5 ± 5.1 56.4 ± 5.3 55.6 ± 4.9Total fibre intake (g) 15.5 ± 3.9 15.4 ± 3.9 15.9 ± 3.5 15.9 ± 3.9 15.4 ± 3.8

Total physical activity (hrs/week) 9.4 ± 4.4 9.3 ± 4.4 9.7 ± 4.1 9.6 ± 4.7 9.3 ± 4.3Total Household size 4.3 ± 0.8 4 .3 ± 0.7 4.4 ± 0.9 4.3 ± 0.8 4.3 ± 0.8Birth weight (g) 3574 ± 483 3560 ± 481 3624 ± 487 3676 ± 498 3566 ± 481Place and mode of delivery (n, %)

Vaginal delivery at home 219 (47.0) 176 (48.6) 43 (41.8) 16 (41.0) 203 (47.6)Vaginal delivery in the hospital 198 (42.6) 150 (41.5) 48 (46.6) 15 (38.5) 183 (43.0)Caesarean section in the hospital 48 (10.4) 36 (9.9) 12 (11.6) 8 (20.5) 40 (9.4)

Time of last antibiotic course (n, %)b

No antibiotic use 398 (85.4) 310 (85.2) 88 (86.2) 35 (89.7) 365 (85.0)Greater than 4 weeks ago 57 (12.2) 46 (12.6) 11 (10.8) 4 (4.3) 53 (12.4)Less than 4 weeks ago 11 (2.4) 8 (2.2) 3 (3) 0 (0.0) 11 (2.6)

Archaea counts (Log10DNAcopies/g faeces) (median, range)c

4.7 (3.8 -10.8) 5.4 (4.8 - 9.2)

aTotals may not add up to 472 because of missing values (for number of missing see results section)bTime of last antibiotic course at time of faecal collection. cMedian counts were calculated from archaea (M. smithii and/or M. stadtmanae ) positive samples only.

M. smithii M. stadtmanae

Evi-039#142 The vaginal microbiome as a predictor for acquiring Chlamydia trachomatis Robin van Houdt (1), Ma Bing (2), Arjen Speksnijder (3), Jannie van der Helm (4), Henry de Vries (4,5), Jacques Ravel (2) 1. VUmc, Medical Microbiology and Infection Control, Amsterdam, the Netherlands 2. University of Maryland, Institute for Genome Sciences, Baltimore, United States 3. Naturalis, Biodiversity Center, Leiden, the Netherlands 4. Public Health Service, Infectious Diseases, Amsterdam, the Netherlands 5. Academic Medical Centre, Dermatology, Amsterdam, the Netherlands Objectives: Chlamydia trachomatis (Ct) is one of the most prevalent bacterial sexually transmitted infections (STIs) in industrialized countries, that can cause long term complications such as pelvic inflammatory disease, ectopic pregnancy, and infertility. Bacterial communities in the vagina seem to play a protective role in preventing colonization by pathogens. Alterations in the composition of these communities disturb the delicate balance of the vaginal environment and its defence mechanisms. This study is the first to present data on the genomic composition of the vaginal microbiome prior to a Ct infection, making it possible to see whether specific compositions of the vaginal microbiome could be a marker for acquiring Ct. Methods: A nested case control study was performed among women at low risk for acquiring Ct. The cases (N=61) were women who acquiring Ct within a year after their first visit. The controls (N=61) did not acquire Ct and were matched for ethnicity, age, and city of residency. Each woman self collected a mid-vaginal sample, using the Aptima vaginal swab collection kit and filled out a standardized questionnaire about their sexual risk behaviour, medical history, and socio-demographic situation. For taxonomic classification, the V3-V4 hyper variable regions of the 16S rRNA gene were sequenced, using next generation sequencing (paired-end sequencing on the Illumina MiSeq platform). The genomic composition of the vaginal microbiome was then linked to the socio-demographic data and data on sexual risk behaviour and analysed using logistic regression. Results: In total, 122 women were included and 16S rRNA sequencing succeeded in 115 women. The vaginal bacterial communities were grouped according to community composition. Among women in Amsterdam, 5 major community state types (CST) of the vaginal microbiome could be determined. The microbiome of the majority of women was dominated by L. crispatus, 37%. Another group was dominated by L. iners, 33%. The third group was characterized by the absence of Lactobacillus spp and the presence of G vaginalis, 25%. Multivariate analysis revealed CST and type of relationship as independent risk factors. Women with a L. iners dominated CST had an increased risk for acquiring Ct, p=0.04 (OR: 2.58, 95% CI: 1.01-6.61). Women with a steady partner, but not living together also had an increased risk for acquiring Ct, p=0.02 (OR: 14.51, 95% CI: 1.45-145.3). Conclusion: The distribution of the various CST is as expected in a predominantly white cohort, except for the relatively large group of women with a CST lacking Lactobacillus spp. Having a vaginal microbiome that is dominated by L. iners increases the risk for acquiring Ct in the long run. Therefore, the composition of the vaginal microbiome can be used as a predictor for acquiring STIs, even over long time periods. Evi-040#143 The relationship between gut microbiome and hepatocellular carcinoma Weilin Wang1,2,3, Shaoyan Xu1,2,3, Zhigang Ren1,2,3, Shusen Zheng1,2,3 1.Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003, Hangzhou, China. 2.Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, College of Medicine, Zhejiang University,310003, Hangzhou, China. 3.Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003, Hangzhou, China. Purposes：We aim to review and discuss the relationship between gut microbiome and hepatocellular carcinoma(HCC). Methods: Studies published in English and exploring the role of gut microbiome in the initiation and progression of HCC are searched , read in detail , trying to get comprehensive and insightful conclusions. Results: There are more than 1000 kinds of microbiome in the human gut. They not only play an important to keep human healthy, but also are closely related to the initiation and progression of some diseases. Kupffer cells are liver tissue macrophages and express most of the major TLRs. Gut microbiome and its products, especially LPS, can combine with TLRs such as TLR-4, leading to the activation of nuclear factor kB (NF- kB), which resulting in the expression of the inflammatory cytokines such as tumour necrosis factor-alpha, interleukin (IL)-1, IL-6 and IL-8. Consquently, the survival advantages of precancerous or cancer cells will be enhanced. Recent years, increasing number of studies focus on the relationship between gut microbiome and HCC. Rats with HCC induced by DEN treatment showed an imbalance of the gut microflora with a significant decrease of Lactobacillus species, Bifidobacterium species and an increase of Enterococcus species. Induction of enteric dysbacteriosis by penicillin or DSS could markedly promoted tumor formation. Moreover, administration of probiotics dramatically improve enteric dysbacteriosis and decreased liver tumor growth and multiplicity. Interestingly, probiotics inhibited the translocation of endotoxin bearing pathogen associated molecular patterns (PAMPs), which can combine with TLRs such as TLR-4. In addition, removing TLR- 4 of hepatic cells could effectively slow down the growth of HCC. Importantly, TLR4 and the gut microbiota are not required for HCC initiation but for HCC progression. Gut microbiome can also lead to hepatic fibrosis, cirrhosis, obesity, diabetes, alcoholic liver disease, non-alcoholic steatohepatitis, which have proved to be important contribution factors for HCC. So, gut microbiome can also promote the initiation and progression of HCC indirectly. Conclusions：Current studies have shown that HCC induced by DEN is associated with the dysbiosis of gut microbiome. However, studies exploring the distinct mechanisms are still few, and more studies are urgently to be performed. Making clear

the mechanisms how gut microbiome contribute to occurrence and development of HCC has the potential of providing a novel direction for HCC diagnosis, prevention and treatment. Evi-041#163 Microbiota in type 1 diabetes and healthy children, both native and immigrant: the case-control study protocol Deborah Traversi (1), Roberta Siliquini (1), Violetta Andriolo (1), Emanuela Lovato (1), Giorgio Gilli (1), Marilena Durazzo (3), Francesco Cadario (2), Franco Cerutti (1) 1. Department of Public Health and Pediatrics, University of Torino, piazza Polonia 94, 10126 Torino 2. Ospedale Maggiore della Carità Novara, Corso Mazzini 18, 28100 Novara 3. Department of Internal Medicine, University of Torino, via Verdi 8, 10100 Torino Diabetes Mellitus Type I (T1DM) is the chronic metabolic disease of childhood with the highest incidence in developed countries. Over the past 10 years, its incidence has been increased in the north Italy, especially in immigrant children. T1DM is the result of a complex interplay of genetic susceptibility and environmental determinants including autoimmunity against pancreatic islet beta cells. It is suggested that early shaping of the immune system in children is critical for the initiation of the disease leading to factors that include how the host establishes a stable ecosystem with a large cohort of accompanying bacteria. Furthermore previous studies showed substantial alterations in microbial interaction networks in the diseased children and such findings underscored a role of the microbiome in the pathogenesis of T1DM. Also Achaea microorganisms seem to be involved into metabolic diseases ongoing and among these Methanobrevibacter smithii is the dominant in the human gut. Such evidences indicate both the role of microbiota and the methanogen concentrations, in the children gut, during T1DM pathogenesis, as relevant aspects of investigation. Moreover determinants that influence the T1DM course in immigrant and Italian children are leading to nutritional and socio-cultural aspects but such factors are not well-known. We showed the design of a case-control study on the children population of the Piedmont Region in the north Italy, in its first underway phase. The study population will consist of 120 children of which 40 with first diagnosis of T1DM and 80 healthy control children, both diseased and healthy children will be divided by individual origins into two cohorts: Italian native and immigrant. The main objective of the project is to evaluate factors that influence the T1DM course moreover a laboratory based study will be conducted comparing the microbiota of diseases children with healthy children both for immigrants and Italian native. All the included cases and controls in the study will be subjected to specific questionnaires for the collection of three kind of data: nutritional, clinical and socio-cultural. Moreover a stool sample will be collected, bringing to the hospital/laboratory within the 24 hours and then stored at -80°C until the DNA extraction. On each DNA extracts will be performed DGGE analyses focusing both on Bacteria and on Archaea. Moreover a qRT-PCR will be performed to assess the concentration of total methanogens and of Methanobrevibacter smithii. The data will be elaborated and included in a statistical analysis with the nutritional, clinical and socio-cultural data. The project is funded by the Italian Health Ministry and it is submitting to our Health Centre Ethical Committee for the approval. Evi-042#156 Reduction of butyrate- and methane-producing microorganisms in patients with irritable bowel syndrome Marta Pozuelo (1), Suchita Panda (1), Alba Santiago (1), Sara Mendez (2), Anna Accarino (2,3), Javier Santos (1,2,3), Francisco Guarner (1,2,3), Fernando Azpiroz (1,2,3), and Chaysavanh Manichanh (1,3) 1. Digestive Research Unit, Vall d’Hebron Research Institute, Barcelona, Spain 2. Digestive Unit, University Hospital Vall d’Hebron, Barcelona, Spain 3. CIBERehd, Instituto de Salud Carlos III, Madrid, Spain Background: As the irritable bowel syndrome (IBS) has a broad clinical heterogeneity, previous analyses of the gut microbiome on small cohorts of IBS patients failed to uncover consistent patterns. Here we investigated the microbiome of a large cohort of IBS patients to identify specific signatures for IBS subtypes. Methods: We examined the microbiome of 113 patients with IBS and that of 66 healthy controls. A subset of these participants provided two samples one month apart. We analyzed a total of 290 fecal samples, generating more than 20 million 16S rRNA sequences. Results: In patients with IBS, a lower microbial diversity was associated with a lower relative abundance of Firmicutes such as Erysipelotrichaceae and Ruminococcaceae, butyrate-producing bacteria. No differences were found between IBS-C and healthy controls. However, four microbial groups differentiated IBS-A and IBS-D from IBS-C and healthy controls. Furthermore, the comparison of healthy controls with IBS patients who did not receive any treatment revealed a lower abundance of methanobacteria in patients. Finally, significant correlations were observed between several bacterial OTUs and sensation of flatulence and abdominal pain. Conclusions: Our comprehensive characterization of the microbiome shows that IBS-A and IBS-D patients are characterized by a reduction of butyrate producing bacteria, known to improve intestinal barrier function. Furthermore, these patients presented a

reduction of methane producing microbes without a compensating increase of sulfate-reducing bacteria -two major mechanisms of hydrogen disposal in the human colon- an observation that could explain excess of abdominal gas and bloating in IBS. Evi-043#159 Microbiota dysbiosis associated with epizootic rabbit enteropathy occurs after disease onset Ana Djukovic (1), Sandrine Isaac (1), Alejandro Artacho (1), Eugenio Martínez-Paredes (2), Jorge Martínez (3), and Carles Ubeda (1,4) 1 Departamento de Genómica y Salud. Centro Superior de Investigación en Salud Pública – FISABIO. Avenida de Cataluña, 21. Valencia, 46020 Valencia, Spain. 2 Institute for Animal Science and Technology. Universitat Politècnica de Valencia. Camino de Vera, s/n. Valencia, 46022, Valencia, 3 Servei de Diagnòstic de Patologia Veterinària, Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, 08193, Bellaterra (Cerdanyola del Vallés), Spain. 4 CIBER en Epidemiología y Salud Pública Numerous studies have proposed a link between microbiota imbalance (dysbiosis) and disease. In most cases, microbiota composition has been characterized after disease onset, remaining unclear if microbial changes are the cause or the result of the pathology. We studied epizootic rabbit enteropathy (ERE), the most devastating disease in rabbits, as a model of spontaneous pathology associated with dysbiosis. 16s-rRNA high-throughput sequencing revealed a marked imbalance and decrease diversity in the intestinal microbiota of rabbits with ERE. However, longitudinal prospective studies showed that the observed dysbiosis occurred exclusively after disease onset, not being the initial cause of ERE. To our knowledge, our results show for the first time that microbial changes associated with a naturally occurring disease can be the consequence of the pathological process and not its causal origin. Our results highlight the importance of performing prospective/retrospective studies to elucidate the causal relationship between dysbiosis and disease. Evi-044#161 Optimisation of a probiotic therapy in patients with irritable bowel syndrome (ibs) under real life conditions; an observational study. Ingrid Rollinger-Holzinger (1), Kim Burgard (2), Valery Bocquet (3), Bernard Weber (2) 1. Internistische Praxis, Luxembourg, Luxembourg 2. Laboratoires Réunis, Junglinster, Luxembourg 3. Luxembourg Institute of Health, Competence Center in Methodology and Statistics, Strassen, Luxembourg Irritable bowel syndrome (IBS) as the most common digestive disorder in the industrial countries is characterized by recurring symptoms of abdominal pain, bloating, and altered bowel function in the absence of structural, inflammatory, or biochemical abnormalities. The etiology of IBS still remains uncertain but is supposed to be multifactorial including stress, altered gastrointestinal (GI) motility, visceral hypersensitivity, altered intraluminal milieu, immune activation, and changes in the intestinal microflora. It has been described that the gut microflora in patients suffering from IBS is characterized by a significantly lower biodiversity of microbes within fecal samples compared to healthy controls. Treatment options for IBS remain dissatisfactory and current guidelines only focus on relieve of abdominal pain. Probiotics are considered to be an effective therapy for patients suffering from IBS. Several trials have already proven their beneficial effect in pain reduction, bowel habit, flatulence, and distension. Laboratoires Réunis is currently conducting an observational study in collaboration with a luxemburgish physician specialized in internal medicine. During her daily work she has observed that treatment of IBS patients with a standard multi-species probiotic is successful in many patients. However, some patients do not respond to the standard probiotic and, consequently, IBS symptoms do not improve. The microbiota in the stool of patients who do not respond to the standard probiotic treatment is analyzed using a comprehensive stool flora test (Florinscan). According to the test results in the Florinscan these patients receive a customized probiotic treatment. As the main study objective we will evaluate if the treatment of IBS patients can be optimized by a customized probiotic therapy depending on the Florinscan results. First results seem to show that non-responders to the standard probiotic treatment can be positively impacted by the intake of a customized probiotic therapy. The study has been accepted by the “Comité National d’Ethique de Recherche (CNER)” Luxembourg and has furthermore been notified at the Luxembourgish “Ministère de la Santé”.

Evi-045#166 Enterococcal load and indoxyl sulfate levels as predictors of microbiome diversity in patients receving allogeneic stem cell transplantation Daniela Sporrer (1), Andreas Hiergeist (2), Josef Koestler (2), André Gessner (2), Peter Oefner (3), Katja Dettmer (3), Daniel Wolff (1), Wolfgang Herr (1) and Ernst Holler (1) 1. Department of Hematology and Oncology, Internal Medicine III, University Medical Center, Regensburg, Germany; 2. Institute of Clinical Microbiology and Hygiene, University Medical Center, Regensburg, Germany; 3. Institute of Functional Genomics, University of Regensburg, Regensburg, Germany; Previous studies described an intestinal loss of bacterial diversity during allogeneic stem cell transplantation (ASCT) and a correlation between low bacterial diversity and clinical long-term outcome like transplant related mortality (TRM) as well as overall mortality in patients after ASCT. We now asked whether high enterococcal load or low levels of urinary indoxyl sulfate (UIS), a metabolite exclusively produced in the presence of colonic commensal bacteria, as indirect parameters of microbiome disruption are associated with gastrointestinal (GI) Graft-versus Host Disease (GvHD) and outcome after ASCT. A total of 160 patients were included in the analysis separated in two cohorts: in cohort one stool specimens from 77 patients were collected at 4 different time-periods during transplantation and enterococcal strain specific PCR analysis for Enterococcus faecalis and Enterococcus faecium was performed. Enterococcal positivity at day 28 as an endpoint was defined by either an increase of enterococcal load or by conversion to positivity until day 28. In a second cohort of further 83 patients UIS levels were measured weekly using liquid chromatography-tandem mass spectrometry. Positivity of both enterococci until day 28 after ASCT was associated with severe stages of GI GvHD (50.0% versus 6.0%, p<0.0001) and higher TRM (p=0.025) within the first year (Figure 1.A). Mean UIS levels dropped significantly during the course of transplantation (p<0.0001), and severe GI GvHD was more prevalent in patients with low UIS levels (25.9% versus 3.4%, p=0.015). Furthermore one-year TRM was higher in patients with low UIS levels (p=0.005) (Figure 1.B).

Evi-046#171 Gut Microbiome Compositional Differences between Tumor and Matching Adjacent Normal Tissues from the US and Spain Imane Allali (1,2,5), Susana Delgado (3), Aurora Astudillo (4), Hassan Ghazal (5,6), Saaïd Amzazi (2), Jen Jen Yeh (7), Temitope Keku (8),M. Andrea Azcarate-Peril (1) 1. Department of Cell Biology and Physiology, and Microbiome Core Facility, University of North Carolina School of Medicine, Chapel Hill 2. Laboratory of Biochemistry & Immunology, Faculty of Sciences, University Mohammed V, Rabat, Morocco 3. Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Villaviciosa-Asturias, Spain 4. Instituto Universitario de Oncología del Principado de Asturias, Hospital Universitario Central de Asturias, Universidad de Oviedo, Asturias, Spain 5. Laboratory of Genetics and Biotechnology, Faculty of Sciences of Oujda, University Mohammed Premier, Oujda, Morocco 6. Polydisciplinary Faculty of Nador, University Mohammed Premier, Morocco 7. Division of Surgical Oncology, Department of Surgery, University of North Carolina School of Medicine, Chapel Hill 8. Division of Gastroenterology & Hepatology, Department of Medicine, University of North Carolina School of Medicine, Chapel Hill

Colorectal cancer (CRC) is the third most common cancer in the world and the second leading cause of cancer deaths in the US and Spain. The molecular mechanisms involved in the etiology of CRC are not yet elucidated due in part to the complexity of the human gut microbiota. In this study, we compared the microbiome composition of 90 tumor and matching adjacent normal tissue (normal) from cohorts from the US and Spain by 16S rRNA amplicon sequencing in order to determine the impact of the geographic origin on the CRC microbiome. Data showed a significantly (P < 0.05) higher Phylogenetic Diversity (PD) for the US (PD Normal = 26.3 ± 5.3, PD Tumor = 23.3 ± 6.2) compared to the Spanish cohort (PD Normal = 18.9 ± 5.9, PD Tumor = 18.7 ± 6.6) while no significant differences in bacterial diversity were observed between tumor and normal tissues for individuals from the same country. Normal tissues from the Spanish cohort were enriched in Firmicutes (SP = 43.9% and US = 22.2%, P = 0.0001) and Actinobacteria (SP = 1.6% and US = 0.5%, P = 0.0018) compared to US normal tissues, while normal tissues from the US had significantly higher abundances of Fusobacteria (US = 8.1% and SP = 1.5%, P = 0.0023) and Sinergistetes (US = 0.3% and SP = 0.1%, P = 0.0097). Comparison of tumor and normal tissues in each cohort identified the genus Eikenella significantly over represented in US tumors (T = 0.024% and N = 0%, P = 0.03), and the genera Fusobacterium (T = 10.4% and N = 1.5%, P = < 0.0001), Bulleida (T = 0.36% and N = 0.09%, P = 0.02), Gemella (T = 1.46% and N = 0.19%, P = 0.03), Parvimonas (T = 3.14% and N = 0.86%, P = 0.03), Campylobacter (T = 0.15% and N = 0.008%, P = 0.047), and Streptococcus (T = 2.84% and N = 2.19%, P = 0.05) significantly over represented in Spanish tumors. Our study suggests that microbiome compositional dissimilarities by geographic location should be taken in consideration when approaching CRC therapeutic options. Evi-047#174 Associations of gut microbiota with metabolic syndrome and heritability in Korean twins and their families Mi Young Lim (1), Hyun Ju You (1), Hyo Shin Yoon (1), Bomi Kwon (1), Jae Yoon Lee (1), GwangPyo Ko (1, 2, 3) 1. Department of Environmental Health, Graduate School of Public Health, Seoul National University, Seoul, Republic of Korea 2. Center for Human and Environmental Microbiome, Seoul National University, Seoul, Republic of Korea 3. N-Bio, Seoul National University, Seoul, Republic of Korea Metabolic syndrome (MetS), a cluster of abdominal obesity, dyslipidemia, hypertension and hyperglycemia, is becoming pandemic lately. Accumulating evidence has shown that the composition of gut microbiota is closely related to the development of metabolic disorders such as obesity and type 2 diabetes. At the same time, MetS has been known that it was greatly linked to host genetics as well. However, there has been very limited investigation on how much and which specific gut microbiota are related to host genetics and responsible for MetS. Here, we investigated how much gut microbiota are heritable and related to MetS in Korean twins and their families. We collected a total of 655 fecal samples from 153 pairs of MZ twins (n=306), 37 pairs of DZ twins (n=74), and their parents and siblings (n=275), and performed V4-16S rRNA sequencing for their fecal DNA samples using an Illumina MiSeq platform. Approximately 18% (121 individuals) of study participants fulfilled MetS criteria. And MetS status as well as each level of MetS components (blood pressure, triglyceride, fasting blood sugar, waist circumference, and high-density lipoprotein cholesterol) were found highly heritable. Significant difference in the composition and function of gut microbiota between healthy and MetS individuals was observed. Multivariate analysis controlling for age, gender, and family relationship showed that specific gut microbes such as Methanobrevibacter, and Eggerthella were significantly enriched in MetS individuals, while Akkermansia, Alistipes, Parabacteroides, and Bifidobacterium were significantly enriched in healthy individuals. To investigate the influence of host genetics on the gut microbiota further, we measured the heritability of the gut microbiota related to MetS. Among the gut microbes which were found to be associated with MetS status, the highest heritability (45.5%) was recorded for Actinobacteria which Bifidobacterium sp belong to. Additionally, Alistipes, Parabacteroides, and Lactobacillus showed significant heritabilities as well. These results suggest that specific gut microbes are heritable and contribute to MetS. The role of host genetics related to gut microbiota should be further investigated. Evi-048#179 Structural Shift of Intestinal Microbiota during Daily Green Tea Consumption might Contributes to the Decrease of Body Weight in Mice Baohong Wang (1), Qiongling Bao (2), Jiangping Ge (3), Yuejian Mao (4), Xiangyang Jiang (5), Lingling Tang (6), Yu Chen (7), Lanjuan Li (8). Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China. Email: ljli@zju.edu.cn Background and Aims: Despite evidence indicated that the daily Green tea consumption (DTC) contribute to the control of body weight in human, possibly via its impact on the intestinal microbiota (IM), the markers of altered IM induced by DTC has not been reported. This study was aimed to observe whether DTC could control the body weight and modulate the composition of IM during DTC in mice with normal diet. Materials and Methods: Male mice fed normal diet were subjected to either water drink (control, n = 16) or DTC (n = 6) (50 mg of green tea/ kg/ day) for 7 days. The body weight was collected before and after the DTC. And bacterial DNA from the fecal of Day 0, 1, 3, 5 and the cecum of Day 7 during DTC was extracted for microbiota study. The microbial community was profiled by

the denaturing gradient gel electrophoresis (DGGE) profiling and validated by quantitative real-time polymerase chain reaction (qRT-PCR) of the 16S rRNA genes. Results: Mice received DTC demonstrated significant decrease in the body weight before and after the treatment (19.6 ± 0.4 vs. 16.8 ±0.43) compared with the control group (19.8 ± 0.3 vs. 21.6 ±0.2) (p < 0.002). Interestingly, fecal DGGE profiling showed a clear deviation of IM during DTC. And qRT-PCR revealed a significantly increase in Escherichia coli, Enterobacteriaceae, Prevotella, Bifidobacterium after DTC of 5 days, then a marked increase in Bacteroides, C. coccoides and C. leptum, and a reduction in Lactobacillus after DTC of 7 days (p < 0.05 or p < 0.01). There were no differences in the Faecalibacterium prausnitzii and Bacteroides fragilis. Specifically, Lactobacillus supplement with DTC could recover the body weight of mice. Conclusion: Our findings firstly evidenced that the alteration of IM is induced by DTC, which might contribute to its effect of the control of body weight. Acknowledgments: This study was supported by the Natural Science Foundation of China (30901190, 81172702), National Program on Key Basic Research Project (2013CB531401) and the Health Bureau of Zhejiang Province Foundation (2008QN010). Evi-049#180 Study of subgingival microbiome in subjects with periodontal disease and HFE-related hereditary hemochromatosis Sandrine Le Gall-David (1), Vincent Meuric (1), Yves Deugnier (2), Martine Bonnaure-Mallet (1) and Frédérique Barloy-Hubler (3) 1. EA 1254, Université Rennes 1 and CHU Rennes, France. 2. INSERM CIC1414, CHU Rennes, service des maladies du foie, France. 3. CNRS-UMR 6290, IGDR, Université Rennes 1, France. Periodontitis is a chronic inflammatory disease related to bacterial infection. The disease involves the destruction of supporting periodontal tissue, ultimately resulting in tooth loss. Association with systemic diseases such as diabetes, cardiovascular diseases, rheumatoid arthritis or cancer has been found. In a recent study, we show that caucasian patients with HFE-related hereditary hemochromatosis (HFE-HH) have a highly prevalence of periodontitis. HFE-HH is an autosomal recessive disorder characterized by iron overload potentially damaging organs such as the liver, heart and pancreas. To understand why the periodontal disease develops preferably in HFE-HH patients, we examined the subgingival bacterial biodiversity in HFE-HH periodontitis patients by sequencing V3-V4 regions of the 16S rRNA genes. Cleaned reads were taxonomically assigned using the RDP-classifier. Bacterial communities with 4 independent healthy patients (no HFE-HH, nor periodontitis) as well as 4 independent chronic periodontitis patients (no HFE-HH) have been used as controls. A total of 24 periodontal pocket microbial community samples were analyzed, yielded a combined total of 510,238 sequences across all samples (median count of 22,944, max. 30,863, min. 2040). The observed richness, alpha and beta diversity (Shannon and Simpson index) tend to significantly increase with the periodontal disease gravity, evaluate through the depth of subgingival pockets (4 and 5 mm = moderate, 6 mm= intermediate and >7 mm = severe). While interpersonal variability is usually observed, HFE-HH patients could be clustered into 5 microbial profiles only. All controls and HFE-HH samples contains the same 5 major phylum (abundance >1%): Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria and Spirochaetes. However, the distribution of these five phyla varied amongst health and periodontitis samples and we observed a significant reduction in the abundance of Actinobacteria in HFE-HH subgingival community (15 fold compared to healthy and 3 fold compared to periodontitis without HFE-HH samples). The disappearance of Actinobacteria population in hemochromatosis-related periodontitis is accompanied by an absence of TM7. TM7 is an obligate epibiont living in symbiosis with Actinomyces. This candidate division TM7 is described in oral healthy cavity at 1% of the whole oral bacterial community and, found increased in abundance in non-HFE periodontitis pockets (6 to 21%). This change is accompanied by a significant increase in Bacteroidetes and Fusobacteria if compared with healthy and non-HFE samples whereas Proteobacteria population remained stable. At this stage of our analysis, our study shows that periodontitis is caused by ecological disturbances in subgingival communities and is also dependent with other chronic disease such as hemochromatosis. Evi-050#181 Effects Of Lactulose On Acute Phase Proteins And Liver Function In Patients With HBV-related Acute-on-chronic Liver Failure Yumin Xu (1), Hui Wang (2), Wei Cai (3), Qing Xie (4) Department of Infectious Diseases, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Objective : To evaluate the clinical efficacy of lactulose treatment in patients with HBV-related acute-on-chronic liver failure(HBV-ACLF). Method: 160 patients with HBV-ACLF were randomly divided into control and treatment group. The patients in controls (n=80) were treated with conventional therapy and patients in the treatment group were treated with lactulose additionally (n=80, received Lactulose 30mL per day). The blood ammonia, the serum alanine aminotransferase（ALT），aspartate transaminase（AST），total bilirubin(TB),，C-reaction protein (CRP), procalcitonin (PCT) and the psychomotor performance were observed at the 0, 3, 7, 10 days of treatment.

Results: In the treatment group，serum CRP and PCT at the baseline were 19.23±4.65 mg/L and 17.28±4.43µg/L，respectively），however，the CRP and PCT decreased to 6.56±1.38mg/L and 5.35±2.12µg/L 7 days after the treatment （P<0.05）；In the control group，the CRP and PCT levels at baseline were 18.21±4.47mg/L and 16.16±8.37µg/L，respectively，but 7 days later, the CRP and PCT levels（16.28±7.36mg/L or 14.01±8.24µg/L）were almost similar to the baseline; The serum ALT （389±47 IU/L）and AST（371±38 IU/L） decreased to 163±32 IU/L and 115±34 IU/L 7 days after the treatment （P<0.05）; The serum TB in the treatment group decreased from 369±78 µmol/L to 226±68µmol/L 7 days later（P<0.05）. The blood ammonia in the treatment group decreased from 123±32µmol/L to 63±27µmol/L （P<0.05）Conclusions : Lactulose efectively and safely improves liver function and ameliorates inflammatory response in patients with HBV-related acute-on-chronic liver failure. Evi-051#182 Airway dysbiosis is associated with enhancing susceptibility to Allergic rhinitis Xingche Xu1, Xiaopeng Yuan1, Liyuan Pang, Rui Ding, Li Tang , jin Zhang, Wenkai Yu, Yinhui Liu, Yanjie Guo , Jieli Yuan, Shu Wen 1These authors contributed equally to this work. *Correspondence to: Shu Wen, shuwen@dlmedu.edu.cn Department of Microecology, College of Basic Medical Sciences, Dalian Medical University, Dalian, Liaoning, China Background: Allergic rhinitis (AR) is a common, multifactorial disease and its prevalence is on the increase. There is now accumulating evidence that environment factors have a vital role on AR susceptibility and severity. Resident microbiota are now recognized as potent modulator of host immune responses associated with allergic disease, particularly during early infancy. The study trajectory of the respiratory tract microbiota is slightly behind the one of gut microbiota because the health lung was traditionally considered sterile. An improved understanding of the respiratory tract commensal bacteria colonization has fueled interest in the relevance of airway microbiota on allergic airway inflammation. Objective: Here we sought to clarify the correlation between early life antibiotic-driven airway microbiota alteration and AR susceptibility. Methods: AR was induced in control rats and Cefotaxime Sodium-treated rats by sensitization and challenge with ovalbumin(OVA). Upper airway microbial community analyses were performed on NALF samples via PCR-DGGE (Denaturing gradient gel electrophoresis). AR symptoms, nasal cavity inflammation, mucosa pathology, serum level of IgE, cytokine responses and Treg cell population in control rats and antibiotics-treated rats were measured to compare the difference of allergic response and the underlying mechanism. Results: Markedly reduced diversity and increased potentially harmful microorganisms colonization in upper airway and lower Treg population were showed in Cefotaxime Sodium-treated rats compared with control rats. We also found antibiotic-OVA rats have elevated nasal rubbing and sneezing and serum IgE levels, higher numbers of leukocyte in their blood and NALF compared with control-OVA rats. Consistent with that, decreased ratio of IFN-γ/IL-4, increased local production of Th2 and Th17 associated cytokines, and lower number Regulatory T-cell populations were observed in antibiotic-treated rat. Conclusions: These data demonstrated that early life antibiotic-driven changes in airway microbiota induced a defective development of immunologic tolerance, increased susceptibility to allergic rhinitis. Evi-052#183 Antibiotic-driven airway dysbiosis in early life elevate allergic respiratory inflammation Wenkai Yu, Xiaopeng Yuan, Xingche Xu, Li Tang , Rui Ding, Liyuan Pang, Yinhui Liu, Huajun Li, Ming Li, Shu Wen* *Correspondence to: Shu Wen, shuwen@dlmedu.edu.cn Department of Microecology, College of Basic Medical Sciences, Dalian Medical University, Dalian, Liaoning, China Background: Recent studies showed that the incidence and severity of asthma were associated with dysbiosis. The study trajectory of the airway microbiota is slightly behind compared with gut microbiota. It suggests airway microbiota may be important in shaping airway immune response as long as the airway dysbiosis was observed in asthma. Objectives: To investigate the effect of airway dysbiosis on the sensitivity to allergen and elucidate the underlying immune mechanisms. Methods: Antibiotic rats were treated by inhaling ceftriaxone sodium and allergic airway inflammation was induced in control and antibiotic rats by sensitized and challenged with ovalbumin. Alterations in airway microbiota colonization were analyzed by denaturing gradient gel electrophoresis (DGGE), the asthma-like symptoms, the Treg cell population and Th-associated cytokines production were measured to check changes of allergic response and the underlying mechanism. Results: Our results showed that the diversity of airway microbiota in BALF was significantly decreased in antibiotic rats compared with control rats, and potentially harmful microorganisms were expanded. At the same time, the nasal rubbing frequency and sneezing number in antibiotic rats were increased. Elevated thickness of epithelial and number of infiltrating lymphocytes in lung tissue combined with an exaggerated number of leukocyte and eosinophil in BALF in antibiotic rats. Furthermore, the ratio of Th1 associated cytokines IFN-γ/Th2 associated cytokines IL-4 was significantly higher, while the cd25+foxp3+Treg cell population was lower in the antibiotic group compared with the control group.

Conclusion: These data elucidated that antibiotic-driven airway dysbiosis could elevate allergic respiratory inflammation ，enhance the susceptibility to asthma. Evi-053#185 Intestinal dysbacteriosis in mice induces changing of T lymphocyte subpopulations in Peyer’s patches (PPs) Fei Gao, Ming Li, Shu Wen, Yinhui Liu , Li Tang Corresponding to: Professor Li Tang Department of Microecology, Dalian Medical University, 9 Western Section, Lvshun South Street, Lvshunkou District, 116044, Dalian, China Background: The large numbers of microorganisms that inhabit mammalian intestine have a highly coevolved relationship with the immune system. Dysbacteriosis in intestinal microbiota induces alterations on immune responses, and closely relates to health and disease development. Peyer’s patches (PPs) are immune sensors of intestine, they exert essential immune functions during the development of inflammatory disease. However, the interactions between commensal bacteria alteration and changes in PPs have been poorly characterized. Objective: To investigate the relevance of intestinal dysbacteriosis on the changing of lymphocyte subpopulations and cytokines production in PPs . Methods: Intestinal dysbacteriosis mouse model was induced by INN sodium. The changing of lymphocyte subpopulations and cytokines production were measured by flow cytometry and emi-quantitative RT-PCR. Results: By flow cytometry detection, the total CD3+ T cells in PPs of mice with intestinal dysbacteriosis were found significantly increased compared to control. The percentage of CD4+ T cells increased by 7.34% and 5.15% in mild and severe groups, respectively. The CD8+ T cells in PPs of mice with dysbacteriosis decreased, resulted in increased proportions of CD4+/CD8+ compared with healthy mice. The regulatory CD4+CD25+ T cells were found decreased severely from 4.79% to no more than 1.53%. The expression levels of cytokines secreted by immune cells were detected by semi-quantitative RT-PCR, results showed that IL-2, and IFN-γ secreted by Th1 type cells decreased dramatically, while the levels of IL-4 and IL-10 that secreted by Th2 type cells increased. Conclusion: These data demonstrated that intestinal dysbacteriosis in mice significantly reduced the immune tolerance in PPs and orientated immune response to humoral immunity. Evi-054#191 Human cellular miR-1224-3p and miR-1913 seed family microRNAs target on HIV-1 and inhibit virus production Xiangyun Lu, Jin Yang, Changzhong Jin, Linfang Cheng, Juan Wang, Zongxing Yang, Fumin Liu, Tiansheng Xie, Haibo Wu, Xiaorong Peng, Hangping Yao, Nanping Wu State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China Background Increasing evidences indicate that both cellular and viral derived miRNAs are important players of host-virus interaction. We are interested in a de novo search of cellular miRNAs that can directly target on the HIV-1 transcripts and function to repress HIV-1 production. Methods The terminal 1.9kb and subfragments of the 3’-UTR of HIV-1 YU2 strain were cloned to luciferase and GFP-based miRNA reporter vectors, respectively. The recombinant reporter gene expression was measured after transfection of 293T, THP-1 cell lines and primary monocyte-derived dendritic cells. The MicroInspector online tool was used to predict cellular miRNAs that might bind to the fragments and induce the reporter gene repression. Subsequent experiments using miRNA mimics, miRNA inhibitors, and miRNA binding site-mutated reporter constructs were performed to verify the function of the predicted miRNAs. Results Reporter gene assay identified a subfragment that evidently impaired reporter gene expression in all three cell types. MicroInspector analysis revealed a panel of miRNAs that might act on this region. Subsequent reporter gene assays using miRNA mimics, miRNA inhibitors, and miRNA binding site mutation method confirmed the effectiveness of the predicted miR-1224-3p and miR-1913 seed family miRNAs. Moreover, mutation of the two miRNA binding sites on the wild type pNL4-3 HIV-1 plasmid could enhance virus production when transfected into 293T cells. Conclusions We have identified a novel array of host miRNAs that directly targeted on HIV-1 transcripts and interfered with virus production.

