asm conferences 2012

4th ASM Conference on

Beneficial Microbes

October 22 – 26, 2012San Antonio, Texas

Final P

rogram and A

bstracts

© 2012, American Society for Microbiology1752 N Street, N.W.

Washington, DC 20036-2904Phone: 202-737-3600

World Wide Web: www.asm.org

All Rights ReservedPrinted in the United States of America

ISBN: 978-1-55581-887-6

14th ASM Conference on Beneficial Microbes

Table of Contents

ASM Conferences Information ...................................... 2

Conference Organization ............................................... 3

Acknowledgments .......................................................... 3

General Information ....................................................... 4

Travel Grants .................................................................. 5

Scientific Program .......................................................... 6

Abstracts for Speakers ................................................. 12

Abstracts for Posters .................................................... 31

Index .......................................................................... 112

ASM Conferences2

William Goldman, ChairUniversity of North Carolina, Chapel Hill

Sean Whelan, Vice ChairHarvard Medical School

Victor DiRitaUniversity of Michigan

Joanna GoldbergUniversity of Virginia

ASM Conferences CommitteeLora HooperUniversity of Texas Southwestern Medical Center

Christine Jacobs-WagnerYale University

Gary Procop Cleveland Clinic

Curtis SuttleUniversity of British Columbia

Statement of PurposeThe purpose of the ASM Conferences program is to address the needs of

the diverse scientific interests of microbiologists by providing a forum for

international groups of scientists, ranging from 100-400 participants, to

discuss their specific area of concentration.


Conference OrganizersPrincipal Organizers:Joerg GrafUniversity of Connecticut

Janet K. JanssonLawrence Berkeley National Laboratory

Co-organizers:Heidi Goodrich-BlairUniversity of Wisconsin

Lora HooperUniversity of Texas Southwestern Medical Center

Robert KnightUniversity of Colorado at Boulder

Spencer NyholmUniversity of Connecticut

Ned RubyUniversity of Wisconsin

Justin SonnenburgStanford University

Acknowledgements

The Conference Organizers and the American Society for Microbiology would like to acknowledge the following for their financial contributions to this conference:

Burroughs Wellcome Fund

MO BIO Laboratories, Inc.

National Science Foundation

NIGMS, National Institutes of Health*

*Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R13GM103179. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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General Information

GENERAL SESSIONS All general sessions will be held in the Regency Ballroom at the Hyatt Regency San Antonio. A name badge is required for entry into all sessions, meals, and social events. ASM staff will be available during the sessions.

POSTER SESSIONSPoster boards are located in the Regency Ballroom at the Hyatt Regency San Antonio. All posters are displayed for the entire conference. Please check your assigned number in the abstract index. The same number is used for the presentation and board number. Odd-numbered posters will be formally presented in the A session on Tuesday, October 23, and even-numbered posters will be presented in the B session on Thursday, October 25. The posters are to be mounted by the morning coffee break on Tuesday, and should be removed at the end of the poster session on Thursday. The poster area will be open for informal viewing throughout the conference.

CERTIfICATE Of ATTENdANCECertificates of Attendance can be found in the registration packet received at the registration desk. Certificates of Attendance do not list session information.

CAMERAS ANd RECORdINGSDigital recorders, cameras (including camera phones) and video cameras (including video phones) are prohibited in the poster hall and general sessions. Anyone found photographing, videotaping or recording in the prohibited areas will be asked to surrender their badge immediately and leave the conference. No refund will be provided. This rule is strictly enforced.

CHILd POLICYChildren are not permitted in session rooms, poster sessions, conference meals or social events. Please contact the hotel concierge to arrange for babysitting services in your hotel room.

GuEST REGISTRATIONAs noted in the program schedule, certain meals and social events are included in the registration fee for conference participants. Registered participants may also purchase tickets for an accompanying guest (age 16 and older) to attend the welcome reception for an additional fee of $50 and/or the conference dinner party for a fee of $100. Guests are not permitted in the general sessions or poster sessions, lunches or coffee breaks. Guests must present their registration badge for entrance to the welcome reception. Non-registered guests are not permitted to attend any part of the conference or social events.


Travel Grants

STudENT TRAVEL GRANTSASM encourages the participation of graduate students and new postdocs at ASM Conferences. To support the cost of attending the conference, ASM has awarded travel grants of $500 to each of the following individuals:

Allison Banse

Javad Barouei

Roxanne Beinart

Michael Bois

John Brooks

Kristen Earle

Jessica Ferreyra

Silke Heinzmann

Rosanne Hertzberger

Nicole Horne

Zehra Ilhan

Hanne Jensen

Sara Jones

Kevin Kohl

In Hyuk Kwon

Sarah-Lynn Martz

Brittany Ott

Vanessa Ridaura

Jon Sanders

Amy Sheflin

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Scientific Program

Monday, October 22, 2012

4:15 – 5:30 pm Opening SessionRegency Ballroom Conference Welcome and Opening Comments Joerg Graf

Keynote Lecture: Microbiome-wide Association Studies for Hunting Down the

Obesity Bugs Liping Zhao; Shanghai Jiao Tong University, Shanghai,

CHINA.

5:30 – 7:00 pm Welcome MixerRio Grande Ballroom

Tuesday, October 23, 2012

8:30 – 11:30 am Session 1: Analysis of the Human MicrobiomeRegency Ballroom Convener: Rob Knight

8:30 – 9:15 am Plenary Lecture: Placing the Human Microbiome in Context Rob Knight; University of Colorado, Boulder, CO.

9:15 – 9:30 am Metagenomic and Biochemical Analysis of Human Oral Bacterial Biofilms Reveals Bacterial Communities that Contribute Nitric Oxide to the Host

Embriette Alicki, Baylor College of Medicine, Houston, TX.

9:30 – 10:00 am Getting More out of Your Microbiome: Computational Tools for Gut Microbiota Data Analysis

Jeroen Raes; VIB, Brussels, BELGIUM.

10:00 – 10:30 am Coffee Break

10:30 – 10:45 am Microbiota-liberated Host Sugars Facilitate Post-antibiotic Emergence of Enteric Pathogens

Jessica Ferreyra; Stanford University, Stanford, CA.


10:45 – 11:15 am Computational Methods for Meta’omic Microbial Community Characterization

Curtis Huttenhower; Harvard School of Public Health, Boston, MA.

11:15 – 11:30 am Cultivars from Microoxic Conditions Disproportionately Occur in the Intestinal Mucosa and Represent a New Source of Beneficial Microbes

Dongjuan Dai; Michigan State University, East Lansing, MI.

11:30 am – 1:00 pm LunchRio Grande Ballroom

1:00 – 4:45 pm Session 2: Beneficial Microbes in the EnvironmentRegency Ballroom Convener: Heidi Goodrich-Blair

1:00 – 1:45 pm Plenary Lecture: Environmental Importance of the Nematode Symbiosis for Pest Control

Heidi Goodrich-Blair; University of Wisconsin, Madison, WI.

1:45 – 2:00 pm Dynamics and Probiotic Effect of the Egg-associated Microbial Community of Lake Sturgeon

Terence Marsh; Michigan State University, East Lansing, MI.

2:00 – 2:30 pm Fungal Endophytes as Epigenetic Drivers of Plant Adaptation Rusty Rodriguez; United States Geological Survey, Seattle,

WA.

2:30 – 2:45 pm Soil Microbial Diversity Differentially Influences Invasion by Pathogenic and Beneficial Microorganisms

Amy Sheflin; Colorado State University, Fort Collins, CO. 2:45 – 3:15 pm Coffee Break

3:15 – 3:45 pm Bacterial Regulation of Colony Development in the Closest Living Relatives of Animals

Nicole King; University of California, Berkeley, Berkeley, CA.

3:45 – 4:00 pm Gut Microbes Facilitate Consumption of Toxic Diets by Herbivores

Kevin Kohl; University of Utah, Salt Lake City, UT.

Scientific Program

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Scientific Program

4:00 – 4:45 pm Plenary Lecture: Host-microbe Interactions in Hydra Thomas Bosch; Kiel University, Kiel, GERMANY.

4:45 – 7:15 pm Poster Session ARegency Ballroom

Wednesday, October 24, 2012

8:30 – 11:15 am Session 3: Beneficial Microbe-Immune System InteractionsRegency Ballroom Convener: Lora Hooper

8:30 – 9:00 am Identification and Temporal Expression Patterns of Innate Immunity Genes in the Medicinal Leech, Hirudo verbena

Lindsey Bomar; University of Connecticut, Storrs, CT.

9:00 – 9:15 am Bacillus subtilis Exopolysaccharides Prevent Colitis via TLR-dependent Signaling

Sara Jones; Loyola University Medical Center, Maywood, IL.

9:15 – 9:45 am Why You Might Not Want to Starve a Fever if You are a Fly Mimi Shirasu-Hiza; Columbia University, New York, NY. 9:45 – 10:15 am Coffee Break

10:15 – 10:30 am Systems Analysis of Antibiotic-associated Defects in Gut Immunity and Microbiota

Andriy Morgun; Oregon State University, Corvallis, OR.

10:30 – 11:00 am Understanding the Cellular Innate Immune Response and Specificity in the Association Between the Hawaiian Bobtail Squid Euprymna scolopes and the Bioluminescent Bacterium Vibrio fischeri

Spencer Nyholm; University of Connecticut, Storrs, CT.

11:00 – 11:15 am Obligate Symbionts Activate Immune System Development in the Tsetse Fly

Brian Weiss; Yale University School of Medicine, New Haven, CT.


Afternoon off for Informal Interactions


Thursday, October 25, 2012

8:30 – 11:30 am Session 4: Inter-kingdom SignalingRegency Ballroom Convener: Spencer Nyholm

8:30 – 9:15 am Plenary Lecture: Sensing of Microbial and Nutritional Signals by the Intestinal Epithelium

Lora Hooper; University of Texas Southwestern Medical Center, Dallas, TX.

9:15 – 9:30 am Analysis of Trans-kingdom Signalling of Bacterial Small Molecules with Eukaryotes using Highly Resolving Metabolite Analysis and Immunological in vitro-test Systems

Anton Hartmann; Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Oberschleissheim, GERMANY.

9:30 – 10:00 am The Simple, Yet Tangled, Tsetse Microbial Community Rita Rio; West Virginia University, Morgantown, WV.


10:30 – 10:45 am Nighty Night: How Photorhabdus luminescens Turns off the Light to Persist in Host Nematode Intestines

Todd Ciche; Michigan State University, East Lansing, MI.

10:45 – 11:15 am Interkingdom Interactions Facilitate the Fitness of Bacteria on Plants

Steven Lindow; University of California, Berkeley, CA.

11:15 – 11:30 am Microbial Regulation of Intestinal Epithelial Cell Proliferation Allison Banse; University of Oregon, Eugene, OR.


1:00 – 4:30 pm Session 5: Role of Beneficial Microbes in Health and Regency Ballroom Disease Convener: Joerg Graf

1:00 – 1:45 pm Plenary Lecture: Sponge-Microbial Symbioses in Health and Disease

Ute Hentschel Humeida; University of Würzburg, Würzburg, GERMANY.

Scientific Program

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1:45 – 2:00 pm Understanding the Contributions of a Cultured Human Fecal Microbiota to Adiposity in Gnotobiotic Mice

Vanessa Ridaura; Washington University in St. Louis, St. Louis, MO.

2:00 – 2:30 pm The Gut Microbiota of the Healthy Honey Bee Nancy Moran; Yale University, New Haven, CT.

2:30 – 3:00 pm Coffee Break

3:00 – 3:30 pm Functional Analysis of the Human Microbiome Using Community Proteomics or Proteogenomics

Nathan VerBerkmoes; New England Biolabs, Ipswich, MA.

3:30 – 3:45 pm Maternal Probiotic Intervention Protects Against Immune Alterations And Gut Dysfunctions In The Maternally Separated Rat Model Of Irritable Bowel Syndrome

Javad Barouei; The University of Newcastle, Newcastle, AUSTRALIA.

3:45 – 4:15 pm Microbiota: Do you Have the Best Team Working for You? Benjamin Willing; University of British Columbia, Vancouver,

BC, CANADA.

4:15 – 4:30 pm Investigating the Systemic and Gut-specific Mouse Metabolome by Non-targeted Metabolite Profiling

Silke Heinzmann; HelmholtzZentrum Muenchen, Munich, GERMANY.

4:30 – 7:00 pm Poster Session BRegency Ballroom

7:30 – 10:30 pm Conference Dinner PartyRio Grande Ballroom

Scientific Program


friday, October 26, 2012

8:30 – 11:45 am Session 6: Systems Biology of Symbiotic SystemsRegency Ballroom Convener: Edward Ruby

8:30 – 9:15 am Plenary Lecture: A Common Polysaccharide Impacts the Squid-vibrio Symbiosis at Many Levels

Edward Ruby; University of Wisconsin, Madison, WI.

9:15 – 9:30 am Differences in Energy and Nitrogen Metabolism Among Symbionts of Hydrothermal Vent Gastropods Relates to Geochemical Niche

Roxanne Beinart; Harvard University, Cambridge, MA.

9:30 – 10:00 am As Simple as Possible, but No Simpler: The Multi-Organismal Basis of Animal Nutrition

Angela Douglas; Cornell University, Ithaca, NY.


10:30 – 10:45 am Conservation of small RNA expression in reduced genomes of the obligate symbiont Buchnera

Allison Hansen; Yale University, West Haven, CT.

10:45 – 11:15 am New Tools for Exploring the Human Gut Microbiome Andy Goodman; Yale University, New Haven, CT.

11:15 – 11:30 am Detecting Codiversification in an Insect Gut Microbiome Jon Sanders; Harvard University, Cambridge, MA.

11:30 – 11:45 am Conference Closing Remarks Janet Jansson

Scientific Program

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n KS1MICRObIOME-WIdE ASSOCIATION STudIES fOR HuNTING dOWN THE ObESITY buGS

L. Zhao; Shanghai Jiao Tong University, Shanghai, CHINA.

n S1:1PLACING THE HuMAN MICRObIOME IN CONTExT

R. Knight; Chemistry and Biochemistry, Univ. of Colorado, Boulder, CO.

The human microbiome is an extraordinarily diverse ecosystem, especially in the gut. But how does this diversity compare with what is observed in other environments? The avail-ability of new standards for annotating data, together with large-scale projects such as the Human Microbiome Project and the Earth Microbiome Project that collect large datasets using consistent methodologies allow us to place the human microbiome in the context of what is observed in other mammals, other spe-cies, and environmental samples as a whole. Network analyses allow us to understand what is unique and what is shared in each lineage, and how differences in health and disease compare in effect size to inter-individual and inter-population variability, technical vari-ability and to cross-species differences. We are therefore poised to gain a unified perspective on the impact of microbes on human, animal and environmental health.

n S1:2METAGENOMIC ANd bIOCHEMICAL ANALYSIS Of HuMAN ORAL bACTERIAL bIOfILMS REVEALS bACTERIAL COMMuNITIES THAT CONTRIbuTE NITRIC OxIdE TO THE HOST

E. R. Alicki1, F. Andrade2, H. B. Kaplan2, G. Tribble2, N. S. Bryan3, J. F. Petrosino1; 1Alkek Center for Metagenomics and Microbiome

Speaker AbstractsResearch, Baylor College of Medicine, Hous-ton, TX, 2University of Texas Health Science Center-Houston, Houston, TX, 3Texas Thera-peutics Institute, Brown Foundation Institute of Molecular Medicine at the University of Texas Health Science Center-Houston, Houston, TX.