Evi-055#202 ASSOCIATION OF CLASSICAL MICROBIOLOGY AND 16S METAGENETIC ANALYSIS TO EVALUATE THE PRESENCE OF CLOSTRIDIUM DIFFICILE IN A BELGIAN NURSING HOME C. Rodriguez1, B. Taminiau1, N. Korsak1, V. Avesani2, J. Van Broeck2, M. Delmée2, G. Daube1. 1Food Science Department, Faculty of Veterinary Medicine, University of Liège, B43bis, Sart-Tilman, 4000 Liège, Belgium. 2Microbiology Unit, Catholic University of Louvain, Avenue Hippocrate 5490, 1200 Brussels, Belgium. Increasing age, several co-morbidities, environmental contamination, antibiotic exposure and other intestinal perturbations appear to be the greatest risk factors for C. difficile infection (CDI). Therefore, elderly care home residents are considered particularly vulnerable to CDI. The main objective of this study was to evaluate and follow the prevalence of C. difficile in a Belgian nursing home. During a 4-month period, stool samples from a group of 23 elderly care home residents were collected weekly. A C. difficile microbiological detection scheme was performed along with an overall microbial biodiversity study of the feces content by 16S metagenetic analysis. Culture of samples was performed in a selective medium cycloserine cefoxitin fructose cholate. An identification of the isolated colonies was done by PCR detection of tpi, tcdA, tcdB and cdtA genes. Toxic activity was confirmed by a cytotoxic immunoassay. Further characterization was performed by PCR ribotyping. The Metagenetic analysis was targeted on the v1-v3 hyper-variable region of 16S rDNA. The taxonomical assignment of the populations was performed with MOTHUR and Blast algorithms. Seven out of 23 (30.4%) residents were (at least one week) positive for C. difficile. The most common PCR-ribotype identified was 027. Microbiota analysis reveals that each resident has his own bacterial imprint, which is stable during the entire study. Residents’ positives for C. difficile by classical microbiology showed an important proportion of C. difficile sequences. However, metagenetic sequencing can’t substitute targeted protocols. It was not used as a diagnostic tool to detect C. difficile but rather to determine the identification and correlations of the major bacterial populations that are present in the gut microbiota. In conclusion, this unique association of classical microbiology protocol with pyrosequencing allowed to follow C. difficile in patients and to identify several other bacterial populations whose abundance is correlated with C. difficile. Evi-056#203 Complementary feeding of breast-fed infants: impact on gut microbiota and gut inflammation Meghan B. Azad (1,2), Elnaz Azad (3), Wafaa Qasem (4,5), Chenxi Cai (4,5), Ehsan Khafipour (2, 3,6), James Friel (1,2,4,5) 1. Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, Canada 2. Children’s Hospital Research Institute of Manitoba, Winnipeg, Canada 3. Department of Animal Science, University of Manitoba, Winnipeg, Canada 4. Richardson Centre for Functional Foods and Nutraceuticals, Winnipeg, Canada 5. Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada 6. Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Canada OBJECTIVE: The American Pediatric Society recommends that iron-fortified cereal or meat be introduced to all breast-fed infants at six months of age to support iron stores and linear growth. We undertook a randomized controlled trial to assess the impact of these recommended first solid foods on the infant gut microbiota and gut inflammation. METHODS: Ninety exclusively breastfed infants were randomized to 1 of 3 feeding groups: iron-fortified cereal (FeCer), iron-fortified cereal with fruit (FeCer+Fr) or meat (M). Fecal samples were collected before introduction of study foods (4 – 5.5 months) and 3 weeks after introduction of these foods. Fecal iron was measured by photometric assay, reactive oxygen species (ROS) were measured by HPLC, and calprotectin was measured by ELISA. Gut microbiota were characterized by Illumina sequencing of the 16S rRNA (V4 region). Differences in gut microbiota communities were visualized by principal coordiante analysis and tested by permutational analysis of variance (PERMANOVA) and microbiota richness (Chao1 estimator) and diversity (Shannon Index) were evaluated. METAGENassist and LEfSe software packages were used to explore differences in microbiota composition across feeding groups. Correlations between microbiota, iron, ROS and calprotectin were also investigated. RESULTS: Fecal iron content was significantly different across feeding groups (p<0.001); levels were highest in the FeCer group and lowest in the M group, indicating better iron absorption from meat versus fortified cereal. Compared to the FeCer group and after correcting for baseline values, ROS tended to be lower in the M group (p=0.08), and fecal calprotectin was higher in the FeCer+Fr group (p=0.02). There was a significant increase in gut microbiota richness in the FeCer+Fr and M groups after weaning, whereas no change was observed in the FeCer group. There was no clear difference in microbiota community structure across feeding groups (PERMANOVA, p=0.22) and the relative abundance of dominant phyla and families was similar, although some genus-level differences were detected. Before weaning, microbiota richness was positively correlated with ROS (Spearman r=0.51, p<0.001), but there was no correlation after weaning (r=0.15, p=0.29). Both before and after weaning, the relative abundance of family Coriobacteriaceae was positively correlated with ROS (r=0.39, p=0.001), and genus Staphylococcus was negatively correlated with calprotectin (r =-0.61, p<0.001). CONCLUSION: The type of first complementary food introduced to breast-fed infants may influence gut inflammation and the gut microbiota, potentially due to variations in iron absorption from different foods. Further research is warranted to fully characterize these associations and to establish implications for infant health.

Evi-057#204 Frequent antibiotic treatment impacts early development of gut microbiota in infants Meghan B. Azad (1), Tedd Konya (2), David S. Guttman (3), Allan B. Becker (1), Malcolm R. Sears (4), Piushkumar J. Mandhane (5), Padmaja Subbarao (6), Stuart E. Turvey (7), James A. Scott (2), Anita L. Kozyrskyj (5), and the CHILD Study Investigators (8) 1. Department of Pediatrics & Child Health, University of Manitoba, Winnipeg, Canada 2. Dalla Lana School of Public Health, University of Toronto, Toronto, Canada 3. Cell & Systems Biology, University of Toronto, Toronto, Canada 4. Department of Medicine, McMaster University, Hamilton, Canada 5. Department of Pediatrics, University of Alberta, Edmonton, Canada 6. Department of Pediatrics, University of Toronto, Toronto, Canada 7. Department of Pediatrics, University of British Columbia, Vancouver, Canada 8. Canadian Healthy Infant Longitudinal Development Study BACKGROUND: Despite declining trends of antibiotic use in children, there is growing concern over the treatment of infants with antibiotics. Antibiotic use has been linked to amoxicillin-resistant late-onset E. coli infections in infants. As shown by rodent models, antibiotic exposure during critical windows of gut microbiota development during infancy has significant ramifications for the development of immune and metabolic disease. To provide evidence in the context of current antibiotic use patterns, we undertook a study of the impact of antibiotic exposure on the gut microbiota of Canadian infants at 3 and 12 months. METHODS: Accessing data on full-term infants at the Manitoba site from the Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort, fecal samples collected at 3 and 12 months were characterized by high-throughput Illumina sequencing of the V4 region of the 16S rRNA gene. These gut microbiota profiles were linked to infant records of oral antibiotic use obtained from a provincial prescription database, and to intravenous and/or intrapartum treatment reported in the birth record. To detect differences in median microbiota species richness and relative abundance of bacterial taxa, the Kruskal-Wallis test was used. Spearman’s correlations were determined for gut microbiota metrics and number of antibiotic courses. RESULTS: In our population of 190 Canadian infants, over 60% had been exposed to antibiotics by 1 year of age, 32% had received 1 course of antibiotics, 16% had received 2 courses and 12% had received 3 or more courses. Amoxicillin was the most commonly-prescribed antibiotic (68%); 12% had received azithromycin. At 3 months of age, microbiota species richness and relative abundance of Bacteroidaceae declined with an increasing number of antibiotic courses (r=-0.18, p<0.02 and r=-0.32, p<0.0001 respectively), but a positive correlation was found between frequency of administration and the Enterobacteriaceae (r=0.21, p<0.005). These trends were also observed in a subset of vaginally-delivered infants (n=137), undertaken to eliminate confounding by method of birth. Regarding temporality of treatment, relative abundance of Bacteroidaceae at 3 months was lowest with early courses of antibiotics; Enterobacteriaceae were more abundant within 10 days of antibiotic treatment. Colonization with the Veillonellaceae family was also significantly suppressed following a recent course of antibiotics. At 1 year of age, Bacteroidaceae abundance remained lower following any antibiotic exposure but few other statistical associations were found for microbial composition at the family level. CONCLUSION: Frequent antibiotic treatment of infants has the greatest impact on gut microbiota early in its development. Affected microbiota include the Bacteroidaceae and Enterobacteriaceae families, which amongst other functions, have important roles in the programming of the immune and metabolic systems. Evi-058#214 Isolation and characterization of adherent-invasive Escherichia coli in Crohn's disease patients in Brazil Rafaella Ferreira Avelar Costa (1,2), Maria de Lourdes de Abreu Ferrari (3), Marie-Agnès Bringer (1), Arlette Darfeuille-Michaud (1), Flaviano dos Santos Martins (2), Nicolas Barnich (1) (1) M2iSH, UMR Inserrm Université d’Auvergne U1071, USC INRA2018, Clermont-Ferrand, France (2) Laboratory of Biotherapeutic Agents, Federal University of Minas Gerais, Belo Horizonte, Brazil (3) Alfa Institute of Gastroenterology, University Hospital, Belo Horizonte, Brazil Crohn's disease (CD) is an inflammatory bowel disease (IBD) characterized by chronic inflammation of the intestine in humans. The etiology of CD remains unknown, however, the most common hypothesis is that chronic inflammation results from an abnormal inflammatory response against intestinal microbiota in a genetically susceptible host. Several studies have demonstrated that the intestinal mucosa of CD patients is abnormally colonized by adherent-invasive Escherichia coli (AIEC) strains. However, to date, no studies have focused on the involvement of such E. coli strains in CD patients in Brazil. The aim of this study was to isolate and characterize the E. coli strains associated from intestinal mucosa in CD patients in Brazil. Biopsies were performed on 35 subjects: 10 CD patients in active phase, 15 CD patients in remission phase and 10 controls (without intestinal disease). Although the difference was not significant, the colonization level of the ileal mucosa by adherent bacteria is higher in CD patients than in the control group. Among 270 isolates strains, 241 were identified as E. coli strains. Mark from different phylogenetic groups of E. coli was carried out by PCR. In controls patients, 47.9% of the strains belong to group A, 2.1% in the B1 group, 6.3 % in the B2 group and 43.7% in group D. In CD patients, 36.8% of the strains belong to group A, 30% in the B1 group, 10.9% in the B2 group and 22.3% in group D. In CD patients, a difference between the classification of E. coli strains was observed related to disease activity, especially among groups B2 and D. CD patients in active phase (20.2%) harboured 10-fold more E. coli belonging to the B2 group compared to CD patients in remission (2.0%) and 2.4-fold less strains of group D (12.8% CD active vs 31.3% CD remission). The adhesion and invasion ability of E. coli strains isolated were determined using human intestinal epithelial cells (I-407) and we observed that 26.9% of the isolated strains from CD patients are invasive. For each patient, the E. coli strain with the greater capacity of invasion was selected to analyze its ability to survive and multiply in human macrophages (THP-1), and we observed that 76.19% of the selected strains can survive and multiply

within human macrophages. This preliminary study on a small cohort of Brazilian CD patients suggests that the ileal mucosa of CD patients in Brazil is also colonized by E. coli strains having adherent and invasive properties. Evi-059#215 Bacterial composition of the infant gut is shaped by maternal prenatal weight Petya T Koleva (1), Ji-Sun Kim (2), David S Guttman (3), Malcolm R Sears (4), Allan B Becker (5), Piush J Mandhane (1), Padmaja Subbarao (6), Stuart E Turvey (7), James A Scott (2), Anita L Kozyrskyj (1), and the CHILD Study Investigators (8) 1. Department of Pediatrics, University of Alberta, Edmonton, Canada 2. Dalla Lana School of Public Health, University of Toronto, Toronto, Canada 3. Cell & Systems Biology, University of Toronto, Toronto, Canada 4. Department of Medicine, McMaster University, Hamilton, Canada 5. Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, Canada 6. Department of Pediatrics, University of Toronto, Toronto, Canada 7. Department of Pediatrics, University of British Columbia, Vancouver, Canada 8. Canadian Healthy Infant Longitudinal Development Study, Canada Obesity in pregnancy alters women’s gut and breast milk microbiota and potentially interferes with the transmission of maternal bacteria to the infant gastrointestinal tract. Elucidation of the influence of maternal obesity on the development of infant gut microbiota and, in turn, as a modifier of child health is a research priority. The aim of this study was to assess the impact of maternal pre-pregnancy overweight status on infant meconium and fecal microbiota. Specific emphasis was put on the Lactobacillales an order of bacteria whose members become more abundant in intestine and vagina during the third trimester of pregnancy. This study comprised a sub-set of 57 mothers and their full-term infants from the Winnipeg site of the Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort. The sub-set of 57 samples was selected based on delivery mode and antibiotic exposure. Microbiota of meconium (infant first stool) and feces collected at 3-4 months were characterized by Illumina high-throughput sequencing of the hyper-variable V4 region of the 16S rRNA gene as a signature locus. Measures of the mother’s anthropometry pre-pregnancy and infant diet were obtained from standardized questionnaires and hospital records. To detect differences in meconium and fecal bacterial populations according to maternal pre-pregnancy overweight status, the Wilcoxon test was used. Spearman’s analysis was employed to investigate correlations in taxon abundance between infant gut microbiota at birth and 3-4 months of age. Of 57 infant meconium samples, sufficient amplification product to permit sequencing was obtained from 13. The prevalence of pre-pregnancy overweight mothers was 61 % (n=8). Forty six percent of infants were exclusively breastfed at 3 months. Abundance of members of the Lactobacillales increased from birth to the 3-4 month fecal sample (median 0.22 vs 1.06, p=0.03). These taxa were strongly correlated with the abundance of the genus Ruminoccocus (r=0.64 and p=0.02) and the family Veillonellaceae (r=0.73 and p=0.01) in meconium and in fecal samples 3-4 months (r=0.81 and p=0.002, and 0.67 and p=0.01, respectively). Significantly higher ratios of Lactobacillales to Bacteroidaceae (0.04), Lactobacillales to Ruminococcaceae (p=0.04) and Lactobacillalles to Lachnospiraceae (p=0.04) were observed in meconium microbiota following pre-pregnancy overweight compared to normal weight. These differences were not observed in samples at the 3-4-month time point. To conclude, this study highlights the influence of pre-pregnancy weight on the microbiota of meconium. Members of the order Lactobacillales were strongly correlated with the abundance of butyrate-producing gut microbial communities in meconium samples. #220 Short Talk Distinct fecal microbiome associations with breast cancer and colorectal pre-cancer: Population-based studies James J. Goedert (1), Ying Zheng (2), Heather S. Feigelson (3), Yangming Gong (2), Gieira Jones (1), Yimin He (4), Xing Hua (1), Peng Peng (2), Xia Xu (5), Roni T. Falk (1), Wenjing Wang (2), Mitchell H. Gail (1), Huanzi Zhong (4), Jianxin Shi (1), Jacque 1. National Cancer Institute, NIH. 2. Shanghai Municipal Center for Disease Control. 3. Kaiser Permanente Colorado. 4. BGI. 5. Leidos Biomedical Research, Inc. 6. University of Maryland Medical School. Background: Fecal microbiome profiling in defined, general populations may provide insights on carcinogenesis and opportunities for prevention. Breast cancer in postmenopausal women and high-risk, precancerous colorectal adenoma (CRA) are informative, but contrasting examples. Methods: We conducted fecal microbiome profiling (Illumina sequencing of 16S rRNA genes amplified from DNA extracted from feces self-collected in RNAlater) of defined populations ages 50-74 – a cancer case-control study in Denver and a screening program in Shanghai. All statistical tests were two-sided. Antibiotics did not affect the associations. Denver results: In 48 postmenopausal breast cancer cases versus 48 controls, urinary estrogens were quantified by liquid chromatography/tandem mass spectrometry. Total estrogens correlated with microbiome alpha diversity in controls [phylogenetic diversity (PD)_whole tree Spearman R=0.37, P=0.009] but not in cases (R=0.04, P=0.77). Compared to controls,

cases had significantly lower alpha diversity (PD_whole tree Padj=0.004). Adjusted for estrogens and other covariates, per tertile PD_whole tree odds ratio of cancer was 0.50 (confidence interval 0.30–0.84, Figure panel A). Cases also had altered microbiome composition (unweighted UniFrac overall P=0.0055, principal component (PC1) P=0.01, Figure B). Differences in specific taxa were not significant with adjustment for multiple comparisons. Shanghai results: Of 61 occult blood-positive residents, colonoscopy revealed 24 completely normal, 20 high-risk CRA, 3 cancers, and 14 other conditions. Phylum-level fecal community composition differed significantly between CRA and normal participants (permutation P=0.02). Rank phylum-level abundance distinguished CRA from normal (area under the curve 0.767, permutation P=0.006, Figure C). CRA prevalence was 59% in phylum-level cluster B versus 20% in cluster A (Fisher’s exact P=0.01). Median relative abundance of Proteobacteria taxa was 3-fold higher in CRA and accounted for most of the difference (Wilcoxon P=0.03, positive predictive value 67%). Conclusions and implications: Fecal microbial composition differed significantly between general population adults and those with postmenopausal breast cancer or pre-cancerous CRA. Breast cancer cases also had substantially reduced alpha diversity, whereas CRA cases did not. The Denver study suggests that the gut microbiota may affect breast cancer risk, perhaps through estrogen-dependent and -independent pathways. The Shanghai study suggests that classification of fecal microbial communities might be employed to improve screening for CRA and ultimately to reduce mortality from colorectal cancer. Evi-060#221 The bidirectional stress-obesity relation: mediating role of inflammation and gut microbiota. Nathalie Michels (1) 1. Department of Public Health, Ghent University, Belgium Background: From a biopsychosocial perspective, disease is not only influenced by biological factors but also by psychosocial determinants like psychosocial stress. Researchers still struggle to understand the complex processes by which chronic stress increases vulnerability to disease. One of the main physiological stress outcomes is the highly prevalent obesity and its metabolic comorbidities. First, the stress-induced cortisol levels might increase adiposity directly by interaction with lipid homeostasis at several levels. Second, distress might indirectly facilitate obesity through behavioural pathways such as maladaptive coping behaviours leading to emotional eating of ‘comfort food’ rich in sugar and fat. These two pathways have recently been shown by our group (Michels Psychosom Med). More recently, low-grade inflammation and disturbed gut microbiota (GMB) composition have been suggested as possible biological factors in this metabolic tilt. Suggested theoretical framework: We suggest the theoretical framework in Figure 1 showing a bidirectional relation between stress and obesity with central roles for gut microbiota and inflammation. Overall, this is a model of a metabolism in homeostatic imbalance: a feedforward mechanism in overdrive without negative feedback resulting in changed physiological set-points as evidenced by increased stress sensitivity and adiposity. Hypotheses: H1 Distress stimulates emotional eating, disturbs the GMB balance and increases low-grade inflammation H2 Inflammation and GMB disturbance increase adiposity and related comorbidities H3 Based on H1 and H2, a psychological intervention is able to decrease emotional eating, restore the GMB balance, decrease inflammation and consequently restore lipid and glucose homeostasis. H4 Adiposity increases inflammation and disturbs the GMB balance H5 Diet (including emotional eating), inflammation and the GMB interact with each other H6 Inflammation and GMB disturbance increase stress sensitivity H7 Based on H5 and H6, GMB alteration by probiotics will change stress sensitivity and inflammation Sample: We will verify all these hypotheses in children. Children are more vulnerable to stressors with even effects during later adulthood or permanent brain changes and also childhood obesity is very likely to persist into adulthood. Experiments and trials in children will be highly informative as they are not confounded by other potential triggers of inflammation and drugs. In addition, childhood and adolescence seems to be a more critical time for microbiota modulation of behaviours and children are still very flexible for psychological treatment. Study design: (1) Pure observational: cross-sectional and longitudinal analyses on existing dataset in children and adolescents. (2) Contrasting groups: 2A depressed versus non-depressed children; 2B: Obese versus normal-weight children. (3) Intervention: 3A Probiotic treatment in depression; 3B Emotion-regulation in the obese.

Evi-061#224 Bacterial diversity and community types in saliva and oral environment Toru Takeshita(1), Michiko Furuta(1), Hidenori Tsuboi(1), Shinya Kageyama(1),Yoshihiro Shimazaki(2), Toshiharu Ninomiya(3), Yutaka Kiyohara(4), Yoshihisa Yamashita(1) 1. Section of Preventive and Public Health Dentistry, Kyushu University Faculty of Dental Science, Fukuoka, Japan 2. Department of Preventive Dentistry and Dental Public Health, School of Dentistry, Aichi-Gakuin University, Nagoya Japan 3. Division of Research Management, Center for Cohort Studies, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 4. Department of Environmental Medicine, Graduate School of Medial Sciences, Kyushu University, Fukuoka, Japan Oral environmental changes including disease development are assumed to drive a microbiota shift toward a disease-associated community. This large-scale population-based study investigated oral environmental factors associated with bacterial assemblage in saliva, focusing on the alpha diversity and community types. Saliva was collected from 2,343 participants (aged 40−79 years) of the Hisayama cohort study in Japan. The microbiota composition was determined by Ion Torrent 16S rRNA gene amplicon (V1–2 region) sequencing. A higher phylogenetic diversity of the salivary microbiome represented a more diverse array of minority members, especially subgingival plaque-related species. Multivariate analysis revealed that it was significantly associated with poor oral hygiene, presence of decayed teeth, gingival bleeding, deepen periodontal pockets, less residual teeth and current smoking. On the other hand, the community types were classified based on relative abundances of dominant members, especially tongue microbiota-related species. The community type characterized by a higher ratio of Prevotella and Veillonella species to Neisseria and Porphyromonas species were significantly associated with a greater age, female, less residual teeth, presence of decayed teeth, more caries-experienced teeth, deepen periodontal pockets and current smoking. These results suggest that various oral environmental conditions are reflected in alpha diversity and community types of salivary microbiome.

General and clinical condition of subjects in each community type.

Community type Adjusted

Type I Type II odds ratio

(n = 1,075) (n = 1,149) (95% CI)

Age, yr 63.7 ± 11.5 59.7 ± 10.6 1.02 (1.01–1.03)***

Sex, no. of female (%) 609 (56.6) 646 (56.2) 1.54 (1.19–2.00)**

No. of teeth 21.2 ± 7.3 24.1 ± 6.0 0.93 (0.91–0.94)***

Dental caries status

Presence of DT, no. (%) 374 (34.7) 311 (27.0) 1.27 (1.04–1.55)*

No. of DFT 14.4 ± 6.0 13.4 ± 5.5 1.10 (1.07–1.12)***

Periodontal condition

Mean PPD 1.97 ± 0.70 1.76 ± 0.63 1.22 (1.03–1.46)*

% teeth with BOP 22.3 ± 23.1 16.7 ± 19.8 0.88 (0.50–1.53)

Mean plaque index 0.83 ± 0.69 0.63 ± 0.60 1.10 (0.92–1.32)

Smoking, no. (%)

No 576 (53.8) 707 (61.5) 1

Past 219 (20.3) 296 (25.7) 1.17 (1.07–1.12)

Current 280 (26.0) 146 (12.7) 4.37 (3.20–5.98)***

Phylogenetic diversity 10.6 ± 1.9 10.5 ± 1.6 1.02 (0.96–1.08)

Multivariate logistic analysis was used to examine the association between community types

of salivary microbiome and environmental conditions.

Values with errors are means ± standard deviation.

Abbreviations: CI, confidence interval; DT, decayed teeth; DFT, decayed and filled teeth;

PPD, periodontal pocket depth; BOP, bleeding on probing.

Evi-062#228 Correlation between either Cupriavidus or Porphyromonas and primary pulmonary tuberculosis found by analysing the microbiota in patients’ bronchoalveolar lavage fluid Yuhua Zhou1,3, Feishen Lin2, Zelin Cui1, Xiangrong Zhang2, Chunmei Hu2, Tian Shen4, Chunyan Chen1, Jinhong Qin1* and Xiaokui Guo1* 1. Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China 2. Department of Tuberculosis, Nanjing Chest Hospital, Nanjing, Jiangsu, 210029, China 3. Department of Emergency Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China 4. Department of Preventive Medicine, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China Abstract Pulmonary tuberculosis (TB) has gained attention in recent decades because of its rising incidence trend; simultaneously, increasing numbers of studies have identified the relationship between microbiota and chronic infectious diseases. In our work, we enrolled 32 patients with primary TB characterised by unilateral TB lesion formation diagnosed by chest radiographic exam. Bronchoalveolar lavage fluid was taken from both lungs. Twenty-four healthy people were chosen as controls. Pyrosequencing was performed on the V3 hypervariable region of 16S rDNA in all bacterial samples and used as a culture-independent method to describe the phylogenetic composition of the microbiota. Through pyrosequencing, 271,764 amplicons were detected in samples and analysed using tools in the Ribosomal Database Project (RDP) and bioinformatics. These analyses revealed significant differences in the microbiota in the lower respiratory tract (LRT) of TB patients compared with healthy controls; in contrast, the microbiota of intra/extra-TB lesions were similar. These results showed that the dominant bacterial genus in the LRT of TB patients was Cupriavidus and not Streptococcus, which resulted in a significant change in the microbiota in TB patients. The abundance of Mycobacteria and Porphyromonas significantly increased inside TB lesions when compared with non-lesion-containing contralateral lungs. From these data, it can be concluded that Cupriavidus plays an important role in TB’s secondary infection and that in addition to Mycobacteria, Porphyromonas may also be a co-factor in lesion formation. The mechanisms underlying this connection warrant further research. Evi-063#231 Evaluation of the lower airway microbiome in Rheumatoid Arthritis. Leopoldo N. Segal, M.D.; Carles Ubeda, Ph.D.; William Wikof, Ph.D.;Anca Catrina, M.D., Ph.D; Jose U. Scher, M.D. Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, New York University Langone Medical Center, New York, USA; Institute for Research in Public Health, Valencia, Spain; Department of Molecular and Cellular Biology & Genome Center, University of California, Davis, USA; Rheumatology Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden; Division of Rheumatology, Department of Medicine, New York University Langone Medical Center, New York, USA RATIONALE Rheumatoid arthritis (RA) is a multifactorial disease in which several genetic and environmental factors have been implicated. Recent data suggest that the microbial environment present in the gut and oral mucosa might potentially contribute in the priming of an aberrant systemic immune response characteristic of RA. Among them, both P. gingivalis in the oral cavity and P. copri in the gut have been described. Imaging abnormalities seen in at-risk individuals for the development of clinical RA suggest that the lung might be yet another site of autoimmunity generation in RA. We therefore tested whether there were distinct features of the RA lung microbiome. METHODS Bronchoalveolar lavage (BAL) samples from 20 subjects with RA and 10 with sarcoidosis were obtained by bronchoscopy. 16S rRNA sequencing of barcoded amplicons targeting the V4 region was used to define microbiota. Levels of arginine and citrulline were measured using gas chromatography mass spectrometry (GC-MS), while CCP2 antibodies were measured in serum and BAL of RA subjects. Spearman correlations were performed. RESULTS There were no differences in demographic or clinical characteristics (including smoking status) between groups. 16S data show similar alpha and beta diversity between groups (Figure 1, Panel A). Comparison of taxonomic data between RA and sarcoidosis was performed using LEfSe and showed significant differences (LDA score>2, Figure 1, Panel B and C). Specifically, while RA BAL samples were enriched with Sphingobacteria, sarcoidosis BAL was enriched with Bacteroidia, Rhizobiales, Nitrospirales, and Campylobacter. GC-MS showed similar levels of arginine and citrulline in BAL (69.2[54.2-11.8] vs. 49.7[37.1-88.7] and 12.1[6.9-20.9] vs. 12.4[8.0-25.4] respectively for the sarcoidosis and RA group). For the sarcoidosis group, Streptococcus and Neisseria correlated with levels of citrulline (rho=-0.91 and -0.67, p=0.0002 and 0.03 respectively). For the RA group, Prosthecobacter and Herbaspirillum significantly correlated with citrulline levels in BAL (rho=0.54 and 0.46, p= 0.013 and 0.038 respectively). Serum levels of anti-CCP2 antibodies had a negative correlation with Escherichia and Bdellovibrio (rho=-0.47 and 0.45, p=0.03 and 0.04 respectively), and a positive correlation with Porphyromonas, Rahnella and Chryseobacterium (rho=0.46, 0.46 and 0.45, p=0.03, 0.03 and 0.04 respectively). CONCLUSIONS Despite the small number of samples analyzed, several taxonomic differences were noted between RA and Sarcoidosis. Correlations between relative abundance of specific taxa in BAL with BAL citrulline and serum anti-CCP2 antibodies supports an association between the lower airway microbiome and the host immune phenotype in RA. Evaluation of functional aspects of the lower airway microbiome (e.g. metagenome/metabolomics) may provide further insights into a possible contribution of active microbial metabolism to the immunological priming of an aberrant immune system in RA. Funding: UL1 TR000038; 5U01CA086137-13; 3R01HL090316-05; AI080298

Evi-064/#234 Exploring the human breast microbiome in benign and malignant disease states Tina J HIeken (1), Nicholas Chia (2,3), Krishna R Kalari (2), Tanya L Hoskin (2), Kevin Thompson (2), Sheri Ramaker (1), Larry M Baddour (4), Karla V Ballman (2), Amy C Degnim (1) 1. Department of Surgery, Mayo Clinic, Rochester, MN, USA 2. Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA 3. Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA 4. Division of Infectious Diseases, Mayo Clinic, Rochester, MN, USA Indirect evidence of a microbiome in the human breast is suggested by the recent description of the presence of immune effector cells within breast tissue. However, there is a paucity of data on deep sequencing of sterilely obtained human breast tissue and no data investigating differences in the microbiota of adjacent normal breast tissue from patients with benign versus malignant disease. With IRB approval, we initiated a prospective study to collect sterile samples from women undergoing lumpectomy for benign or malignant breast disease. After intraoperative pathology confirmation of complete removal of the targeted lesion, we collected three sterile specimens from each subject consisting of a skin swab adjacent to the incision, skin tissue and putatively normal breast tissue adjacent to the resected lesion. We studied 10 women aged 44 to 77 years, with a mean BMI 28.9 (range 25 to 37.25). Nine of 10 women were postmenopausal; 5 had malignant tumors (all ER+, 1 HER2+) and 5 had benign disease. The mean distance of the breast tissue sample to the nipple was 6.7 cm (range 3-11 cm). Study specimens were immediately snap-frozen in liquid nitrogen in the operating room. All samples were then sent to the Microbiome Laboratory at Mayo Clinic for 16S rRNA sequencing using a high-throughput next-generation Illumina MiSeq (250 PE, San Diego, CA) sequencing platform. The average coverage was more than 526,000 reads per sample, with samples from 10 subjects used for the analysis. Tornado and Kraken pipelines were used to identify relative abundance (OTU reads/total bacterial OTU reads) with core OTUs defined as those with a mean relative abundance > 0.1% at the cohort level. Analysis of these 10 sterile specimen triplets showed demonstrable differences in the microbiome of normal adjacent breast tissue and skin tissue, while confirming effective skin antisepsis as no bacterial DNA was retrieved from swabs of the sterilely prepped skin at the operative site. Significant shifts at the taxonomic order level were observed as Lactobacillales, Selenmonadales and Bacillales in the breast tissue compared to the skin tissue. Further, we identified Bacillus, Salinbacilus, Brochothrix, Sinovbaca and Seinonella at genus level among the Bacillales in increased relative abundance in the adjacent normal breast tissue from patients with malignant tumors, while Ardenticatenaceae (represented by Adenticatena) were seen more frequently in patients with benign disease as shown in the figure. These data confirm the presence of a human breast microbiome distinct from that of the breast skin. Further, our pilot evaluation suggests that the microbiome of normal adjacent breast tissue is demonstrably different in women with malignant versus benign disease states.

Figure 1: Evaluation of distinct features of the lower airway microbiome in RA. (A) PCoA based on UniFrac distances showed overlap of the lung microbiome of RA (red) as compared with sarcoidosis (green). LEfSe analysis shows taxonomic differences in Cladogram (B), several with a significant LDA (>2, C).

Evi-065#241 Seminal microbiome in the men with and without prostatitis Reet Mändar (1,3), Eleri Lapp (1,3), Ave Ahelik (1,3), Paul Korrovits (1,3,4), Margus Punab (1,4), Andres Metspalu (5), Kaarel Krjutškov (1,5), Hiie Nõlvak (1,2), Jens-Konrad Preem (1,2), Kristjan Oopkaup (1,2), Andres Salumets (1), Jaak Truu (1,2) 1. Competence Centre on Health Techonologies, Tartu, Estonia 2. Faculty of Science and Technology, University of Tartu, Tartu, Estonia 3. Department of Microbiology, Faculty of Medicine, University of Tartu, Tartu, Estonia 4. Andrology Center, Tartu University Hospital, Tartu, Estonia 5. Estonian Genome Center, University of Tartu, Tartu, Estonia Rapidly developing sequencing methods and analytical techniques are enhancing our ability to understand human microbiome, however, characterization of male genital tract microbiome has always lagged behind investigations in other body sites. Culture-based studies have indicated differences in seminal microbiota of prostatitis patients and healthy men. We profiled the seminal microbiome applying Illumina sequencing. Methods. Altogether 67 men were involved in the study, of them 21 with inflammatory prostatitis (NIH IIIA or NIH IV category) having >1 M WBC/ml semen, and 46 men without inflammation in their semen. Seminal microbiomes were profiled applying the method that uses combinatorial sequence tags attached to PCR primers that amplify the rRNA V6 region (967–985 and 1078–1061, Escherichia coli 16S rRNA segment). Amplified PCR products were sequenced using an Illumina paired-end protocol on HiSeq2000 platform. Overlapping segments of the forward and reverse reads was performed with PEAR software. Mothur and CROP softwares were applied to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units and obtain distance matrices. Results. The most abundant phylum in the semen was Firmicutes comprising nearly half of the sequences found (median 41.7%, quartiles 28.5-47.2%). The other numerous phyla included Bacteroidetes (19.5% [14.8-26.8%]), Proteobacteria (15.9% [12.7-24.2%]) and Actinobacteria (9.7% [7.1-13.0%]) while Fusobacteria (0.05% [0.02-0.1%]) were less abundant. The most numerous species were Lactobacillus iners (median proportion 12.9%, quartiles 6.5-18.3%), followed by Lactobacillus crispatus (9.5% [4.3-13.0%] and genus Gillisia (8.0% [5.1-13.6%]). The most remarkable difference between the groups appeared in the counts of lactobacilli that were higher in healthy men than inflammatory prostatitis patients (27% [20.2-34.6%] vs 20.2% [4.9-25.0%]; p=0.05), especially on behalf of Lactobacillus iners (14.2% [8.8-19.4%] vs 9.8% [3.2-14.3%]; p=0.013). Proteobacteria comprised higher proportions in prostatitis patients than healthy men – 19.6% [15.2-27.7%] vs 15,5% [12.0-21.5%], especially on behalf of genera Oxalobacter, Curvibacter and Pseudomonas, however, this difference did not reach the significance level. Conclusions. Semen of inflammatory prostatitis patients contains less health-supporting lactobacilli than that of healthy men. Firmicutes (especially lactobacilli), Bacteroidetes, Proteobacteria and Actinobacteria comprise the highest proportion of seminal microbiome. According to our best knowledge this is the first study comparing seminal microbiome in inflammatory prostatitis patients and healthy men applying Illumina sequencing.

LEfSe Analysis of Adjacent Normal

Breast Tissue in Benign versus

Malignant Disease States

Evi-066#244 Gut microbiota, obesity, adipose tissue inflammation and Toll-like receptor 5 Eveliina Munukka (1), Satu Pekkala (2), Anniina Rintala (1), Petri Wiklund (2), Pentti Huovinen (1), Sulin Cheng (2) 1. Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland 2. Department of Health Sciences, University of Jyväskylä, Jyväskylä, Finland Recent evidence suggests that metabolically unbalanced i.e. dysbiotic gut microbiota (GM) composition may affect the onset and progression of fat accumulation and related comorbidities. GM regulates and controls the host’s metabolic functions such as de novo lipogenesis and triglyceride storage in adipose and hepatic tissue. Gut-derived bacterial fragments such as lipopolysaccharide and flagellin might have an important role in the development and progression of fat accumulation, and may contribute to the onset of inflammation and further insulin resistance (IR). Despite the accumulating evidence that highlights the role of GM in obesity and related metabolic disorders, the underlying mechanisms in humans are still unclear. The aim of this study was to elucidate the the possible role of bacterial flagellin-recognizing Toll-like receptor 5 (TLR5) -mediated metabolic changes in humans by characterizing subjects having higher expression TLR5 signaling pathway genes in their adipose tissue and further link their dysbiotic gut microbiota composition to obesity and unfavorable metabolism. An adipose tissue microarray database was used to compare women who had the highest (n=4, H-TLR group) and lowest (n=4, L-TLR group) expression levels of several TLR5 signaling pathway genes. The GM composition was profiled by 16S rRNA fluorescence in situ hybridization and obesity-related metabolic variables were determined with standard laboratory techniques. The in vivo findings were verified using cultured human adipocytes and flagellin. The H-TLR group had higher fecal Clostridium cluster XIV abundance and Firmicutes-to-Bacteroides ratio compared to the L-TLR group. They also had obese phenotype characterized by greater waist circumference, higher fat%, blood pressure, serum leptin, but lower adiponectin levels (p<0.05 for all) compared to the L-TLR group. In addition, 668 metabolic and immunedefence-related adipose tissue genes were differentially expressed between the groups. In vitro studies using cultured human adipocytes confirmed that flagellin activated TLR5 inflammatory pathways and signaling kinases, decreased insulin signaling, and increased glycerol secretion. The in vivo findings suggest that among Clostridium cluster XIV that harbors flagellated bacterial members contribute to the development of obesity and metabolic changes through adipose tissue inflammation. The in vitro studies in adipocytes show that the underlying mechanisms of the human findings may be due to flagellin-activated TLR5 signaling. Evi-067#249 Epidemiological investigation of the cognitive level of hepatitis B and its influence factors among the general residents and cases infected with hepatitis B virus Yang Shigui (1,2), Yu Chengbo (1,2), Xu Kaijin (1,2), Ren Jingjing (1,2), Wang Bing (1,2), Li Yiping (1,2), Chen Ping (1,2), Xie Tiansheng (1,2), Deng Min (1,2), Wang Chencheng (1,2), Li Jing (3), Yao Jun (3), Ruan Bing(1,2), Li Lanjuan (1,2) 1 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003, China 2 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, 310003, China 3. Zhejiang Provincial Center for Disease Control and prevention, Hangzhou, 310003, China. Objective: hepatitis B is still a serious threat to human health, especially in the Southeast Asian countries such as China. This study is to conduct epidemiological investigation of the cognitive level of hepatitis B and its influence factors among the general residents and cases infected with hepatitis B virus, in order to provide scientific basis for the prevention and control of hepatitis B. Methods: a cross-sectional survey was designed with stratified cluster sampling from 12 counties of Zhejiang Province during the period from January to June 2013. Ten thousand general population over the age of 15 and 10 000 cases infected with hepatitis B virus were selected. Results: a total of 17 389 valid questionnaires were collected with average age of 49.8 + 13.9 years, including 10 330 cases of male and 7 059 female. Among the 9 264 general individuals, 5 568 (60.1%) were male, 3696 were female (39.9%), with average age of 49.1 + 14.8 years; among 8125 cases of hepatitis B infection, 4762 were male (58.6%), 3363 were female (41.4%), with average age of 50.6 + 12.7 years. The proportion of correct cognition in many aspects of hepatitis B among the general population were significantly higher than that of cases with HBV infection, and the proportion of comprehensive cognitive status of "know" was significantly higher than that of cases with HBV infection (74% vs 71.5%). Logistic regression model showed that gender, age, culture level, and marital status were the influence factors of the cognitive level of hepatitis B. The cognitive level of male is better than female with the adjusted value of OR was 1.17 (95%CI:1.07-1.27). In terms of age, compared with the 15-29 years old group, the cognitive level among the 30-45 years old group were no significant difference, but significant higher than that of the two group of the age 45-59 and 60 years. With the cultural degree improvement, hepatitis B cognitive level showed an increasing trend, with an adjusted value OR of 1.41 (95%CI:1.32-1.50). In addition, marital status was also an important factor that the cognitive level among married group was significantly higher than that of unmarried group, with an adjusted value OR of 1.54 (95%CI:1.24-1.91). Conclusion: Hepatitis B infection is related with the cognitive level about hepatitis B, and gender, age, educational level and marital status are the influence factors the cognitive level of hepatitis B. health education about hepatitis B should be further strengthened, especially for women, under 45 years of age or unmarried populations.