Nitric oxide (NO) plays a critical role in vari-ous physiological processes, including vascular homeostasis and host defense mechanisms. In mammals, NO synthases generate endogenous NO from the amino acid L-arginine and oxy-gen. Thus, NO production is inhibited when oxygen is absent, such as during hypoxia or ischemia. The nitrate-nitrite-NO pathway for NO production is an oxygen-independent pathway that has gained recent attention as an alternative pathway to NO production. The first step of this pathway is the reduction of dietary nitrate to nitrite through the action of oral bacterial nitrate reductases. As humans cannot reduce nitrate, the oral microbiota appears to function in a symbiosis with the host that could be exploited to improve human health. The aim of this study was to dissect the nitrate reducing capacity of oral bacterial communities through biochemical and metagenomic analyses with the goal of optimizing nitrate reduction to improve NO production in the host. Tongue scrapings from six healthy volunteers were inoculated onto poly-methyl-methacrylate (PMMA) biofilm discs in biofilm medium and incubated anaerobically. Spent medium was collected every 24 hours for 96 hours and me-dium nitrate levels measured via HPLC. One PMMA disc was also collected at each time point for bacterial DNA extraction for metage-nomic analysis. Based on HPLC results, samples were divided into three groups: best, intermediate, and worst nitrate reducers. 16S pyrosequencing identified specific genera that were significantly more abundant in the best nitrate reducing samples compared to the worst samples. To provide species-level classification and gene detection, whole genome shotgun (WGS) sequencing was performed on a subset


of samples. Interestingly, although many of the candidate species identified through WGS are known oral nitrate reducers, other taxa not previously associated with oral nitrate reduc-tion or whose genomes do not encode nitrate reductase genes were identified. These results suggest that the community dynamics leading to efficient nitrate reduction are complex, with non-nitrate reducers likely acting as “helper” species to enhance nitrate reduction by other bacteria. Supporting this hypothesis, four oral species were isolated and grown individu-ally or as a consortium and quantification of nitrate reduction revealed that although the consortium was proficient at reducing nitrate, two species were efficient nitrate reducers and two were poor nitrate reducers. Metatranscrip-tomics studies to further dissect the complex inter-bacterial interactions leading to nitrate reduction are pending. Together, these studies have enabled us to begin to dissect the com-plex oral bacterial interactions leading to ni-trate reduction and indicate that bacterial con-sortiums optimized for nitrate reduction with in vivo probiotic potential can be generated.

n S1:3GETTING MORE OuT Of YOuR MICRObIOME: COMPuTATIONAL TOOLS fOR GuT MICRObIOTA dATA ANALYSIS

J. Raes; VIB, Brussels, BELGIUM.

n S1:4MICRObIOTA-LIbERATEd HOST SuGARS fACILITATE POST-ANTIbIOTIC EMERGENCE Of ENTERIC PATHOGENS

J. A. Ferreyra, K. M. Ng, J. L. Sonnen-burg; Stanford University, Stanford, CA.

The human intestine, colonized by a dense community of resident microbes, is a frequent target of bacterial pathogens. Undisturbed, this intestinal microbiota provides protection from bacterial infections, and disruption of the microbiota with oral antibiotics often precedes the emergence of several enteric pathogens.

The mechanisms used by pathogens to expand and capitalize upon the post-antibiotic dis-turbed intestinal environment remain unclear. To untangle the complex microbiota-pathogen-host interactions that lead to antibiotic-associ-ated pathogen emergence, we have colonized germ-free mice with antibiotic-associated pathogens in the presence and absence of model human gut symbionts, such as Bacte-roides thetaiotaomicron. We show that two antibiotic-associated pathogens, Clostridium difficile and Salmonella typhimurium, employ a common strategy of consuming microbiota-liberated host mucosal carbohydrates during their emergence within the gut. Our data reveal that antibiotic-induced disruption of the resi-dent microbiota and subsequent alteration in mucosal carbohydrate availability are similarly exploited by these two distantly related enteric pathogens. Our data offer mechanistic insight into the selective forces that have shaped en-teric pathogen adaptation to antibiotic-induced vulnerability of the gut ecosystem. These findings suggest new therapeutic approaches for preventing diseases caused by antibiotic-associated pathogens.

n S1:5COMPuTATIONAL METHOdS fOR META’OMIC MICRObIAL COMMuNITY CHARACTERIzATION

C. Huttenhower; Harvard School of Public Health, Boston, MA.

Linking large-scale diversity to microbial and biomolecular details is key to understanding metagenomes, metatranscriptomes, and the human microbiome. An important resource for doing so is the catalog of thousands of se-quenced microbial isolates and their millions of annotated, and often characterized, genes. I will discuss the PhlAn tools for phylogenetic analysis, which leverage information from these genomes to better characterize microbi-omes. These include MetaPhlAn, which uses hundreds of taxonomically unique marker sequences to quantify specific species and

Speaker Abstracts

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subspecies within communities; PhyloPhlAn, which uses hundreds of ubiquitous markers for phylogenetic reconstruction and taxonomic labeling of newly sequenced genomes; and GraPhlAn, a visualization tool. In conjunc-tion with the HUMAnN system for metabolic profiling, these have established an overview of microbial metabolism and function core to the healthy human microbiome, along with insights into how disruption of these functions in the gut microbiota contributes to Crohn’s disease and ulcerative colitis.

n S1:6CuLTIVARS fROM MICROOxIC CONdITIONS dISPROPORTIONATELY OCCuR IN THE INTESTINAL MuCOSA ANd REPRESENT A NEW SOuRCE Of bENEfICIAL MICRObES

D. Dai1, K. Kim1, E. B. Chang2, M. Sogin3, V. B. Young4, T. M. Schmidt1; 1Michigan State University, East Lansing, MI, 2the University of Chicago, Chicago, IL, 3the Marine Biological Laboratory, Woods Hole, MA, 4the University of Michigan, Ann Arbor, MI.

Microbes associated with the intestinal mucosa are positioned for direct communication with host epithelial cells and provide a barrier for invasion by pathogens. We developed a low-oxygen strategy to cultivate the mucosal mi-crobiota and screened cultivars for the capacity to inhibit two common intestinal pathogens: Clostridium difficile and Candida albicans. Given the low but constant flux of oxygen that permeates the habitat adjacent to the intestinal epithelium, we introduced a microoxic atmo-sphere (2% oxygen), in addition to traditional anoxic and oxic incubation conditions, to cul-tivate bacteria from biopsy or brush samples of the intestinal mucosa from 10 healthy individuals. We isolated 58 bacterial species, covering the four most abundant phyla in the gut microbiome. Microoxic cultivation yielded numerous species that were not recovered from traditional conditions. If the microoxic cultivars have a competitive growth advantage in the mucosa, they should be more frequently

detected in the mucosa than in feces. This hypothesis was supported by the observation that species of microoxic cultivars occurred more frequently in 16S rRNA-based surveys of mucosa than in feces. Among the thousand cultivars from the mucosa, we screened for strains that inhibited the growth of C. difficile or the expression of virulence genes in C. albi-cans. To date, we have identified cultivars from three species (Enterococcus, Streptococcus, and Propionibacterium) that inhibit the growth of C. difficile. It is noteworthy that most of the strains that are antagonist of C. difficile were recovered from microoxic cultivation, even though they also grow anaerobically. Cultivars from one species interfered with the expression of virulence genes in C. albicans. This study highlights the importance of a low-oxygen atmosphere in cultivating mucosal microbes, which may be a rich source of beneficial mi-crobes.