Evi-068#256 Mucosa-associated biohydrogenating microbes protect the simulated gut microbiome from stress by a Western-style fat consumption Rosemarie De Weirdt (1), Emma Hernandez-Sanabria (1), Bruno Vlaeminck (2), Eva Mees (1), Ruy Jauregui (3), Dietmar H. Pieper (3), Annelies Geirnaert (1), Florence Van Herreweghen (1), Ramiro Vilchez Vargas (1), Veerle Fievez (2) and Tom Van de Wiele (1) (1) Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Ghent, Belgium. (2) Laboratory for Animal Nutrition and Product Quality (LANUPRO), Ghent University, Melle, Belgium. (3) Microbial Interactions and Processes Research Group. Helmholtz Centre for Infection Research, Braunschweig, Germany. A Western high fat/low fibre diet is increasingly evidenced to disrupt gut microbiome homeostasis and these changes have been associated with the incidence of chronic illnesses such as type-2 diabetes, obesity and IBD. The direct effects of increased levels of dietary fat on the colon microbiome are not well understood. Fat, and in particular poly-unsaturated fatty acids (PUFA), may affect colon microbiome homeostasis in two ways. They may exert (specific) antimicrobial effects, and act as detergents at the gut mucosa disrupting the establishment of a mucosa-associated microbial community. On the other hand, colon microbes may gradually saturate PUFA in a process named biohydrogenation. Here, we investigated how linoleic acid (LA), the main PUFA in the Western diet, and its biohydrogenation to vaccenic acid (VA) and stearic acid (SA) affected the activity and composition of the human colon microbiome in vitro. First, standardized batch incubations of faecal microbiota of a healthy volunteer were supplemented with 1 g/L LA, VA or SA to screen for general metabolic effects (SCFA production) and biohydrogenation activity. Second, the dynamic and validated SHIME-model was used to investigate how daily exposure to 1 g/L LA affects the microbial composition (Illumina-based 16S rRNA gene profile) and functionality (SCFA, biohydrogenation), either in the absence (L-SHIME) or presence (M-SHIME) of a mucosal environment. In the absence of a mucosal environment, LA specifically inhibited two health-promoting gut microbial parameters: butyrate production and levels of Faecalibacterium prausnitzii. In batch, VA and SA did not or barely inhibit butyrate production. In the presence of a mucosal environment, LA did not affect butyrate production and F. prausnitzii levels. Interestingly, the mucosal environment of the M-SHIME appeared to be a hotspot for LA biohydrogenation, with LA:VA:SA profiles of about 50:10:40 in the mucus compared to 80:10:10 in the lumen. In accordance, 16S rRNA gene profiling showed that the two most important biohydrogenating genera of the human gut – Roseburia and Pseudobutyrivibrio – specifically colonized the mucosal environment of the M-SHIME. Correlation network mapping furthermore revealed that LA supplementation stimulated these genera to shift from a strict mucosal niche (Fig. 1A) towards a more central position in the SHIME microbial network (Fig. 1B), indicating an increased functional interaction between distinct mucosal and luminal microbes. In a final co-culture experiment, we confirmed that, also in the absence of a mucosal environment, biohydrogenating R. hominis could directly protect F. prausnitzii from LA stress. Hence, we concluded that mucosal biohydrogenating species in the M-SHIME were responsible for protection against LA. Overall, these results demonstrate the importance of a healthy mucus layer harbouring biohydrogenating species to provide resilience for the gut microbiome upon exposure to high levels of LA.

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Dialister

Eisenbergiella

Eubacterium

Faecalibacterium

Flavonifactor

Fusicatenibacter

Lachnospira

Lactobacillus

Odoribacter

Oscillibacter

OscillospiraParabacteroides

Parasutterella Prevotella

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Evi-069#258 Are you what you eat? Associations of diet and lifestyle with the gut microbiome Emily Vogtmann (1), Jianxin Shi (1), Georg Zeller (2), Jim Goedert (1), Peer Bork (2), Rashmi Sinha (1) 1. Division of Cancer Epidemiology & Genetics, National Cancer Institute, Bethesda, MD, USA 2. Structural and Computational Biology, European Molecular Biology Laboratory, Heidelberg, Germany Introduction Diet and lifestyle factors have been observed to be associated with gut microbial communities, but these studies have generally been conducted in highly motivated, health conscious groups. Therefore, we considered the associations between typical dietary habits and lifestyle factors with the gut microbiome assessed using shotgun metagenomics technology within a group of adults from the general population. Methods Freeze-dried fecal samples were collected as a part of a colorectal cancer case-control study in the Washington DC area in the 1980s. Participants in this study completed questionnaires which included data about demographics, behavior, medical history, and typical dietary intake. For this study, fecal samples from 52 non-colorectal cancer cases (i.e., controls) were selected and genomic DNA was extracted from these samples. Using the Illumina HiSeq, whole-genome shotgun sequencing of the extracted DNA was performed. Taxonomic relative abundance profiles were created using MOCAT and clusters at the species level were identified. The Shannon diversity index and species richness and evenness were determined from the cluster data. We calculated Spearman correlations between the relative abundances of the clusters with the continuous variables and logistic regression models for the dichotomous variables. Results Out of the 52 participants, 71% were male and the majority of them were non-Hispanic white (90%). The mean age was 61.2 (standard deviation (SD) 11.0) years and the mean body mass index (BMI) was 25.3 (SD 4.3) kg/m2. Over 42% of the participants had never smoked cigarettes and the average number of alcohol drinks consumed weekly was 6.1 (SD 10.4). From the Spearman correlation models, age was positively correlated with two Streptococcus clusters and negatively correlated with Odoribacter splanchnicus. BMI was positively correlated with Campylobacter concisus, Streptococcus pseudopneumoniae, and Leuconostoc gelidum. Daily consumption of dietary fiber was positively correlated with the relative abundance of Fusobacterium periodonticum, Bifidobacterium longum, and Streptococcus infantarius, while it was negatively correlated with Bacterioides finegoldii. Conclusions In this study, associations were detected between some typical dietary components and lifestyle factors with species level clusters detected from fecal samples within a group of adults from the general population in the Washington DC area during the 1980s. Future work should consider whether specific genes or gene functions are associated with dietary and lifestyle factors in this population and whether these associations have changed over time.

Akkermansia

Alistipes

Anaerostipes

BacillusBacteroides

Barnesiella

BifidobacteriumBilophilaBlautia

ClostridiumCollinsella

Coprococcus

Dialister

Eisenbergiella

Eubacterium

Faecalibacterium

FlavonifactorFusicatenibacter

Lachnospira

Lactobacillus

Odoribacter

Oscillibacter

Oscillospira

Parabacteroides

Parasutterella

PrevotellaPseudobutyrivibrio

Roseburia

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Evi-070#260 Alterations of oral microbiome in early childhood caries Hui Chen, Yuan Wang, Xi Chen, Wen Jiang, Ying Wang, Yunjie Zhu, xiaolong Lin , Xiangyu Hu Department of Conservative Dentistry and Periodontics, Affiliated Hospital of Stomatology, College of Medicine, Zhejiang University, No. 395 Yan’ an Road, Hangzhou, Zhejiang 310006, China Background: Caries is one of the most prevalent chronic diseases in the world. Some studies have revealed that alterations in the oral microbiome are important in the progression of dental caries, but definitive associations of oral microbiome and caries are still unclear. The aim of this study was to evaluate the microbiome of severe early childhood caries (ECC) by means of species clustering analysis and gene function analysis based on the next-generation sequencing. Methods: 25 children with severe ECC and 19 caries-free children aged 3-5 years were selected according to the International Caries Detection and Assessment System-Ⅱ. Samples of Saliva were collected . Deep metagenomic sequencing on Hiseq2000 platform was applied to study the phylogenetic and functional profiles of these cohorts. Results: (1) An average of 3.01 Gb clean data was generated for the 44 samples, from which a non-redundancy oral gene set containing 2.48 million genes was constructed. And there were 1,042,382 genes annotated to KEGG KO orthologous.(2) The most abundance phylum was Proteobacteria, followed by Firmicutes, Bcteroidetes, Actionobacteria and Fusobacteria. These five phyla represent 99% of all the community. Streptococcus mutans, Veillonella and Prevotella spp. such as V.parvula, P.nigrescens, P.oris, P.oulorum were enriched in caries children. However, Neisseria lactamica was the only species enriched in healthy children. Principle component analysis of phylogenetic profile showed overlap and shift community structure between healthy and caries children. (3) For the 2,477,091 genes in the oral microbial gene catalogue, 6178 and 833 were enriched in carious and healthy people, respectively (wilcox-test fdr<0.1). Genes with higher caries abundance were significantly enriched in pathways such as glycosaminoglycan degradation, nitrogen metabolism and lipotic acid metabolism. Glycosaminoglycan degradation and lipotic acid metabolism maybe related to the sugar utility, while, nitrogen metabolism may be related to the PH change during caries development. Conclusions: (1) Compared to traditional 16S rDNA approaches, oral microbiome was evaluated at species and functional level with whole genome metagenomic sequence. (2) An oral saliva microbial gene catalogue containing 2,477,091 genes was established. (3)Caries enriched genes should be considered as biomarkers of caries for further research. These data may facilitate improvements in the prevention and treatment of severe early childhood caries. Key words: dental caries, oralmicrobiota, metagenomic, functional gene (Supported by grants from the National Natural Science Foundation of China 81371142 and 2011 China State Key Clinical Department Grants.) Evi-072/#267 Intestinal pro-inflammatory disbyosis in HIV-1 infection Muntsa Rocafort (1,2), Marc Noguera-Julian (1,2,3), Yolanda Guillén (1,2), Mariona Parera (1,2), Maria Casadellà (1,2), Rocío Bellido(1,2), Cristina Rodríguez(1,2), Javier Rivera-Pinto (1,3), Isabel Bravo (4), Carla Estany (4), Josep Coll (1,4), Julià Bla 1. IrsiCaixa AIDS Res Inst., Badalona, Catalonia, Spain 2. Univ. Autònoma de Barcelona, Bellaterra, Catalonia, Spain 3. Univ. de Vic-Univ. Central de Catalunya, Vic, Catalonia, Spain 4. Unitat VIH, Hosp. Univ. Germans Trias i Pujol, Badalona, Catalonia, Spain BACKGROUND Recent studies suggest a role of the gut microbiome on HIV/AIDS pathogenesis, but it is unknown if the gut microbiota differs by HIV-1 control and immune status, or if antiretroviral treatment (ART) affects the intestinal microbial content to any extent. METHODS This cross-sectional study compared HIV-1-negative (HIVneg) subjects with HIV-1-infected individuals with the following phenotypes: late presenters (LP: no ART, CD4≤200 cells/mm3), elite controllers (EC: no ART, HIV-1 RNA (VL)<50 copies/mL for 1 year), viremic controllers (VC: no ART, VL 50-2000 copies/mL for 1 year), ART-naïve (AN: no ART, CD4≥500 cells/mm3, VL>2000 copies/mL), early treated (ET: on ART started ≤6 months from HIV-1 infection, VL<50 copies/mL), immune concordant (IC: on ART≥2 years, CD4≥500 cells/mm3, VL<50 copies/mL), and immune discordant (ID on ART≥2 years, CD4≤300 cells/mm3, VL<50 copies/mL). Participants were 18-60 years old, had body mass index 18.5-30 and, except for LP, had no antibiotic usage during the previous 3 months. The fecal microbiota was characterized by massive 16S rRNA sequencing (MiSeqTM). Richness (Chao1 estimator), diversity (Shannon index) and microbial taxonomic analyses were performed using Mothur and R/Vegan software packages and the SILVA database. The LEfSe algorithm was used to identify which bacterial taxa contributed to differences between HIV-infected and HIVneg individuals. RESULTS The study included 80 individuals: 16 HIVneg and 64 HIV-1-infected (5 LP, 3EC, 6 VC, 7 AN, 5 ET, 27 IC and 11 ID). The gut microbiome of HIV-infected subjects was significantly less rich and diverse than that of HIVneg individuals (Figure). HIV phenotypes corresponding to more advanced HIV disease stages, ie: LP, ID, but also IC subjects, had the lowest richness and diversity, whereas AN and ET subjects had similar richness and diversity to HIVneg individuals. HIV-1-infected subjects in the LP, ID, and IC groups also showed the lowest Firmicutes:Bacteroidetes phyla ratio. Using LEFse, the only unbalanced phylum between HIV positive and negative subjects was Lentisphaerae, which was enriched in HIVneg individuals. At the family level, HIV-1 positive subjects were enriched in Bacteroidaceae and Porphyromonadaceae, and depleted in Victivallaceae, Peptostreptococcaceae, Clostridiaceae and Prevotellaceae. At the genus level, HIV-infected individuals were enriched for Bacteroides, Bilophila, Parabacteroides, Alistipes and Odoribacter, and depleted for Dialister, Butyrivibrio, Coprococcus,

Catenibacterium, Desulfovibrio, RC9, Clostridium, Oribacterium, Prevotella and 3 uncultured or unclassified genus (one Ruminococcaceae, one Victivallaceae and one Peptostreptococcaceae) (Figure). CONCLUSIONS HIV-1 infection is associated with significant decreases in gut microbiome richness and diversity as well as to an intestinal pro-inflammatory dysbiotic profile. Our findings thus suggest a central role of the intestinal microbiome on HIV pathogenesis.

Evi-073#269 Human gut microbial modulation of insulin sensitivity Helle Krogh Pedersen (1), Valborg Gudmundsdottir (1), Trine Nielsen (2), Matej Oresič (3), Oluf Pedersen (2), MetaHIT consortium, Henrik Bjørn Nielsen (1) 1. Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark 2. The Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark 3. Steno Diabetes Center, Gentofte, Denmark Insulin resistance (IR) and metabolic syndrome (MS) are established risk factors for both type 2 diabetes (T2D) and cardiovascular disease. Mounting evidence suggests a link between the gut microbiome and human metabolic health and microbiome transplant studies in both humans and mice have demonstrated the transferability of metabolic phenotypes. These effects may partly be driven by metabolite mediated host-microbe interactions. Here we describe the largest study to date combining gene repertoire obtained by deep shotgun sequencing of the fecal microbiome with untargeted serum metabolomic profiling across 291 nondiabetic and 75 T2D Danish individuals. We observe clear metabolic signatures of IR and MS that extend into the T2D population and show, for the first time, that several critical serum metabolites, including branched chain amino acids (BCAA), are indeed correlated with the corresponding metabolic potential of the gut microbiome gene repertoire.

Figure. (A) Diversity (Shannon) and richness (Chao1) estimates for HIV-1 positive and HIV negative subjects. (B) LEfSe algorithm results showing

which bacterial genus contribute to the differences in taxonomic composition between HIV-1 positive and HIV negative gut microbial populations.

Evi-074#273 Nasopharyngeal as compared to the ear microbiota in toddlers with acute otitis media: a proxy for the diagnosis? Julia G. Kraemer (1,2), Moana Mika (1), Anne Oppliger (2) and Markus Hilty (1, 3) 1. Institute for Infectious Diseases, University of Berne, Switzerland 2. Institute for Work and Health, Lausanne-Epalinges, Switzerland 3. Department of Infectious Diseases, University hospital, Bern Switzerland Acute otitis media (AOM) is a leading cause of antibiotic prescription in primary health care. We previously showed that AOM has an effect on the composition of the nasopharyngeal microbiota in children under two years old as compared to healthy individuals (Hilty et al. The Journal of infectious diseases. 2012;205(7):1048-55) In this study, we investigated if the nasopharyngeal microbiota is a proxy for detecting AOM as compared to the ear. Furthermore we examined whether the age influences the nasopharyngeal microbiota of individuals affected by AOM. Nasopharyngeal swabs from 228 AOM patients (mean age=2.0, SD=1.9) and ear swabs from 21 AOM patients (mean age=2.3, SD=1.5) were taken, whereas all specimens were collected between 2004 and 2010. The characterization of the microbiota was accomplished by using multiplexed pyrosequencing (454 platform) of the variable regions V3 to V5 of the 16S rDNA. In total 125 bacterial families were identified and the families Moraxellaceae (mean=38.3%, SD=36.7), Streptococcaceae (mean=25.2%, SD=31.3), Pasteurellaceae (mean= 17.7%, SD=30) and Staphylococcaceae (mean=5.1%, SD=19) were the most frequent. However, Moraxellaceae was significantly more abundant in the nasopharynx than in the ear (P<0.001) and beta diversity measurements revealed that the two different sampling sites (nasopharynx and ear) are significantly different, too. In addition, we found that the colonization density (estimated by measuring the DNA concentration) in AOM patients ranging from under one to six years old (n=228, mean age= 1.9, SD=2.0) significantly increases with age. Looking at alpha and beta diversity indices, toddlers aged < 2 years show a statistically significant different composition of the microbiota as compared to infants > 2 years. This study shows that the microbiota of nasopharynx as compared to the ear is not a proxy for suspected AOM cases, because the microbial composition differs strongly compared to the ear. Furthermore the nasopharyngeal, microbiota changes in young children when they become older, indicating that age but not the diagnosis of AOM is a main driving factor for the nasopharyngeal, microbial composition. Evi-075#276 Dominant genera of the stool microbiome reflect arginine metabolism in a multi-national cohort of women J.L. Cope (1,2), J.W. Hsu (3), P. Dwarkanath (4), J. M. Karnes (3), C.C. Kao (3), R.A. Luna (1,2), M.M. Thame (5), A.V. Kurpad (4), J. Versalovic (1), E.B. Hollister (1,2), F. Jahoor (3) 1. Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA 2. Texas Children’s Microbiome Center, Department of Pathology, Texas Children’s Hospital, Houston, Texas, USA 3. United States Department of Agriculture, Agricultural Research Station, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA 4. St John’s Research Institute, Bangalore, India 5. University of the West Indies, Mona, Kingston, Jamaica Despite adequate dietary intakes of energy and protein, Indian women give birth to low birth weight (LBW) babies at high rates. Previous research has shown that, although women from other nations increase arginine production during pregnancy, Indian women do not, and there is a strong association between the prevalence of LBW babies and reduced arginine production. In these cases, reduced bioavailability of arginine was not due to inadequate dietary intake but rather to increased catabolism by arginase, possibly by the gut microbiota. To investigate whether the gut communities of Indian women differ in composition and arginine utilization and metabolism from American and Jamaican we recruited a multi-national cohort. Their stool microbial communities, dietary habits, and metabolic characteristics were evaluated, including total carbohydrate and energy intakes, gut absorptive capacity, and the kinetics of several arginine metabolic pathways. 454 pyrosequencing was used to sequence the V3V5 region of the 16S rRNA gene from stool. Sequences were assigned to operational taxonomic units (OTUs), and the OTU assignments were used to generate functional predictions, which were compared among subjects. Three dominant community types were identified among the stool specimens and were characterized by increased abundances of genera Prevotella, Bacteroides, or Bacteroides with Clostridium, respectively. These dominant states tended to reflect the geographic origin of their samples, but not exclusively so. Subjects harboring Prevotella-dominant communities had lower gut absorptive capacity and a lower endogenous arginine flux, while communities containing higher abundances of Clostridium showed a marked increase in both gut absorptive capacity and endogenous arginine flux. Subjects with greater body weight and gut absorptive capacity were more likely to have a Bacteroides-dominated community even with a converse low energy intake and low total carbohydrate intake possibly due to high gut absorptive capacity values. Prevotella-dominant or the mixed Bacteroides with Clostridium-dominant type occurred in subjects with lower body weight, higher energy and total carbohydrate intake, and lower gut absorptive capacity. Congruent with physiological measurements of arginine flux and the three dominant fecal community types detected among the members of this multi-national cohort, predictions of metagenomic function suggest that the conversion of arginine to ornithine is likely to be lower in the non-Bacteroides-dominant communities. Likewise, the further conversion of ornithine to citruline is likely to be lower in the Prevotella-dominated communities.

We demonstrate that the gut microbiota vary in conjunction with arginine metabolism and have the potential to impact arginine availability in the human host. Modulation of the gut microbiota may represent a future treatment for inadequate arginine availability during pregnancy and decrease the risk for LBW babies. Evi-076#279 Metagenomic and metatranscriptomic evidence suggest that altered amino acid and carbohydrate metabolism are associated with pain frequency and intensity in children with irritable bowel syndrome Emily B. Hollister (1,2) , Ruth Ann Luna (1,2), Kevin Riehle (3,4), Xiangjun Tian (5), Nadim Ajami (5), Erica M. Weidler (6,7,8), Sabeen Raza (1,2), Michelle Rubio-Gonzalez (1,2), Toni-Ann Mistretta (1,2), Richard Gibbs (9), Joseph Petrosino (5, 9, 10), R 1. Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX 2. Texas Children’s Microbiome Center, Department of Pathology, Texas Children’s Hospital, Houston, TX 3. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 4. Bioinformatics Research Laboratory, Baylor College of Medicine, Houston, TX 5. Alkek Center For Metagenomics and Microbiome Research, Baylor College of Medicine, Houston, TX 6. Department of Pediatrics, Baylor College of Medicine, Houston, TX 7. Children’s Nutrition Research Center, Houston, TX 8. Pediatric Gastroenterology, Hepatology, and Nutrition, Texas Children’s Hospital, Houston, TX 9. Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 10. Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder characterized by abdominal pain and altered bowel habits and is estimated to affect 10 to 20% of the world’s population. IBS is among the most common GI complaints among children, and many who experience IBS as a child continue to suffer from it as adults. Although the cause of IBS has not been identified definitively, there is evidence to suggest that altered structure and function of the gut microbial community may be involved in its etiology. Using shotgun metagenomic sequencing we sought to characterize the composition and functional potential of the gut microbiome in preadolescent, school-aged children (ages 7-12 years) with and without IBS. Relationships between metagenomic features, including KEGG ortholog, module, and pathway abundances, were evaluated relative to IBS symptom frequency and severity; and metatranscriptomic sequencing was used to determine if microbiome gene expression patterns were consistent with the distribution and abundance of functional genes observed among the shotgun metagenomic profiles. Predictions of function based on shotgun metagenomic sequencing suggest that, relative to healthy controls, children with IBS harbor increased abundances of genes belonging to the KEGG pathway for phenylalanine, tyrosine, and tryptophan biosynthesis (ko00400), including genes involved in the production and conversion of pathway intermediates, such as chorismate, phenylpyruvate, phenylalanine, and tyrosine; and the relative abundances of genes associated with the KEGG module for tyrosine biosynthesis were found to be significantly correlated with pain frequencies and intensities reported by subjects. In contrast, the gut communities of children with IBS harbored fewer genes associated with the KEGG pathway for starch and sucrose metabolism (ko00500), and the relative abundances of genes associated with KEGG modules involved in carbohydrate metabolism, including glycolysis and gluconeogenesis, were negatively correlated with pain intensity. These predictions were verified, in part, by metatranscriptomic sequencing, which confirmed decreased expression, based on log-2 fold changes, of the majority of genes involved in the KEGG pathway for starch and sucrose metabolism in children with IBS. Although the findings of our metagenomic and metatranscriptomic profiling were not completely congruent with one another, their combined results suggest that altered carbohydrate metabolism within the gut microbiome may contribute to pain symptoms experienced by children with IBS, and they provide support for the use of dietary interventions and other therapies directed at microbiome modulation in individuals with IBS. Evi-077#283 Functional profiling of the gut microbiome in HIV infection Yolanda Guillén (1,2), Marc Noguera-Julian (1,2,3), Muntsa Rocafort (1,2), Mariona Parera (1,2), Maria Casadellà (1,2), Rocío Bellido (1,2), Cristina Rodríguez (1,2), Javier Rivera-Pinto (1,3), Isabel Bravo (4), Carla Estany (4), Josep Coll (1,4), Julià B 1. irsiCaixa AIDS Res. Inst., Badalona, Catalonia, Spain 2. Univ. Autònoma de Barcelona, Bellaterra, Catalonia, Spain 3. Univ. de Vic-Univ. Central de Catalunya, Vic, Catalonia, Spain 4. Unitat VIH, Hosp. Univ. Germans Trias i Pujol, Badalona, Catalonia, Spain Background. The gut microbiome plays an essential role in human physiology. We investigated to what extent HIV infection could modify its functions. Methods. We used PICRUSt to infer the functional profile from 16S rRNA MiseqTM data, which was obtained in a cross-sectional study comparing the intestinal microbiome of HIV-negative (HIVneg) and HIV-1-infected subjects with different phenotypes, i.e., late presenters (LP: no ART, CD4≤200 c/mm3), elite controllers (EC: no ART, HIV-1 RNA (VL)<50 c/mL for 1 year), viremic controllers (VC: no ART, VL 50-2000 c/mL for 1 year), ART-naive (AN: no ART, CD4≥500 c/mm3, VL>2000 c/mL), early treated (ET: ART started ≤6 months from HIV-1 infection, VL<50 c/mL), immune concordant (IC: on ART≥2 years, CD4≥500 c/mm3, VL<50c/mL), and immune discordant (ID: on ART≥2 years, CD4≤300 c/mm3, VL<50c/mL). Gene abundance

data predicted by PICRUSt was analyzed with HUMann to reconstruct the metabolic pathways and modules. Then we ran the LefSe algorithm in order to identify which pathways significantly contributed to differences among different HIV phenotypes. Results. The parent study included 80 subjects: 58 men, 21 women and 1 transgender woman. Men and women showed statistically significant differences (p-value log (LDA score) < 0.05) in 38 metabolic pathways. Within men, HIV+ subjects (5 LP, 1 EC, 3 VC, 6 AN, 5 ET, 17 IC, 8 ID) showed enrichment in genes implicated in biotin metabolism; alanine, aspartate and glutamate metabolism and sulfur metabolism relative to HIVneg (n=13), and were depleted in genes related to synthesis and degradation of ketone bodies and fatty acid biosynthesis. Most differences among HIV+ men involved the ID group (Figure 1), whose microbiome was enriched for genes related to biotin metabolism, RNA degradation and beta-lactame resistance. The microbiome of VC patients contained more genes involved in Alanine metabolism as well as in the citrate cycle (TCA cycle). ET patients showed an increase in ABC transporters relative to other phenotypes, whereas AN showed an enrichment in genes implicated in the atrazine degradation. Conclusions. HIV infection is associated with bidirectional unbalances in intestinal metabolic activity affecting mucosal barrier integrity and function, including local and systemic inflammatory processes and energy storage and consumption. Keywords. ART (Antiretroviral treatment), CD4 (Cluster of differentiation 4), VL (Viral load).

Evi-078#287 A Possible Role Of IL-17 During Salmonella Typhimurium Infection WL Ling, LJ Wang, Godfrey CF Chan, and James CB Li Department of Paediatrics and Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong Interleukin 17 is a crucial immunomodulator in various chronic immunological diseases and infectious diseases including inflammatory bowel disease and tuberculosis. The action of IL-17 in chronic disease could be detrimental through the overproduction of proinflammatory cytokines but IL-17 can also be protective during microbial infection by suppressing the overwhelmed immune responses. Therefore, the balance between the population of IL-17 producing cells (Th17) and other T cell subset plays an important role in maintaining the homeostatic of the immunity. Previous studies showed that the gut

microbes and its colonies could enhance the Th17 cells differentiation and induce the production of IL-17 in intestine. During the pathogenic bacteria infection, such as Salmonella typhimurium infection, the colonies of the microbiota will be affected. These changes may activate the T cells and macrophages to produce proinflammatory cytokines to combat both the commensals and pathogenic bacteria. As IL-17 is crucial in both innate and mucosal immunity, we hypothesize that IL-17 could affect the immune cells to fight against the bacterial infection in intestine. Human macrophages were isolated and differentiated from peripheral blood mononuclear cell of healthy donors. In order to mimic the condition at the intestine that IL-17 is induced by commensals, the macrophages were primed with interleukin-17A for 3 days, followed by Salmonella typhimurium infection for 24 h. Culture supernatant was collected for enzyme-linked immunosorbant assay to quantify proinflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6. We observed that IL-17A-treated macrophages exhibited suppressed productions of TNF-α and IL-6 in response to Salmonella typhimurium infection. The reduction of cytokines production was not associated with cell death. These data suggested IL-17 primed macrophage could limit the proinflammatory cytokine production during pathogenic bacterial infection. As the microbiota could induce the Th17 differentiation, this could be a protective mechanism for commensal bacteria to fight against other bacterial infection in intestine. Evi-079#288 Pregnant Women Contain a Unique Urinary Microbiome Krystal Thomas-White (1), Kristin Jacobs (2), Evann Hilt (1), James Grayczyk (1), Thaddeus P. Waters (2), and Alan J. Wolfe (1). 1. Loyola University Chicago, Department of Microbiology & Immunology 2. Loyola University Medical Center Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine Historically, urine has been considered sterile. However, several investigations have noted this to be a false assumption. Most women have detectable bacterial DNA and/or cultivatable bacteria in urine obtained by transurethral catheter, indicating an overlooked microbial niche in the female urinary tract. However, no data exists regarding the composition of the urinary microbiome in pregnant women. At the initial prenatal visit, up to 10% of pregnant women will lack symptoms, but have evidence of uropathogens (e.g E. coli) by traditional culture assessment. This is known as asymptomatic bacteriuria (ASB). If untreated, up to 50% of these women will develop pyelonephritis, which is associated with maternal sepsis and preterm labor. Untreated ASB is also associated with spontaneous preterm labor (sPTB) and low birth weight newborns. Even though the presence of ASB is known to be a risk factor for poor pregnancy outcomes, nothing is known about the normal urinary flora of pregnant women. Here, we set out to characterize the maternal urinary microbiome (MUM) and to determine the normal microflora in a population of pregnant women. To do this, we used two complementary approaches, 16S rRNA gene sequencing and a novel expanded quantitative urine culture (EQUC) technique. We compared our pregnant population to that of a control population from a separate study. Although the non-pregnant controls tended to be older women, we saw similar levels of diversity in each cohort. However, the distribution of distinct bacterial communities (urotypes) differed by both sequencing and culture. For example, the cohort of pregnant women had a greater number of Gardnerella isolates and fewer Streptococcus when compared to controls. The majority of sequence-positive women were dominated by Lactobacillus species with a marked decrease in urotypes that are considered to be “diverse.” When EQUC was compared to standard clinical urine culture, we found a 97.14% false negative rate, with standard culture missing common uropathogens, including E. coli. Upon separating the samples into women who carried to full term versus pre-term, we observed that full term women tended to be dominated by Lactobacillus and Gardnerella, measured by both sequencing and culture. The women who delivered pre-term (less than 37 weeks) tended to have a more diverse microbiome with fewer Lactobacillus dominant profiles. These data support the hypothesis that pregnant women have a unique urinary microbiome, which varies by gestational age at delivery. Evi-080#289 Urine is Not Sterile: A Description of the Female Urinary Microbiome Alan J. Wolfe (1), Krystal Thomas-White (1), Travis Price (1), Cynthia Fok (2), Meghan Pearce (), Evann Hilt (1), Michael J. Zilliox (2), Elizabeth R. Meuller (4), Kristin Jacobs (4), Xioawu Gai (3),and Linda Brubaker (4) 1. Loyola University Chicago, Department of Microbiology & Immunology 2. University of Minnesota, Department of Urology 3. Loyola University Chicago, Center for Biomedical Informatics 4. Departments of Obstetrics/Gynecology & Urology Contrary to medical dogma, urine is not sterile. Most women have detectable bacterial DNA and/or cultivatable bacteria in urine taken directly from the bladder, indicating that the human urinary tract is a previously overlooked microbial niche. Here, we demonstrate techniques to characterize this previously unexplored microbiome and present two clinical applications. To characterize the female urinary microbiome (FUM), we use a combination of 16S rRNA sequencing and an expanded quantitative urine culture (EQUC) technique. Previously, we showed that urine taken directly from the bladder (bypassing vulvo-vaginal contamination) has a distinct microbiome and determined that catheterization is the optimal collection method. Sequencing identified five urotypes named after the dominant genus or family (Lactobacillus, Gardnerella, Corynebacterium, Staphylococcus, Enterobacteriaceae) and two more called “Low biomass” (PCR negative urines) or “diverse” (microbiomes no dominant taxon).

Understanding the FUM can help us understand, and potentially treat, urinary disorders, such as urinary incontinence (UI) and urinary tract infections (UTI). UI affects 30-60% of middle-aged and older women and accounts for $20 billion of medical costs per year in the US. There are two major subtypes of UI, stress urinary incontinence (SUI) and urge urinary incontinence (UUI). The FUM of women with UUI differs from the FUM of asymptomatic women by both culture and sequencing. Culturing has detected the presence of certain organisms that correlate with particular UI subtypes. For example, Lactobacillus crispatus ST1 is most commonly associated with asymptomatic women, whereas Gardnerella vaginalis 594 is associated with UUI. Furthermore, an understanding of the FUM shows promise for predicting Post-Operative (Post-O) UTI susceptibility. 25-50% of women develop a Post-O UTI following surgery, but the risk factors are poorly understood. We have found that FUM compositions containing high and low biomass can indicate prevalence for Post-O UTI. However, mid-level biomass, containing certain urotypes, has a low rate of PostO-UTIs, suggesting the presence of a protective microbiome. Evi-081#291 Delivery mode and feeding method shape infant microbiome composition in a US birth cohort Anne G. Hoen (1, 2, 3), Juliette C. Madan (2, 4), Kathryn L. Cottingham (2, 5), Hongzhe Li (6), Shohreh Farzan (1, 2, 7), Hilary Morrison (8), Mitchell Sogin (8), Jason H Moore (3), Margaret R. Karagas (1, 2) 1. Department of Epidemiology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA 2. Children’s Environmental Health & Disease Prevention Research Center at Dartmouth, Hanover, New Hampshire, USA 3. Computational Genetics Laboratory, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA 4. Division of Neonatology, Department of Pediatrics, Children’s Hospital at Dartmouth, Lebanon, New Hampshire, USA 5. Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, USA 6. Department of Biostatistics and Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA 7. Department of Population Health, New York University School of Medicine, New York, New York, USA 8. Josephine Bay Paul Center, Marine Biological Laboratory, Woods Hole, Massachusetts, USA The intestinal microbiome in early infancy may have a role in mediating protection from a range of health outcomes in early childhood and later in life. Previous studies suggest that delivery mode is a critical factor in the patterns of initial acquisition of the infant microbiome, but the importance of this effect beyond the hours after birth is not clear. Feeding method (infant formula vs. human milk) has been implicated as another factor in microbiome composition in infants. The relative contributions of these two factors and the potential interactions between them have not been established. We aimed to evaluate the relationships between these two potentially critical factors and the composition of the infant intestinal microbiome in a large US cohort of healthy infants and their mothers. We characterized the infant intestinal microbiome at the genus level using high throughput sequencing targeting the V4-V5 region of microbial 16s rDNA using infant stool samples collected at 6 weeks of life. Subjects were followed as part of the New Hampshire Birth Cohort Study, a large, ongoing cohort of mother-infant dyads in the state of New Hampshire, USA. We abstracted delivery mode from the delivery medical record and ascertained feeding at the time of stool sample collection through a 2-day feeding diary that was confirmed with a telephone survey. We observed a diverse intestinal microbiome among six-week-old infants, dominated by Bacteroides, Bifidobacterium, and Streptococcus. Feeding method, but not delivery mode, was associated with microbial diversity, with breastfed infants harboring a less diverse intestinal microbiome than formula-fed infants. Generalized UniFrac analyses identified distinct microbial community composition by both delivery mode and feeding method, independent of the other. In linear models, enrichment of Bacteroides was associated with vaginal delivery in exclusively breast fed infants but not infants exposed to formula. Future investigations of the inter-relationships between the microbiome, feeding and delivery mode, and common health outcomes will serve to inform feeding choices and shed light on the mechanisms behind the lifelong health consequences of factors that shape the microbiome in early life. Detailed, longitudinal evaluations of the relationships between the intestinal microbiome composition and common prenatal and postnatal infant exposures will provide a needed foundation for understanding the potential role of the microbiome in mediating the influence of these factors health outcomes in children. Evi-082#292 Oral microbiome dysbiosis in HIV-associated periodontal disease Marc Noguera-Julian(1,2,3), Yolanda Guillén(1,2), David Reznik(4,5), Minh Nguyen(4), Roger Paredes(1,2,3,6), Timothy Read(4), Vincent C. Marconi(4,7) 1. IrsiCaixa AIDS Res. Inst., Badalona, Catalonia, Spain 2. Univ. Autònoma de Barcelona, Bellaterra, Catalonia, Spain 3. Univ. de Vic-Univ. Central de Catalunya, Vic, Catalonia, Spain 4. Division of Infectious Diseases, Emory University School of Medicine, Atlanta, USA 5. Infectious Diseases Program, Grady Health System, Atlanta, USA 6. Unitat VIH, Hosp. Univ. Germans Trias i Pujol, Badalona, Catalonia, Spain 7. Department of Global Health, Emory University Rollins School of Public Health, Atlanta, USA Background. HIV infection increases the risk of periodontal diseases (PD). The oral microbiome structure differs between HIV-infected (HIV+) and uninfected patients (HIV-), even when Antiretroviral Therapy (ART) successfully suppresses viral replication

and can be related to an increased risk of developing PD. Here, we characterize the oral microbial signatures of HIV+ and HIV- at different levels of PD. Methods. This cross-sectional study included both HIV+ and HIV- with varying degrees of PD, as assessed by oral exam using CDC/AAP criteria (severe (S-PD), moderate (M-PD), mild/none (N-PD)). A total of 5 oral samples (2 tooth, 2 cheek, 1 saliva) were used for sequencing with Illumina/MiSeq standard protocols. Mothur phylotype pipeline and SILVADB were used to classify sequences at the genus level. R/Bioconductor (Vegan, PhyloSeq and DESeq2) were used for statistical analysis. Redundancy analysis (RDA) and Bray-Curtis distances were used both for constrained and unconstrained ordination on genus proportions. Adonis was used to assess overall microbiome structure differences between groups. A negative binomial fit and Wald test were run on every genus to assess differential abundance. P-values were adjusted using Benjamini-Hochberg correction. Results. 250 samples from 50 patients were included. Of these, 40 (80%) were HIV-infected patients. 18 (36%), 16 and 16 (32%) patients presented N, M and S-PD, respectively. Sequencing data were available for 242 (96.8%) samples. Globally, unconstrained ordination analysis showed clustering by anatomic site, [Adonis test (p<0.001)]. Constrained ordination analysis showed that sampling site explained 25% of the total variance, while HIV infection and PD status explained only ~1% of such variance. Genera Abiotrophia (0.017), Neisseria (adj-p=0.003), Kingella (p=0.04) and unclassified Neisseriaceae (p=0.001) were enriched in HIV+ samples while Leptotrichia (p=0.04) and Selenomonas (p=0.04) were depleted. Neisseria genus enrichment was detected in cheek and teeth samples, but not in saliva, where no difference between HIV+ and HIV- was found. PD was also associated with enrichment of several genera. Of these, only Veillonella was depleted in S-PD samples both from cheek (p<0.001) and saliva (p=0.006). Interestingly, samples from HIV+ patients with N- PD showed a strongly significant enrichment in genus Aggregatibacter (p<0.0001) along with an unclassified Neisseriaceae family genus when compared to HIV- N-PD patients. Kingella (p=0.006) and Neisseria (p<0.001) genera enrichment was also found for HIV+ samples with M-PD. Conversely, those HIV- samples within the M-PD showed an enrichment of Treponema genus (p=0.005). Differential abundance between HIV+/HIV- of these genera was not found for samples within the S-PD group. Conclusions. HIV infection-driven changes on oral microbiome structure result in subtle but distinct microbial signatures along different stages of PD. Evi-083#296 Malleability of the human gut microbiome Edi Prifti (1,2), Shobha Potluri (3), Gonneke Willemsen (4), Emmanuelle Le Chatelier (1), Nicolas Pons (1), Florence Levenez (1), Benoit Quinquis (1), Nathalie Galleron (1), Sean Kennedy (1), Steven Pitts (3), Marina Sirota (3), Jean-Michel Batto (1), Pie 1. INRA, Metagenopolis, Jouy-en-Josas, France 2. Institute of Cardiometabolism and Nutrition (ICAN), Paris, France 3. Rinat-Pfizer, South San Francisco CA, USA 4. Department of Biological Psychology, VU University, Amsterdam, Netherlands 5. Center for host-microbiome interactions, King’s College, London, United Kingdom Background. The important role of gut microbiota in human health and disease has been increasingly demonstrated over the past few years. This complex and mostly unknown ecosystem can reach relatively stable states and be resilient to abrupt environmental changes. Nevertheless, studies have shown that some interventions such as for instance fecal transplantation or long-term dietary challanges can durably alter the gut microbial ecosystem by shifting it towards other alternative states. Moreover, recent evidence indicates the existence of a genetic component in shaping gut microbiome, but the part it takes compared to that of the environment remains unknown. Microbial richness as an important and straightforward property characterizing ecosystems was shown to be associated with health and disease. Individuals with high gut richness are healthier compared to individuals with lower gut richness who also are more prevalent among patients suffering from different diseases. Understanding the malleability of gut microbiome is important with regards to both more complete insight in human biology and the capacity to intervene for preventing and treating human diseases. Methods. We recruited 275 healthy study participants from the Netherlands Twin Register (NTR), including 234 monozygotic twins (MZ; 117 pairs, 16 singletons and 25 spouses, and deep sequenced their gut microbiome. Quantitative metagenomic analyses using Meteor and Metaominer pipelines yielded a high-resolution dataset allowing us to better understand gut microbial malleability. Results. We found that genetically identical twins of our cohort had highly correlated gut microbial richness (r=0.67) whereas that of the randomly constituted control pairs was not correlated (r=0). However, spouses, who are presumably as genetically different as randomly paired individuals, had significantly correlated richness (r=0.42). Our data show that cohabiting MZ twins, who share the same environment, are closer than MZ twins living apart. Their resemblance decreases with age. This highlights the importance of the environment in shaping the microbiome, not only early in life but also in adulthood. We identified smoking as an environmental factor that impacts gut microbial richness. Smokers have lower richness than non-smokers and there is a negative correlation between the richness and blood cotinine level. However, richness of individuals who quit smoking is comparable to those who never smoked. Our results indicate that gut microbiota is a complex and malleable ecosystem that can change, at least a certain extent, in response to the environment. This encourages development of interventions to prevent and possibly better treat disease that are impacted by the gut microbiome, such as the risk to develop metabolic syndrome related co-morbidities or complication of liver cirrhosis (Le Chatelier et al. Nature, 2013; Cotillard et al., Nature, 2013; Qin et al, Nature, 2014).