n S2:1ENVIRONMENTAL IMPORTANCE Of THE NEMATOdE SYMbIOSIS fOR PEST CONTROL

H. Goodrich-Blair; University of Wisconsin, Madison, WI.

n S2:2dYNAMICS ANd PRObIOTIC EffECT Of THE EGG-ASSOCIATEd MICRObIAL COMMuNITY Of LAkE STuRGEON

T. L. Marsh, M. Fujimoto, B. Lovett, P. Niren-berg, J. Bauman, K. Scribner; Michigan State University, East Lansing, MI.

Egg laying amphibian and fish deposit rela-tively sterile eggs into an environment heavily populated by microbes. These eggs are rapidly colonized by a subset of bacteria from the water column. Egg-associated microbes can be pathogenic, mutualistic or symbiotic. Clearly an evolutionary balance has been struck whereby eggs are populated by bacteria, yet there is sufficient survival through larval stages

Speaker Abstracts


for successful recruitment of the species. We hypothesized that i.) bacterial load can have an adverse affect on egg mortality, ii.) systematic characterization of bacterial strains populating egg surfaces will reveal symbiotic strains that can be used in a probiotic manner to decrease mortality, iii.) the egg-associated community changes over the developmental time of the egg, and iv.) interactions between community members on the eggs surface are complex and contribute to community structural shifts. Our manipulations of the aquatic community with 0.2 µm filtration and UV treatment revealed that diminishing the aquatic community size and composition decreased egg mortality. Comparative microbial community analysis with T-RFLP, clone libraries and 454 pyro-sequencing indicate that the egg-associated community is substantially different from the aquatic community and changes over the devel-opmental time of the egg, suggesting that the initial attachment requirements are followed by selective forces that reshape the community. The phylogenetic census of microbes on the egg’s surface revealed populations that are con-sistently associated with healthy eggs and are hence candidate probiotics. We have isolated over 100 strains from the colonized egg and have tested their influence on egg mortality. When added to the egg fertilization medium of 7 different mating pairs of sturgeon, an Acidov-orax isolate reduced the egg mortality by 20%. Characterization of the egg-associated isolates using soft agar overlays to evaluate strain in-teractions identified 8 strains that produce an-timicrobials and are antagonistic to 80% of the remaining strains as well as 7 known Flavobac-terium fish pathogens. Several putative cases of mutualistic interactions between isolates were also observed. Using a standard microtiter plate assay for biofilm formation we have identified several isolates from the surface of the egg including Pseudomonas sp., Acidovorax sp. and Caulobacter sp. that produce robust biofilm under at least one of several metabolic condi-tions. These data suggest a dynamic, relatively short-lived microbial community on the egg’s surface that can be manipulated in ways that

improve egg mortality. Moreover comparative phylogenetics offers a rationale approach to the search for probiotics.

n S2:3fuNGAL ENdOPHYTES AS EPIGENETIC dRIVERS Of PLANT AdAPTATION

R. Rodriguez; United States Geological Sur-vey, Seattle, WA.

Symbiotic associations span a continuum from parasitism to mutualism and the outcome of specific associations is context driven based on inter-genomic interactions and environmental factors. These factors will determine the abil-ity plants and animals to adapt to a chang-ing climate. For example, plants in natural ecosystems adapt to abiotic stress by forming symbiotic associations with fungal endophytes that confer stress tolerance. Without the endo-phytes, the plants are not stress tolerant and do not survive in the habitats to which they are adapted. Symbiotically conferred stress tolerance typically occurs in a habitat-specific manner, a phenomenon we designate Habitat Adapted Symbiosis (HAS). Although several biochemical processes have been correlated to plant stress tolerance, few processes correlate with symbiotically conferred stress tolerance. I will present a mechanism for symbiotically conferred stress tolerance, how endophytes adapt plants across environmental gradients and a symbiotic strategy to mitigate impacts of climate change.

n S2:4SOIL MICRObIAL dIVERSITY dIffERENTIALLY INfLuENCES INVASION bY PATHOGENIC ANd bENEfICIAL MICROORGANISMS

A. Sheflin1, D. K. Manter2, J. Vivanco1; 1Colo-rado State University, Fort Collins, CO, 2Soil-Plant-Nutrient Research, USDA-ARS, Fort Collins, CO.

Studies focusing on the role that the indig-enous soil community plays in plant growth

Speaker Abstracts

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promoting rhizobacteria (PGPR) inoculant success or failure are limited. Soil microbial diversity has been shown to play a role in pathogen invasions, but is not yet tested on invasion of soil communities by beneficial microorganisms. In this study, six distinct soil communities were collected and characterized via pyrosequencing to determine relative diver-sity for each soil microbial community. These communities were extracted into naturalized assemblages and applied to alfalfa plants growing in a greenhouse environment. Either a PGPR or a pathogenic fungal microorganism was applied and effects on plant productivity were measured. Soil microbial diversity aug-mented beneficial effects with added PGPR, Pseudomonas putida. Conversely, disease incidence caused by Phytophthora medicaginis had an inverse relationship to soil microbial di-versity. These results support an important role in soil microbial community composition and its functional relationship with the invading organism in predicting effects of an introduced PGPR inoculant or soil pathogen.

n S2:5bACTERIAL REGuLATION Of COLONY dEVELOPMENT IN THE CLOSEST LIVING RELATIVES Of ANIMALS

N. King; University of California, Berkeley, Berkeley, CA.

The evolution of animals from their single celled ancestors represents one of the major transitions in life’s history. By studying cho-anoflagellates, the closest living relatives of animals, my lab aims to reconstruct the biology of the last common ancestor of animals. I will describe our development of a colony-forming choanoflagellate, Salpingoeca rosetta, as a new model for investigating host-microbe interac-tions and the origin of animal multicellularity. Through our study of S. rosetta, we have dis-covered that a developmental switch in choano-flagellates is regulated by a secreted signal from environmental bacteria. In collaboration with Jon Clardy’s lab, we have isolated the inducing

molecule, named RIF-1 for “Rosette Induc-ing Factor,’ and determined that it is a member of the little-studied group of sulfonolipids. RIF-1 has extraordinary potency (femtomolar, or 10^-15 M) and S. rosetta can respond to it over a broad dynamic range - nine orders of magnitude. This study provides a prototypical example of bacterial sulfonolipids triggering eu-karyotic morphogenesis and suggests molecular mechanisms through which bacteria may have contributed to the evolution of animals.

n S2:6GuT MICRObES fACILITATE CONSuMPTION Of TOxIC dIETS bY HERbIVORES

K. D. Kohl, R. B. Weiss, C. Dale, M. Dear-ing; University of Utah, Salt Lake City, UT.

For decades, ecologists have hypothesized that herbivorous mammals might host beneficial microbes that facilitate the ingestion of diets containing toxic plant secondary compounds (PSCs). However, this idea has never been sufficiently tested in wild herbivores. We studied a small herbivorous rodent, the des-ert woodrat (Neotoma lepida) that naturally feeds on a toxic shrub, creosote bush (Larrea tridentata). Creosote leaves produce large quantities of a phenolic-rich resin that is that is lethal to lab mice in the doses consumed by woodrats. Woodrats were fed either a control diet of rabbit chow or rabbit chow plus 2% extracted creosote resin. Following acclima-tion, animals were dissected and we conducted metagenomic sequencing of the contents of the woodrat foregut. Additionally, a subset of ani-mals were given a broad-spectrum antibiotic (neomycin); food intake and body mass were monitored. When feeding on creosote resin, the woodrat foregut metagenome was notably enriched in genes associated with the metabo-lism of aromatic compounds, stress responses, protein metabolism, carbohydrate metabolism, and membrane transport. An important detoxi-fication enzyme, aryl-alcohol dehydrogenase, was only detectable in the metagenomes of creosote-fed animals. Woodrats given antibiot-