Evi-084#300 The effect of smoking cigarettes on the oral microbiota Simone S Stahringer (1), Robin P Corley (1), Daniel McDonald (1), Matthew B McQueen (1), Matthew Simonson (1), Se Jin Song (1), Sophie Weiss (1), John K Hewitt (1), Rob Knight (1), Kenneth S Krauter (1) 1. University of Colorado, Boulder, USA According to the World Health Organization, oral health is an integral part of overall human health. Oral diseases such as periodontitis and tooth decay are likely caused by an imbalance of the oral microbiota. The composition of human associated microbial communities is influenced by external environmental factors such as diet, hygiene, and exposures to chemicals, for example through smoking. The oral microbiota is directly exposed to tobacco smoke and it is known that smokers exhibit a more severe form of periodontitis compared to their non-smoking peers. Furthermore, smoking is the number one risk factor for periodontitis. However, it is currently unclear whether a global shift in the composition of oral bacteria might be the underlying cause. In this research, we compared the salivary microbiota of 136 smokers and 52 non-smokers (age 12 to 65) with culture-independent high throughput sequencing of parts of the bacterial 16S rRNA gene (Roche 454). By analyzing the data set using the unweighted UniFrac, a similarity measure based on the whole bacterial community, we observed an increase in diversity between smokers compared to non-smokers. Furthermore, non-smokers shared more bacterial genera than smokers. These findings suggest that non-smokers exhibit a common group of bacteria, whereas smokers exhibit a more diverse microbiota, which may be more prone to cause disease. As a proof a concept, we successfully identified a known tobacco sensitive bacterial genus, Neisseria, which we commonly found in saliva of non-smokers, but was drastically reduced in smokers. In contrast, the genus Filifactor is rarely observed in non-smokers, but was commonly observed in smoking individual and might be a signature organism for an altered oral microbiota. This research sheds light on the effects of a common environmental variable, smoking tobacco, on the oral microbiota and suggests that smoking causes a shift away from a shared more similar, healthy microbiota towards multiple possible diseased states. In addition, the presentation will cover a possible effect of host age. Funding was provided by NIH grants HD-010333 and DA-011015. Evi-085#302 The effects of a whole grain rich diet on the community structure of the gut microbiome Lea Benedicte Skov Hansen (1), Marlene Danner Dalgaard (1), Martin Iain Bahl (2), Henrik Bjørn Nielsen (1), Tine Rask Licht (2), Ramneek Gupta (1). 1. Department of Systems Biology, Center for Biological Sequence Analysis, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark. 2. Division of Food Microbiology, Technical University of Denmark, National Food Institute, 2860 Søborg, Denmark. The complex interplay between host, diet and the gut microbiome is a key process to understand the development of lifestyle related diseases. The intake of whole grains has been shown to induce positive effects on human health compared a gluten rich diet, which seems to affect negatively. Here we investigating the impact of whole grains on the human gut microbiome by introducing a dietary whole grain addition (≥ 75 g/day) in a randomized cross over intervention study, where the control diet consisted of elevated gluten levels (> 20 g/day) and lower content of whole grains (< 10 g/day). The dietary intervention periods lasted 8 weeks, separated by a 6 weeks wash out period and 50 participants were recruited for this intervention, who were apparently healthy but with a risk of developing metabolic diseases. To investigate the microbial composition of the fecal samples, 16S rRNA gene amplicon profiles were obtained. By analyzing the trajectories of the different operational taxonomic units (OTUs) over time, the changes in community structure of the gut microbiome in response to the specific diets were analyzed. Furthermore, we identified the specific OTU clusters that were most affected by the diet intervention and investigated the interpersonal differences in response to the diet intervention. Evi-086#303 The invasive microbiome Dries Budding (1), Martine Hoogewerf (1), Paul Savelkoul (1,2) 1. Department of medical microbiology and infection control, VU University medical center, Amsterdam, the Netherlands 2. Department of medical microbiology, Maastricht University medical center, Maastricht, the Netherlands Invasion of indigenous microbes to normally sterile locations in the host is a well-known phenomenon. An invasion may be asymptomatic when the invasive microbes are rapidly cleared by an immunocompetent host, but can also lead to serious clinical complications such as formation of an abscess or empyema. While this phenomenon has long been recognized, the identity of the invading microbes has been studied almost exclusively by bacterial culture. We now know that this is a serious technical limitation, as many species of the human indigenous microbiota are refractory to standard cultivation techniques.

To gain a better understanding of our invasive microbiome, we prospectively collected 160 pus samples from abscesses and empyemas and subjected them to routine bacterial culture and analysis by IS-pro, a molecular bacterial profiling tool designed for microbiota analysis in a clinical setting. We found that the current dogma which states that only a limited number of endogenous anaerobic species (mainly Bacteroides fragilis group) is invasive in humans is not correct. We found many more species deriving from our indigenous microbiota than only the Bacteroides fragilis group and identified highly diverse microbial communities in abscesses. Overall, we found more bacterial species than were found with culture in over 60% of samples (including 30% of samples that were negative in culture). In conclusion, we found that the number of endogenous bacterial species with invasive potential has been highly underestimated. We suggest that a better knowledge of the composition of abscess related microbiota may not only lead to better choice of antibiotic treatment regimens, but also to a better understanding of abscess formation and propagation. Evi-087#304 The microbiome of the aerodigestive tract as a tool in the diagnosis of aspiration in children Ruth Ann Luna, PhD (1,2), Abria Haynes PhD (1,2), Jessica Runge (1,2), Michelle Rubio-Gonzales (1,2), Eric Howard Chiou, MD (3), Julina Ongkasuwan, MD (4), Julia Cope, PhD (1,2), Emily B. Hollister, PhD (1,2), James Versalovic MD, PhD, (1,2,3), and Timoth 1. Department of Pathology & Immunology, Baylor College of Medicine 2. Texas Children’s Microbiome Center, Department of Pathology, Texas Children’s Hospital 3. Department of Pediatrics, Baylor College of Medicine 4. Department of Surgery, Baylor College of Medicine Diagnosis of aspiration in children is currently based on the cumulative clinical impression following review of symptoms, videofluoroscopic swallow study (VFSS), and the presence of lipid laden macrophages (LLM) in bronchoalveolar lavage (BAL) specimens. This approach is not regarded as sensitive or specific, and there remains a clinical need for a robust qualitative test for confirmation of aspiration. Chronic aspiration causes significant damage to the respiratory system, and due to the mechanism of aspiration, crosstalk between the gastrointestinal (GI) and respiratory microbiome is inevitable. Pediatric subjects (n=47) were recruited from the aerodigestive program at Texas Children's Hospital. A bilateral buccal swab (obtained prior to manipulation of the airway or GI tract), a 1 mL sample of BAL fluid, and a 1-2 mL stomach wash sample (gastric fluid) were collected during routine endoscopic evaluation. Following thorough review of diagnostic criteria, patients were categorized into 4 groups (aspiration (n=8), likely aspiration (n=11), unlikely aspiration (n=14), indeterminate (n=14)). Bacterial DNA was extracted from all specimens. Amplification and subsequent 454 sequencing targeted the V3V5 region of the 16S rRNA gene, and operational taxonomic units (OTUs) were assigned and evaluated based on aspiration status, location within the aerodigestive tract, and other clinically relevant data. The aerodigestive tract was characterized by a predominance of Streptococcus, Prevotella, Neisseria, Haemophilus, Porphyromonas, and Veillonella, with subtle variations in average relative abundance of each genera based on specimen type. As expected, gastroesophageal reflux and fundoplication were associated with changes in the aerodigestive microbiome. In gastric fluid, Capnocytophaga, Fusobacterium, Aggregatibacter, and Eikenella spp. were noted in the aspiration/likely aspiration groups but were absent or decreased in the unlikely aspiration group. Flavobacteriaceae was present in the gastric fluid of the majority of subjects in all groups, but these OTUs were only present in the BALs of the aspiration/likely aspiration groups. The trend with Flavobacteriaceae was upheld upon evaluation of the buccal swabs, where again Flavobacteriaceae was observed in the aspiration/likely aspiration groups but absent in the unlikely aspiration group. Aggregatibacter and Eikenella spp. were also identified in the buccal swabs of subjects suspected of aspiration but not in the unlikely aspiration group. Current diagnostic approaches to aspiration require invasive testing and are highly subjective. A buccal swab based diagnostic test could serve as a screening tool prior to endoscopic procedures, where BAL-based testing could confirm aspiration based on the presence of key organisms found in the GI tract. Further investigation is needed to confirm these findings in a larger cohort and to determine the core group of bacteria clinically relevant in aspiration. Evi-088#306 Specific microbiome changes after antibiotic treatment enable vancomycin-resistant Enterococcus intestinal colonisation. Sandrine Isaac (1) Ana Djucovik (1) Carles Ubeda (1) 1.Departamento de Genómica y Salud, Centro Superior de Investigación en Salud Pública ,Fundación para el fomento de la investigación Sanitaria y Biomédica de la comunitat Valenciana (FISABIO), Valencia, Spain Vancomycin-resistant-Enterococccus (VRE) is one of the most important causes of bacteremia in hospitalized patients following antibiotic treatment. Infections by VRE frequently start by colonization of the intestinal tract. In normal conditions, the hundreds of commensal bacteria inhabiting our gut suppress VRE colonization. However, antibiotic therapy, by disrupting the intestinal microbiota, enables VRE intestinal colonization. Once VRE has colonized the intestine, it reaches extremely high numbers, which promotes its dissemination to the bloodstream, putting in serious danger the life of the patient. Thus, understanding which

changes in the microbiota allow VRE colonization and which commensal bacteria are key for suppressing it is crucial to combat infections produced by VRE. Here, using a mouse model of infection and high-throughput 16s rRNA sequencing analysis, we have investigated how changes in the microbiota induced by antibiotics of different spectrum affect VRE intestinal colonization. We demonstrate that different antibiotics impact to a different extend on VRE colonization. Ampicillin, vancomycin, clindamycin and ceftriaxone highly promote VRE intestinal colonization while low levels of VRE were observed after neomycin and ciprofloxacin treatment. On the other hand, allowing the murine microbiota to recover for two weeks after ampicillin or clindamycin treatment before infection diminish the ability of VRE to colonize the intestine, while VRE still efficiently colonize mice that recovered from vancomycin treatment. Analysis of the microbiota of mice treated with these antibiotics showed marked differences in their intestinal microbiota composition. Importantly, correlation analysis of the changes in the microbiota composition with the levels of VRE colonization showed that antibiotic depletion of specific anaerobic bacterial genera (Alistipes, Allobaculum, Barnesiella) and families (Ruminococcaceae and Lachnospiraceae) increase the risk of VRE intestinal colonization. In addition, PICRUSt in silico analysis of microbiota encoded functions showed that VRE levels negatively correlate with the presence of metabolic pathways involved in catabolism of certain amino acids (tryptophan, phenylalanine, tyrosine and glycine), suggesting nutrient competition as a mechanism of microbiota protection against VRE. Our results may be useful for the development of novel therapies or biomarkers to combat VRE infections. Evi-089#308 Gut microbiota in diverticulosis; looking for signs of an inflammatory precursor stage of diverticulitis M.L.M. van Doorn-Schepens (1), A.E. Budding (1) , N. de Korte (2) , A. Eck (1), H.B.A.C. Stockmann (3) 1 Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands 2 Department of Surgery, Spaarne Medical Center, Hoofddorp, the Netherlands 3 Department of Surgery, Kennemer Gasthuis Medical Center, Haarlem, the Netherlands Objectives: It was previously shown that gut microbiota composition is different in patients with diverticulitis, compared to controls. It has long been hypothesised that all forms of diverticulitis are the result of a colonic (micro) perforation of a fecolith in the diverticular lumen. Recently it has been hypothesised that chronic subclinical inflammation may be a precursor stage to the clinically manifest stages of acute diverticulitis. This research aims to look for signs of a chronic subclinical inflammatory state in diverticulosis, reflected in differences in gut microbiota composition in diverticulosis patients versus controls. Methods: We aimed to enroll 20 patients with diverticulosis and 20 patients without diverticulosis. a total of five biopsies were taken from the mucosa around diverticula in the sigmoid colon and five biopsies were taken from the transverse colon as a control location. In the control group, five biopsies were taken from the sigmoid colon at random and from the transverse colon at random. Mucosal microbiota profiles were obtained by means of IS-pro. IS-pro involves bacterial species differentiation by the length of the 16S–23S rDNA interspace region with taxonomic classification by phylum-specific fluorescent labeling of PCR primers. A correlation matrix was generated by means of cosine correlation, then clustering was done with the unweighted pair group method with arithmetic mean (UPGMA). A partial least squares discriminant analysis (PLS-DA) regression model was used for the prediction of clinical status of samples. Results: In total 43 patients were enrolled, of which 19 patients had diverticulosis and 24 had no diverticulosis. For a per-sample analysis of all data, we generated a clustered heat map. IS-profiling showed that there is no clustering for diverticulosis or control samples. PLS-DA indicated the absence of truly disease-specific bacterial species in this data set. So there was no predictive power. Conclusions: We demonstrated that there are no evident changes in gut microbiota composition in individuals with diverticula compared to a control group. This makes the hypothesis that a chronic subclinical inflammatory state in patients with diverticulosis proceeds diverticulitis less plausible. Evi-090#318 The microbiome of type 1 diabetes autoimmunity: The Environmental Determinants of Diabetes in the Young (TEDDY) study Joseph F. Petrosino (1,2,3), Nadim J. Ajami (1,2), Matthew C. Wong (1,2), Daniel P. Smith (1,2), Ginger Metcalf (3), Donna Muzny (3), Richard Gibbs (3), Richard Lloyd (2), Beena Akolkar (4), William Hagopian (5), Marian Rewers (6), Jin-Xiong She (7), Jor 1. Alkek Center for Metagenomics and Microbiome Research (CMMR), Baylor College of Medicine, Houston, TX, USA 2. Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA 3. Human Genome Sequencing Center (HGSC), Baylor College of Medicine, Houston, TX, USA 4. National Institute of Diabetes & Digestive & Kidney Diseases, Bethesda MD, USA 5. Pacific Northwest Diabetes Research Institute, Seattle, WA, USA 6. Barbara Davis Center for Childhood Diabetes, University of Colorado, Aurora, CO, USA 7. Center for Biotechnology and Genomic Medicine, Medical College of Georgia, Georgia Regents University, Augusta GA, USA 8. Department of Pediatrics, Turku University Hospital, Turku, Finland 9. Institute of Diabetes Research, Helmholtz Zentrum München, and Klinikum rechts der Isar, Technische Universität München, and Forschergruppe Diabetes e.V. Neuherberg, Germany 10. Department of Clinical Sciences, Lund University, Malmö, Sweden 11. University of Tampere and Tampere University Hospital, Tampere, Finland

12. Health Informatics Institute, Department of Pediatrics, Morsani College of Medicine, University of South Florida, Tampa FL, USA The incidence of type 1 diabetes (T1D) and other immunity-related diseases has increased dramatically in the world in the last 50 years while infectious diseases have declined. These trends cannot be explained by genetic factors alone, but suggest that the modern environment has changed leading to this increased risk. The link between our genetic blueprint, in utero exposures, and the development of our microbiome in early life sets our baseline health state. Increased gut permeability, intestinal inflammation and deregulated oral tolerance have all been observed in children with T1D. Furthermore, data support the hypothesis that an infectious trigger may be responsible for the emergence of autoantibodies that ultimately lead to the decline to T1D. In the largest clinical microbiome study to date, including virus culturing and mycobiome components, we explore the comprehensive taxonomic and functional changes in the microbiome between birth and T1D onset in over 13,403 stool and 6,380 plasma samples from a subset of cases and controls (1:1; n=820) from the TEDDY international prospective cohort. Preliminary results examining 16S rRNA gene, and bacterial/viral metagenomic data exploring the developmental microbiome in this cohort, including the impact of country of origin and breastfeeding have identified a significant difference in microbial richness and evenness across countries (p<0.001). In addition, samples collected from infants while being breastfed presented with an increased relative abundance of Bifidobacterium spp. (p<0.001), which were found to markedly decrease upon cessation of breastfeeding. At this time, a significant increase in Blautia species was observed (p<0.001). As the study continues, integrated analyses of multiple “-omics” data and extensive TEDDY metadata including autoantibody exposure will begin to provide a complete prospective profile of the microbiome associations with autoimmunity and T1D. These data will hopefully lead to better therapeutics and diagnostics for treatment and prevention of T1D. Evi-091#320 Specific microbiome changes after antibiotic treatment enable vancomycin-resistant Enterococcus intestinal colonisation. Sandrine Isaac (1) Ana Djucovik (1) Carles Ubeda (1) 1.Departamento de Genómica y Salud, Centro Superior de Investigación en Salud Pública ,Fundación para el fomento de la investigación Sanitaria y Biomédica de la comunitat Valenciana (FISABIO), Valencia, Spain Vancomycin-resistant-Enterococccus (VRE) is one of the most important causes of bacteremia in hospitalized patients following antibiotic treatment. Infections by VRE frequently start by colonization of the intestinal tract. In normal conditions, the hundreds of commensal bacteria inhabiting our gut suppress VRE colonization. However, antibiotic therapy, by disrupting the intestinal microbiota, enables VRE intestinal colonization. Once VRE has colonized the intestine, it reaches extremely high numbers, which promotes its dissemination to the bloodstream, putting in serious danger the life of the patient. Thus, understanding which changes in the microbiota allow VRE colonization and which commensal bacteria are key for suppressing it is crucial to combat infections produced by VRE. Here, using a mouse model of infection and high-throughput 16s rRNA sequencing analysis, we have investigated how changes in the microbiota induced by antibiotics of different spectrum affect VRE intestinal colonization. We demonstrate that different antibiotics impact to a different extend on VRE colonization. Ampicillin, vancomycin, clindamycin and ceftriaxone highly promote VRE intestinal colonization while low levels of VRE were observed after neomycin and ciprofloxacin treatment. On the other hand, allowing the murine microbiota to recover for two weeks after ampicillin or clindamycin treatment diminished but not abolish the ability of VRE to colonize the intestine, while the observed impairment on VRE colonization was less pronounced after vancomycin recovery. Analysis of the microbiota of mice treated with these antibiotics showed marked differences in their intestinal microbiota composition. Importantly, correlation analysis of the changes in the microbiota composition with the levels of VRE colonization showed that antibiotic depletion of specific anaerobic bacterial genera (Alistipes, Allobaculum, Barnesiella) and families (Ruminococcaceae and Lachnospiraceae) increase the risk of VRE intestinal colonization. In addition, PICRUSt in silico analysis of microbiota encoded functions showed that VRE levels negatively correlate with the presence of metabolic pathways involved in catabolism of tryptophan, phenylalanine, tyrosine and glycine, suggesting a possible role of competition for amino acids in colonization resistance against VRE. Our results expand our previous knowledge on the microbiome features that associate with VRE protection, which may increase our ability to design novel therapies to combat infections against antibiotic resistant pathogens. Evi-092#322 Gut microbes provide carbohydrate-derived metabolites that fuel polyp formation in APCMin/+MSH2-/- mice. Antoaneta Belcheva (1), Thergiory Irrazabal (1), Susan J. Robertson1, Catherine Streutker (2), Heather Maughan (3), Stephen Rubino (4), Eduardo H. Moriyama (5), Julia K. Copeland (6), Sachin Kumar (1), Blerta Green (1), Kaoru Geddes (1), Rossanna C. Pezo 1. Department of Immunology, University of Toronto, Toronto, Ontario, Canada. 2. Department of Laboratory Medicine, St. Michael’s Hospital, Toronto, Ontario, Canada. 3. Ronin Institute, Montclair, New Jersey, USA. 4. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. 5. Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada. 6. Centre for the Analysis of Genome Evolution & Function, University of Toronto, Toronto, Ontario, Canada.

7. Department of Medical Oncology, University of Toronto, Toronto, Ontario, Canada. 8. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. 9. Department of Radiation Oncology, Princess Margaret Hospital, Toronto, Ontario, Canada. 10. Department of Nutritional Sciences, University of Toronto, Toronto, Ontario, Canada. 11. Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA. Colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammation, and gut microbiota. However, the mechanism through which the microbiota synergizes with these factors to promote CRC is unknown. We found that altering the microbial composition through antibiotics treatment reduces CRC in APCMin/+MSH2-/- mice, and that a diet reduced in carbohydrates phenocopies this effect. We found that, in these mice, the main mechanism by which gut microbes induce CRC is not inflammation. Our analysis revealed that the composition of inflammatory cells of the lamina propria was within normal limits in all the genotypes and treatments, and genetic manipulations of the immune system could not recapitulate the effect of the antibiotics treatment or the low carbohydrate diet (LC diet). In addition, gut microbes did not induce CRC through the production of DNA mutagens since neither the mutation frequencies nor the rate of double-stranded DNA breaks were affected by the antibiotic treatment or the LC diet. Owing to the enormous metabolic potential of the gut microbiota, we hypothesized that gut microbes might facilitate CRC development by providing diet-derived metabolites. We found that gut microbes induce CRC in APCMin/+MSH2-/- mice by providing carbohydrate-derived metabolites that fuel the aberrant hyperproliferation of MSH2-/- colon epithelial cells. Furthermore, we found that the mismatch repair pathway has a role in regulating β-catenin activity, which is a key factor for colon epithelial proliferation. When we analyzed the colon contents from mice under the antibiotics treatment or LC diet we found that butyrate, a metabolite necessary for colon epithelial proliferation, was reduced compared to controls. Furthermore, when butyrate was directly administered in the colon of antibiotic-treated mice by rectal instillation, we found that physiological concentrations of butyrate stimulated proliferation of colon epithelial cells in APCMin/+MSH2-/- mice but not in controls. Thereby providing an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC. Evi-093#325 Insights on the lipid metabolic potential of the gut microbiome in different human and animal groups Stephanie Schnorr (1), Simone Rampelli (2), Silvia Turroni (2), Elena Biagi (2), Clarissa Consolandi (3), Patrizia Brigidi (2), Alyssa Crittenden (4), Marco Candela (2), Amanda Henry (1) 1. Plant Foods in Hominin Dietary Ecology Research Group, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany 2. Department of Pharmacy and Biotechnology, University of Bologna, Italy 3. Institute of Biomedical Technologies, Italian National Research Council, Segrate, Milan, Italy 4. Metabolism, Anthropometry, and Nutrition Laboratory, Department of Anthropology, University of Nevada, Las Vegas, USA Lipid metabolism in bacteria is considered an important process both for energy production and cellular growth and maintenance. Lipids are rich in carbon and so are dense sources of energy. Phospholipids are also synthesized by bacteria as they comprise roughly ten percent of the total cellular dry weight. Therefore, significant investment by the cell is put towards manufacture and tight regulation of lipid production. Furthermore, the lipid metabolic pathways in prokaryotes are catalyzed by discrete proteins rather than more complex and integrated mammalian polypeptides, making it much easier to define intermediates in a step-wise process of lipid synthesis and degradation in bacteria. Recently, significant focus has been placed on the nuances of microbial accessible carbohydrates and metabolism in metagenomic communities, and this has gained us much insight to the microbe-host mutualism. However, the role of lipid manufacture and processing in bacteria as it relates to host diet and health is by comparison less well understood beyond the scope of medical research on the microbial role in cholesterol production. Here we aim to contribute a focused analysis of the lipid metabolic profiles from gut microbiome communities in two very different human populations, as well as other animals from different dietary niches. Using shot-gun sequencing on fecal microbiota, we obtained metagenomic sequences of bacteria from individuals in two different human communities to use for comparison: the Hadza hunter-gatherers of Tanzania, and urban living Italian controls. Reads were assembled into contigs for gene mapping and analysis of subsequent protein coding regions. We assembled databases of curated protein families implicit in lipid metabolic pathways and searched for correspondent sequences among our samples. The lipid metabolic profile is very different for both human microbial communities, and we explore this difference further, detailing the distribution of genes for both synthesis and degradation of lipids as well as activities related to bile acid metabolism and recycling. We infer a host dietary origin of influence on the observed protein coding distribution, and for illustration, we make comparative models from other metagenomic communities found within other herbivores, carnivores, and omnivores. This work attempts to understand the dietary influence on lipid metabolic specializations in different host microbial communities. This has important implications for understanding how the gut microbiota are affected in a carbohydrate resource limiting environment, and how differences in diet, especially for dietary non-specialists such as humans and human ancestors, may reshape microbial activities in lipid processing as they relate to energy capture and maintenance of the microbiome and host health.

Evi-094#331 Microbial communities in the ileal tissues of healthy Koreans and patients with Crohn’s disease or ulcerative colitis Min-Jung Kwak (1,2), Chang Soo Eun (3), Ar Reum Lee (3), Byung Kwon Kim (1), Dong Soo Han (3), Jihyun F. Kim (1) 1. Department of Systems Biology and Division of Life Sciences, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Republic of Korea 2. Biosystems and Bioengineering Program, University of Science and Technology, 217 Gajung-ro, Yuseong-gu, Daejeon 305-350, Republic of Korea 3. Department of Internal Medicine, Hanyang University Guri Hospital, 153 Gyeongchun-ro, Guri 471-701, Republic of Korea Gut microflora plays important roles in human health. Especially, dysbiosis of the intestinal microbiota is reportedly related with the occurrence and development of the inflammatory bowel disease (IBD). To analyze the alteration of the gut microbial community structure in Korean patients of Crohn’s disease (CD) and ulcerative colitis (UC), we performed a comparative metagenomic analysis between healthy persons and patients with Crohn’s disease or ulcerative colitis using biopsy samples of the terminal ileum. At the class level, relative abundance of Fusobacteria and Gammaproteobacteria was higher, whereas that of Clostridia was lower, in CD patients as compared to healthy control (HC). This observation was more obvious in patients of active CD or L3/B2 categories according to the Montreal classification. In the case of UC patients, changes in the relative abundance of bacteria at the phylum or class levels in comparison to HC were less significant. At the genus level, however, abundance of Faecalibacterium, Streptococcus, Sutterella, and Sphingomonas was relatively higher in UC patients. Also, abundance of Fusobacterium in new UC patients was higher than established patients or HC. Through the comparative analysis of the mucosal bacterial communities between healthy people and IBD patients, we demonstrate the alteration in the patterns of the bacterial dynamics. Our data also alludes to the probable causative bacteria of CD or UC. Evi-095#332 Microbiome analysis of human gestational tissues in normal and complicated pregnancies Lydia Leon (1), Ronan Doyle (1), Nigel Klein (1), Philip Stanier (1), Gudrun Moore (1) 1) Institute of Child Health, University College London, London, UK Preterm birth (before 37 completed weeks gestation) affects around 5% of births in developed countries and rates in developing countries are thought to be significantly higher. Being born preterm is the leading cause of neonatal death globally and is associated with a range of morbidities and mortalities that extend beyond infancy, into adulthood. My PhD project uses molecular genetic techniques to quantify and characterise the presence of bacterial DNA in placental parenchyma and placental basal plate tissue from 364 deliveries (187 term and 177 preterm). Participants were recruited as part of the large Baby Bio Bank, a biological repository of maternal, infant and paternal samples collected from three hospitals across London. Quantitative PCR is being used to identify which samples have evidence of broad range bacterial presence above an experimentally defined threshold of ‘infection’. These samples are analysed using the absolute quantification method. Preliminary data from these experiments indicate that bacteria are present across all types of pregnancies, but levels appear to be especially raised among the spontaneous labour and spontaneous rupture of membranes subgroups. Furthermore, the proportion of preterm placenta from spontaneous labour that exceed the qPCR threshold of 'infection' is significantly higher than the proportion of preterm placenta that were not from spontaneous preterm labour groups (p=0.03) - an association that is line with much recent research. This trend is also observable with reference to spontaneous membrane rupture, although with only about half the cohort analysed so far, this is not significant. Following the qPCR experiments, ‘infected’ samples will be taken forward for next generation sequencing of the V5-V7 region of the 16S rRNA locus, exploring the make up of the 'placental microbiome' across the cohort. This process will enable a description of the species composition and relative abundance of the bacterial communities within the placenta, identifying any associations between community structure and pregnancy outcome or clinical characteristics in the cohort. Future work is planned in which the above process will be repeated for a group of women with pre-eclampsia from the same Baby Bio Bank cohort, to see whether bacterial infection also has a role in this common and serious complication in pregnancy. Finally, I will link these results with those from a high throughput human cytokine ELISA panel that will be conducted in Virginia, USA, in autumn 2015, testing the hypothesis that the maternal immune response is an important causal factor in infectious mediated complications in pregnancy.

Evi-096#336 Cultural Practices Influence Tsimané Gut Microbiome Maturation Daniel Sprockett (1), Melanie Martin (2), Elizabeth Costello (1), Michael Gurven (2), and David Relman (1,3) 1. Microbiology & Immunology Department, Stanford University, Palo Alto, CA, USA 2. Anthropology Department, University of California at Santa Barbara, Santa Barbara, CA, USA 3. Department of Medicine, Stanford University, Palo Alto, CA, USA Although recent investigations have begun characterizing the ecological processes that determine the structure and assembly patterns of human-associated microbial communities, little is known about the influence of non-western cultural practices. Here we characterize the gut and oral microbiome of the Tsimané people, an indigenous hunter/gatherer society living along the Maniqui River in the lowlands of Bolivia. Stool and saliva samples were collected from 47 mother/child pairs over the first two years of the child’s life. Detailed surveys were also collected at the time of sampling, including information on the parent and child’s diet (weaning status, meal frequency, access to processed foods, etc.), health (height/weight, history of illness, recent medications, etc.), and lifestyle (family structure, travel history, activity level, etc.). We then used 16S rRNA profiling on the Illumina MiSeq to quantify taxonomic diversity found in 600 samples taken from six different Tsimané villages with varying levels of exposure to western cultural, agricultural, and medical practices. These data show that the factors we examined influence the diversity and structure of the Tsimané microbiome in complex ways. Typical of non-western populations, Tsimané infant gut communities are dominated by Bifidobacterium, while the adults possesses a relatively high abundance of Prevotella species and high alpha diversity. After accounting for body site and age, the major sources of variation in this dataset, we found that associations with diet are more robust than other health or lifestyle factors. Furthermore, we compare the Tsimané to other published gut microbiome datasets from Venezuela, Malawi, Bangladesh, and The United States. The trajectory of gut maturation in the Tsimané is more similar to other non-western populations than those from the US. These findings broaden our understanding of the way behaviors normally associated with maternal and child wellbeing can also influence the ecological processes that structure the microbiome. Evi-097#348 Serological, Virulence, Molecular Characteristics and Antimicrobial Susceptibility of Clinical Vibrio parahaemolyticus Strains in Southeastern China: 2009 to 2013 Yu Chen (1), Xiao Chen (1), Lanjuan Li (1) State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases，The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China Vibrio parahaemolyticus is a leading cause of foodborne bacterial diarrhea in coastal countries. Although the infection was first reported as early in 1962 in China, the prevalence and population structure of the pathogen remains unclear. During July 2009 to June 2013, we collected 6,951 faecal specimens for pathogen detection, from which 563 (8.1%) V. parahaemolyticus strains were isolated. Then, we chose 501 V. parahaemolyticus strains that isolated as sole pathogens for the following etiological investigation. As many as 21 serotypes were identified from all the strains, among which O3: K6 was the most common and important serotype (65.0%), followed by O4:K8, O4:K68 and O1: K36 serotypes. A newly discovered serotype O4: K18 was first identified from clinical patients. The hemolysin tdh genes (466 strains, 93.0%) was the key characteristic of the virulent strains, but few strains carried trh gene, indicating that tdh was the most prevalent hemolysin gene of V. parahaemolyticus. The positive ratio of pandemic markers toxRS/new and orf8 was 64.9% (325/501) and 65.1% (326/501), suggesting that pandemic and pathogenic strains occupied for 62.1% and 93.6% of all the tested isolates, respectively. Furthermore, pandemic O3: K6 clones were predominant in this region, accounting for 69.3% of all the pandemic strains. It was the first time that O3:K8 reported as a pandemic serotype, but not only pathogenic. We also confirmed that T3SS1 genes were amplified from all strains and T3SS2 possessed in almost all pathogenic strains. Subsequent multilocus sequence typing (MLST) split isolates into 16 sequence types (STs) and found ST3 (n=414, 82.6%) and ST88 (n=46, 9.2%) were two STs that most prevalent in Zhejiang province. STs of clinical V. parahaemolyticus was concentrated in the pandemic groups, but dispersed in non-pandemic strains. The majority of pandemic strains belonged to ST3 (n=289), and the remaining pandemic isolates were categorized into ST88 (n=25) and ST 672 (n=3). More than 95% of the isolates were sensitive to the antibacterials tested in our study, however, a large amount (87.1%) of strains were resistant to ampicillin. To conclude, pandemic isolates became the most prevalent strains and surveillance should be emphasized to monitor epidemic trends, antimicrobial control and discover more reliable predictors for virulence.

Evi-098#350 Risk Factors for Invasive Fungal Infections in Patients with Hematopoietic Malignancies Chen Li (1), Zhang Wei-li (2), Ye Xian-fei (3), Ge Chao-rong (1) , Chen Yu (1,2,4), Li Lan-juan (2*) 1. Department of clinical Laboratory, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China 2. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, College of Medicine, Zhejiang University, Hangzhou, China 3. Wenzhou Medical University, Wenzhou, China 4. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China *Corresponding Author Hematopoietic malignancy has been a common cancer with a high mortality and increasing incidence in recent years. Accompanying of fungal infection is a focus topic since it usually increases the mortality and medical expenses and has the increasing incidence rate for patients with hematopoietic malignancies. To draw up efficient measures for preventing and treating invasive fungal infections (IFI), we investigate the incidence and risk factors of IFI occurred in patients with hematopoietic malignancies. A retrospective cohort study was performed to 495 patients with hematopoietic malignancies, who were admitted to our hospital in the period between January to December 2013. The risk factors of IFI were identified by chi square test and multivariate logistic regression analysis.16.4% (81/495) of total patients were infected by funguses, and among which, the incidence of IFI in patients with acute leukemia, chronic leukemia, myelodysplastic syndrome, lymphoma and multiple myeloma were 31.1% (66/212), 0% (0/19), 31.2% (5/16), 4.8% (10/207) and 0.0% (0/41) respectively. According to the chi square test, with chronic underlying diseases (yes), the category of hematopoietic malignancy, being treated with glucocorticoid (yes), the number of category for antibiotics treating, the length of time for antibiotics treating (≥8 days), the lowest counts of WBC in peripheral blood (≤1.0E+9/L), the length of neutropenia (≥8 days) and being treated with antifungal drugs (yes) were correlate with the incidence of IFI. There was no significant difference of IFI incidence between patients treated without antibiotics and that treated with one kind of antibiotic (χ2=0.774, P=0.379), and also no significant difference between patients treated with three kinds of antibiotics and more than three (χ2=3.232, P =0.072). It had higher IFI incidence in patients treated with two kinds of antibiotics than one (χ2=4.743, P=0.029), and, similarly, it also had higher IFI incidence in patients treated with three kinds of antibiotics than two (χ2=10.368, P=0.001.The risk factors of IFI in patients with hematopoietic malignancies were as follows：1. The lowest counts of WBC in peripheral blood was less than 1.0E+9/L; 2. The length of neutropenia was longer than 8days; 3.Treated with glucocorticoid; 4. Treated with more than two kinds of antibiotics. The protective factor was prophylactic treating with antifungal drugs. As opportunistic pathogens, fungi seldom cause infection in a normal condition. But IFI sometimes occur in the patients with hypoimmunity or/and dysbacteriosis or/and mucosal damage just as the patients with hematopoietic malignancies after immunosuppressant and long-term antibiotics treating. So, maybe we can consider from the perspective of reconstruction of intestinal microflora for preventing and treating the patients with hematologic malignancies accompany with IFI.

Table 1 Demographic character istics and risk factors of I F I in patients with hematologic malignancies

Factors No I F I

Total number

Incidence of

I F I (%)

1.196 0.274

＜65yesrs 331 69 400 17.3

83 12 95 12.6

Gender

0.022 0.882

male 249 48 297 16.2

female 165 33 198 16.7

Chronic undelying disesaesa

5.554 0.018

yes 119 34 153 22.2

no 295 47 342 13.7

M ain diagnosis

78.263 0

AL 146 66 212 31.1

CL 19 0 19 0

MDS 11 5 16 31.2

NHL 197 10 207 4.8

MM 41 0 41 0

Glucocorticoid

therapy(yes/no) 4.058 0.044

yes 292 66 358 18.4

no 122 15 137 11

Antifungal drugs therapy

5.478 0.019

yes 74 6 80 7.5

no 340 75 415 18.1

Category number of

antibiotics being treated b

110.906 0

0 86 2 88 2.3

1 183 10 193 5.2

2 68 10 78 12.8

3 46 25 71 35.2

31 34 65 52.3

Duration of antibiotics

therapy b

80.5 0

< 8days 295 15 310 4.8

119 66 185 35.7

The lowest counts of W B C c

150.633 0

65 66 131 50.4

＞1.0E+9/L 349 15 364 4.1

Length of neutropenia

113.215 0

0day 272 10 282 3.5

1day 23 5 28 17.9

2days 23 3 26 11.5

3days 23 5 28 17.9

4days 10 7 17 41.1

5days 10 7 17 41.1

6days 12 3 15 20

7days 8 2 10 20

33 39 72 54.2

a:The chronic underlying diseases include diabetes, hypertension, chronic hepatitis B and COPD；b:Not include antifungal drugs;

c:count of WBC in peripheral blood

Table 2 Logistic regression analysis of risk factors for fungal infection!

Factors OR P value

The lowest counts of WBC in peripheral blood ≤1.0E+9/L 15.83 0.000

Length of neutropenia ≥8days 41.667 0.011

Glucocorticoid therapy (yes) 3.745 0.035 Category number of antibiotics being treated ≥3 8.264 0.007

Antifungal drug therapy (yes) 39.085 0.000

Evi-099#355 Intestinal function and bacterial translocation after cerebral ischemia in mice Naoki Oyama (1), Katarzyna Winek (1), Priscilla Koduah (1), Tian Zhang (1), Yasmin Beckers (1), Yvonne Amoneit (1), Sabine Kolodziej (1), Andreas Meisel (1, 2), Ulrich Dirnagl (1, 2) (1) Department of Experimental Neurology, Charité Universitätsmedizin Berlin, Berlin, Germany (2) Center for Stroke Research Berlin, Charité Universitätsmedizin Berlin, Berlin, Germany Background and Purpose: Intestine and gut microbiota play an important role in nutrient absorption, mucosal barrier function, and immune regulation. Accumulating evidence suggests that alterations in microbiota and/or intestinal function influence brain function and behavior through neural and humoral pathways, and vice versa over the microbiota-gut-brain axis. Cerebral ischemia leads to immunodepression and infectious complications. However, it is not fully investigated whether and how the brain lesion affects intestinal function. Our aim is to clarify intestinal function after cerebral ischemia and whether there is bacterial translocation from gut to extraintestinal organs. Methods: Male 12-week-old C57BL/6J mice were used for experiments. To assess the intestinal barrier integrity, we isolated epithelial cells from ileum and colon, and evaluated expression of tight junction proteins (occludin, claudin-1 and zonula occludens-1) 1 and 3 days after 60 min middle cerebral artery occlusion (MCAO). Lactulose/mannitol/sucralose test was performed for assessment of intestinal permeability 24 to 48 and 48 to 72 hours after MCAO. Local immune cell populations in Peyer´s patches were also assessed by fluorescence assisted cell sorting (FACS). Finally, we evaluated bacterial translocation in extraintestinal organs (lung, liver, spleen and mesenteric lymph nodes) by culture and fluorescence in situ hybridization (FISH) with a eubacterial 16S rRNA probe (EUB338) 1 and 3 days after MCAO. Results: Three days after MCAO expression of occludin and claudin-1 was significantly decreased in the epithelial cells of ileum but not in the colon. Results of sugar permeability test indicate that the intestinal permeability may be increased in the MCAO animals. We did not observe significant differences in immune cell populations of Peyer´s patches among the groups. Bacterial translocation to extraintesinal organs was seldom observed in MCAO group. Conclusions: These findings indicate that stroke may cause intestinal dysfunction although that rarely induces bacterial translocation. We need further study to investigate how intestinal dysfunction after stroke could influence in terms of nutrient absorption, metabolism and immune system as well as bacterial translocation. Evi-100#356 Risk Factors for Invasive Fungal Infections in Patients with Hematopoietic Malignancies Chen Li (1), Zhang Wei-li (2), Ye Xian-fei (3), Ge Chao-rong (1), Chen Yu (1, 2,4), Li Lan-juan(2*) 1. Department of clinical Laboratory, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China 2. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, College of Medicine, Zhejiang University, Hangzhou, China 3. Wenzhou Medical University, Wenzhou, China 4. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China *Corresponding Author Hematopoietic malignancy has been a common cancer with a high mortality and increasing incidence in recent years. Accompanying of fungal infection is a focus topic since it usually increases the mortality and medical expenses and has the increasing incidence rate for patients with hematopoietic malignancies.

3days 23 5 28 17.9

4days 10 7 17 41.1

5days 10 7 17 41.1

6days 12 3 15 20

7days 8 2 10 20

33 39 72 54.2

Factors OR P value

To draw up efficient measures for preventing and treating invasive fungal infections (IFI), we investigate the incidence and risk factors of IFI occurred in patients with hematopoietic malignancies. A retrospective cohort study was performed to 495 patients with hematopoietic malignancies, who were admitted to our hospital in the period between January to December 2013. The risk factors of IFI were identified by chi square test and multivariate logistic regression analysis.16.4% (81/495) of total patients were infected by funguses, and among which, the incidence of IFI in patients with acute leukemia, chronic leukemia, myelodysplastic syndrome, lymphoma and multiple myeloma were 31.1% (66/212), 0% (0/19), 31.2% (5/16), 4.8% (10/207) and 0.0% (0/41) respectively. According to the chi square test, with chronic underlying diseases (yes), the category of hematopoietic malignancy, being treated with glucocorticoid (yes), the number of category for antibiotics treating, the length of time for antibiotics treating (≥8 days), the lowest counts of WBC in peripheral blood (≤1.0E+9/L), the length of neutropenia (≥8 days) and being treated with antifungal drugs (yes) were correlate with the incidence of IFI. There was no significant difference of IFI incidence between patients treated without antibiotics and that treated with one kind of antibiotic (χ2=0.774, P=0.379), and also no significant difference between patients treated with three kinds of antibiotics and more than three (χ2=3.232, P =0.072). It had higher IFI incidence in patients treated with two kinds of antibiotics than one (χ2=4.743, P=0.029), and, similarly, it also had higher IFI incidence in patients treated with three kinds of antibiotics than two (χ2=10.368, P=0.001.The risk factors of IFI in patients with hematopoietic malignancies were as follows：1. The lowest counts of WBC in peripheral blood was less than 1.0E+9/L; 2. The length of neutropenia was longer than 8days; 3.Treated with glucocorticoid; 4. Treated with more than two kinds of antibiotics. The protective factor was prophylactic treating with antifungal drugs. As opportunistic pathogens, fungi seldom cause infection in a normal condition. But IFI sometimes occur in the patients with hypoimmunity or/and dysbacteriosis or/and mucosal damage just as the patients with hematopoietic malignancies after immunosuppressant and long-term antibiotics treating. So, maybe we can consider from the perspective of reconstruction of intestinal microflora for preventing and treating the patients with hematologic malignancies accompany with IFI.

Table 1 Demographic character istics and risk factors of I F I in patients with hematologic malignancies

Factors No I F I

Total number

Incidence of

I F I (%)

1.196 0.274

＜65yesrs 331 69 400 17.3

83 12 95 12.6

Gender

0.022 0.882

male 249 48 297 16.2

female 165 33 198 16.7

Chronic undelying disesaesa

5.554 0.018

yes 119 34 153 22.2

no 295 47 342 13.7

M ain diagnosis

78.263 0

AL 146 66 212 31.1

CL 19 0 19 0

MDS 11 5 16 31.2

NHL 197 10 207 4.8

MM 41 0 41 0

Glucocorticoid

therapy(yes/no) 4.058 0.044

yes 292 66 358 18.4

no 122 15 137 11

Antifungal drugs therapy

5.478 0.019

yes 74 6 80 7.5

no 340 75 415 18.1

Category number of

antibiotics being treated b

110.906 0

0 86 2 88 2.3

1 183 10 193 5.2

2 68 10 78 12.8

3 46 25 71 35.2

31 34 65 52.3

Duration of antibiotics

therapy b

80.5 0

< 8days 295 15 310 4.8

119 66 185 35.7

The lowest counts of W B C c

150.633 0

65 66 131 50.4

＞1.0E+9/L 349 15 364 4.1

Length of neutropenia

113.215 0

0day 272 10 282 3.5

1day 23 5 28 17.9

2days 23 3 26 11.5

3days 23 5 28 17.9

4days 10 7 17 41.1

5days 10 7 17 41.1

6days 12 3 15 20

7days 8 2 10 20

33 39 72 54.2

Factors OR P value

Evi-101#357 Impact of elimination of the microbiota on the outcome of experimental stroke. Katarzyna Winek (1), Odilo Engel (1), Priscilla Koduah (1), Markus Heimesaat (2), André Fischer (2), Stefan Bereswill (2), Olivia Kershaw (3), Claudia Dames (4), Caterina Curato (5), Naoki Oyama (1), Christian Meisel (4), Andreas Meisel (1), Ulrich Dirnag (1) Department of Experimental Neurology, Charité-Universitätsmedizin Berlin, Germany (2) Department of Microbiology and Hygiene, Charité-Universitätsmedizin Berlin, Germany (3) Department of Veterinary Medicine, Freie Universität, Berlin, Germany (4) Institute for Medical Immunology, Charité-Universitätsmedizin Berlin, Germany (5) Deutsches Rheuma-Forschungszentrum (DRFZ), Charité-Universitätsmedizin Berlin, Germany Signaling over bidirectional brain-gut-microbiota axis is thought to be important in the pathogenesis of several diseases, including these of the nervous system. Stroke is one of the most frequent causes of death and the number one cause of adult disability. In our experiments, we hypothesized that elimination of microbiota has an impact on the outcome of focal cerebral ischemia. We aimed to assess outcome parameters in C57Bl6 mice rendered gnotobiotic by 5-fold antibiotic cocktail treatment, subjected to experimental stroke (Middle Cerebral Artery Occlusion, MCAo). Several experimental groups were included in our studies: gnotobiotic mice with antibiotics stopped before surgery, gnotobiotic animals with continuous antibiotic treatment, gnotobiotic mice recolonized with Specific Pathogen Free (SPF) flora from littermates and SPF mice (MCAo and sham operated). Infarct volumes assessed on day 1 by Magnetic Resonance Imaging did not differ between the groups. Surprisingly, we observed that the withdrawal of antibiotics before operation resulted in severe colitis in sham and MCAo animals 5-6 days after surgery. We found significantly increased mortality in the gnotobiotic MCAo group with stopped antibiotic treatment (95%, compared to 56% sham operated mice, Log-rank Test p=0,002). We have shown that this phenotype can be prevented by continuous antibiotic treatment or recolonization with microbiota from SPF littermates. Additionally, we investigated main immune parameters (CD11b+CD11c+, CD3+, CD8+, CD8+ TCRgd+, CD4+, CD19+, TCRgd+ cells and secretion of IFNg and IL17 in spleen, mesenteric lymph nodes and Peyer's Patches) in gnotobiotic animals at two time points (day 3 and day 5) after MCAo and concluded that impaired immune responses after ischemic brain lesion (stroke-induced immunodepression syndrome, SIDS) in combination with lack of protection from physiological flora or continuous protection from antibiotics, may promote the poor outcome in our model (possibly due to the spontaneous recolonization with potentially harmful bacteria). These results may have direct clinical importance, since stroke patients are often treated with several broad-spectrum antibiotics due to post-stroke infections promoted by SIDS. The impact of this treatment on the intestinal microbiota and its possible consequences have not been assessed so far.

3days 23 5 28 17.9

4days 10 7 17 41.1

5days 10 7 17 41.1

6days 12 3 15 20

7days 8 2 10 20

33 39 72 54.2

Factors OR P value

Evi-102#359 Studying the role of gut microbiome in type 1 diabetes using OMICS approaches Ramana Madupu (1), Andrey Tovchigrechko(1), Vishal Thovarai(1), Moojin Suh(1), Karen Nelson(1), Rembert Pieper(1) J.Craig Venter Institute, 9704 Medical Drive, Rockville, MD 20850 Type 1 Diabetes (T1D) is an autoimmune disorder characterized by loss of function of insulin producing pancreatic beta cells leading to insulin insufficiency. It is estimated that T1D affects approximately 1 in every 300-500 children in the United States and has been rising rapidly in the past two decades with an average rate of 3% per year. The pathogenic mechanisms of T1D implicate complex interactions between genetic and environmental determinants. Our hypothesis: Due to the interconnectedness of the gut microbiome and the immune system, changes in the gut microbiota result in inflammatory responses mediated by cytokines which also influence the activation of apoptotic pathways. Increased activities of pro-apoptotic molecules in pancreatic beta cells are a consequence, and mediators of apoptosis and inflammation are persistently augmented in these cells, including reactive oxygen species. We hypothesize that these autoimmunity-associated molecular patterns can be detected in blood plasma, and, by lack of retention during glomerular filtration, are excreted in urine. Urine is therefore a body fluid in which surrogate markers of autoimmune T1D can be discovered. To test our hypothesis we designed a case-control study to investigate the distal gut microbial diversity using metagenomic approaches and the urinary proteome and metabolome profiles of T1D patients, their healthy siblings, and individuals at-risk of T1D. The main objective is to identify molecular signatures predictive or indicative of disease onset. Results of these analysis will be presented.

MODEL SYSTEMS IN MICROBIOME RESEARCH

Mod-001#16 The historical origin of microbiome : Pierre Joseph Van Beneden (1809-1894) and commensalism Brice POREAU, MD, PhD/MA (1) 1. Laboratoire S2HEP, Université Lyon1, Lyon, France Commensalism is a biological association in which one partner (the commensal) benefits while neither harming nor benefitting the host. Parasitism and mutualism were well defined during the nineteenth century and commensalism was theorized during the second part of that century. Pierre- Joseph Van Beneden (1809-1894), a Belgian professor at the University of Louvain, developed this concept of commensalism. In his 1875 publication Animal Parasites and Messmates, Van Beneden presented 264 examples of commensalism. His conception was widely accepted by his contemporaries and commensalism has continued to be used as a concept right up to the present day. In our presentation, we examine the development of commensalism during the nineteenth century and the use of the concept in contemporary science. We have used hitherto unpublished archival material for Pierre-Joseph Van Beneden to explore the pertinence of his concept. From an epistemological point of view, commensalism can be seen as a marker of the new domains in the life sciences such as microbiology and genetics. Through their use of different models of the concept, these two sciences gave a new sense to commensalism with the new concept of microbiome. We propose to establish the historical and epistémological links between the past concept of commensalism and the microbiome. Beyond being simply scientific concepts, commensalism and microbiome illustrate the complexity of life. Mod-002#38 Immuno-biological effects of different probiotic preparations and bacterial secondary metabolites on a human organo-typical (HOT) co-culture system: HOT-Co gut Schmolz MW(1), Venema K (2) ( 1) HOT Screen GmbH, Aspenhaustr. 25 D-72770 Reutlingen GERMANY Tel.: +49 7121 434103 Fax: +49 7121 491074 (2) TNO, Zeist, NL Abstract A major drawback of traditional cell culture models is their substantial lack in complexity compared to the local situation of the different tissues in vivo. This limits considerably the predictive value of results obtained with such test systems when, for example, immunologically active substances have to be screened or profiled in vitro. To overcome these problems, we developed a novel co-culture system that combines differentiated human intestinal epithelia with cultures of human whole-blood: HOT-Co gut. In this complex, organo-typical environment the gut epithelium not only serves as a natural barrier, separating non-absorbable substances from the immune cells (thereby preventing false positive results), but also actively or passively transfers absorbable ingredients from its luminal side to the basolateral (immune cell) compartment, as it does happen in vivo. Equally important, these epithelia contribute significantly to a cytokine cross-regulation similar to what is known from the human gut. Such a regulatory environment allows the generation of results of much higher relevance than when using any other, more simple model system in vitro. As an example, a series of well-known bacterial metabolites, as well as two different probiotic E.coli products ("A" and "B") were tested. From product B two individual production lots were examined and exhibited clearly different activities in the HOT-Co gut model. It is postulated that these differences may be the result of an altered metabolic profile due to manufacturing variabilities.

Mod-003#91 Minimal pathway enumeration to predict gene knockout effects in microbial pathways Steve Pastor(1), Yemin Lan(1), Gail Rosen(2) 1. School of Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia, PA 2. Electrical and Computer Engineering Department, Drexel University, Philadelphia, PA We hereby introduce a software that simulates the effect of one or multiple gene knockouts on the microbiome’s ability to generate a particular metabolic product. Studies on the functional composition in human microbiomes have revealed diverse metabolic activities performed by our microbial residents. However, tracing the metabolic activities back to the contributing organisms or genes has been challenging. To understand how one or a set of genes would contribute to the overall metabolism in a microbiome, we try to simulate the effect of gene knockouts, which was typically employed to elucidate gene functionality in one or only a few organisms of interest. We construct a supraorganism model encompassing a metabolic network from the enzymes, intermediates, products, and genes of all members in the community using existing databases (e.g. KEGG and MetaCyc). Our software requires two inputs from the user: (1) an end product metabolite of interest and (2) a list of gene knockouts. The program utilizes a minimal pathway enumeration algorithm to count metabolic pathways contributing to the end product, and outputs the number of pathways that are eventually affected under the specified gene knockouts. The software allows researchers to observe how gene knockouts may perturb the system’s ability to generate a user-specified product, provides experimental guidance for gene knockout experiments in a complex microbial environment and provides further insights into the division of labor in metabolism exhibited in community metabolism. Mod-004#96 correlation of hepatitis B e antigen levels and HBVDNA levels in asymptomatic carriers with HBeAg-positive Ping Chen, Jinghua Wang, Chengbo Yu, Wei Wu, Shigui Yang, Jingjing Ren, Bing Ruan and Lanjuan Li State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qing-Chun Road, Hangzhou, People´s Republic of China Abstract Objective:This study was designed to clear the quantitatively relationship between HBeAg and HBV DNA, and verify whether quantitative measurement of HBeAg could be used as a marker of viral replication in asymptomatic HBsAg-positive carriers. Methods: A cross-sectional, community-based survey was carried out in 12 communities of two counties. 724 asymptomatic carriers with HBeAg-positive were divided into four groups by HBV DNA levels. Cases in groups I (n=99), II(n =79), III (n = 98) and IV (n = 448) were reported HBV DNA levels of < 103 (PCR undetectable),103 to 105 (PCR detectable), > 105 copies/ml(hybridization detectable) and > 107 copies/ml, respectively. Results: The HBeAg levels have a good correlation with HBV DNA (r=0.765; P < 0.001) on a log scale. The mean log HBeAg (S/CO) of group I, II, III, IV were 0.66±0.54, 0.98±0.81,1.49±0.92,3.10±0.89, respectively. The mean log HBeAg level of group IV was significantly higher than that of the other groups. the best cut-off value of HBeAg in differentiating group IV from the other groups was 750 S/CO with a sensitivity of 97.3% and a specificity of 98.2%. Conclusions: Quantitative measurement of HBeAg titres may be an easy and economical reference for HBV replication in HBV carriers. Mod-005#102 Cholesterol-lowering probiotics combined with anthraquinone of cassia ameliorate non-alcoholic fatty liver in diet-induced rats through gut-liver axis Lu Mei(1,2), Zhi-Qiang Liu(3),You-Cai Tang(1), Ping-Chang Yang(4),Jie-Li Yuan(2*), Peng-Yuan Zheng(1*) 1. Department of Gastroenterology, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou,China 2. Department of Microecology, School of Basic Medical Science, Dalian Medical University, Dalian, China 3. Longgang Central Hospital, ENT Hospital, Shenzhen ENT Institute, Shenzhen, China 4. Department of Pathology&Molecular Medicine, McMaster University, Hamilton, Ontario, Canada Background and Aims: Non-alcoholic fatty liver disease (NAFLD) is becoming a common liver disease in recent decades in the world. Probiotics and Chinese herb medicine may be promising therapeutic approaches. The present study aims to investigate how efficient the total anthraquinone from cassiae together with efficient cholesterol-lowering probiotics could improve high-fat diet (HFD)-induced NAFLD rat model and explore the underlying mechanism as well. Methods: Cholesterol-lowering probiotics were screened out by MRS-cholesterol broth with ammonium ferric sulfate method in vitro test. Then the experiment employed male Sprague–Dawley (SD) rats which fed on HFD for 150 consecutive days to build NAFLD animal models. Rats were intragastric administrated with total anthraquinone from cassiae(TAC) and/or cholesterol-

lowering probiotics (P). After conducting the last intragastric administration, the lipid metabolism, expression of fat synthesis genes in rat livers and the diversity of intestinal flora were evaluated. Results: Successfully screened out two cholesterol-lowering probiotics used in experiments. Compared with the NAFLD rats control, after the administration of probiotics and total anthraquinone from cassia, the indicators in rat blood serum of total cholesterol (TC) level, triglyceride (TG) level, free fatty acid (FFA) level and low density lipoprotein (LDL) level decreased noticeably while high density lipoprotein (HDL) level increased greatly. Besides, cholesterol 7a-hydroxylase (CYP7A1) low density lipoprotein receptor (LDL-R) in the liver and farnesoid X receptor (FXR) in the gut was up-regulated while the expression of 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGCR) in the liver reduced (P < 0.05). And accordingly, the expression of peroxisome proliferator activated receptor (PPAR)-α protein in the liver increased significantly while the expression of PPAR-γ and sterol regulatory element binding protein-1c (SREBP-1c) was down regulated (P < 0.05). In addition, compared with HFD rats, in TAC group, P group and TAC+P group, the expression of intestinal tight junction protein occludin and zo-1 in the NAFLD rats were all up regulated (P < 0.05). What’s more, an altered intestinal flora diversity was observed after the treatment of probiotics and total anthraquinone from cassiae. Conclusions: Cholesterol-lowering probiotics combined with total anthraquinone from cassiae have a therapeutic effects for non-alcoholic fatty liver disease in rats by up-regulating CYP7A1,LDL-R,FXR mRNA and PPAR-α protein produced in the process of fat metabolism while down-regulating the expression of HMGCR mRNA, PPAR-γ and SREBP-1c protein, also through normalizing the intestinal dysbiosis and improve the intestinal mucosal barrier function. Mod-006#107 Changes of the Gut Microbiome in Acute Liver Failure Pigs Yimin Zhang(1,2), Ning Zhou (1,2), Jianzhou Li(1,2), Juan Lu(1,2), Ermei Chen(1,2), Jie Wang(1,2), Zhongyang Xie(1,2), Lanjuan Li(1,2) 1.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Department of Infectious Diseases, First Affiliated Hospital, School of Medicine,Zhejiang University, Hangzhou, Zhejiang 310003, P. R. of China 2.Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, Zhejiang 310003, P. R. of China Aim：To manifest the changes of gut microbiome in acute liver failure pigs. Methods: Acute liver failure was induced with D-galactosamine in experimental pigs. The general conditions of pigs were observed while haematological and biochemical parameters of pigs were tested to confirm the liver failure. The faecal samples were collected before D-galactosamine administration and the situation of liver failure. Denaturing gradient gel electrophoresis (DGGE) was used to manifest the microbiome changes in two situations. Results: The acute liver failure pig model was successfully established. After drug administration, appetite decrease and drinking reduction were manifested in all pigs following with ataxia, somnolence, and coma. All pigs showed marked increases in prothrombin time and a increase in levels of liver enzymes, bilirubin and ammonia. The DGGE results showed that the intestinal microbial diversity increased in liver failure situation. Homogeneous analysis showed uniqueness and stability of predominant fecal microbiome composition in both situations. The gut microbiome in both situations can be distinguished differentiated by MDS and PCA analysis. Conclusions: The microbiome of pigs are significantly different between the situation of liver failure and the healthy situation. Specific DGGE profile may be a powerful tool for diagnosis of liver failure. Mod-008#145 eGUT: Predictive Tool to Understand Gut Microbiota and Host Interactions Robert Clegg (1,2), Jan-Ulrich Kreft (1,2) 1. Centre for Systems Biology, School of Biosciences, University of Birmingham, UK 2. Institute of Microbiology and Infection, University of Birmingham, UK There has been an explosion of research into the gut microbiota, driven by developments in experimental technology and the realisation of the profound effects the microbiota has on the host. As awareness increases, animal experiments where the diet or microbiota are manipulated may rise markedly, making efforts to replace such experiments with complementary predictive tools urgent. Performing in silico experiments using computational tools has advantages over animal studies: the conditions are fully controlled, the consequences of assumptions and hypotheses concerning the microbiota can be studied, and investigations can be performed that may not be possible due to ethical, technical or resource constraints. We are in the process of constructing eGUT (Electronic Gut), a predictive simulation of gut microbiota and host interactions. Initially, eGUT may only reduce animal studies, but in the long-term eGUT may ultimately replace these experiments in studies similar to those previously validated. We have adopted an individual-based modelling approach that allows us to capture activities of individual microbes of various species, to determine how microbial and environment dynamics emerge from interactions between the host and microbiota. Through modelling a hierarchical structure of compartments representing host organs (Figure), we can capture exchange of metabolites, signals, and microbes, to determine how this is affected by mucosal activities including nutrient uptake, excretion of digestive enzymes and mucin, and effects of probiotics and prebiotics. Simulated behaviour will be validated against that observed in the laboratory.

eGUT will be a freely available platform that researchers can use to design and run virtual experiments without the need for complex mathematics or programming skills. We welcome collaboration to inform the initial design of eGUT and feeding future iterations, ensuring the creation of a tool fulfilling our objective of reducing animal experiments and improving science.

#187 Short Talk A microfluidics-based co-culture platform to systematically investigate host-microbe molecular interactions Pranjul Shah (1), Joëlle V. Fritz (1), Mahesh Desai (1), Enrico Glaab (1), Matthew Estes (2), Frederic Zenhausern (2), Paul Wilmes (1) 1. Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Luxembourg 2. Center for Applied Nanobioscience and Medicine, University of Arizona, USA There is growing consensus that the human microbiome plays an active role in modulating human health and disease. High-throughput multi-omic analyses have revealed that altered microbiomes, primarily along the gastrointestinal tract, are hallmarks of a range of idiopathic medical conditions including diabetes, cancer and inflammatory disorders. Such studies have generated important hypotheses regarding disease causation which now require experimental testing. The lack of representative in vitro models allowing the systematic manipulation of variables to test the multitude of hypotheses, which arise from high-throughput molecular studies, represents a major bottleneck in the field. Microfluidic cell culture approaches offer exciting prospects for first-pass experiments aimed at proving cause-and-effect relationships prior to further hypothesis testing in animal models. We have developed a modular microfluidics-based co-culture model (HuMiX) which allows systematic multi-omic investigations of human-microbial molecular interactions. The model consists of three microchambers: (1) a medium perfusion chamber, (2) a human epithelial cell culture chamber, and (3) a microbial culture chamber. Through perfusion of dedicated culture media through the respective chambers, representative oxygen gradients are established. In particular, human cells are cultured aerobically whereas microorganisms are exposed to anaerobic conditions. Molecular interactions are guaranteed between both cell contingents through separatory semi-permeable membranes. Finally, the model allows monitoring of important parameters including concentration of metabolite, cytokine, oxygen and tight junctions. We present proof-of-concept results demonstrating the representative co-culture of polarized epithelial cells (Caco-2) and anaerobically growing Lactobacillus rhamnosus GG (LGG) followed by systematic multi-omic (transcriptomics, metabolomics) analyses of the effects of the induced co-culture on human cell physiology. Our transcriptomic results reflect gene expression data from human clinical trials with LGG which provide validation of our approach to mimic host-microbe interactions occurring in the human gut. Furthermore, a differential transcriptomic analysis comparing LGG co-culture under aerobic and anaerobic conditions, respectively, provides unique insights into the anaerobic microbial metabolism-inducible effects on human epithelial

cells. In particular, metabolomic analyses demonstrate elevated levels of TCA cycle metabolites (fumarate, isocitrate and citrate), in line with previously described findings in conventionalised animals. Additionally, we have discovered a novel metabolite produced under co-culture conditions which highlights the ability of HuMiX to provide novel insights into the complex dynamics of human-microbial crosstalk. In summary, the HuMiX model provides an important research tool for investigating the molecular mechanisms of host-microbe interactions. Mod-009#193 Glycerol supplementation enhances Lactobacillus reuteri’s protective effect against Salmonella Typhimurium colonization in a 3-D model of colonic epithelium Rosemarie De Weirdt (1), Aurélie Crabbé (2), Stefan Roos (3), Sabine Vollenweider (4,5), Christophe Lacroix (4), Jan Peter van Pijkeren (6), Robert A. Britton (6), Shameema Sarker (2), Tom Van de Wiele (1), Cheryl A. Nickerson (2) 1. Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Ghent, Belgium 2. Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, USA 3. Department of Microbiology, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden 4. Institute of Food, Nutrition and Health, ETH, Zürich, Switzerland 5. Current address: Flavour Science & Technology, Natural Flavour Ingredients, Givaudan Schweiz AG, Dübendorf, Switzerland 6. Department of Microbiology & Molecular Genetics, Michigan State University, East Lansing, USA The probiotic effects of L. reuteri have been speculated to partly depend on its capacity to produce the antimicrobial substance reuterin during the reduction of glycerol in the gut. In this study, the potential of this process to protect human intestinal epithelial cells against infection with Salmonella enterica serovar Typhimurium was investigated. We used a three-dimensional (3-D) organotypic model of human colonic epithelium that was previously validated and applied to study interactions between S. Typhimurium and the intestinal epithelium that lead to enteric salmonellosis. The model was expanded with L. reuteri supernatant or a live L. reuteri population according to the set-up depicted in Figure 1. We show that L. reuteri protects the intestinal cells against the early stages of Salmonella infection and that this effect is significantly increased when L. reuteri is stimulated to produce reuterin from glycerol. More specifically, the reuterin-containing ferment of L. reuteri caused a reduction in Salmonella adherence and invasion (1 log unit), and intracellular survival (2 log units). In contrast, the L. reuteri ferment without reuterin stimulated growth of the intracellular Salmonella population with 1 log unit. The short-term exposure to reuterin or the reuterin-containing ferment had no observed negative impact on intestinal epithelial cell health. However, long-term exposure (24h) induced a complete loss of cell-cell contact within the epithelial aggregates and compromised cell viability. Collectively, these results shed light on a potential role for reuterin in inhibiting Salmonella-induced intestinal infections and may support the combined application of glycerol and L. reuteri. While future in vitro and in vivo studies of reuterin on intestinal health should fine-tune our understanding of the mechanistic effects, in particular in the presence of a complex gut microbiota, this is the first report of a reuterin effect on the enteric infection process in any mammalian cell type. Note that the successful incorporation of high densities of a commensal gut bacterium in the 3-D model was a first and crucial step in the development of an integrated in vivo-like model of intestinal epithelium to study complex host-microbiota interactions.

Mod-010#217 The intricate relationships of bifidobacterial communities within the mammalian’s gut microbiota Francesca Turroni (1), Sabrina Duranti (1), Marta Mangifesta (1), Christian Milani (1), Alice Viappiani (1), Gabriele Andrea Lugli (1), Laura Gioiosa (2), Paola Palanza (2), Douwe van Sinderen (3) and Marco Ventura (1) 1. Laboratory of Probiogenomics, Department of Life Sciences, University of Parma, Italy; 2. Dipartimento di Biologia Evolutiva e Funzionale, Universita’ degli Studi di Parma; 3. Alimentary Pharmabiotic Centre and Department of Microbiology, Bioscience Institute, National University of Ireland, Western Road, Cork, Ireland The levels of cooperation and competition between microorganisms are poorly investigated for particular components of the gut microbiota. In order to obtain insights into the manner by which different bifidobacterial species can coexist in the mammalian gut, we studied the interaction between the human gut commensals Bifidobacterium bifidum PRL2010, Bifidobacterium adolescentis 22L, Bifidobacterium breve 12L and Bifidobacterium longum subsp. infantis ATCC15697 in the intestine of conventional mice. Simultaneous whole genome transcription profiling coupled with metagenomics analyses of single, dual or multiple-associations of these strains revealed an expansion of the mice gut glycobiome toward dietary as well as host-glycans polysaccharides. Furthermore, these simplified bifidobacterial communities evoked major changes in the host metabolomics profiles consisting in shifts in SCFAs and carbohydrate composition in the mice cecum. Thus, revealing the ecological strategies followed by bifidobacteria to colonize and persist in the mammalian gut. Mod-011#229 Systems biology of a defined, simplified gut microbiome Greg Medlock (1), Matthew Biggs (1), Glynis Kolling (2), Jonathan Swann (3), Martin Wu (4), Jason Papin (1), Richard Guerrant (2) 1. Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA 2. Department of Internal Medicine/Division of Infectious Diseases, University of Virginia, Charlottesville, VA, USA 3. Food Microbial Sciences Unit, School of Chemistry, Food and Pharmacy, University of Reading, Reading, United Kingdom 4. Department of Biology, University of Virginia, Charlottesville, VA, USA The gut microbiome is an immensely complex community of microbes that is integrally associated with host health in part through metabolic interactions. Computational models can integrate complex interspecies and host-microbe interactions and enable useful therapeutic predictions. Building and understanding a system that is smaller and less complex than an in vivo gut microbiome may help guide the development of clinically relevant models with predictive capabilities. For this purpose, we present a simplified, defined microbial community consisting of gut-derived bacteria. Genome-scale metabolic network reconstructions have been drafted for these species using an automated method that leverages the phylogenetic relationship between species to create maximally parsimonious reconstructions. We are contextualizing and analyzing the community using growth data, metabolomics, and transcriptomics in combination with these reconstructions. We hope to enhance our ability to explore host-microbe interactions by understanding interspecies interactions in this well-defined community. Mod-012#253 Phenotypic differentiation of gastrointestinal microbes is reflected in their metabolic repertoire Eugen Bauer (1), Cedric Christian Laczny (1), Stefania Magnusdottir (1), Paul Wilmes (1) and Ines Thiele (1) 1. University of Luxembourg, Luxembourg Centre for Systems Biomedicine, L-4362 Esch-sur-Alzette, Luxembourg The human gastrointestinal tract harbors a diverse microbial community, in which metabolic phenotypes play important roles in health/disease of the human host. Recent developments in meta-omics attempt to unravel metabolic roles of individual microbes/microbial populations by linking genotype with phenotype. This connection, however, still remains poorly understood with respect to its evolutionary and ecological context. Here, we automatically reconstructed and refined genome scale metabolic models of 301 representative intestinal microbial organisms in silico. We applied a combination of unsupervised machine learning and systems biology techniques to study individual and global differences on the genomic and metabolic level. Based on the global metabolic differences, we found that energy metabolism and membrane synthesis play important roles in delineating different taxonomic groups in the gastrointestinal tract. Furthermore, we found an exponential relationship between metabolic capabilities and phylogenetic distance, meaning that closely related microbes can exhibit pronounced differences with respect to their metabolic traits while at a certain phylogenetic distance only marginal metabolic differences can be observed. This finding was further substantiated by the metabolic divergence within different genera. In particular, we could distinguish three sub-type clusters within the Lactobacilli as well as two clusters within the Bifidobacteria and Bacteroides. The differences between those subclusters could be explained by differing pathways for energy metabolism and membrane synthesis. Based on our results, we demonstrate that phenotypic differentiation within closely related species may be explained by the metabolic

potential/activity of those microbes rather than their phylogenetic relationships. These results have important implications in our understanding of the ecological and evolutionary complexity of the human gastrointestinal microbiome. Mod-013#259 Fate of arabinoxylan and beta-glucan during simulated digestion by human duodenal and stomach fluids and during in vitro fermentation with human faecal slurry Ida Rud (1), Anne Berit Samuelsen (2), Birgitte Moen (1), Anne Rieder (1), Ingrid Måge (1), Gerd Vegarud (3), Svein Halvor Knutsen (1) 1. Nofima, The Norwegian Institute of Food, Fisheries and Aquaculture, Aas, Norway 2. School of Pharmacy; University of Oslo, Oslo, Norway 3. Norwegian University of Life Sciences, Aas, Norway Beta-glucans (BG) and arabinoxylans (AX) are typical fibre constituents found in common cereals such as oat and barley. Their contribution to health is related to their abilities to increase viscosity in the small intestine as well as being substrates for the microbiota in the large intestine. Starch free isolated polysaccharides were studied in two different in vitro systems respectively representing the upper and lower human gastrointestinal tract. It was initially shown that AX and BG were not depolymerized during a simulated digestion based on sequential treatment with human saliva, gastric and duodenal juice. In subsequent designed batch fermentation, the inoculum was based on mixed human faecal materials from at least 6 different healthy subjects and the cereal fibres were used as the sole carbohydrate substrate. The cereal fibres were either run individually or as mixtures with different ratios. Fermentations were followed for 24h by analysing the remaining polymeric carbohydrate, pH reduction, production of gas and short chain fatty acids (SCFA) and analysing the microbiota composition (16S rRNA gene deep sequencing). Furthermore two specific chromatographic approaches were used to trace the specific fate of BG in the complex mixtures. During the first 8h of fermentation, a faster fermentation rate of pure AX compared to pure BG was evident. However, after 24h BG was fermented to an almost comparable degree. Interestingly, higher amounts of butyric acid were produced after 24h with BG compared to AX, which might be correlated to the increase of Roseburia (known butyrate-producing bacteria) observed with BG. Both fermentation of AX and BG were associated with increase of Bacteroides. Fermentation of the AX-BG mixtures resulted in a BG/AX ratio-dependent response on the fermentation parameters measured. Mod-014#261 Circadian rhythm and stability of salivary microbial communities Lena Takayasu(1), Wataru Suda(1), Tageyasu Takanashi(1), Erica Iioka(1), Misa Kiuchi(1), Rina Kurokawa(1), Chie Shindo(1), Yasue Hattori(1), Naoko Yamashita(1), Sangwan Kim(1), Kenshiro Oshima(1), Suguru Nishijima(1), Misako Takayasu(2), Hideki Takayasu(3 1. Center for Omics and Bioinformatics, Graduate School of Frontier Sciences, University of Tokyo, Japan 2. Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Japan 3. Sony Computer Science Laboratories, Inc., Japan It is known that the fluctuation of human microbiota is deeply associated with host physiological conditions and vice versa. Quantification of microbiota fluctuations will not only deepen our understanding of bacterial ecosystem traits, but also make it possible to predict microbial composition change against perturbation. We collected time series DNA data of human saliva for many subjects by 16S rRNA analysis. We elucidated the circadian rhythm of the saliva microbiota and the oxygen concentration is indicated to play an important role on it. We also observed fluctuation of population share for each bacterial species both for time series of individual subjects and for inter-individual fluctuation. It is found that histograms of fluctuation ratios of population share follow long tail distributions approximated by power laws. This result indicates that the magnitude of fluctuation of population share is small for majority of cases, however, there is a non-negligible probability of huge fluctuations, and such stability properties can be characterized by the power law exponent of the distribution. We confirmed that temporal fluctuation distributions of saliva microbiota are quite similar among individuals. We introduced a mathematical model of human microbiota ecosystem and compared resulting fluctuation ratio distributions with those of real ones, discussing about the origin of the power law.