Speaker Abstracts


ics consumed less food and lost more weight compared to woodrats not given antibiotics, but only when the diet contained creosote resin. Metagenomic results revealed that di-etary toxins strongly alter the functional profile of woodrat gut microbes, which may have im-pacts on host homeostasis. The antibiotic study represents the first experimental evidence that microbes enhance the consumption of PSCs in wild herbivores. These results suggest that beneficial microbes play a large role in enhanc-ing dietary niche breadth in herbivores by al-lowing them to consume toxic plants. This may have implications for wild and domesticated herbivores facing rapid changes in plant com-munities due to changes in global climate or land-use practices.

n S2:7HOST-MICRObE INTERACTIONS IN HYdRA

T. C. Bosch; Kiel University, Kiel, GERMANY.

For a long time, the main purpose of host-as-sociated microbiology was to study pathogenic bacteria and infectious disease; the potential benefit of good bacteria remained unrecog-nised. However, individuals from Hydra to man are not solitary, homogenous entities but consist of complex communities of many spe-cies that likely evolved during a billion years of coexistence. The hologenome theory of evo-lution considers the holobiont with its hologe-nome as a unit of selection in evolution. Defin-ing the individual microbe-host conversations in these consortia, therefore, is a challenging but necessary step on the path to understand-ing the function of the associations as a whole. Here I present the Hydra holobiont which in-volves at least three types of organisms that all share a long coevolutionary history and appear to depend on each other as model for examin-ing a microbe/symbiont-dependent life style and its evolutionary consequences. Recent work has shown that Hydra has an elaborate innate immune system for sensing bacterial invaders and shaping the community of as-sociated beneficial microbes. Because these

immune responses function much the same way in vertebrates, findings derived from the in vivo context of the Hydra model may provide one of the simplest possible systems to address questions of how a stable host-microbe com-munity is established and remains in balance over time.

n S3:1IdENTIfICATION ANd TEMPORAL ExPRESSION PATTERNS Of INNATE IMMuNITY GENES IN THE MEdICINAL LEECH, Hirudo verbena

L. Bomar; University of Connecticut, Storrs, CT.

n S3:2bACILLuS SubTILIS ExOPOLYSACCHARIdES PREVENT COLITIS VIA TLR-dEPENdENT SIGNALING

S. E. Jones, K. L. Knight; Loyola University Medical Center, Maywood, IL.

Beneficial microbes can limit disease caused by invading pathogens; however, the mecha-nisms and genes utilized by these select microbes to inhibit pathogenesis are poorly understood. Citrobacter rodentium is used to model the human pathogens, enterohemor-rhagic E. coli and enteropathogenic E. coli in mice. C. rodentium is an attaching and effac-ing mouse pathogen that associates intimately with the intestinal epithelium and infections result in acute colitis. We show that Bacillus subtilis, a spore-forming bacterium found in the gut of mammals, prevents C. rodentium-associated disease when administered 24 hr prior to pathogen infection. Disease was as-sessed by determining the extent of colonic epithelial hyperplasia, splenomegaly, diarrhea, and pathogen colonization. Interestingly, B. subtilis treated mice were successfully colo-nized by the pathogen, yet they did not show any evidence of disease. Importantly, a B. subtilis epsH mutant, defective for exopolysac-charide (EPS) production, did not protect mice from C. rodentium-associated disease, and

Speaker Abstracts

ASM Conferences18

purified EPS from B. subtilis was sufficient to prevent disease. We hypothesize that B. subtilis EPS modulates host TLR signaling pathways to mediate their protective effects, because MyD88 KO mice, deficient in TLR signaling, are not protected by B. subtilis from the enteric pathogen. To elucidate the host TLR pathways modulated by B. subtilis, we are testing if TLR2 and TLR4 KO mice are susceptible to C. rodentium after B. subtilis treatment. Prelimi-nary data suggest that B. subtilis EPS utilize both TLR2 and TLR4 to mediate protection. Our data demonstrate that wildtype B. subtilis prevents disease caused by C. rodentium infec-tion through a mechanism that requires bacte-rial production of EPS and we hypothesize that EPS utilizes host TLR signaling pathways to suppress inflammation and colitis. By ex-panding our current mechanistic knowledge of bacterial protection, we can better utilize beneficial microbes to prevent intestinal dis-ease caused by pathogenic bacteria, ultimately reducing human disease.

n S3:3WHY YOu MIGHT NOT WANT TO STARVE A fEVER If YOu ARE A fLY

M. Shirasu-Hiza; Columbia University, New York, NY.

n S3:4SYSTEMS ANALYSIS Of ANTIbIOTIC-ASSOCIATEd dEfECTS IN GuT IMMuNITY ANd MICRObIOTA

N. Shulzhenko1, A. Dzutsev2, J. Ravel3, P. Matz-inger2, W. Hsiao4, A. Morgun1; 1Oregon State University, Corvallis, OR, 2National Institutes of Health, Bethesda, MD, 3Institute for Ge-nome Sciences, Baltimore, MD, 4University of British Columbia, Vancouver, BC, CANADA.

Antibiotics are essential for the treatment of bacterial infections and are widely used. How-ever, more than 10% of people who receive this medication suffer from adverse effects ranging from antibiotic associated diarrhea and

colitis to allergies and autoimmune diseases. One of the primary factors contributing to these disorders is the depletion of commensal microorganisms (microbiota). Intestinal micro-biota provide several valuable physiological functions for their hosts, including the key role in the induction of different aspects of intes-tinal immunity. This has led to the emerging concept that the use of antibiotics creates de-fects in local immunity due to the depletion of commensal microbiota, and that these immune alterations facilitate antibiotic associate disor-ders. Despite a general consensus in the field regarding the influence of antibiotics on the immune system, the extent of immune altera-tions beyond some readouts is not clear. We obtained a comprehensive picture of changes in intestinal gene expression induced by a cocktail of antibiotics commonly used to gen-erate a surrogate model of germfree mice. By comparing these changes with those of antibi-otic-treated and untreated germfree mice, we defined a profile that was related to the deple-tion of microbiota and not to the direct effect of antibiotics on intestinal tissues. The major-ity of the genes in the profile (400 out of 500) were downregulated by antibiotics and were highly represented by immune response genes, including those related to intraepithelial and lamina propria T lymphocytes, plasma cells, myeloid cells and innate immune properties of epithelial cells. Analysis of 16S rRNA gene by Phylochip showed that antibiotics significantly decreased amounts of ~5500 bacterial species with concomitant increase in ~320 species of Gammaproteobacteria and a few other related taxa. To identify which bacterial members might be the key drivers of mucosal immunity, we integrated microbiome and host transcrip-tome data by reconstruction of trans-kingdom (host-microbiome) network. The network that emerged has specific topological features known to be characteristic of regulatory bio-logical networks with some bacterial species being highly connected to multiple T and B lymphocyte genes suggesting that they might be strong inducers of adaptive immunity in the gut. Thus, this new systems approach reveals