Mod-015#272 Systematic analysis of the metabolic interactions in a model gut microbiota community and its impact on host metabolism Almut Heinken (1), Ines Thiele (1) 1. Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Belval, Luxembourg Genome-scale metabolic reconstructions (GENREs) represent a knowledge base of the reconstructed organism and are powerful tool for contextualizing high-throughput data. Here, we present the first genome-scale model of a gut microbe community, consisting of 11 manually curated and validated gut microbe reconstructions spanning three phyla. The model was joined with the human host to systematically predict host-microbe and microbe-microbe interactions in silico. The model gut community was joined with a manually curated reconstruction of a global human metabolism, Recon2 (1). We then systematically explored the effects of the different microbes on host metabolic tasks while simulating four different dietary regimes. A variety of human body fluid metabolites were influenced by microbial presence, including many that have been measured in vivo and found to be affected by the microbiota. The secretion of amino acid derivatives including hormones by the host was increased by up to 100-fold in the presence of the microbes, in agreement with the role of the microbiota as an additional organ. When comparing two five-member model communities, the more diverse group had a significantly more pronounced effect on host metabolism. The microbes further provided a variety of metabolic products relevant for host health, e.g., energy sources, vitamins, and potentially toxic metabolites. Specific microbes combined with dietary fatty acid supply were able to increase host production of pro-inflammatory leukotrienes as well as increased production of taurine-containing bile acids. Moreover, the 11 microbes were joined pairwise in all possible combinations with and without a reconstruction of the small intestine enterocyte (2) as background. A variety of interactions including commensalism, mutualism and competition was predicted depending on the nutrient environment. Notably, anoxic conditions promoted mutualistic behavior in pairs including the lactic acid bacterium L. plantarum. The availability of oxygen abolished these syntrophic interactions, resulting in competition. Recurring cross-feeding patterns between microbes were identified, providing insight into the mechanisms behind the observed microbe-microbe interactions on a molecular level. Taken together, we present a comprehensive computational modeling approach to investigate systematically host-microbiome and microbe-microbe interactions. The framework can readily incorporate any number of microbes and should prove useful in future efforts to elucidate the metabolism of the complex ecosystem in the human gut and its effects on human health. 1. I. Thiele et al., Nat Biotechnol 31, 419-425 (2013). 2. S. Sahoo et al., Human molecular genetics 22, 2705-2722 (2013). Mod-016#278 Mouse model for studying microbiota and metabolic changes induced by different diets Peter Falck, Eva Nordberg Karlsson, Patrick Adlercreutz. Biotechnology Department of Chemistry, Lund University, Lund, Sweden An imbalanced gut microbiota can increase the risk of obesity, diabetes, coronary heart disease, hypertension and gastrointestinal disorders such as colorectal cancer. Certain bacteria belonging to bifidobacteria or lactobacilli can improve the gut microbiota balance towards homeostasis and are used as markers for a healthy microbiota. Their presence limits the growth and activity of disease-causing bacteria and can reduce inflammation. Using a mouse as a model system it is possible to see how the diet impacts the composition of the microbiota and specifically on certain bacterial genus related to health or disease. A C57BL/6 mouse on a high-fat diet was used in this particular study as a model for obesity and insulin resistance associated with microbial dysbiosis. The aim of this study was to observe the impact of different cereal diets rich in fibers on the microbial composition and metabolic products related to health. It was specifically aimed at increasing the number of bifidobacteria and lactobacilli together with short-chain fatty acids produced from microbial fermentation. Using this model system it was possible to see changes in the microbial population with diets rich in soluble fibers, containing mainly poly- and oligosaccharides, with indications of improved metabolic functions compared with the high-fat diet control. Soluble fibers from Rye increased the number of bifidobacteria with indications of improved liver function. Oat derived products caused an increase in lactobacilli and propionic and butyric acid associated with beneficial metabolic effects. Akkermansia muciniphila, known to enhance the gut integrity through increased mucin production, was increased with diets supplemented with Guar gum. A main conclusion was that soluble fibers from different sources have different impact on the gut microbiota and important metabolic markers (Berger et al., 2014). Mouse model systems are important for studies of the gut microbiota interactions with the host and the impact of different diets on the composition and metabolic activity. Using a mouse model system it is possible to learn more how the diet impacts the microbiota for improving health through dietary interventions. Reference Berger, K., Falck, P., Linninge, C., Nilsson, U., Axling, U., Grey, C., Stalbrand, H., Nordberg-Karlsson, E., Nyman, M., Holm, C., Adlercreutz, P. (2014). Cereal Byproducts Have Prebiotic Potential in Mice Fed a High-Fat Diet. Journal of Agricultural and Food Chemistry, 62(32), 8169-8178.

Mod-017#290 The Athlete Microbiome Project: The search for the golden microbiome. Lauren M Petersen (1), Erica Sodergren (1), Shana Leopold (1), Purva Vats (1), Benjamin Leopold (1), Blake Hanson (1), Lei Chen (1), Sai Lek (1), George M Weinstock (1) 1. The Jackson Laboratory for Genomic Medicine, 10 Discovery Drive, Farmington, CT 06030 USA. Recent advances in sequencing technology and bioinformatic analysis have led to a tremendous increase in understanding the human microbiome. Most studies have focused on identifying what a ‘healthy’ microbiome is by comparing the bacterial communities of diseased individuals to those of healthy individuals in order to understand how microbes influence human physiology, metabolism, nutrition, and immune function. However, few studies have looked at the microbiomes of highly fit athletes to answer questions regarding how the genetic and metabolic potential of the microbiome may play a pivotal role in performance during competition and recovery. In this study, The Athlete Microbiome Project, 23 highly fit cyclists, who race at or one level below the professional level, were recruited to provide stool and saliva samples to have their gut and oral microbiomes analyzed. By utilizing 16S rRNA gene sequencing, whole genome sequencing, RNA-Seq, and other cutting edge technologies, the goal is to characterize each athlete’s microbial community, identify genetic capabilities of those communities, measure gene expression patterns, and identify characteristics associated with extraordinary fitness. The overall aim of this project is to gain a better understanding of the complexity of host-microbe interactions that contribute to high levels of fitness and robust health. Sequencing and bioinformatic analysis is currently taking place and results of this study will be presented. Mod-018#294 Rapid and profound shifts in the vaginal microbiota Bryan T. Mayer (1), Sujatha Srinivasan (1), Tina L. Fiedler (1), Jeanne M. Marrazzo (3), David N. Fredricks (1,2,3,4), Joshua T. Schiffer (1,2,3) 1. Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America 2. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America 3. Department of Medicine, University of Washington, Seattle, Washington, United States of America 4. Department of Microbiology, University of Washington, Seattle, Washington, United States of America Bacterial vaginosis (BV) is a common polymicrobial disease associated with negative reproductive health outcomes and increased risk of HIV acquisition. BV is treatable with antibiotics but relapse is common. A more detailed understanding of vaginal microbiome dynamics is needed to identify conditions that favor establishment, maintenance and eradication of BV. We applied mathematical models to daily quantitative measurements of eleven key bacterial species during metronidazole treatment for 15 cases of BV. We identified complete reorganization of vaginal bacterial composition within a day of initiating therapy. Though baseline bacterial levels predicted longer time to clearance, all anaerobic species were eliminated with median clearance half-lives of less than 8 hours. However, re-emergence of BV-associated species was common following treatment cessation. Gardnerella vaginalis, a facultative anaerobe, was cleared more slowly than other BV-associated species and often rebounded during treatment. We observed gradual Lactobacillus spp. growth, indicating that untargeted microbes fill the transient vacuum formed during treatment. Under antibiotic pressure, we demonstrate that the human microbiome undergoes dramatic, rapid shifts. When treatment is stopped, BV-associated bacteria quickly re-emerge, suggesting a possible role for intermittent prophylactic treatment. Overall, these data suggest that polymicrobial environments can undergo massive reorganiazation over time frames of only hours. Mod-019#298 Analysis of the skin microbiota in house mice using genetic and evolutionary approaches Meriem Belheouane (1, 2), Yask Gupta (3), Saleh Ibrahim (3), John F. Baines (1, 2) 1. Institute for Experimental Medicine, Christian Albrechts University, Kiel, Germany 2. Max Planck Institute for Evolutionary Biology, Plön, Germany 3. Dermatology Department, Lübeck University, Lübeck, Germany Various factors including the environment and host genetics influence skin microbial community structure. However, the relative extent to which these forces shape skin microbial diversity and how they interact with each other remain unclear. We addressed these questions using three complementary approaches. First, we identified host genomic regions influencing variation in the abundances of skin bacterial taxa using quantitative trait locus (QTL) mapping in an advanced intercross between mouse strains. Second, we examined patterns of skin microbial diversity in 217 wild-caught individuals from different natural locations. Together, these studies reveal a strong influence of the environment in accounting for variation in community structure and indicate that gene-by-environment interactions likely play an important role in understanding the origins and consequences of variation in the skin microbiota. Finally, to gain more insight into possible co-evolutionary dynamics, we surveyed the skin

microbiota of several house mouse subspecies and related species and genera to test for patterns of “phylosymbiosis” in skin microbial communities. Mod-020#305 Pneumococcal vaccination and the nasopharyngeal microbiome: a longitudinal study in The Gambia Blake Hanson (1), Brenda Kwambana (2), Archibald Worwui (2), Yanjiao Zhou (3), Maze Bi Ndukum-Ndonwi (3), Ma Ansu Kinteh (2), Martin Antonio (2), Erica Sodergren (1), Ed Clark (2), Sulayman Bah (2), Ebou Bah (2), Kally Sanneh (2), Mohammed Kijera (2), Isa 1. The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA 2. Medical Research Council Unit, Fajara, The Gambia 3. Department of Pediatrics, Washington University School of Medicine, St Louis, MO, USA Streptococcus pneumonia is the most common cause of pneumonia in children under 5 worldwide, killing an estimated 1 million each year with the majority of deaths occurring in developing countries. To mitigate the morbidity and mortality of this disease, pneumococcal conjugate vaccine is administered. Most validation and investigative studies of these conjugate vaccines were performed in developed countries, and now national vaccine programs are being implemented throughout the world. In The Gambia, such a national program began several years ago and this study aims to determine the impact of the vaccine on a naïve population, in particular the effect on the nasopharyngeal microbiota. We conducted a longitudinal study, enrolling 102 newborns in The Gambia between December 1st, 2008 and January 31st, 2009 and following them for the first 12 months of life. Over the 12-month period we collected risk factor data and nasopharyngeal samples at 17 time points: every two weeks for the first six months and every two months for the last six months. Participants were broken down into three different vaccination groups: non-vaccinated infants in non-vaccinated villages (n=33), vaccinated infants in non-vaccinated villages (n=30), and vaccinated infants in vaccinated villages (n=39). Vaccinated infants received the pneumococcal conjugate vaccine 7 (PCV7), a pneumococcal conjugate vaccine that protects against 7 strains of Streptococcus pneumonia. Vaccinations associated with the study occurred at 8, 12, and 16 weeks, and many infants received additional doses of PCV7 after 6 months during a national vaccination campaign. Participant risk factor data was collected at 1640 time points, with nasopharyngeal samples collected at 1594. DNA was extracted from the nasopharyngeal samples and the V1-V3 region of the 16S gene was sequenced using the Roche 454 platform. With this rich dataset, we will describe the overall characteristics of the nasopharyngeal microbiome among our cohort, as well as describe the variability of the microbiome during the first year of life. We will assess how the PCV7 vaccination affects the composition of the microbiome, focusing on the entire community structure as well as the effect on individual bacterial taxa. Additionally, we will identify any associations between the collected risk factors and the bacterial diversity and community structure of the nasopharynx. Results of the project will be presented at the conference. Mod-021#307 A multiomics approach to study the effects of diet on the intestinal microbiome of pre-diabetic patients Shana Leopold (1), Wenyu Zhou (2), Daniel Spakowicz (1), Blake Hanson (1), Eddy Bautista (1), Erica Sodergren (1), Lauren Petersen (1), Purva Vats (1), Sai Lek (1), Lei Chen (1), Denis Salins (2), Brian Piening (2), George Weinstock (1), Michael Snyder (2 1. The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA 2. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA The Integrative Human Microbiome Project (iHMP, http://hmp2.org), the second phase of the NIH Human Microbiome Project, consists of three separate three-year longitudinal studies (inflammatory bowel disease, preterm birth and type 2 diabetes) exploring the host-microbiome interaction and microbiota changes at the onset of human disease. Our type 2 diabetes (T2D) longitudinal study will profile subjects who are at risk for T2D through different perturbations such as healthy vs. respiratory infection, the effects of over-feeding, and other dietary pertubations. We will analyze the host and several microbiomes (e.g., fecal, nasal, blood) by omics profiling (genomic, transciptomic, proteomic and metabolomic). Here we will focus on the over-feeding cohort of this study. This cohort consists of nine subjects who are pre-diabetic. Each subject undergoes a period of weight gain through increased, controlled caloric intake, followed by weight loss. Stool samples are collected at each of these time points (base, peak weight, weight loss), and 16S targeted sequencing, meta-whole genome sequencing, and RNA-Seq was performed on the extracted nucleic acids. A preliminary analysis shows a pattern of association of Bacteroidetes and Firmicutes abundance with insulin-sensitive and –resistant status.

Mod-022#339 Towards The Development Of An In Vitro Screening Platform For Upper Respiratory Tract Microbiota Bart Keijser (1), Hakim Rahaoui (1), Mariska Gröllers-Mulderij (1), Ingeborg Kooter (2), Heleen Wortelboer (1), Debby Bogaert (3), Roy Montijn (1) 1. TNO Microbiology and Systems Biology, Zeist, The Netherlands 2. TNO Applied Environmental Chemistry, Utrecht, The Netherlands 2. Department of Pediatric Immunology, UMC Utrecht, Utrecht, The Netherlands The upper respiratory tract is colonized by a diverse group of bacterial species, of which several potential respiratory pathogens. Recent work has indicated that in infants eight distinct nasopharyngeal microbiota profiles can be identified (1). Stable microbiota profiles seem marked by early presence and high abundance of Moraxella and Corynebacterium/Dolosigranulum and are positively associated with exclusive breastfeeding in the first months of life and with lower rates of parental-reported respiratory infections in the first years of life. Less stable profiles are marked by high abundance of Haemophilus or Streptococcus. To further examine the functional impact of the distinct microbiota community types, we have developed an in vitro multispecies cultivation protocol to study behavior of the upper respiratory microbial community. This model allows us to stabilize a representative multispecies microcosm at abundance representative for the in vivo state, and enables the testing of various compounds, including blood plasma, iron and human milk oligosaccharides. We found that exposure to selected compounds resulted in changes in the in vitro microbiota composition towards different stable state profiles reflecting one of the eight microbiota profiles found in vivo. We next tested the subsequent impact of different microbiota communities as well as selected species for their effects in a 3D human respiratory epithelial model. Bacterial suspensions were applied directly onto the epithelial cells for twenty-four hours followed by a challenge with copper oxide nanoparticles for another twenty-four hours, assessing cytotoxicity (LDH) and inflammatory markers (IL8). This showed that microbial colonization attenuated the pro inflammatory effects of the nanoparticles in a dose and species dependent manner. We hope the test model will allow functional and mechanistic insight in the contribution of commensal microbial species in resilience of the upper respiratory ecosystem and identification of strategies to improve respiratory health. 1) Am J Respir Crit Care Med. 2014 Dec 1;190(11):1283-92

STATUS OF CURRENT INTERVENTIONS FOR MICROBIOME DISEASES AND DISORDERS

Stat-001#125 Quantification of the butyryl-CoA: acetate-CoA transferase gene in the faecal microbiota of overweight and obese pregnant women at 16 weeks gestation Luisa.F Gomez-Arango (1,2), Mark Morrison (3), Alicia Kang (3), Helen Barret (1,2), Shelley Wilkinson (1), H.David McIntyre (1), Leonie K. Callaway (1,2) , Marloes Dekker Nitert (1) 1 School of Medicine, 2 UQCCR 3 Diamantina Institute The University of Queensland. St Lucia QLD Australia Women that are overweight or obese during pregnancy can experience adverse outcomes, some of which have been linked to alterations in the gut microbiome and dysbiosis. The short-chain fatty acid butyrate is produced by the gut microbiota and widely viewed as a biomarker of healthy gut function because of its effects on gut homeostasis, epigenetic regulation and pancreatic beta cell function. Here we compare the butyrate-producing capacity of intestinal bacteria and gut microbial profiles between 39 overweight (BMI 25-29.9 kg/m2), 61 obese (BMI 30-39.9 kg/m2) and 8 morbidly obese (>40 kg/m2) pregnant women at 16 weeks gestation from the SPRING cohort. Methods: Fecal microbiota profiles were assessed by rrs gene amplicon sequencing using the Illumina MiSeq system. The relative abundances of butyrate producers from Clostridium clusters IV (Faecalibacterium prausnitzii) and XIVa (Eubacterium/Roseburia spp.) were compared. Quantification of the gene encoding butyryl-CoA:acetate CoA-transferase (BCoATscr) was assessed by quantitative PCR. Dietary intake of protein, dairy, and fiber was stratified by tertiles and correlated to the BCoATscr gene copies determined for the stool sample from each subject. Results: The BCoATscr copy number was variable in pregnant women (range 1.48 x 105 to 6.68 x 106 copies/15 ng DNA) and could not be correlated with BMI category, Gestational Diabetes Mellitus (GDM) status (16/108), protein, or dairy intake. Although the relative abundances of the known butyrate producers from Clostridium clusters IV and XIVa were positively associated with BCoATscr copy number (Spearman’s rho 0.221, p=0.022) there was also a trend for a negative correlation between total fiber intake and BCoATscr copy number (Pearson’s r= -0.188, P= 0.052). The Firmicutes:Bacteroidetes ratio (F:B) was 4:1 and interestingly, while the F:B ratio was not correlated to maternal BMI, there was a positive correlation with infant birth weight (Spearman’s rho 0.222, P=0.031). Additionally, there was no significant alteration in F:B observed for women who later developed GDM (2.4 ± 0.4:1 vs. 4.2 ± 0.7:1, P=0.37). Conclusion: These preliminary studies suggest that the butyrate producing capacity of overweight and obese women at 16 weeks gestation appears to be independent of their BMI, and not different in women who did or did not develop GDM. However, the F:B at 16 weeks gestation does appear to be positively associated with infant birth weight, suggesting a role for the maternal gut microbiota in affecting fetal growth Stat-002#33 The gut microbiota and the liver diseases Zhang Xiaoqian, Li Lanjuan State Key Laboratory for Diagnosis and Treatment of Infectious Disease, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou 310003, China With increasing evidences for the microbiota in the precipitation of infectious and non-infectious complications of liver diseases, the role of the gut microbiota in the disease of the liver has been estimated. Many studies of hypothesis of the role for our microbial flora in health and disease has been reported since the early years of the 20th century. On the basis of the acknowledge of paramount importance of the gut microbiota in the development and regulation of human homeostasis, numerous treatment such as prebiotics, probiotics, non-absorbable antimicrobial agents has been proposed to deal with the hepatic disorders. In this review, we describe the role of the microbiota in some of these disorders, such as non-alcoholic fatty liver disease (including obesity), non-alcoholic steatohepatitis, alcoholic liver disease, and liver cirrhosis and its complications and refine these related treatment options thus to improve their efficacy in the next few years. Furthermore, we proposed a novel model in which gut microbiota are co-cultured with microencapsulated hepatocytes often used in the bioartificial liver support system that could imitate the 3D culture model in order to exactly show the liver function in vivo. In this model we hope to test the candidated prebiotics or probiotics that have a positive effect on the hepatocytes in vitro. This conceived model, however, haven’t been experimented further work will be need done.

Stat-003#37 Characterisation of Novel Biomarkers in HBV-related Acute-on-chronic Liver Failure and Their Predictive Value for Mortality Qian Zhou（1）, Jiaojiao Xin（1）, Wenchao Ding（2）, Shaorui Hao（1）, Longyan Jiang（1）, Dongyan Shi（1）,Hongcui Cao（1）, Lanjuan Li（1）, Jun Li（1） 1.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,The First Affiliated Hospital, Zhejiang University School of Medicine. 79 Qingchun Rd., Hangzhou, China. 2. Systems Biology Division, Zhejiang-California International Nanosystems Institute,Zhejiang University. Background Hepatitis B-related acute-on-chronic liver failure (HBV-ACLF) has a high mortality rate in the Asia-Pacific region, and the mechanisms of its development and progression remain unclear. Objective Searching for new biomarkers to diagnose and predict the severity and early treatment. Novel serological biomarkers of HBV-ACLF were screened by cytokine antibody array. Significant biomarkers were then confirmed by enzyme-linked immunosorbent assay (ELISA) in 440 HBV-ACLF patients in the experimental group and an external validation group. Results The initial screen of an antibody array showed that 15 cytokines were significantly differentially expressed in patients with HBV-ACLF and chronic hepatitis B (CHB). Six of these cytokines, including hepatocyte growth factor (HGF), macrophage inflammatory protein 3α(MIP-3 alpha), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), growth differentiation factor 15 (GDF15), E-selectin and osteopontin, were significantly increased in HBV-ACLF subjects compared to CHB subjects. These results were confirmed by ELISA in 304 HBV-ACLF patients, 40 CHB patients and 20 normal adults. Moreover, high HGF and GDF15 expression levels could distinguish the HBV-ACLF and CHB patients, and 116 CHB patients could be accurately distinguished from 304 HBV-ACLF patients. The MIP-3 alpha level was closely related to the mortality of HBV-ACLF patients, as confirmed using the external validation group. Immunohistochemistry showed that HGF, GDF15 and MIP-3 alpha were positive in HBV-ACLF-derived liver tissues and negative in CHB-derived and normal liver tissues. Conclusions HGF and GDF15 represent potential novel biomarkers for the early diagnosis of HBV-ACLF, and MIP-3 alpha might be useful to predict the mortality of HBV-ACLF. Stat-004#43 Liver disease and intestinal flora Chenxia Hu(1),LanJuan Li(2) Collaborative innovation center for diagnosis and treatment of infectious diseases；State Key Laboratory for Diagnosis and Treatment of Infectious Diseases; School of Medicine; First Affiliated Hospital; Zhejiang University; Hangzhou, Zhejiang, PR China Gut barrier is consist of intestinal tract normal bacteria flora, mucous layer, epithelial cell layer, immune system, gut-liver axis and defensins and intestinal ecosystem contains trillions of microorganisms including bacteria, archaea, yeasts and viruses. Obligate anaerobic bacteria is the major kind of intestinal flora which can protect intestinal and maintain homeostasis. In various severe liver diseases, the number, composition, location of probiotics will change and the homeostasis are easy to be damaged, this can be attributed to that gut and liver are connected by the portal venous system, which makes the liver more vulnerable to translocation of bacteria, bacterial products, endotoxins or secreted cytokines. The direct cause of death in severe hepatopathy is always infection, which can result in severer endoxemia and systemic inflammatory response syndrome (SIRS), accompany with overgrowth of bacteria, drug-resistance bacteria plantation and bacterial translocation. Gram negative bacilli increased but bifidobacterium and other probiotics decreased, then intestinal dysbacteriosis aggravate hepatic function disorders, the two process can be mutually promoted and result in deterioration of liver disease. Generally hepatopath with intestinal microecology disorder is treated by narrow-spectrum antibiotic, probiotic products, prokinetic agent, microelement, antioxidant and other drugs which can regulate intestinal microecology. In this part, probiotic products can be used to improve hepatic function because of their engraftment antagonism, inhibition of pathogenic bacteria and trophic action. The products in clinical usage are probiotics, prebiotics and synbiotics. In this review, we list various liver diseases such as liver cirrhosis, liver failure, the products are nonalcoholic fatty liver disease, alcoholic liver disease, hepatic encephalopathy and liver tumor, which are associated with intestinal dysbacteriosis. We summarized researchs in recent years and try the best to demonstrate the potential mechanism of interaction between liver disease and intestinal flora, mechanism of how probiotic products repair liver damage, and treatment of probiotic products in each kind of liver disease.

Stat-005#48 Post-partum antibiotic treatment disturbs development of gut microbiota Anat Eck (1), Nicole Rutten (2), Maartje.Singendonk (3), Ger Rijkers (4), Clemens Meijssen (5), Clarissa Crijns (6), Annemarie Oudshoorn (7), Arine Vlieger (2), Paul Savelkoul (1), Dries Budding (1) 1. Department of Medical Microbiology and Infection Control, VU University medical center, Amsterdam, the Netherlands 2. Department of Pediatrics, St Antonius Hospital, Nieuwegein, the Netherlands 3. Department of Pediatric Gastroenterology and Nutrition, Emma Children’s Hospital/AMC, Amsterdam, the Netherlands 4. Department of Sciences, University College Roosevelt Academy, Middelburg 5. Department of Pediatrics, Meander Medical Center, Amersfoort, the Netherlands 6. Department of Pediatrics, Tergooi Hospital, Blaricum/Hilversum, the Netherlands 7. Department of Pediatrics, Gelre Hospitals, Apeldoorn, the Netherlands. Objectives: The growing recognition that intestinal microbiota may contribute fundamentally to childhood development and immunity is creating an impetus to understand the dynamics that lead to colonization of intestinal microbiota in infants. Evidence is growing that administration of antibiotics peripartum can lead to the development of an aberrant microbiota composition. However, studies on the effects of antibiotics on the establishment and recovery of the gut microbiota of infants are limited. The aim of this study was to determine the impact of antibiotic use in the first week of life on microbial colonization in a well-defined homogenous group of infants. Methods: Forty-five newborns, of which 21 who received antibiotics in the first week of life (AB) and 24 healthy controls, were sampled at three time points: T1, one week; T2, one month; T3, three months. All infants were vaginally term-born and exclusively breastfed infants. We used IS-pro, a high-throughput PCR-based profiling technique that was specifically optimized for the complex microbiota of the human intestinal tract. IS-pro combines bacterial species differentiation by the length of the 16S-23S rDNA interspace region with instant taxonomic classification by phylum specific fluorescent labeling. Stability was calculated as within-individual between- time points cosine distances. Results: Infants were clustered into two distinct sub-populations based on T1 sample, Bacteroidetes-dominant (subgroup B) or Firmicutes-dominant (subgroup F) microbiota, regardless of their treatment group. We observed a significantly reduced Bacteroidetes abundance and diversity at all time points in AB Infants of subgroup B. A delayed Bacteroidetes colonization was observed over time in AB infants of subgroup F. Escherichia coli, selected beforehand as a marker specie for antibiotics effect, was more prevalent at T1 and T3 and its colonization was more stable over time in control individuals compared to the AB group, in which it was detected in consecutive time points in fewer individuals. AB infants had a significantly less stable microbial composition when followed over time compared to controls, reflected by increased intra-individual cosine distances in AB infants. Conclusions: We identified significant effects of antibiotic treatment on the development of the infant gut microbiome. Our findings show a less diverse and less robust, and therefore less stable, microbiota in antibiotic treated infants when compared to healthy controls. We conclude that post-partum antibiotics administration disturbs the microbiota development in infants, mostly pronounced in species of the Bacteroidetes phylum. As the long-term health implications of these effects remain unknown, long-term follow-up studies are warranted. Stat-006#54 Dietary modulation of gut microbiota alleviates human genetic obesity Chenhong Zhang(1), Aihua Yin(2), Hongde Li(3), Ruirui Wang(1), Guojun Wu(1), Jian Shen(1), Menhui Zhang(1), Linghua Wang(1), Yaping Hou(2), Haimei Ouyang(2), Yan Zhang(2), Yinan Zheng(2), Jicheng Wang(2), Xiaofei Lv(2), Yulan Wang(3), Feng Zhang(1), Feiya 1. State Key Laboratory of Microbial Metabolism and Ministry of Education Key Laboratory of Systems Biomedicine, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China 2. Medical Genetic Centre and Maternal and Children Metabolic-Genetic Key Laboratory, Guangdong Women and Children Hospital, Guangzhou, Guangdong 510010, China 3. CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, China Gut microbiota has been causatively linked with diet-induced obesity; however, its role in genetically predisposed obesity remains elusive. To tackle the question of whether dysbiosis of the gut microbiota contributes to metabolic deteriorations in human genetic obesity, we recruited children morbidly obese with PWS and simple obesity (SO) for a hospitalized intervention study to assess the effect of the WTP diet (14) on composition of the gut microbiota and host metabolic parameters. By using a top-down, systems strategy to combine metagenomic characterization of the gut microbiota, metabonomic profiling of co-metabolites between host and gut bacteria, and transplantation of human gut microbiota to germfree mice, we demonstrate a significant contribution of dysbiotic gut microbiota to the metabolic deteriorations associated with genetically predisposed obesity, as with simple obesity. Here we show that a diet rich in non-digestible carbohydrates induced structural changes of the gut microbiota and concomitant alleviation of metabolic deteriorations in children morbidly obese with Prader-Willi Syndrome. The diet-modulated gut microbiota caused reduced inflammation and fat accumulation when transplanted into germ-free mice. Co-abundance network analysis of 161 prevalent bacterial genomes de novo assembled from metagenomic datasets showed diet-induced enrichment of Bifidobacterium spp. Metabonomic profiling of urine metabolites identified significantly reduced trimethylamine N-oxide and indoxyl sulfate, host-bacteria co-metabolites known to induce metabolic deteriorations. Specific bacterial genomes, which were correlated with the decreased co-metabolites, harbored genes for fermenting choline or tryptophan into their toxic precursors. Hence, the dysbiotic gut microbiota contributes significantly to the metabolic deteriorations in PWS, serving as an effective target for alleviation of obesity regardless of its origin.

Stat-007#59 The hollow fiber bioartificial liver support system based on mircroencapsulated primary porcine hepatocytes could reduce the risk of Porcine Endogenous Retrovirus transmission Qian Yang,Fei Liu state key lab of infectious diseases,Zhejiang University,China Bioartificial liver support system plays an important part in treating the liver failure patients, but due to the widely used cell source: the primary porcine hepatocytes, the risk of Porcine Endogenous Retrovirus transmission is always be concerned. Recently Fruhauf indicated that short-term contact of primary porcine liver cell supernatants with human cells could result in PERV transmission. Here we combined the mostly used hollow fiber reactor with microencapsulated primary porcine hepatocytes by put the microencapsulated primary porcine hepatocytes in the extraluminal compartment of hollow fiber cartridges(HFC) and evaluated the bioasafetey of this kind of bioartificial liver support system. We collected the cycle media of extraluminal and intraluminal compartment of HFC and then infected the HEK-293T cell, finally we used the RT-PCR to detect the PERV-specfic sequence of the cycle media and the infected cells’ supernatants. As a result, the intraluminal cycle media of the HFC and the infected HEK-293T cell culture supernatant could not be detected the PREV-specifc sequence. Our findings suggested that the hollow fiber bioartifical liver support system based on microencapsulated primary porcine hepatocytes could reduce the risk of PERV transmission. Stat-008#63 sepsis in patients of cirrhosis after endoscopic variceal ligation Xuan Zhang,Xiaoli Liu State Key Laboratory for Diagnosis and Treatment of Infectious Diseases ,First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China Endoscopic variceal ligation is an effective procedure for the treatment and prevention of esophageal variceal bleeding.We observed two patients of cirrhosis had sepsis after endoscopic variceal ligation,the pathogen were both streptococcus.One of the patients was diagnosed infective endocarditis.Therefore,it suggest that it is necessary to preoperative anti-infection treatment. Stat-009#67 Study of Rat Intestinal Microecology after Liver Transplantation Chunlei Chen, Xiuli Yu, Haifeng Lu, Weiling Mao, Lanjuan Li* State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China. Introduction:Orthotopic liver transplantation (OLT) is the best way to cure the end-stage liver diseases, and infection is the main cause of the mortality of liver transplantation. Our work was focused on the changes of intestinal micro-ecology after liver transplantation (LT) on the basis of steady rat model. Result showed that disturbance of intestinal microflora was observed in rat after LT, simultaneity with the damage of the intestinal mucosal barriers. The unbalance of intestinal flora in isogenic LT group was alleviated after a period of time, whereas no remarkable improvement was observed in allogenic LT group. Conclusion: Disturbance of intestinal micro-flora was observed in rat after liver transplantation, which is related to the chemical/reperfusion (I/R) liver injury and the chronic rejection occurred in the allogenic LT group. Outlook: There is a potential that the plasma endotoxin level and the rate of bacterial translocation after LT might be effectively lowered by micro-ecological preparations.

!Figure.1 A. the intestine before surgery；B. the intestine during surgery (intestinal congestion was observed); C. the intestine 5 min after surgery (partial recovery was observed)

Table 1. The micro-ecology condition of rat cecum 50 days after LT (Lg C F U/g, Mean±SD)!

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Figure.2 The change curves of ALT content in blood after LT for each group!

Figure.3 The change curves of AST content in blood after LT for each group!

Stat-010#69 Efficacy of Oseltamivir–Paramivir combination compared to Oseltamivir monotherapy for H7N9 influenza Yan Zhang(1), Hai-nv Gao(1),Xiao-xin Wu(1),Weifeng-Liang(1),Ling-ling Tang(1),Ji-fang Sheng(1),Lan-juan Li(1),Qin Gu(2),Jian-He Gan(3),Hong-Zhou Lu(4) 1.Department of Infectious Diseases,The First Affiliated Hospital of Zhejiang University,HangZhou,China 2.Intensive Medicine,Nanjing Drum Tower Hospital,NanJing,China 3.Department of Infectious Diseases,The First Hospital Affiliated to SuZhou University,SuZhou,China 4.Department of Infectious Diseases, Shanghai Public Health Clinical Center,Shanghai,China The novel H7N9 avian influenza occurred in China in 2013, neuraminidase Inhibitors(NAIs) such as Oseltamivir,Paramivir turned out effective in treating H7N9 virus.But for the critical patients whose course of disease were more than 5 days,it is unclear weather the antiviral treatment can block the occurrence of Acute Respiratory Distress Syndrome(ARDS)，and whether Oseltamivir–Paramivir combination is superior to Oseltamivir monotherapy.In order to solve the above problems，a retrospective medical chart review of 104 patients was conducted in 6 Centrals in China.Their clinical information was collected systematically, and analyzed.And reached to the conclusion：Oseltamivir-Paramivir combination is not superior to Oseltamivir monotherapy for H7N9 influenza；Taken NAIs in or after 3 days after symptom onset does not much affect the process of virus turn negative probably, but earlier initiation of NAIs administration after symptom onset significantly may prevent the development of severe disease;How fast of H7N9 influenza turn negative may foresee the prognosis.

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Figure.4 The change curves of TBIL content in blood after LT for each group!

Figure.5 The endotoxin content in blood of each group!

Stat-011#71 Epidemiology of Clostridium difficile (sequence type 17 [ST37]) in a tertiary care hospital of China Si-Lan Gu (1),Yun-Bo Chen (1), Tao Lv (1), Ping Shen (1), Lan-Juan Li (1) 1. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China Clostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea and is endemic in hospitals. Research on C. difficile infection has primarily focused on hypervirulent strains such as the C. difficile ST-1 (ribotype 027) and ST-11 (ribotype 078) emerging in North America and Europe. However, other new emerging ribotypes in some countries have attracted attention, such as variant toxin A-negative/toxin B-positive strains of ST-37 (ribotype 017) in several Asian countries. ST-37 is the predominant type in China according to previous studies, which is quite different from other STs. In this study, we report the molecular epidemiology and ward distribution of C. difficile ST-37 in a tertiary hospital of China during three years. A total of 41 toxigenic ST-37 strains were isolated from 41 patients originating from different wards. This ST-37 showed higher resistant rate to clindamycin, fluoroquinolones (such as moxifloxacin, levofloxacin), erythromycin and rifampin. Variable isolation rates were observed in different wards and the occurrence was higher in infection ward and haematology ward. Today, China is facing a severe test as a heavy usage of antibiotics combines with an ageing increasingly hospitalised population. Further epidemiological studies across the country as well as improving surveillance should be seen as essential in preventing unnecessary morbidity and mortality. Stat-012#82 comparision of real-time PCR with pp65 antigenemia assay for mesuring viral load of CMV in liver transplant recipients Xuan Zhang,Yaping Huang,Jun Fan State Key Laboratory for Diagnosis and Treatment of Infection Diseases,the first affiliated hospital,school of medicine,Zhengjiang university,Hangzhou,China CMV is still important cause of morbility and occasional mortality in liver transplant recipients.The CMV pp65 antigenemia assay has been widely used,and often considered as a golden stardant.The real-time PCR could provide sensitive data for detecting CMV.In this study,we have described with pp65 antigenemia assay and real-time PCR in 56 liver transplant recipients.CMV DNA positive was 48.2％,the positive antigenemia was51.8％.The rate by both assays was46.4％,the negative rate in both assays was 46.4％.The cocidience was92.9％. Therefore, real-time PCR was able to detect CMV reactivation in liver transplant recipients as well as pp65 antigenemia assay. Stat-013#83 Effects of changed gut microflora on the plasmic metabolic profiles in rats liyongtao, chenyunbo, wuzhongwen, lilanjuan Infection of microecology State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou , China. Background Metabolomics studies hold promise for the discovery of pathways linked to disease processes. In clinical practice, many cases often cause disruption of the gut microflora. The aim of this work was to evaluate the effects of microflora variations on the body’s metabolic profiles. Methods Sprague-Dawley rats received either saline, probiotics, Escherichia coli, Salmonella enteritidis, Gentamicin or MgSO2 via daily gavage for 7 days, GC/MS analysis the serum metabolic profiles and intestinal bacteria was studied. Results Probiotics can markedly reduce the content of Alanine ,and in the nonpathogenic E. coli group, it significantly increase in alanine; orally administered S. enteritidis caused many of these amino acids (alanine, leucine , isoleucine, serine) significantly reduce; an overall reduction in gut microflora due to orally administered Gentamicin resulted in a meaningful reduction in proline and butyric acid. Conclusion(s) The changed gut microflora have diverse effects on the serum metabolic profiles ,especially on the Ammonia.

Figure. PCA three-dimensional scatter Title of Figure

Stat-014#90 Contribution of efflux pumps to tigecycline resistance Klebsiella pneumonia in China Guoping Sheng , Huihui Dong , Yonghong Xiao, Jifang Sheng, Weihang Ma, Laijuan Li State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University The emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) bacteria can lead to the disastrous results for infectious disease patients, and some of them are lethalethal. The drug resistant Klebsiella pneumoniae is one of the most popular strain in gram negative bacteria, which is rising so quickly that the antibiotics discovery could not keep the steps with the new antibiotics discovery. Tigecycline is a new intravenous antibiotics, and it has a broad spectrum of antibacterial activity. It is an important option for MDR/XDR K. pneumoniae. However, tigecycline resistance has increasingly been reported in K. pneumoniae recently, and the existence of drug-resistance gene always leads to the high rate of treatment failure. Our study was to assess the epidemiology and mechanisms of tigecycline resistance in clinical isolates of K. pneumoniae collected from a hospital in China. A total of 183 K. pneumoniae isolates were collected, including 139 ESBL+ and 44 ESBL- isolates. K. pneumoniae ATCC 27799 was chosen as control. The susceptibility to tigecycline was measured by micro dilution method. The range of MIC was from 0.03ug/ml to 4ug/ml in the ESBL- group, and 0.125 ug/ml to 8ug/ml in the ESBL+ group, including 26 tigecycline-resistant isolates, which were chosen as typical strains to detect the Multi Locus Sequence Typing and PFGE. 15 ST types were found, and type 11 was the most popular one. The efflux pump genes, such as AcrA and oqxA were detected. AcrA and oqxA were both related with the resistance to tigecycline. RT-PCR of AcrA and oqxA showed the expression had the linear dependence with the MIC value. The efflux pump inhibitors (EPIs) could partially reverse the resistance pattern of tigecycline. So our data suggested that ESBL+ isolates had higher rate of tigecycline resistance. AcrA and oqxA over expression contributed to it. And we have to increase the dose to the ESBL+ isolate patients to improve the outcome.

Stat-015#95 Distribution of pharyngeal flora in adults Hongyu Jia(1,2),Dairong Xiang(1),Jiangshan Lian(1),Jun Liu(1),Zhi Chen(1,2) 1.State key laboratory for diagnosis and treatment of infectious diseases, the first affiliated hospital, college of medicine, Zhejiang University. 2.Collaborative innovation center for diagnosis and treatment of infectious diseases Objective To investigate the flora distribution in the upper respiratory tract of adults in order to treat respiratory infectious diseases scientifically by micro-ecological methods by searching for dominant microflora. Methods 100 adults were selected voluntarily and randomly from the Health Examination Center of the First Affiliated Hospital of Zhejiang University. Bacteria from the pharyngeal specimens were collected for isolation and culturing and biochemical identification. Results Flora from the pharyngeal wall of the adults were detected in 20 genus, 43 species. The 95 % confidence interval for aerobic bacteria was ( 5.1 x 106，7.0 x 106) CFU/mL；that for anaerobic bacteria was ( 6.3 x 106，8.2 x 106) CFU/mL；Aerobic bacteria flora density was 5.2476±1.3609，anaerobic bacteria flora density was 5.5279±1.0941. Streptococcus had the highest detection rate ( 99% ) in the aerobic bacteria，followed by Neisseria( 79% ) and Staph ( 37% ). The detection rate and constituent ratio of Streptococcus mitis，Streptococcus o ralis and Neisseria cinerea were higher. In the anaerobic bacteria，the higher detection rate and constituent ratio were Veillonella, Bacteroides, Gemella and Actinomyces. Conclusion The floras in the upper respiratory tract of adults are complicated. The dominant aerobic and anaerobic floras are Streptococcus mitis，Streptococcus oralis，Neisseria cinerea，Veillonella，Bacteroides and Gemella，which may play an impor tant role to maintain the micro ecological balance in the upper respiratory tract. Stat-016#110 Effects of a Bifidobacterium Strain Administration in an Acute Liver Injury Rat Model Ren Yan, Long-Xian Lv, Fang-Qiong Dai, Ding Shi, Xia-Wei Jiang, Lan-Juan Li State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases，The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003 Hangzhou, China Since probiotics exhibit good abilities in enhancing intestinal health and influencing the gut-liver axis, they have been proposed in the treatment and prevention of multiple gastrointestinal diseases, including the liver diseases of different etiologies. In this study we investigated the effect of intragastric supplementation of a Bifidobacterium strain from healthy human on acute liver injury induced by D-galactosamine in rats. Bifidobacterium spp LI106 was administered intragastrally to Sprague-Dawley rats once daily for 8 days. Acute liver injury was induced on the 8th day by intraperitoneal injection of 1.1 g/kg body weight D-galactosamine, and samples were collected after 24 h. Liver enzymes and serum bilirubin levels, histology of the terminal ileum and liver, bacterial translocation (to arterial and portal blood, liver and mesenteric lymph nodes), and intestinal microflora were evaluated. The results indicated that Bifidobacterium spp LI106 pretreatment was highly effective in prevention of liver injury and bacterial translocation. Bifidobacterium spp LI106 significantly reduced the elevation of alanine aminotransferase and total bilirubin, reduced the histological abnormalities of both the terminal ileum and liver, prevented bacterial translocation, modulated the cecal microbiome composition, and decreased the Enterobacteriaceae count in the cecum and colon. Based on the above, we figured that Bifidobacterium spp LI106 is a promising probiotic candidate in acute liver failure treatment. Stat-017#119 Liver ischemic preconditioning (IPC) delays acute rejection following liver transplantation through enhancing Tregs and modulating intestinal microbiota Zhigang Ren1,2,3, Haifeng Lu3,4, Shaoyan Xu1,2,3, Jianwen Jiang1,2,3, Yong He1,2,3, Haiyang Xie1,2,3, Lin Zhou1,2,3, Weilin Wang1,2,3, Lanjuan Li3,4*, and Shusen Zheng1,2,3* 1Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. 2Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. 3Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China; 4State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. Background: Acute rejection (AR) remains a life-threatening complication after liver transplantation (LT). Our previous research has proved that liver ischemic preconditioning (IPC) can improve intestinal microbiota and intestinal microbial variation is closely associated with AR after LT. This study aims to explore whether liver IPC improve AR after LT and the possible mechanisms.

Methods: The AR models of liver IPC before LT in rats were established and dynamically observed. Hepatic graft function was assessed by graft histology, ultra-structure and serum ALT/AST levels. The percentages of CD3+, CD4+ and CD8+ T cells and Treg cells in both liver and serum were assessed by flow cytometry. Liver CD8+ cells were also observed by IHC. Intestinal microbial community was analyzed by way of 454 pyrosequencing of the 16S ribosomal RNA V3 region followed by real-time quantitative PCR. Intestinal microbial variation was observed between AR and Liver IPC. Principal Findings: Hepatic graft suffered from the I/R injury on day 1, initial AR on day 3 and severe AR on day 7 after LT. Liver IPC before LT could improve the I/R injury and delay AR development after LT by observation of graft histology, ultra-structure and liver function. Notably, rejection still developed after liver IPC corresponding to severe rejection in AR group. After liver IPC, inflammation response was attenuated, CD8+ T cells were decreased and Treg cells were enhanced in both liver and serum at the initiation of AR. Also, IL-10 secreted by Treg cells was increased. Importantly, intestinal microbiota significantly changed between liver IPC and AR groups. PCA analysis indicated that intestinal microbial composition after liver IPC approached Control group, but obviously deviated from AR group. At bacterial phylum level, Firmicutes and Bacteroidetes were predominant in all groups, but liver IPC significantly increased Bacteroidetes, Proteobacteria and Cyanobacteria, and decreased Firmicutes, Tenericutes and Verrucomicrobia. At genus level, 12 bacteria including Oscillospira and Paraprevotella were increased, and 9 bacteria including Flavobacterium and Paludibacter were decreased in liver IPC versus AR group. RT-qPCR results also verified intestinal predominant bacterial variation. Conclusions: Liver IPC before LT can delay acute rejection development following liver transplantation through enhancing Tregs cells and modulating intestinal microbial variation. This finding provided the effective strategy preventing acute rejection after LT. Stat-018#120 The imbalance of intestinal microbiota promotes liver diseases Zhigang Ren1,2, Shaoyan Xu1,2, Haiyang Xie1,2, Lin Zhou1,2, Weilin Wang1,2, Shusen Zheng1,2* 1 Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, P. R. China. 2 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, P. R. China. Abstract: Intestinal microbiota is closely associated with the initiation and development of liver diseases. This article reviewed the alterations of intestinal microbiota in liver diseases including alcoholic liver disease, non-alcoholic fatty liver disease, viral-induced liver disease, liver cirrhosis and liver cancer. We also indicated that the imbalance of intestinal microbiota promotes liver diseases. Due to the intimate anatomical and functional relationship between the gut and the liver, intestinal organisms and their metabolites interact with the host and play an important role in liver inflammation, chronic liver fibrosis, liver cirrhosis and cancer progression through liver-gut circulation and microbiota-liver axis. The key functional bacteria in intestinal microbiota may become a novel biomarker for liver diseases progression, and fecal microbial transplantation may become a potential and effective therapy for liver diseases. Key words: Intestinal microbiota; Liver cirrhosis; Liver-gut circulation; Alcoholic liver disease; Stat-019#121 Effects of Lactobacillus sp. LI208 in Rats with Liver cirrhosis Xia-Wei Jiang, Ding Shi, Long-Xian Lv, Dai-Qiong Fang, Ren Yan, Lan-Juan Li State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases，The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003 Hangzhou, China Liver cirrhosis is caused by liver diseases and can lead to loss of liver function and other serious complications. The damage caused by liver cirrhosis is irreversible and the treatment can prevent or delay the further damage. Previous studies showed patients with liver cirrhosis harbor different fecal microbial communities compared to the healthy controls and certain microorganisms can provide health benefits to humans. These microorganisms known as probiotics were believed to be benefical in the treatment and prevention of various gastrointestinal diseases including liver diseases. In this study we investigated the effects of a lactic acid bacteria, Lactobacillus sp. LI208, on liver cirrhosis in rats. Lactobacillus sp. LI208 was isolated from healthy human. The rat models of liver cirrhosis were produced by carbon tetrachloride and Lactobacillus sp. LI208 was administered intragastrally once daily. The body weight and fatality rate were recorded and samples were collected after 12 weeks. Liver enzymes, liver function, bacterial translocation and composition of the gut microbiome were examined. The results showed pretreatment with Lactobacillus sp. LI208 can prevent the body weight loss and reduce the fatality rate of rats. The liver function was improved and the bacterial translocation was decreased. The cecal microbiome was differed from that of the controls. Based on the study, we proposed Lactobacillus sp. LI208 may be useful in the treatment of liver cirrhosis.

Stat-020#123 A prebiotic and glutamine intervention modifies the host immunity in the HIV gut-associated dysbiosis Jorge F. Vázquez-Castellanos 1,2, Sergio Serrano-Villar 3, Amparo Latorre 1,2, Nadia Madrid 3, Talía Sainz 4, Sara Ferrando-Martínez 5, Alejandro Artacho 1, Santiago Moreno 3 , Vicente Estrada 7, Andrés Moya 1,2, María José Gosalbes 1,2. 1. Unidad Mixta de Investigación en Genómica y Salud de la Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO-Salud Pública) y el Institut Cavanilles de Biodiversitat y Biología Evolutiva (Universitat de València), 46020 València, Spain. 2. CIBER en Epidemiología y Salud Pública (CIBERESP), 28029 Madrid, Spain. 3. Department of Infectious Diseases, University Hospital Ramón y Cajal and Ramón y Cajal Health Research Institute (IRYCIS), 28034 Madrid, Spain. 4. Laboratory of Immune Biology, University Hospital Gregorio Marañón, 28007 Madrid, Spain. 5. Laboratory of Immunovirology, Biomedicine Institute of Seville (IBIS), Service of Infectious Diseases, University Hospital Virgen del Rocío, 41013 Sevilla 6. HIV Unit, Department of Internal Medicine, University Hospital Clínico San Carlos, 28040 Madrid, Spain. Recent studies in metagenomics have confirmed a dysbiosis in HIV+ subjects that correlates with the T-cell activation and an increment in IL-6 plasma level, which are all associated with disease progression in HIV infection. Current knowledge is limited to correlations between immune activation markers and the abundance of single bacterial taxa in order to determine microbial targets for the treatment of immune dysfunction. However such an approximation does not take into account the effect of the whole microbial community structure on the clinical response. In this study, a cohort of 44 individuals from four different HIV status and antiretroviral treatment (HIV untreated, immune recovery, inmune not recover and HIV- ) completed a 6-week study treatment with prebiotics and glutamine (PG), 33 in the active arm and 11 in the placebo arm. The microbiome for the cohort was determined by pyrosequencing the 16S rRNA gene and by shot-gun sequencing applying Illumina technology, using state-of-the-art software to analyze the taxonomic and functional composition. Likewise the activation of the adaptive immunity, innate immunity and the expression level of several genes involved in pathogenic pathways were measured for each individual of the cohort before and after the PG intervention. In this work we used a probabilistic directed acyclic graphical model approach and generalized linear models (GLM) in order to find significant relationships between the decay of the host health and the modulation of the bacterial population. We found a markov blanket that associated changes in the markers of activation of the adaptive immunity and the increase of Gram-negative bacteria. Also, the markers for bacterial translocation and systemic inflammation showed positive relationships with several Gram-negative bacterial species. The magnitude of the prebiotic intervention was studied by means of the GLM, which showed that the maximal viral load (an estimator of the HIV harassment to the gut associated lymphoid tissue (GALT)) is one of the main factors to determine the effect of the prebiotic treatment. Communities in which the virus has not damaged considerably the GALT, we observe an increase of Firmicutes phylum bacteria that are able to produced short chain fatty acids. These bacteria correlate negatively with the expression level of the gene coding for the APOBEC3G, a protein that is related with the innate anti-viral immunity, and the gene expression of the CCL2 and CCR2 proteins that are involved in the monocyte chemotaxis related with inflammatory diseases. Then, these results suggest that serious damage of GALT produced by HIV at early infection might allow the overgrowth of Gram-negative bacteria species which are able to trigger the systemic immune response. Also, the degree of deterioration of the GALT seems to be the main factor determining the effectiveness of the prebiotic treatment. Stat-021#124 Study on effects of CYP for the dysbacteria animals by PCR-DGGE Yu Lian(1),Xu Xin(1), ,Su Jin(1),Sun Wei-tong(1),Li Shou-jun(1),Hu Yan-qiu(1),Ping Yang(1) ,Zhang Lei(1),Zhou Tong(1),Ma Shu-xia(2),ZhaoTao(2),Meng De-xin(2) 1.School of Pharmaceutical Sciences, Jiamusi University ,Jiamusi ,China 2.School of Basic Medical Sciences,Jiamusi University ,Jiamusi ,China Abstract:objective To separate PCR products in 16SRNA V3 variable region of intestinal bacteria useing PCR-DGGE. The data were analyzed by DGGE profile strips to probe CYP affect on dysbiosis modelrole mice.Method The intestinal microbe dysbiosis mice were established by intragastric administration of linconycin,and them treated with CYP,At the same time,normal control,Live Bifidobacterium Preparation and natural recovery group were set up,were given the appropriate medication. All mice were sacrificed after ten days gavage, ileocecal stool specimens collected,and then the DNA was extracted and PCR-DGGE method was used for detection for the specimens ntestinal flora DNA.Finally, all data collection.The get DGGE gel using Quantity One software to analyze similarity and diversity,observation of the effect of CYP on the treament of intestinal dysbiosis mice.Recust PCR-DGGE results showed that :a stool sample total DNA concentration measured value(ng/µL) the CYP group(0.18±0.005) and the Live Bifidobacterium Preparationgroup(0.175±0006)comparedwiththenatural recovery group(0.072±0.001),significantly increase the DNA concentration(p<0.05) and retural recovery group(0.182±0.006).DGGE diversity index analysis that the CYP group（23.95±2.36) and the Live Bifidobacterium Preparation group（24.00±1.68）is greater than the natural recovery group（19.57±2.52）and return to recovery group,(25.87±2.10),the difference was statistically significant(p<0.05).Conclusion The experiment results show that CYP group containing DNA quantity is higher than natural recovery group,suggets that CYP group in the group total DNA content is richer,to verify the diversity of targeted agents in the CYP group .DGGE gel electrophoresis can separate DNA bands that the same length but sequence is differenece,bands that biodiversity,the more abundant the more the brighter the stripe said to strip the more the number of bacteria,which can reflect

the number and variety of microorganism.Verified by PCR-DGGE experiment at the genetic level targeting perparation by adjusting the bactcterium group richness to treat to dysbacteriosis.PCR-DGGE results show that the CYP group targeted agents for intestinal microecological imbalance has significant adjustment in wistar mice. Key word: Nano CYP ;Colon targeting preparation ;PCR-DGGE Stat-022#130 Gut microbiome in organ and cell transplantation Shaoyan Xu(1,2,3), Zhigang Ren(1,2,3), Jianwen Jiang(1,2,3), Weilin Wang(1,2,3), Shusen Zheng(1,2,3) 1.Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003, Hangzhou, China. 2.Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, College of Medicine, Zhejiang University,310003, Hangzhou, China. 3.Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003, Hangzhou, China. Purposes：We aim to review and discuss the alteration of gut microbiome after organ and cell transplantation, as well as the relationship between complications after organ and cell transplantation and gut microbiome dysbiosis. The role of intervention methods including administration of antibiotic or probiotics in complications after transplantation are also discussed. Methods: Studies exploring the role of gut microbiome in organ and cell transplantation and published in English are searched , read in detail , trying to get insightful and helpful conclusions. Results: Animal and clinical studies have revealed that the population and diversity of gut microbiome in patients after transplantation such as liver transplantation, small intestine transplantation, hematopoietic stem cell transplantation and kidney transplantation, altered and had a tendency restoring to normal status. However, when complications such as infection, rejection and graft versus host disease(GVHD) occured, the population and diversity of gut microbiome presented a significant dysbiosis mainly with a decrease of commensal bacteria and an increase of pathogenic bacteria. The distinct gut microbial profiling could be a potential diagnostic biomarker of complications such as acute rejection after transplantation. Taking probiotics and prebiotics could effectively regulate the gut microbiome and reduce the incidence of complications after transplantation. However, the role of intestinal decontamination in organ or cell transplantation is controversial. There are still few researches on the mechanism between gut microbial dysbiosis and complications and more studies are urgently to be performed. Conclusions: The distinct gut microbial profiling could be a potential diagnostic biomarker of complications after transplantation. Gut microbiome have the potential of being a novel therapeutic target to restrict, improve and even reverse complications after liver transplantation. Studies should not only be performed in-depth, but also should be extended to other types of organ and cell transplantation, and then the development of transplantation has the potential of a new breakthrough. Stat-023#134 Lactobacilli sp. LI56 selectively inhibits growth of Staphylococci by nutrition competition Longxian Lv， Ren Yan，Daiqiong Fang，Ding Shi，Xiawei Jiang, Haiyan Shi, Lanjuan Li State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases，The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003 Hangzhou, China Staphylococci are gram-positive aerobic organisms that can cause a multitude of diseases including boils, impetigo, food poisoning, cellulitis, and toxic shock syndrome in humans and animals through either toxin production or penetration. For many years, antibiotics have been used as routine and good treatments. However, recently, staphylococcal infections by drug-resistant strains, especially those by methicillin-resistant Staphylococcus aureus (MRSA), are becoming not only a thorny problem that must be faced by clinicians, but also a threat to human health. It is reported that about 19,000 deaths per year are due to hospital acquired MRSA, which is more deaths than HIV causes per year in the United States. Thus, it is essential to develop alternative strategy to prevent and treat Staphylococcal infections. Probiotics can prevent and treat infections by secreting organic acid, antibacterial peptide or by nutrient competition. From tens of lactobacilli, we discovered a strain, named lactobacilli sp. LI56, can significantly inhibit the growth of most staphylococci. As that of most probiotics, stain LI56 produces lactic acid and several kinds of antibacterial peptides to exclude staphylococci. However, when these acids or peptides were removed, strain LI56 still had good ability to inhibit the growth of staphylococci. Using a synthetic medium and the UPLC-MS method, we found that lactobacilli sp. LI56 shared some essential nutrients with several staphylococci such as staphylococcus epidermidis, staphylococcus aureus. Then, we sequenced the whole genome of strain LI56 and compared with those of several strains of staphylococci. Some similar pathways related to nutrient competition between strain LI56 and staphylococcus epidermidis or staphylococcus aureus were detected. Then these pathways were validated by the above metabolic analysis. This study shows an alternative way to prevent and treatment of staphylococcal infections. Furthermore, the similar nutrient pathway between these strains indicates interesting research Directions in both microbial evolution and micro ecology.

Stat-024#137 Expression and significance of B7-H4 and HBx in HBV related hepatocellular carcinoma Hang-Ping Yao, Xiao-Xin Wu, Chang-Zhong Jin, Hai-Bo Wu, Lin-Fang Cheng, Nan-Ping Wu State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University Objective: Hepatitis B virus (HBV) is a major public health problem, and Hepatitis B virus-related hepatocelular carcinoma (HBV-HCC) has an extremely poor prognosis due to a lack of effective treatments. B7-H4 is a novel member of the B7 superfamily that are actively involved in regulating the pathogenesis of tumors. However, the intrahepatic expression of B7-H4 in HBV-HCC patients has not been described. In this study, we investigated the expression and clinical significance of B7-H4 and Hepatitis B Virus X (HBx) protein in HBV-HCC. Methods: The expression of B7-H4 in the human HCC cell lines HepG2 and HepG2215 was detected by western blotting, flow cytometry and immunofluorescence analysis. The expression of B7-H4 and HBx in 83 HBV-HCC was detected by immunohistochemistry, and the relationship with clinicopathological features was analyzed. Results: B7-H4 was significantly up regulated in HepG2215 cells than in HepG2 cells. The positive rates of B7-H4 and HBx in 83 HBV-HCC tissues were 68.67%（57/83）and 59.04%（49/83） respectively. The expression of HBx was correlated with TNM staging. The expression of B7-H4 was positive correlated with HBx (rs=0.388, P<0.01). The expression level of B7-H4 in HBx-positive HBV-HCC tissues was substantially higher than that in HBx-negative HBV-HCC tissues, which was negative related to tumor TNM stage. Conclusion: The higher expression of HBx and B7-H4 were correlated with tumor progression of HBV related hepatocelular carcinoma. B7-H4 may be an effect molecule in HBV related hepatocarcinogenesis. Stat-025#138 Clinical significance of inflammatory cytokine and chemokine expression in hand foot and mouth disease Hang-Ping Yao, Hui-Lin Ou, Xiao-Xin Wu, Chang-Zhong Jin, Hai-Bo Wu, Lin-Fang Cheng, Tian-Shen Xie, Nan-Ping Wu State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University Objective: To determine the relationship of cytokine/chemokine expression with the clinical presentation of hand, foot and mouth disease (HFMD). Methods: This study involved 28 mild HFMD patients, 44 severe HFMD patients and 26 healthy children. Venous blood was tested for cytokines (IL-4, IL-12, IL-18, TNF-α, IFN-γ) and chemokines (IL-8, RANTES, MCP-1, IP-10). Stool samples from the patients were tested for EV71 nucleic acid using reverse transcription polymerase chain reaction. Results: All cytokine/chemokine levels were higher in severe HFMD patients than in mild HFMD patients or controls (P < 0.01). RANTES, MCP-1, IL-4, IL-12 and IL-18 levels were higher in mild HFMD patients than in the controls (P < 0.05). In severe HFMD, all levels (except IL-8 and IL-4) were higher in patients with encephalitis plus pulmonary edema than in those with encephalitis alone (P < 0.05). All levels (except IL-8) were higher in EV71-positive patients than in EV71-negative patients (P < 0.05). In mild HFMD, all levels (except IL-8 and IL-4) were higher in EV71-positive patients than in EV71-negative patients (P < 0.05). In severe HFMD, only RANTES, IP-10 and IFN-γ levels were higher in EV71-positive patients than in EV71-negative patients (P < 0.05). In the EV71-negative group, all levels were higher in severe HFMD than in mild HFMD (P < 0.01). In the EV71-positive group, all levels (except IL-8) were higher in severe HFMD than in mild HFMD (P < 0.01). Conclusion: Cytokines/chemokines participate in HFMD pathogenesis, and may be useful to monitor disease progression and predict prognosis. Stat-026#140 Distribution of multidrug resistant bacteria and the analysis of treatment of children with severe pneumonia in PICU. Lina Mao(1), Chunlan Song(2), Yu Luo(3) 1.Department of Pharmacology, Henan Medical College, Zhengzhou 451191, China 2.Department of Intensive Care Unit, Children's Hospital of Zhengzhou ，Zhengzhou 450053，China 3.Department of Immunology Research，Academy of Medical and Pharmaceutical Sciences，Zhengzhou University，Zhengzhou 450052，China Corresponding author: Yu Luo，Email: 1289299548@qq.com

Background Pneumonia is a common disease in infants and young children. The multidrug resistant bacteria can cause severe pneumonia. The treatment of severe pneumonia is difficult. Understand the pathogen of severe pneumonia and resistance is of great significance for treatment. Method The children with severe pneumonia were admitted in our hospital from November 2014 to December 2014. The ductal lavage fluid specimens of children with severe pneumonia in PICU were cultured,the bacteria separated was identified,and their drug susceptibility were determined by fully automatic instrument（BD Phoenix 100）. The anti-infective drugs were chosen according to the patient's condition and results of drug sensitivity. After 3 days treatment, the condition changes was observed. Result There were 22 strains multi-resistant pathogens isolated from the 22 cases，which contained 8 strains of Acinetobacter baumannii, 8 strains of Pneumonia klebsiella pneumonia subspecies, 4 strains of Escherichia coli , 2 strains of Pseudomonas aeruginosa. The sensitive medicine were polymyxin, SMZ-TMP and ceftazidime for Acinetobacter baumannii,the 2/3 cases were effective by choosing ceftazidime, the 5/5 cases were effective by choosing cefoperazone/sulbactam. The sensitive medicine were SMZ-TMP and tetracycline for Pneumonia klebsiella pneumonia subspecies, the 2/4 cases were effective by choosing cefoperazone/sulbactam, the 2/2 cases were effective by choosing cefoperazone/sulbactam + fluconazole; the 2/2 cases were effective by choosing cefoperazone/sulbactam + vancomycin. The sensitive medicine were amikacin, gentamicin,imipenem, meropenem, amoxicillin/clavulanic acid, ampicillin/sulbactam, piperacillin/tazobactam,SMZ-TMP, ciprofloxacin,levofloxacin and tetracycline for Escherichia coli , the 2/2 cases were effective by choosing meropenem,the 2/2 cases were effective by choosing piperacillin/tazobactam. The sensitive medicine were aztreonam and polymyxin for Pseudomonas aeruginosa, the 2/2 cases were effective by choosing aztreonam. The effective rate was 18/22 cases. Conclusion Acinetobacter baumannii and Pneumonia klebsiella pneumonia subspecies were main pathogens in 22 strains multidrug resistant pathogens. The effective rate was 18/22 cases by choosing cefoperazone/sulbactam and other drug from the sensitive drugs. Key words Severe pneumonia; Multidrug resistant bacteria; Drug resistance;treatment Stat-027#150 The construct of Computer-Aid Microbiome Intervention Platform Tiansheng Xie(1),Zhehao He(2) 1. State Key Laboratory for Infectious Disease Diagnosis and Treatment, The First Affiliated Hospital, College of Medical Sciences, Zhejiang University, Hangzhou 310003, China 2. Department of Cardiothoracic Surgery, The First Affiliated Hospital, College of Medical Sciences, Zhejiang University, Hangzhou 310003, China As it known to all microbiome is a systemic issue to our health, it's intervention also include biological and non-biological strategy, the optimize way to microbiome intervention is to strengthen the education and behavioral intervention in all population. We focus on the individual cognitive level, cultural background, and the pattern of activities, with the web and database technology, through two-dimension personalized way develop a suitable personalized prevention platform for all kinds of people. Web-based architecture design, includes user systems, management system, learning, surveys, information, consulting .User system was designed to collect the individual information (education background, occupation, sexual orientation, marital status etc.) and microbiome (gut microbiota, oral microbiota, etc.). Exploring and optimizing personalized intervention measures is the key point for microbiome, so such a personalized and integrated intervention platform can inevitably enhance Users’ initiative of participation. It is practical and innovative will play an important role in the microbiome intervention area. Professor Liu Depei proposed “6P” as a new concept of Medicine which conclude promotive medicine, protective medicine, predictive medicine, prewarning medicine, preventive medicine, personalize medicine. struggle for exploring and optimizing personalized preventive measures is the key point for microbiome intervention,so such a personalized and integrated prevention platform can inevitably enhance Users’ initiative of participation. It is practical and innovative. And will surely play an important role in the microbiome intervention area. Stat-027#153 Clostridium butyricum in the prevention of antibiotic-associated diarrhea in patients infected with the novel avain influenza A (H7N9) virus Guanjing Lang (1), Hainv Gao (1), Lanjuan Li (1) 1.State Key Laboratory Diagnosis and Treatment for Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, the First Affiliated College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, China Background: Antibiotic-associated diarrhea (AAD) is the most frequent side effect of antibiotic therapy, especially occurs in those who are exposed to board-spectrum antibiotics. Treatment of board-spectrum antibiotics is most needed for patients with avian influenza virus subtype H7N9 infection. Methods: We use retrospective analysis of patients with avian influenza virus subtype H7N9 infection who received probiotic treatment with Clostridium Butyricum Tablets or not to evaluate the effectiveness. Results: There were total 44 patients infected with avian influenza virus subtype H7N9 in our hospital. And 36 patients were recruited to the study during their admission, of which 31 received Clostridium Butyricum Tablets. 17 patients manifested AAD among those who received probiotic treatment, while there was 1 patient occurs AAD in the no probiotic treatment group. There was no significant difference in occurrence of AAD between the two groups. Conclusion: We identified

no evidence that the clostridium butyricum was effective in prevention of AAD in patients infected with the novel avain influenza A (H7N9) virus.

Stat-029#164 Early-life antibiotic use and the intestinal microbiota Katri Korpela (1), Anne Salonen (1), Lauri J. Virta (2), Riina A. Kekkonen (3), Kristoffer Forslund (4), Peer Bork (4) & Willem M. de Vos (1,5,6) 1. Immunobiology Research Programme, Department of Bacteriology and Immunology, University of Helsinki, Helsinki, Finland 2. Research Department, Social Insurance Institution, Turku, Finland 3. Valio Limited, R&D, Helsinki, Finland 4. European Molecular Biology Laboratory, Heidelberg, Germany 5. Laboratory of Microbiology, Wageningen University, Wageningen, the Netherlands 6 Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland Early-life antibiotic use is associated with increased risk for metabolic and immune-related diseases, potentially by disturbing the intestinal microbiota. In adults and neonates antibiotics dramatically affect the intestinal microbiota. In children this is thought to interfere with the development of the immune system, although little is known about the long-term impacts of antibiotics on the developing microbiota of children. Using phylogenetic and metagenomic approaches combined with individual drug purchase records; we show that antibiotic use in 2-7 year old Finnish children (N=142; most sampled at two time points) is associated with long-lasting changes in the intestinal microbiome. In particular, the effects of repeated macrolide use – low diversity, altered composition and metabolism, and increased antibiotic resistance – accumulate with age and are associated with later asthma development and excessive weight gain. Penicillins appear to be a more microbiota-friendly treatment option. Without compromising clinical practice, the impact on the intestinal microbiota should be considered when prescribing antibiotics.

Stat-030#165 Structural modulation of gut microbiota during alleviation of type 2 diabetes with a Chinese herbal formula Jia Xu (1), Fengmei Lian (2), Linhua Zhao (2), Yufeng Zhao (3), Xinyan Chen (2), Xu Zhang (1), Yun Guo (2), Chenhong Zhang (1), Qiang Zhou (2), Zhengsheng Xue (1), Xiaoyan Pang (1), Liping Zhao (1,3), Xiaolin Tong (2) 1. State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, PR China. 2. Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, PR China. 3. Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, PR China. The gut microbiota is hypothesized to play a critical role in metabolic diseases, including type 2 diabetes (T2D). A traditional Chinese herbal formula, Gegen Qinlian Decoction (GQD), can alleviate T2D. To find out whether GQD modulates the composition of the gut microbiota during T2D treatment, 187 T2D patients were randomly allocated to receive high (HD, n=44), moderate (MD, n=52), low dose GQD (LD, n=50) or the placebo (n=41) for 12 weeks in a double-blinded clinical trial. Patients that received the HD or MD demonstrated significant reductions in adjusted mean changes from baseline of fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) compared with the placebo and LD groups. Pyrosequencing of the V3 regions of 16S rRNA genes revealed a dose-dependent deviation of gut microbiota in response to GQD treatment. This deviation occurred before significant improvement of T2D symptoms was observed. Redundancy analysis identified 47 GQD-enriched species level phylotypes, 17 of which were negatively correlated with FBG and 9 with HbA1c. Real-time qPCR confirmed that GQD significantly enriched Faecalibacterium prausnitzii, which was negatively correlated with FBG, HbA1c and 2-h postprandial blood glucose levels and positively correlated with homeostasis model assessment of beta-cell function. Therefore, these data indicate that structural changes of gut microbiota are induced by Chinese herbal formula GQD. Specifically, GQD treatment may enrich the amounts of beneficial bacteria, such as Faecalibacterium spp. In conclusion, changes in the gut microbiota are associated with the anti-diabetic effects of GQD.

Figure 1. Dose-dependent alterations of the gut microbiota in T2D patients treated with different

doses of GQD.

Figure 2. Biplot of RDA of the microbiota composition responding to GQD therapy.

Stat-031#172 The clinical experience of probiotics uesd in IBS patients Jingjing Wu (1) Feifei Guo(2) The FIrst Affiliated Hospital, College of Medicine, Zhejiang University Irritable bowel syndrome is a common disorder and its pathogenesis is still unclear.Due to limited efficacy of available therapies in the clinical ,probiotics ,because of its fewer adverse effects ,has been fouced on great enthusiasm and more and more trails reported the feasibility.This study aims to investigate the clinical experience of probiotics that used in IBS patients oral administration of Lactobacillus or Bifidobacterium are the most common probiotics taken by TBS.Responce to therapy such as the severity and frequency of abdominal pain /discomfort,bloating /abdominal distention ,or the bowel moverment difficulty was recorded and collected weekly through a questionaire designed properly .Quality of life assessment,stool microbiologic studies and blood samples are the main indices in the study.At the end of the study ,we reach the conclusion that probiotics have shown efficacy for improvement of IBS symptoms.The individual score for abdominal pain/discomfort,bloating/distention,and bowel movement difficulty was significantly lower than before oral administration of probiotics. Stat-032#176 Administration of probiotics on the intestinal microbiota: current clinical applications and future perspectives. Wen-Rui Wu State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases，The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China There are a body of researches focusing on the human microbiota.And it is indicated that varieties of human diseases have an intimate relationship with gut flora. Consequently, an increasing emphasis is being attached on a new product-the probiotics, which are live microorganisms that can reach the human intestine and modulate the gut microbiota, as well as the immune system, generating a beneficial effect on the health of host. The related mechanisms of these roles involve competing with pathogenic microorganisms, inhibiting their growth, and promoting intestinal barrier function. Currently, probiotics most commonly used are lactic acid-producing bacterium, mainly referring to Bifidobacteria and Lactobacilli. It is also reported that certain yeasts can be used as probiotics, especially Saccharomyces boulardii. This review primary highlights recent literature findings on potential therapeutic characteristics of these probiotics in human diseases and their contributions to health. KEYWORD: human microbiota; probiotics; Bifidobacteria; Lactobacilli; Saccharomyces boulardii. Stat-033#177 The gut microbiota vary in autoimmune liver disease Feiei Guo (1,2), Ding Shi(1,2) , Daiqiong Fang(1,2) , Wenrui Wu(1,2), Lanjuan Li(1,2) * 1.State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, Zhejiang University, Hangzhou, PR China. 2.Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China Primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH) are the the three major representatives for autoimmune liver diseases (AILD). Innate immunity seems to play a significant role to the pathogenesis of autoimmune liver disease. The cause of autoimmune liver disease is unclear and the diagnosis mainly depends on the detection of serum autoantibodies. A study suggests that the gastric parietal cell antibodies and intrinsic factor antibodies have a significant proportion in primary biliary cirrhosis. In patients with PBC, intestinal permeability was found to be increased. However, the small intestinal bacterial overgrowth and increased intestinal permeability may not have important contributor to the primary sclerosing cholangitis pathogenesis. An aberrant intestinal T lymphocyte derive from the liver may induce immune hepatic damage. Growing evidence demonstrates the close interaction of the gastrointestinal tract and the live and the fact that the intestinal flora disorder, especially the migration of bacteria or bacterial products across the intact intestinal epithelium, seems to be involved in the progression of liver cirrhosis. Reciprocal screening for those studies is recommended, the alteration of gut microbiota may associate with the autoimmune response in AILD, which can improve acknowledge for this disease further and may offer a powerful tool for their detection.