Speaker Abstracts


microbial candidates that might regulate host immune functions affected by antibiotics and can be tested in colonization experiments.

n S3:5uNdERSTANdING THE CELLuLAR INNATE IMMuNE RESPONSE ANd SPECIfICITY IN THE ASSOCIATION bETWEEN THE HAWAIIAN bObTAIL SquId EuPRYMNA SCOLOPES ANd THE bIOLuMINESCENT bACTERIuM VIbRIO fISCHERI

S. V. Nyholm; Molecular and Cell Biology, University of Connecticut, Storrs, CT.

The establishment of the light organ sym-biosis between the Hawaiian bobtail squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri depends on a molecu-lar “dialogue” between the partners that leads to the selection of V. fischeri and exclusion of non-symbiotic bacteria from the environ-ment. Current evidence suggests that the host’s cellular innate immune system, in the form of macrophage-like hemocytes, helps mediate tol-erance to V. fischeri. To further understand the role of hemocytes in this association, we have analyzed circulating adult hemocytes by high-throughput 454 transcriptomic and liquid chro-matography/mass spectrometry (LC-MS/MS) proteomic analyses. 650,686 transcripts total-ing 279.9 Mb and 702 unique putative proteins were identified. A number of pattern receptors involved with the recognition of microbe-associated molecular patterns were identified. Among these was a complete open reading frame to a peptidoglycan recognition protein (EsPGRP5) with conserved residues for pre-dicted amidase activity. EsPGRP5 was among the most abundant hemocyte transcripts de-tected and results from immunocytochemistry suggest the protein is present in intracellular vacuoles. Other transcripts and proteins identi-fied included members of the conserved NF-κB signaling pathway, predicted members of a complement-like pathway, the carbohydrate binding protein galectin and a putative cepha-lotoxin. Quantitative PCR of complement-like

genes, cephalotoxin, EsPGRP5, and a nitric oxide synthase showed differential expression in circulating hemocytes isolated from adult squid with colonized light organs compared to those from cured hosts. Further analyses using quantitative proteomic techniques also revealed differences in proteins thought to be involved with cell adhesion and calcium signaling. These data from adults suggest that colonization state influences gene expression of the cellular innate immune system of the host. Current efforts are underway to charac-terize changes in gene and protein expression of hemocytes challenged with symbiotic and non-symbiotic bacteria and to understand the juvenile hemocyte response during initial colo-nization.

n S3:6ObLIGATE SYMbIONTS ACTIVATE IMMuNE SYSTEM dEVELOPMENT IN THE TSETSE fLY

B. Weiss, M. Maltz, S. Aksoy; Yale University School of Medicine, New Haven, CT.

Beneficial microbial symbionts serve impor-tant functions within their hosts, including maintenance of immune system homeostasis. However, little is known about the mecha-nisms that enable these bacteria to induce host immunity-related phenotypes during develop-ment and into adulthood. Tsetse flies (Glossina spp.) house 3 distinct endosymbiotic bacteria that are vertically transmitted from mother to offspring during this insect’s unique viviparous mode of reproduction. In an effort to learn more about the relationship between tsetse immunity and its associated symbionts, we developed a line of flies that undergo larval de-velopment in the absence of their endogenous microbiota. As adults, these ‘aposymbiotic’ flies present a severely compromised immune system that is characterized by the absence of phagocytic hemocytes and atypical expression of immunity-related genes. Correspondingly, these flies quickly succumb to infection with normally non-pathogenic E. coli. The suscepti-ble phenotype exhibited by aposymbiotic tsetse

Speaker Abstracts

ASM Conferences20

can be reversed when they receive hemocytes transplanted from wild-type donor flies prior to infection. Furthermore, the process of immune system development can be restored in intra-uterine aposymbiotic larvae when their moms are fed a diet supplemented with cell extracts of tsetse’s obligate symbiont, Wigglesworthia. Our finding that molecular components of Wigglesworthia exhibit a ‘trans-generational’ immune priming response within tsetse is rep-resentative of a novel evolutionary adaptation that steadfastly links an obligate symbiont with it’s host.

n S4:1SENSING Of MICRObIAL ANd NuTRITIONAL SIGNALS bY THE INTESTINAL EPITHELIuM

L. V. Hooper; Center for Immunology, Uni-versity of Texas Southwestern Medical Center, Dallas, TX.

The mammalian intestine is home to ~100 tril-lion bacteria that perform important metabolic functions for their hosts. Intestinal epithelial cells are an important physical interface be-tween host tissues and the vast populations of luminal bacteria, and play a central role in directing immune responses to the microbiota. We are investigating how gut epithelia sense bacterial as well as nutritional signals and how they integrate these signals to modulate im-mune responses that protect against bacterial invasion of mucosal tissues. We have shown that intestinal bacteria direct expression of key antimicrobial proteins that are essential for maintaining physical separation between the microbiota and the small intestinal epithelial surface. In addition, we are investigating bacte-rial influences on vitamin A metabolism and transport within the intestinal epithelium, and how these influences impact the development of adaptive immunity against the microbiota. Together, our findings set the stage for a de-tailed understanding of how epithelial cells integrate microbial and nutritional signals to direct innate and adaptive immunity to the microbiota.

n S4:2ANALYSIS Of TRANS-kINGdOM SIGNALLING Of bACTERIAL SMALL MOLECuLES WITH EukARYOTES uSING HIGHLY RESOLVING METAbOLITE ANALYSIS ANd IMMuNOLOGICAL IN VITRO-TEST SYSTEMS

A. A. Hartmann1, I. Kepert1, K. Hoch-wind1, J. Fonseca1, M. Schmid1, S. van Hemert2, P. Schmitt-Kopplin1, S. Krauss-Etschmann1; 1Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Oberschleissheim, GERMANY, 2Win-clove, Amsterdam, NETHERLANDS.

We followed the hypothesis, that small mole-cules produced by bacteria are good candidates for inter-kingdom signaling to eukaryotic or-ganisms. We investigated culture supernatants and fractions from a series of Gram-positive probiotic bacteria, using in vitro test systems as screening tools for immune-modulatory activity, like human dendritic cells and a Hodg-kin lymphoma cell line, which secretes high levels of the allergy-related thymus and activa-tion regulated chemokine (TARC). Key meth-ods of our highly resolving chemical analysis were ultra-performing liquid chromatography (UPLC), fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and NMR-analysis. Culture supernatants of several Lactobacillus spp. and Bifidobacterium spp. were tested for their ability to prevent the lipopolysaccharide-induced maturation of hu-man monocyte-derived dendritic cells (DC). The bacterial supernatants were also tested to inhibit TARC secretion of Th2-polarised KM-H2 Hodgkin Lymphoma cells (ELISA, R&D). After further separation using UPLC, immune-active fractions were chemically characterized by FT-ICR-MS and NMR-analyses. Super-natants of strains were found to significantly down-regulate the percentages of LPS-matured DCs expressing the activation markers CD83, CD86 and CD40. They also significantly reduced TARC secretion of KM-H2 cells. After liquid chromatography fractionation of