Stat-034#195 Early dynamics of the gastrointestinal microbiome in infants at risk of metabolic disease in adulthood Linda Wampach (1), Anna Heintz-Buschart (1), Angela Hogan (2) Lutz Bindl (3), Jean Bottu (3), Jochen Schneider (1), Carine de Beaufort (1,3), Paul Wilmes (1) 1. Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Luxembourg, Luxembourg 2. Integrated BioBank of Luxembourg, Luxembourg, Luxembourg 3. Centre Hospitalier de Luxembourg, Luxembourg, Luxembourg Recent studies have led to a growing body of evidence that diet-related chronic diseases may have their origins in a microbial disequilibrium in the gastrointestinal tract, which is potentially already established in early infancy. It has been observed that children, who are born small for gestational age (SGA) or who have other known risk factors with negative modulatory effects on the composition of the colonising microbiome (e.g. born by caesarean section), are at increased risk for developing chronic metabolic disorders in adulthood, notably metabolic syndrome, obesity and type 2 diabetes. These infants typically show a rapid increase in body weight in the first weeks after birth, which has been linked to their propensity to develop metabolic disorders in adulthood (Barker hypothesis). We hypothesise that the developing gastrointestinal microbiome, which is integrally important for energy harvest from food, plays an important role in this weight catch-up. To detect potential differences in the dynamic processes of colonisation, succession and evolution of the gastrointestinal microbiome between infants with no known risk factors (controls) and neonates at risk of developing metabolic disease in adulthood, a deeply sampled longitudinal study called ‘COSMIC’ has been set up in Luxembourg in 2012. The study specifically aims to generate basic knowledge about the colonising gastrointestinal microbiome for the future development of potential interventions in early paediatric care in infants at risk for developing metabolic disorders in adulthood. In a pilot phase, biomolecular extractions of faecal samples from the first 18 infants (8 controls; 10 at risk), ranging from day 1 to six months postpartum, were carried out to obtain metabolite and DNA fractions. Using deep amplicon sequencing of the 16S and 18S rRNA genes, we were able to retrace the dynamics of microbial and eukaryotic populations in infants over time. Infants, who are at risk for metabolic disease in adulthood (being born SGA or/and by caesarean section), showed a significantly (P<0.01) higher weight gain between day 5 and 28 postpartum compared to the control group. The 16S rRNA gene sequencing data in infants that were born by caesarean section showed a nearly complete depletion in the bacterial phylum Bacteroidetes around day 5 postpartum and a significantly higher Firmicutes/Bacteroidetes ratio at days 5 and 28 (P<0.05), suggesting a higher energy harvesting capacity of the gastrointestinal microbiome in these infants according to recent microbiome studies of obese individuals. Furthermore, infants born by caesarean section showed a delayed colonisation by Bacteroides spp. and a higher level of diversity inside the Bacteroidetes phylum. The COSMIC study provides a detailed longitudinal overview of the gastrointestinal microbiome in infants who are born with known risk factors for metabolic disorders in adulthood and may inform preventative early paediatric care in the future. Stat-035#199 Long term analysis of gut microbiota in at-risk atopic children: impact of probiotics Nicole Rutten (1), Monique Gorissen (2), Anat Eck (3), Arine Vlieger (1), Isolde Besseling-van der Vaart (4), Dries Budding (3), Kors van der Ent (2), Ger Rijkers (5) (1) Department of Paediatrics, St Antonius Hospital, Nieuwegein, the Netherlands; (2) Department of Paediatric Pulmonology and Allergology, Wilhelmina Children’s Hospital, University Medical Centre, Utrecht, the Netherlands; (3) Department of Medical Microbiology and Infection Control, VU University Medical Centre, Amsterdam, the Netherlands; (4) Winclove Probiotics BV, Amsterdam, the Netherlands; (5) Department of Sciences, University College Roosevelt Academy, Middelburg and Department of Medical Microbiology and Immunology, St Antonius Hospital, Nieuwegein, the Netherlands Introduction: Imbalance of the human gut microbiota in early childhood is suggested as a risk factor for immune-related disorders. Probiotic supplementation might be a potential approach for prevention of allergic diseases in infants by modification of the intestinal microbiota and establishing and/or restoring the physiological balance between Th1 and Th2 cells. The aim of this study was to assess the composition and microbial diversity in stool samples of infants at high-risk for atopic disease, from birth onwards to six years of age, who were treated with probiotics or placebo during the first year of life. Methods: In a double-blind, randomized, placebo-controlled follow-up trial, a probiotic mixture consisting of B. bifidum, B. lactis and Lc. lactis (Ecologic® Panda) was perinatally administered to expecting mothers during the last 6 weeks of pregnancy and their offspring daily during the first year of life. During follow-up, faecal samples were collected from 99 children over a 6-year period with the following time points: first week, second week, first month, three months, first year, eighteen months, two years and six years. Bacterial profiling was performed by IS-pro[1]. Differences in bacterial abundance and diversity were assessed by conventional statistics. Results: The presence of the probiotic strains was confirmed, and the probiotic strains had a higher abundance and prevalence in the probiotic group during the supplementation period of one year. Otherwise, composition of the microbiota did not differ between the probiotic and placebo group, nor between the atopic and non-atopic group,although the diversity of Bacteroidetes was significantly higher after two weeks in the placebo group, and at the age of two years atopic children had a significantly higher Proteobacteria diversity (p < 0.05). Children had a shared microbiota development over time, determined by age, that got on to the age of six years. At that time, no differences in microbiota composition were found between the probiotics and placebo

group. The clinical effects of the intervention were a significant reduction of atopic eczema at 3 months of age but no difference in the incidence of asthma at six years. Conclusions: Perinatal supplementation with Ecologic® Panda probiotics to children at high-risk for atopic disease had minor effects on gut microbiota composition during the supplementation period. No prolonged effects on gut microbiota composition were identified. Regardless of intervention or atopic disease status, children had a shared microbiota development over time, determined by age. [1] Budding AE, Grasman ME, Lin F, et al. IS-pro: high-throughput molecular fingerprinting of the intestinal microbiota. FASEB J. 2010;24(11):4556–64. Stat-036#201 An Important Follow-up Point for Predicting Long-term Efficacy after Treatment for Bacterial Vaginosis with Metronidazole Bingbing Xiao(1), Ben Wang(1), Xiaoxi Niu (1), Risu Na (1), Qinping Liao (1)* Department of Obstetrics and Gynecology, Peking University First Hospital, Xi'anmen Street, Beijing 100034, PR China Purpose:To determine whether a special predictive follow-up point (6-8 days after metronidazole use) can detect the final treatment outcomes by using 454-pyro sequencing. Methods:We recruited 192 women (aged 18–53 years) with regular menstrual cycles to undergo screening for vaginitis at Peking University First Hospital. Two vaginal samples (one for gram staining, one for bacterial genomic DNA extraction) were collected at the initial visit, 6-8 days and 1 month after a 5-day treatment with intravaginal metronidazole gel. Bacterial vaginosis was diagnosed if the Nugent score was 7–10 and the patient was positive for the modified Amsel criteria. We used 454 pyro sequencing to examine the 16S rRNA gene hypervariable V1V3 region in the extracted bacterial DNA. Statistical analysis with one-way analysis of variance and chi-square tests was used to determine the relationship between the bacterial components at the visit after 6-8 days and treatment outcomes after one month. Results:70 women were diagnosed with BV by the Nugent score and the modified Amsel criteria. All of these women turned to nomal after a 5-day treatment with intravaginal metronidazole gel at 6-8 days, while 22 of them relapsed eventually and 48 of them had been cured at 1month. According to microscopic examination of the first visit for a test of early treatment efficacy (Day 6-8), 70 women were divided into 3 groups, which belonged to normal flora group, flora suppression group and abnormal flora group. The types of group were related to the treatment outcomes. Normal flora group had the highest cure rate at the test of cure visit after one month with cure rate surpassing 70%, while flora suppression group was 42.86% and abnormal flora group was only 10%. The cardinal bacteria in normal flora were always lactobacillus whether at the visit after 6-8 days or at the visit after one month. The bacteria can be divided into 3 clusters at 6-8 days, each of which was related to different treatment outcomes respectively. It has been proved that the bacterial structure of all samples at 6-8 days is meaningful to predict the outcomes by bioinformatics analysis. Conclusion:6-8 days after treatment with metronidazole can be a special follow-up point to predict the final treatment outcomes after one month. Stat-037#207 Identification of a gut microbial signature linked to severity of irritable bowel syndrome Julien Tap1, Lena Ohman2, Hans Törnblom2, Boris Le Nevé3, Rémi Brazeilles3, Joël Doré1, Muriel Derrien3, Magnus Simren2 1. INRA MetaGenoPolis, Jouy en Josas, France. 2.Department of Internal Medicine & Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 3. Danone Nutricia Research, Avenue de la Vauve, Palaiseau, France Introduction: Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder affecting around 10-15% of the western population. Alterations in gut microbiota composition have been reported in IBS patients (Rajilić-Stojanović et al, 2011; Jeffery et al, 2012), but low sample size and heterogeneity of outcome measures may explain observed differences between studies. Material and methods: In our study, 130 subjects including 95 IBS (ROME III, all subtypes) and 35 healthy controls were thoroughly characterized for symptom severity (IBS-SSS, GSRS, VSI), psychological comorbidities (HAD, FIS, PHQ-15) and quality of life (IBSQoL). A nutrient and lactulose challenge test (Le Nevé at al AJG 2013) with symptom assessment (eight digestive symptoms and digestive comfort) and measurement of exhaled hydrogen and methane was performed in all subjects, and unprepared sigmoid colon biopsies and stool samples were obtained. Paired fecal and mucosal microbiota were then analyzed by 16S rRNA targeted pyrosequencing with QIIME v1.6 using CD-HIT to identify Operational Taxonomical Units (OTUs, 97% identity), and GreenGenes database (release version 13.8 August 2013). Microbial enterotype stratification was identified using previously described methods with the Dirichlet multinomial bayesian statistics (Holmes et al, 2012). Machine learning procedure to identify microbial IBS signature was carried out using L1 regularized logistic regression using the liblinear library (Fan et al, 2008) validated through a ten-fold independent cross-validation.

Results: 16S rRNA microbiota data complexity were reduced using a machine learning procedure into a “species-specific IBS severe signature”, consisting of 34 bacterial OTUs (species) linked to IBS severity as assessed by IBS-SSS. Notably, this specific signature is also linked to previously described enterotypes driven by Bacteroides, Prevotella and Clostridiales groups as well as exhaled CH4. High CH4 producers (> 10 ppm), Prevotella and Clostridiales enterotypes associated positively with mild and moderate IBS symptoms (IBS-SSS < 300). This IBS severity microbial signature has been further confirmed in sigmoid mucosal microbiota (n=59, AUC=0.80) and external validation stool set (n=47, AUC=0.7), allowing to discriminate severe IBS (IBS-SSS > 300) from mild/moderate IBS patients and healthy controls. Conclusions: Overall, our study indicates that IBS symptom severity is associated with a distinct signature at the fecal microbiota level. Exhaled CH4, enterotype stratification and mucosal microbiota are linked with this signature. Whether this indicates a causal role of gut microbiota alterations in the genesis of IBS remains to be investigated. Stat-038#211 Fecal microbiota transplantation in inflammatory bowel disease Marie Joossens (1,2,3), Severine Vermeire (4), Kristin Verbeke (4), Jun Wang (2,3), Kathleen Machiels (4), João Sabino (4), Marc Ferrante (4), Gert Van Assche (4), Paul Rutgeerts(4), Jeroen Raes (1,2,3) 1. Department of Microbiology, VUB, Belgium 2. KU Leuven, Laboratory of Molecular Bacteriology, Department of Microbiology and Immunology, Rega institute, 3000 Leuven, Belgium 3. VIB Center for the Biology of Disease, Belgium 4. Translational Research Center for Gastrointestinal Disorders (TARGID), University Hospitals Leuven, KU Leuven, Leuven, Belgium Introduction: Fecal microbiota transplantation (FMT) is a successful therapy for patients with refractory Clostridium difficile infections. Given role of the gut microbiota in triggering gut inflammation and the observed dysbiosis in inflammatory bowel disease (IBD), FMT has also been suggested as a treatment option. We assessed the influence of FMT on the diversity and the composition of the microbiota in IBD patients and studied predictors of (non)response to FMT in the microbial profiles of the donors and/or patients. Methods: Fourteen patients refractory to standard medical therapy for IBD (8 patients with ulcerative colitis (UC) and 6 patients with Crohn's disease (CD); 7 females, median disease duration of 18 years) consented to undergo FMT. Fecal samples of the patients were collected at baseline and 1, 2, 4, 6, 8 and 24 weeks after FMT. The baseline samples of the donors that were used to perform the FMT were also collected. After extracting the microbial DNA, the fecal microbiota composition of all samples was analyzed by 16S rRNA gene pyrosequencing. Bacterial community comparisons were done using the "Vegan" R package. Clinical efficacy of FMT was assessed by endoscopy, clinical activity scores and blood values at week 8. Results: There was no clinical improvement among the 6 CD patients week 8 after FMT. One patient with UC-like CD had a temporary clinical remission for 6 weeks with recurrence of symptoms at week 8. In contrast, 2/8 UC patients entered endoscopic remission following FMT at week 8 and of the 6 non-responding UC patients, one reported temporary remission for 6 weeks. Overall donor microbiota richness and successful transfer of Roseburia were significantly associated to treatment success in IBD. Positive C-reactive protein values, two weeks post-FMT were predictive for failure of response to FMT. Significant fever developed in 4/14 (28%) patients. Increased Acidaminococcus genus was significantly associated with post-FMT fever. Conclusion: FMT led to endoscopic and long-term (> 2 years) remission in 2/8 patients with UC. Higher baseline donor richness was associated with successful FMT. Based on our data, FMT with donor pre-screening could be a potential treatment for selected UC patients that are refractory to standard medical therapy. Stat-039#213 Changes of the human gut microbiome induced by a fermented milk product Patrick Veiga (1), Nicolas Pons (2), Anurag Agrawal (3), Raish Oozeer (1), Denis Guyonnet (1), Rémi Brazeilles (1), Jean-Michel Faurie (1), Johan E.T. van Hylckama Vlieg (1), Lesley A. Houghton (3,4), Peter J. Whorwell (3), S. Dusko Ehrlich (2,5) and Sea 1. Danone Research, Palaiseau, France 2. Metagenopolis, Institut National de la Recherche Agronomique, Jouy-en-Josas, France 3.Centre for Gastrointestinal Sciences, University of Manchester, University Hospital of South Manchester, Manchester, UK 4.Division of Gastroenterology and Hepatology, Mayo Clinic, Florida, USA 5. Centre for Human Microbiome Interactions, King's College London, London, UK The gut microbiota (GM) consists of resident commensals and transient microbes conveyed by the diet but little is known about the role of the latter on GM homeostasis. Here we show, by a conjunction of quantitative metagenomics, in silico genome reconstruction and metabolic modeling, that consumption of a fermented milk product containing dairy starters and Bifidobacterium animalis potentiates colonic short chain fatty acids production and decreases abundance of a pathobiont Bilophila wadsworthia compared to a milk product in subjects with irritable bowel syndrome (IBS, n=28). The GM changes parallel improvement of IBS state, suggesting a role of the fermented milk bacteria in gut homeostasis. Our data challenge the view that microbes ingested with food have little impact on the human GM functioning and rather provide support for beneficial health effects.

Stat-040#216 The endogenous gut microbiota dictates ecosystem permissivity to food-borne bacteria Chenhong Zhang (1), Muriel Derrien (2), Florence Levenez (1), Sonia A. Ballal (3), Jason Kim (3), Marie-Christine Degivry (2), Peggy Garault (2), Johan van Hylckama Vlieg (2), Wendy S. Garrett (3), Patrick Veiga (2,3) and Joel Doré (1) 1.Metagenopolis, Institut National de la Recherche Agronomique, Jouy-en-Josas, France 2.Danone Nutricia Research, Palaiseau, France 3. Harvard School of Public Health, Boston, MA Colonization resistance is a common function of a stable gut ecosystem. To test the implication of the resident gut microbiota in colonization resistance towards food-borne bacteria, we fed rats (n=52) a fermented milk product (FMP) containing live bacteria and quantified the fecal abundance of the FMP bacteria during and after the intervention. Repeatedly, approximatively half of the FMP-fed rats (hereafter called “permissive”) showed a slower clearance of one of the FMP-strain, Lactococcus lactis CNCM I-1631, compared to other rats (hereafter called “resistant”). 16S rDNA pyrosequencing analyses showed (p < 0.05) that the gut microbiota of permissive rats was: i) enriched in Lachnospiraceae,ii) more susceptible to FMP-induced changes and iii) less resilient. Post-hocanalyses of available human data showed similar patterns. Additionally, we demonstrated that the permissive phenotype was transferrable to germ-free rats and abolished when L. lactis CNCM I-1631 was devoid of surface proteins as shown with a sortase-negative mutant. In conclusion, we showed that a significant fraction of individuals shared unique gut microbial signatures associated with a higher susceptibility to shifts induced by food-borne bacteria. Future probiotic studies should interrogate the links between gut microbiota permissivity and the host’s susceptibility to positively respond to a probiotic intervention. Stat-041#223 Inflammatory signature in the tissue-associated microbiota in pediatric eosinophilic esophagitis. Christian Hoffmann (1,5), Alain J. Benitez (2), Amanda B. Muir (2,4), Jonathan M. Spergel (3,4), Frederic D. Bushman (1), and Mei-Lun Wang (2,4). 1. Department of Microbiology, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA. 2. Division of Gastroenterology, Hepatology and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, USA. 3. Division of Allergy and Immunology, The Children’s Hospital of Philadelphia, Philadelphia, USA. 4. Department of Pediatrics, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA. 5. Department of Food and Experimental Nutrition, School of Pharmaceutical Sciences, University of Sao Paulo, Brazil. Eosinophilic esophagitis (EoE) is an allergic disorder characterized by eosinophil-predominant esophageal inflammation, which can be ameliorated by food antigen restriction. Though recent studies suggest that changes in dietary composition may alter the distal gut microbiome, little is currently known about the impact of a restricted diet upon microbial communities of the oral and esophageal microenvironments in the context of EoE. We hypothesize that the oral and esophageal microbiomes of EoE patients are distinct from non-EoE controls, that these differences correspond to changes in esophageal inflammation, and that targeted therapeutic dietary intervention may influence community structure. Using 16S rRNA gene sequencing, we characterized the bacterial composition of the oral and esophageal microenvironments using oral swabs and esophageal biopsies from 35 non-EoE pediatric controls, and compared this cohort to samples from 33 pediatric EoE subjects studied in a longitudinal fashion before and after defined dietary changes. Firmicutes were more abundant in esophageal samples compared to oral. Proportions of bacterial communities were significantly different comparing all EoE esophageal microbiota compared to non-EoE controls, with enrichment of Proteobacteria, including Neisseria and Corynebacterium in the EoE cohort, and predominance of the Firmicutes in control subjects. We detected a statistically significant difference between actively inflamed EoE biopsies to non-EoE controls. Overall, though targeted dietary intervention did not lead to significant differences in either oral or esophageal microbiota, reintroduction of highly allergenic foods led to enrichment in Granulicatella and Campylobacter genera in the esophagus. We conclude that the esophageal microbiome in EoE is distinct from that of non-EoE controls, with maximal differences observed during active allergic inflammation. Stat-042#245 Long-Term Effects of Faecal Transplantation to the Intestinal Microbiota of Patients with recurrent Clostridium difficile infection Jonna Jalanka(1), Eero Mattila(2), Jarkko Salojärvi(1), Willem M. de Vos(1)(3)(4), Perttu Arkkila(5), Reetta Satokari(1) 1)Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland 2)Department of Infectious Diseases, Helsinki University Central Hospital, Helsinki, Finland 3) Immune Biology Research Program, Department of Bacteriology and Immunology, University of Helsinki, Helsinki, Finland 4) Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands

5)Department of Gastroenterology, Helsinki University Central Hospital, Helsinki, Finland The incidences of Clostridium difficile infections (CDI) have been rising worldwide and the infection have had the tendency to be more severe and refractory. Moreover, CDI has been shown to be the leading cause of nosocomial diarrhea worldwide and is associated with substantial morbidity. The first line of treatment is antibiotics, which significantly alter the intestinal microbiota composition and e.g. lower its capability to resist colonization of pathogens. In up to 30% of cases the CDI recurs after the antibiotics treatment. However, by reestablishing the patients’ microbial composition to resemble that of a healthy individual the recurrent infections can be resolved. Reestablishing a healthy-like intestinal microbiota can be achieved with faecal microbiota transplantation (FMT). Although this treatment has been proven to be highly effective, clearing >90% of the CDI cases, the long-term stability of the FMT-introduced microbiota has not been investigated. In the current study we aimed to investigate the how the FMT treatment affected the intestinal microbiota of the patients and how stable the reinstated microbiota remained over one year. All together 14 CDI patients received a single faecal transplant from one of the 3 healthy donors. Microbiota composition was from the pre-treatment samples and as well as from seven follow-up faecal samples in duration of one year. The intestinal microbiota composition was determined with a validated and benchmarked phylogenetic microarray, HITChip. The patients’ microbial composition differed from the healthy controls significantly before the acquiring the FMT. However, this compositional difference was abolished already 2 weeks after the treatment when the microbiota of patients resembled that of the donors. It was also shown that the microbial similarity was greater between the patient and his/hers donor than what was observed between healthy controls and moreover, this high similarity was retained for one year. The results suggest that the transplanted inoculum altered the microbiota towards a donor-like composition and that this transition was stable for at least one year. Stat-043#319 Gut dysbiosis and phage therapy in Bangladeshi children hospitalized with acute bacterial diarrhea Shafiqul Alam Sarker (1), Shamima Sultana (1), Gloria Reuteler (2), Deborah Moine (3), Patrick Descombes (3), Florence Charton (2), Gilles Bourdin (2), Shawna McCallin (2), Catherine Ngom-Bru (2), Tara Neville (2), Mahmuda Akter (1), Sayeeda Huq (1), Mich 1. International Center for Diarrheal Diseases Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212, Bangladesh 2. Nestlé Research Center, Nestec Ltd, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland 3. Nestlé Institute of Health Science, EPFL Innovation Park, CH-1015 Lausanne, Switzerland Escherichia coli accounts for a third of diarrhea cases in children from developing countries where diarrhea represents a major cause of childhood mortality. The involved E. coli strains are largely antibiotic-resistant and no specific vaccine is available. Alternative treatment methods are thus urgently needed and we explored the use of oral bacteriophages infecting E. coli as treatment option. Phage therapy has been developed in the former Soviet Unit and commercial phage preparations are commonly sold in Russian pharmacies. However, their therapeutic use has never been demonstrated in controlled clinical trials. At the International Center for Diarrheal Diseases Research in Dhaka/ Bangladesh, the world’s largest diarrhea research hospital, 120 children hospitalized with acute bacterial diarrhea were enrolled into a randomized, double-blinded, placebo-controlled clinical test to conduct a proof of concept trial. A total dose of 108 to 109 phages was given orally in divided doses over 4 days. No adverse events were noted clinically and when children were evaluated with a large panel of clinical chemistry or hematology tests that could be attributed to oral phage. Neither an oral T4-like phage cocktail developed at our research center nor a commercial Russian E. coli phage cocktail led to an amelioration of quantitative diarrhea parameters in the patients. Fecal phage excretion, but no substantial intestinal phage amplification was observed. Daily stool samples were collected and the microbiota composition was determined by 16S rRNA gene sequencing. On the average E. coli represented less than 5 % of the total fecal bacteria. Surprisingly, their titer development did not systematically decrease with recovery from diarrhea. However, we observed a marked dysbiosis in the acute stool samples showing in comparison with healthy control children an increased proportion of streptococci and decreased proportion of bifidobacteria. The proportion of bifidobacteria gradually normalized with resolution of diarrhea. The fecal streptococci belonged to two closely related species complexes: Streptococcus bovis and S. salivarius. Genome sequencing of Streptococcus bovis isolates from five patients identified S. bovis, S. gallolyticus, S. infantarius and S. pasteurianus lacking known virulence genes. Apparently, streptococci experienced a growth advantage in the diarrhea-damaged gut. The data raise doubts about the value of phage therapy of E. coli diarrhea. Corrections of the streptococcal/bifidobacterial dysbiosis in diarrhea children with nutritional interventions might represent a more promising alternative approach. Stat-044#270 The Gut Flora Changes under Immunosuppression after Liver Transplantation in Rats Jianwen Jiang, M.D., Zhigang Ren, M.S., Xinhua Chen, M.D., Lin Zhou, Ph.D. and Shusen Zheng, Ph.D.,M.D.. Department of Hepatobiliary Pancreatic Surgery, Key Lab of Combined Multi-organ Transplantation, Ministry of Public Health; First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang province, China, 310003. Objective: To investigate the gut flora changes under immuopsuppression after liver transplantation（LT） in rats .

METHODS: The whole experiment was divided into three groups: (1) Normal group (n = 12): normal BN rats ; (2) SGT group (syngeneic transplantation of BN-BN, n = 12): both donors and recipients were BN rats; and (3) AGT group (allogeneic transplantation of Lewis-BN, n = 12): Donors were Lewis and recipients were BN rats. In the AGT group, all recipients were subcutaneously injected by Cyclosporin A after LT. Survival time was observed ,All the dying rats were sampled, Twenty-one d after LT, 8 rats were selected randomly in each group for sampling. the plasma endotoxin, cytokines , and faeces flota were analyzed. RESULTS: all rats in each group were still alive in the next 2 week. On 21 d after LT, compared with the normal group and SGT group , plasma endotoxin in the AGT group was remarkably increased. Plasma tumor necrosis factor-α and interleukin-6 were also significantly elevated in the AGT group vs the normal and SGT groups. Furthermore, The analysis of intestinal microflora was performed. Compared to the normal group and SGT group , the numbers of Enterococcus and Enterobacteria in the AGT group were significantly increased. Meanwhile, compared to the normal group, the numbers of Bifidobacterium and Lactobacillus in the AGT group were remarkably reduced . CONCLUSION: The numbers of Enterococcus and Enterobacteria significantly increased and Bifidobacterium and Lactobacillus significantly decreased under immunosuppression after liver transplantation in rats, which maybe related with infection and intestinal barrier function injury after LT. Stat-045#271 The Establishment of Operational Immune Tolerance Model of Liver Transplantation and Gut Flora Investigation in Rats Yixin Zhu(1), M.D., Jianwen Jiang(2), M.D., Lin Zhou(2), Ph.D., ShuSen Zheng(2), Ph.D.,M.D.，Lanjuan Li(1)，MD. 1:State Key Laboratory for Diagnosis and Treatment of Infectious Diseases; 2:Department of Hepatobiliary Pancreatic Surgery, Key Lab of Combined Multi-organ Transplantation, Ministry of Public Health; First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang province, China, 310003. Objective: It was found that gut flora changed when liver graft suffers acute rejection, infection or chronic graft dysfunction after liver transplantation(LT). the immune tolerance is the ideal state for LT,we want to establish LT operational immune tolerance model in rats and investigate gut flora change. Methods: Donors were inbred Lewis （n=24 ）and recipients were inbred BN rats（n= 36）.the experiment was divided into three groups(n= 12): (1) normal rats (N); (2) allogeneic transplant group (AGT of Lewis-BN,n= 12); (3) Cyclosporin treatment group (CsA of LEW-BN,n= 12): In the CsA group, all rats were subcutaneously injected by CsA at 1 mg/kg daily for 30 d, and at 2 mg/kg daily for the next 100 d after LT，then removal of CsA. Survival time was observed for 2 year. All the rats were sampled faeces for gut flora analysis by real-time fluorescent quantitative PCR (RT-PCR). Results: 12 normal BN rats survived more than 2 years, 12 rats die of acute rejection in AGT group on 35-56 days after LT, in the CsA group, 8 rats died of liver necrosis(n=2),infection(n=2) and chronic biliary obstruction hyperplasia (n=4), after removal of CsA, 4 rats live more than 2 years with normal plasm alanine aminotransferase (ALT) and aspartate aminotransferase (AST),the operational immune tolerance（OPT） LT model was established, the gut flora investigation by RT-PCR showed that:for the ten species of bacteria in the stool including Bacteroides,Enterococcus,enterobacter,Lactobacillus,lactic acid bacteria,Faecalibacteriumprausnitzii,Clostridium，butyricum,Clostridium,Clostridiumtwo,Bifidobacterium，there were no difference between the OPT Rats after LT and long-term healthy rats. Conclusion: The operational immune tolerance model of LT in rats could be established, for the ten gut flora,there were no difference between the OPT rats after LT and long-term healthy rats. This finding is worthy of clinical doctor and patients of LT for reference. Stat-046#316 MyNewGut Project: Understanding the role of the gut microbiome in nutrient metabolism and metabolic health Alfonso Benítez-Páez (1), Kevin Portune (1), Eva Maria Gómez-del-Pulgar (1), Lesli Hingstrup-Larsen (2), Francoise Blachier (3), Max Nieuwdorp (4), Patrizia Brigidi (5), Yolanda Sanz (1). 1. Microbial Ecology, Nutrition and Health Laboratory. Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), C/ Catedràtic Agustín Escardino 7, 46980 Paterna-Valencia, Spain. 2. Department of Nutrition, Exercise and Sports (NEXS). University of Copenhagen. Rolighedsvej 26, DK-1958 Frederiksberg C, Denmark. 3. French National Institute for Agricultural Research - INRA-AgroParisTech. Physiologie de la Nutrition et du Comportement Alimentaire. 16 rue Claude Bernard, 75005 Paris, France. 4. Department of Internal Medicine, Academic Medical Center – AMC. Meibergdreef 9, room F4-159.2, 1105, AZ Amsterdam, The Netherlands. 5. Laboratory of Microbial Ecology of Health. Department of Pharmacy and Biotechnology. University of Bologna. Via Belmeloro 6, 40126 Bologna, Italy. The MyNewGut Project, (Microbiome influences on energy balance and brain development/function put into action to tackle diet-related diseases and behaviour), which receives funding from the European Union’s Seventh Framework Programme, will research the composite lifestyle factors that influence the human gut microbiota and its genome (microbiome) and their impact on diet-related disorders, such as obesity and the associated co-morbidities. One of the specific objectives of the project is to

progress in the identification of the consortium of bacteria and pathways involved in nutrient metabolism and, thereby,in energy balance in humans. The role of the microbiota in the breakdown of indigestible carbohydrates is the best characterized to date but controversy exists about the physiological consequences of their fermentation and the specific role of the generated products (e.g. short-chain fatty acids). Gut microbiota is also likely to participate in the catabolism of peptides and amino acids and in the utilization of ammonium for amino acid biosynthesis. The gut microbiota could also participate indirectly in lipid metabolism by interfering with the enterohepatic circulation of bile acids and directly by utilizing dietary fat that escapes digestion as reflected in metagenomics studies. Nevertheless, the understanding of the role of the microbiota in protein and lipid metabolism and their physiological consequences is by far more limited. To progress in the understanding of diet-gut microbiota interactions, we will develop a multidisciplinary research approach, using functional omics-technologies and systems biology, in well-controlled human trials. This information will be used to develop microbiome-based dietary recommendations and interventions to ultimately reduce the socioeconomic burden of diet-related disorders in the EU. #326 Short Talk Phase I trial on FMT in autistic children Lara Hudy (1), Chaysavanh Manichanh (2), Alba Santiago (2), Marta Pozuelo (2), David Campos (2), Yeire Pezzotti (1), Beatriz Cordova (1), Yasel Lee (1), Manuel Abood (1), Francisco Guarner (2), Dolly de Motta (1) 1. Biomed Therapeutic, Panama City, Panama 2. Digestive System Research Unit, Vall d’Hebron Research Institute, Barcelona, Spain BACKGROUND Dysfunction of the microbiome-brain-gut axis has been implicated in neurodevelopmental disorders such as autism. Indeed, experiments with germ-free animals showed an increase anxiety-like behavior, which was then compensated by microbial colonization. We aimed to modulate the gut microbiome dysbiosis by a fecal transplant in autistic children and evaluate clinical outcome. METHODS Sixteen children (10 males) with moderate to severe autistic disorders, and average age of 5.7 year (1.8-13; SD=2.6), were transplanted with freshly diluted fecal specimen from a single donor by duodenal infusion. We collected two sequential fecal samples at baseline (inclusion and after 4 weeks running period), and two samples after transplantation (at 1 and 6 months). Sixty-eight samples were submitted to Illumina sequencing of 16S rDNA gene, generating around 3 millions sequence reads, to compare compared microbial community composition between patients, donor and 12 healthy controls. RESULTS At baseline, autistic children showed changes in microbiome composition compared to healthy controls with a tendency to have higher proportion of porphyromonadaceae and enterobacteriaceae (combined P=0.003; FDR=0.1). Microbial diversity of the patients significantly increased boht at 1 and 5 months after FMT (P<0.05). Microbial community samples clustered with those of the donor up to five months after FMT. Furthermore, patients showed similar microbial community composition to healthy controls at 1 and 5 months post-transplant. All but one patient improved neurological scores such as comprehension (P=0.003) and verbal communication (P=0.009). No significant side effects were reported. CONCLUSION Our findings demonstrate for the first time a long-term modulation of the gut microbiota of autistic children by FMT leading to improvement of their clinical condition, paving the way to larger cohort trials. Stat-047#335 The Present Situation and Prospect of Research on Intestinal Microecology Hua Guo M.D.,Jianwen Jiang, M.D., Weilin Wang, M.D. and Shusen Zheng, Ph.D.,M.D.. Department of Hepatobiliary Pancreatic Surgery, Key Lab of Combined Multi-organ Transplantation, Ministry of Public Health; First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang province, China, 310003. Intestinal microflora is closely related to human health and disease. In the past 10 years, Nature, Science, Cell and other famous magazines have published a number of articles on Intestinal microflora, they thought it equivalent to an important “ organ” . The intestinal microflora and host have the symbiosis and coevolution relationship, plays an important role in regulating host digestion absorption, immune response, metabolism; intestinal microecological imbalance closely related to many human diseases such as infectious diseases, obesity, diabetes, liver disease, coronary heart disease and tumor and other chronic diseases. From the international research view of intestinal flora , the exploration of the relationship between the development of many chronic diseases，infectious diseases and intestinal microecology, the early diagnosis of various diseases from the angle of intestinal microbial biomarkers and potential targets to treat various diseases in view of intestinal microecology for drugs, these findings will present a significant progress on the treatment of serious infections and a variety of chronic diseases . In 2001, professor Li Lanjuan first put forward the infection microecology theory. this theory from the view of overall function of the health body, pay attention to the maintenance of human microecology balance , advocate the reasonable application of antibiotics, protect organ function, and in 2002 professor Li published the first book on "infection microecology”. In recent years, more and more evidence shows that the alteration of intestinal microbiota are closely related to the occurrence and development of obesity, diabetes mellitus. Because the physiology of liver and intestinal special anatomic relationship with

microorganisms through liver- bowel axis, and microbiota-liver axis, intestinal microbiota play an important role in the liver injury, chronic inflammation, fibrosis and tumor development. the disturbance of gut microbiota promote the progression of hepatocellular carcinoma. Darnaud M proposed the scientific ideas on targeted intestinal micro ecological treatment may prevent hepatocellular carcinoma progression，it is one idea worthy of further study . Although the international research on intestinal microecology in the ascendant, the present study on intestinal microecology in the development of a variety of diseases is still in the primary stage, there are a lot of key scientific and technological problems need further exploration and research. Intestinal microecological imbalance and infectious diseases and a variety of major chronic diseases and the interaction between the mechanism has become an international research trends. With metagenomics, genomics,transcriptomics, proteomics, metabolomics technology rapid development, the relationship and the mechanism of intestinal micro ecological imbalance and these diseases are expected to be in-depth study. Stat-048#342 Cyclooxygenase-2 Gene Polymorphisms And Susceptibility To Hepatocellular Carcinoma: A Meta-Analysis Based On 10 Case-Control Studies Wei Xu(1), Yaping Huang(1), Ting Zhang(1), Rong Yang(1), Xutao Hong(2), Lingyun Zhao(2), Jun Fan(1), Lanjuan Li(1) 1.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Medical College, Zhejiang University, Hangzhou, China 2.Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang , China Objective: The association between cyclooxygenase-2 (COX-2) gene polymorphisms and hepatocellular carcinoma (HCC) has been widely reported, but the results are still controversial. To clarify the effect of COX-2 -1195G/A (rs689466), -765G/C (rs20417) and +8473T/C (rs5275) polymorphisms on HCC risk, a meta-analysis was performed. Methods: The PubMed, Embase, Cochrane Library, Web of Science, Chinese BioMedical Literature (CBM), Wanfang and Chinese National Knowledge Infrastructure (CNKI) databases were systematically searched to identify potential studies published up to October 10, 2014. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the strength of association. Results: A total of 8 studies with 2060 HCC cases and 2610 controls for -1195G/A, 6 studies with 1295 cases and 2193 controls for -765G/C, and 4 studies with 1477 cases and 1747 controls for +8473T/C were included in this meta-analysis. Overall, the COX-2 -1195G/A and +8473T/C polymorphisms were both significantly associated with an increased risk of HCC (rs689466 GA+AA vs. GG: OR=1.390, P=0.006, 95% CI: 1.099-1.759, I2=50.7%, Pheterogeneity=0.048; rs5275 CC vs. TT+TC: OR=1.484, P=0.041, 95% CI: 1.017-2.165, I2=0.0%, Pheterogeneity=0.416). In the subgroup analyses stratified by ethnicity, the COX-2 -1195G/A, -765G/C, and +8473T/C were all associated with an increased HCC risk in Asian populations (rs689466 A vs. G: OR=1.346, P=0.001, 95% CI: 1.137-1.595, I2=0.0%, Pheterogeneity=0.869; rs20417 CC vs. GG+GC: OR=3.069, P=0.013, 95% CI: 1.265-7.447; rs5275 CC vs. TT+TC: OR=1.626, P=0.020, 95% CI: 1.079-2.452, I2=0.0%, Pheterogeneity=0.495). Conclusions: Our meta-analysis suggests that -1195G/A, -765G/C, and +8473T/C in COX-2 may contribute significantly to HCC risk. Stat-049#235 Microbiome analysis of the gut microbiota Kazakhstan women Almagul Kushugulova (1), Samat Kozhakhmetov (1), Indira Tynybayeva (1), Saule Saduakhassova, Gulnara Shakhabayeva, Zhanagul Khassenbekova (1), Talgat Nurgozhin (1), Zhaxybay Zhumadilov (1). 1()Center for Life Sciences Background. Microbial community consists of hundreds of forms of microorganisms. This "organ" has a high metabolic capability; it is involved in protection against pathogens, modulation of gastrointestinal development. Normal enterobacterial flora affects the number of functions and plays a key role in nutrition, maintenance of integrity of the epithelial barrier, and the development of parietal immunity in human metabolism. The gut microflora may change depending on different factors, including diet, age, gender, geographic location, health condition, etc. In this study a question about the age influence on the gut microflora composition of women is considered. Methods. Collection of samples is in accordance with the protocol HMP-07-001 Core Microbiome Sampling Protocol A. Subjects are issued leaflets and informed consent on the procedure, screening and collection of samples. Subjects are also given questionnaires of the basic health indicators. Preliminary questionnaire is scheduled prior screening to cut potentially inappropriate subjects. During the implementation phase subjects of different age groups were selected according to WHO age classification: young (up to 44), mean age (45-59), elderly age 60-74, long-lived (90 years and older). Isolation of total DNA was performed using protocol from Furet J-P (2009). To determine the composition of the microflora was used to amplification a fragment of the gene 16S rRNA. Plasmid library with target insertion were obtained on the basis of hight copy plasmid vectors produces high pGem-T. The definition of direct nucleotide sequence was performed by the method of Sanger using a set of "BigDye Terminanor v 3.1 Cycle sequencing Kit with automatic genetic analyzer ABI 3730xl (Applied Biosystems, USA). Results. According to the sample analysis two precise types of the gut microbiome were found with significant differences: in the first case Bacteroidetes type prevailed, and in the second case – Firmicutes type. Percentage differences of the specified taxonomic groups were insignificant in the majority of the samples.

It was found that detected types of gut microbiota composition do not depend on the age factor. Nevertheless, it is necessary to note that such taxonomic groups as Fusobacteria, Chlamydiae, Lentisphaerae, Tenericutes were found only in the group older than 90 years old. Conclusions. In general, these results support that microbiome includes different types, in our case types Bacteroidetes and Firmicutes were detected. The study also demonstrates that the age factor influences insignificantly on gut microbial composition. It is necessary to conduct further studies for more detailed understanding of intestinal microbiota composition in healthy Kazakhstani population.

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