Speaker Abstracts


supernatants, the immune modulatory activity was found in the 20%, 40% and 50% methanol fractions. The immune-active compound in the 20% methanol fraction was finally character-ized as D-tryptophan. D-tryptophan was the only D-amino acid having this immune modu-latory effect at a concentration level of 50nM. Summary: The present work demonstrated that D-tryptophan has strong in vitro immune reactive activity and it occurs in culture su-pernatants of some probiotic Lactobacillus and Bifidobacterium strains. It needs to be demonstrated, which activities this compound has in more complex epidermal assay systems and what the chemical nature of the other more lipophilic compounds is. Conclusions: Other bacterial compounds, like e.g. the autoinducer-molecules N-acyl homoserine lactones of Gram-negative bacteria have also been charac-terized as immune modulating compounds in plants and animal/human systems (Hartmann and Schikora, 2012). Therefore, inter-kingdom signalling research which currently focuses mostly on interactions of microbial associated molecular patterns (MAMPS) like surface macromolecules with the host´s immune sys-tem should be complemented with integrative studies of diffusible small bacterial molecules for a more complete analysis of inter-kingdom interactions. Reference: Hartmann, A. and Schikora, A. (2012) Quorum sensing of bacte-ria and trans-kingdom interactions of N-acyl homoserine lactones with eukaryotes. J. Chem. Ecol. 38, 704-713.

n S4:3THE SIMPLE, YET TANGLEd, TSETSE MICRObIAL COMMuNITY

R. V. Rio; Biology, West Virginia University, Morgantown, WV.

Microbial symbiosis, once regarded as an ecological anomaly, is now recognized as a major contributor to host physiology, nutri-tion, development and evolutionary diversi-fication. The strictly sanguivorous tsetse fly (Diptera: Glossinidae) is of high medicinal

and socioeconomic significance due to its role as the obligate and sole vector of African trypanosomes (Trypanosoma spp.). In addition to potentially harboring trypanosomes, the maternally transmitted tsetse microbial com-munity consists of two Gammaproteobacteria; the ancient obligate mutualist Wigglesworthia spp. and a more recently established commen-sal Sodalis glossinidius, as well as the parasitic Alphaproteobacteria Wolbachia. Annotated genomes of the tsetse resident microbes have spurred research aiming to identify areas of po-tential interactions, and how these may impact community dynamics and contribute towards stability of the relationships across genera-tions. Moreover, the genome sequence of a second Wigglesworthia sp. (Wgm), estimated to have split from a previously annotated basal sister species (Wgb) at the commencement of tsetse radiation, has enabled the examination of symbiont genome diversification through evolutionary time, and how metabolome dif-ferences may influence their particular tsetse host species, such as in vector competence. Transcriptional profiling of B vitamins, as well as chorismate and phenylalanine biosynthetic loci, which are differentially retained by tsetse symbionts, indicates variations in expres-sion through host development, pregnancy, and trypanosome infection. Furthermore, the exogenous provisioning of metabolites was shown to alter the expression of correspond-ing symbiont biosynthetic pathway loci and decrease symbiont populations, most likely host-modulated through the suppression of the Peptidoglycan Recognition Protein (pgrp-lb). Lastly, symbiont genome sequencing has elu-cidated mechanisms enabling symbiont verti-cal transmission. Despite the high fidelity of maternal transmission, host antimicrobial im-mune activation during tsetse early adulthood negatively affects Wigglesworthia milk gland population, responsible for seeding tsetse off-spring. Host immunity coupled with nutritional integration of the microbial community may both be instrumental towards symbiont com-munity regulation.

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ASM Conferences22

n S4:4NIGHTY NIGHT: HOW PHOTORHAbduS LuMINESCENS TuRNS Off THE LIGHT TO PERSIST IN HOST NEMATOdE INTESTINES.

V. S. Somvanshi1, R. E. Sloup2, J. M. Craw-ford3, A. R. Martin2, A. J. Heidt2, K. Kim2, J. Clardy4, T. A. Ciche2; 1Indian Agricultural Institute, New Delhi, INDIA, 2Michigan State University, East Lansing, MI, 3Yale University, New Haven, CT, 4Harvard Medical School, Boston, MA.

Heterorhabditis bacteriophora nematodes are specifically associated with Photorhabdus luminescens bacteria where the pair cooper-ates in insect killing. The bacteria benefit the nematodes by killing insects, preserving the insect cadaver and being essential for nema-tode reproduction. Fluorescent tags facilitate detection of individual cells inside the small and transparent nematode body. Maternal nem-atodes acquire the symbionts as a biofilm that forms on the posterior intestine and transmits these symbionts to offspring developing inside the body cavity. We discovered that cells initi-ating mutualism had changed form, called M form, from the insect pathogenic P-form par-ent. The M-form cells were 1/7 the volume of P form and grew as unstable small colony vari-ants. The switch to the M form was triggered by a single promoter inversion, with the MadO invertase required to switch on, the MadR invertase required to switch off normally, and MadJ likely functioning as the regulatory output of the switch. Locking the promoter in the off and on orientations locked the cells in P form and M form, respectively. P-form cells were pathogenic to insects but unable to initiate nematode mutualism. In contrast, the M-form cells were avirulent to insects. The M form differed from the P form in many other characteristics such decreased production of antibiotics, bioluminescence, crystalline in-clusion proteins, and pigments. The M form was more antibiotic tolerant than the P form and differentially regulated more than 10% of the genome. Reporter studies revealed that a

minority cells in the maternal nematode gut switched to the M form and these were the cells that selectively adhered and dominated further steps of symbiont transmission before the cells switched back to the P form in nema-tode offspring. Observing beneficial microbe interactions develop at single cell resolution revealed an essential function for a phenotypic variant and new mechanism for switching.

n S4:5INTERkINGdOM INTERACTIONS fACILITATE THE fITNESS Of bACTERIA ON PLANTS

S. E. Lindow; Plant and Microbial Biology, University of California, Berkeley, CA.

The production of 3-indole acetic acid (IAA) is a very common trait in bacteria that live on plants but uncommon in those in other habi-tats. While IAA production contributes to the epiphytic fitness of bacteria, the mechanism by which it does so has remained unclear. The concentration of sucrose on leaf surfaces is lower in the presence of IAA while the concentration of fructose is higher. IAA thus appears to activate plant cell wall invertases that results in a conversion of plant-derived sucrose into fructose and glucose, both of which are accessed at lower concentrations by bacteria than sucrose itself. Thus IAA-medi-ated interkingdom signaling leads to resource conversion whereby bacteria can access nu-trients not previously available to them due to their low concentrations. The composition of the epiphytic bacterial communities on plants can be influenced not only by the plant itself, but also by fungal endophytes and epiphytes. Neotyphodium sp., intercellular fungal symbi-onts of cool season grasses that reduce herbiv-ory due to their production of loline alkaloids also modulate epiphytic bacterial communities. The lolines appear to constitute the major source of Carbon and Nitrogen available to bacteria on fungal endophyte-infected grasses; Burkholderia sp. achieve an epiphytic popula-tion size that is as much as 10-fold higher on fescues infected with loline-producing endo-

Speaker Abstracts


phytic fungi compared to that on uninfected plants or plants infected with a loline-deficient fungal variant. A majority of bacteria recov-ered from loline-containing plants catabolize this resource, while such bacteria are absent from non-grasses. Thus fungal colonization apparently selects for a specialized group of bacteria that might be exploited to achieve a biased rhizosphere or phyllosphere to enable enhanced bioremediation.

n S4:6MICRObIAL REGuLATION Of INTESTINAL EPITHELIAL CELL PROLIfERATION

A. V. Banse, J. V. Troll, K. J. Guillemin; Uni-versity of Oregon, Eugene, OR.

The resident microbial community of the intes-tine (intestinal microbiota) profoundly affects homeostasis of the intestinal epithelium in ver-tebrates and is a potential contributor to hyper-proliferative diseases such as colorectal cancer. The microbiota of both mice and zebrafish has been shown to stimulate intestinal epithelial proliferation. Our lab has developed a zebraf-ish model to study the molecular mechanisms by which the microbiota influences prolifera-tion of the intestine. We previously used this model to demonstrate that a resident intestinal bacterium, Aeromonas veronii, can enhance the stability of β-catenin in intestinal epithelial cells and thus promote cell proliferation in the developing zebrafish intestine. It was further shown that A. veronii cell-free supernatant (CFS) purified using a concentrator with a mo-lecular mass cutoff of 10 kDa is sufficient to induce cell proliferation in the zebrafish intes-tine. This observation suggests that A. veronii produces a secreted signal(s) larger than 10 kDa that is able to increase levels of intestinal epithelial cell proliferation. We have initiated further research to determine the identity of the pro-proliferative signal secreted by A. veronii. We have determined that the signal(s) is specif-ically secreted by the type II secretion system (T2SS). Further, using ammonium sulfate frac-

tionation of the CFS we have identified several interesting candidate proteins. Determining the chemical identity of this factor will help us to define and understand mechanistically how microbial signals contribute to intestinal epithelium homeostasis as well as how these signals may contribute to hyperproliferative diseases such as colorectal cancer.

n S5:1SPONGE-MICRObIAL SYMbIOSES IN HEALTH ANd dISEASE

U. H. Humeida; University of Würzburg, Wuerzburg, GERMANY.

Sponges (phylum Porifera) are the most ancient still living metazoan phylum with a fossil record dating back more than 600 mil-lion years in time. Many sponges are known to contain large amounts of phylogenetically diverse microorganisms that are located extra-cellularly in the mesohyl matrix. This presenta-tion will discuss our current understanding of the interactions of marine sponges and their microbial symbiotic consortia and highlight recent insights into these relationships from genomic studies. Recent results obtained by -omics approaches in the author’s laboratory and presented in the literature will be reviewed towards this goal. Much less effort has been undertaken to investigate the interactions of sponges with potentially pathogenic micro-organisms. The presentation will furthermore discuss microbial diversity and function under conditions of sponge disease using two Ca-ribbean sponges as examples. References: - Hentschel U, Piel J, Degnan SM, Taylor MW (2012) Genomic insights into the marine sponge microbiome. Nature Reviews Microbi-ology: doi: 10.1038/nrmicro2839 - Angermeier H, Kamke J, Abdelmohsen UR, Krohne G, Pawlik JR, Lindquist NL, Hentschel U (2011) The pathology of sponge orange band disease affecting the Caribbean barrel sponge Xes-tospongia muta. FEMS Microb Ecol 75(2): 218-230

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ASM Conferences24

n S5:2uNdERSTANdING THE CONTRIbuTIONS Of A CuLTuREd HuMAN fECAL MICRObIOTA TO AdIPOSITY IN GNOTObIOTIC MICE

V. K. Ridaura1, J. J. Faith1, F. E. Rey1, J. Cheng1, A. E. Duncan1, J. R. Bain2, L. K. Ur-sell3, M. J. Muehlbauer2, J. C. Clemente3, A. Gonzalez3, W. A. Walters3, C. B. Newgard4, A. C. Heath1, R. Knight3, J. I. Gordon1; 1Wash-ington University in St. Louis, St. Louis, MO, 2Sarah W. Stedman Nutrition and Metabolism Center, Duke University, Durham, NC, 3Univ. of Colorado, Boulder, CO, 4Duke University, Durham, NC.

Efforts to understand the relationship between the human gut microbiota and obesity have es-tablished correlations between microbial com-munity structure and host phenotype. However, more information is needed to determine whether the microbiota plays a causal role in this disorder. We have established methods for successfully transplanting previously frozen human fecal microbiota samples into gnotobi-otic mice, allowing for reproducible replica-tion of the majority of bacterial diversity and microbiome gene content present in the donor sample in recipient animals. In the present study, we have transplanted uncultured fecal microbiota from 4 twin pairs discordant for obesity, as well as collections of cultured bac-teria created from the microbiota of one repre-sentative twin pair, into groups of gnotobiotic C57BL/6J mice. All recipient animals were fed the same low-fat, plant polysaccharide-rich sterile chow diet. The structure and expressed functions of the transplanted, replicated micro-biota were characterized at the level of DNA (bacterial 16S rRNA amplicon and whole microbial community shotgun sequencing), RNA (microbial RNA-Seq), and metabolites (targeted and non-targeted GC/MS). Adiposity was measured in transplant recipients by quan-titative magnetic resonance imaging, and by epididymal fat pad weights. Both uncultured microbiota, and the derived culture collections, transmit the increased adiposity phenotype of

the obese co-twin donors to gnotobiotic mouse recipients. The differences in adiposity were not associated with statistically significant dif-ferences in daily chow consumption, or signifi-cant differences in oral glucose tolerance tests, or with appreciable differences in the CD4+ and CD8+ T cell compartments in spleen, mes-enteric lymph nodes, small intestine or colon. The transplanted microbiota from lean donors manifested an increased efficiency of carbo-hydrate metabolism. To test if recipients of a lean co-twin’s culture collection (Ln) could rescue the adiposity phenotype of mice harbor-ing the obese co-twin’s culture collection (Ob), we co-housed Ln animals with one another, Ob animals with one another, and Ob and Ln mice together. The ability of one community to invade another was defined using 16S rRNA-based microbial source tracking. Microbial community metabolism was also characterized as a function of co-housing conditions. Our results provide additional insights about the microbial basis for host adiposity phenotypes, and suggest that these pre-clinical models are useful for proof-of-mechanism studies, as well as proof-of-principle tests of therapeutic concepts.

n S5:3THE GuT MICRObIOTA Of THE HEALTHY HONEY bEE

N. A. Moran; Ecology and Evolu. Biology, Yale University, New Haven, CT.

Honey bees are social insects that live in large colonies dominated by female workers who rear young, build comb, clean the hive, and forage for nectar and pollen, the only sources of nutrients. Due to their key role in crop pol-lination, honey bees are one of the important domesticated animals, and colony declines have been a major concern. We are exploring the role of the gut microbiota in bee health and disease. Recent non-culture based studies of 16S rRNA diversity in studies of the microbio-ta of the guts of adult worker bees has revealed a highly consistent set of eight species (or spe-

Speaker Abstracts


cies clusters). These are present in honey bees worldwide, in which they make up 95-99.5% of the bacteria present in individual bees, based on pyrosequencing studies. Some of these spe-cies (or close relatives) are present in related social bees, but all are absent from other en-vironments. Of these 8 species, 6 are in every bee sequenced, including 3 Gram negative species and 3 Gram positive species located mostly in the rectum. Two species, Candidatus Gilliamella apicola (Gammaproteobacteria) and Candidatus Snodgrassella alvi (Betapro-teobacteria) live in characteristic locations and in close proximity in the ileum, where waste products are delivered by the Malphigian tubules. Strains of these two species also are found in bumblebees, in which they have been linked to resistance to trypanosome pathogens. In honey bees, G. apicola and S. alvi occur as multiple strains even within a single bee, and colonies differ in strain composition. Metagenomic sequence data and experiments with cultured isolates indicate that strains vary in the presence of genes likely to affect host functions. For example, some but not all G. apicola strains possess a genetic island encod-ing pectate lyase, which enables breakdown of macromolecules in plant cell walls and which is otherwise known only from plant pathogenic bacteria. This likely plays a role in breakdown of cell walls of pollen, a highly refractory ma-terial that is the only source of protein for bees. Other potential roles, in digestion, nutrition, detoxification, and disease resistance, remain to be explored.

n S5:4fuNCTIONAL ANALYSIS Of THE HuMAN MICRObIOME uSING COMMuNITY PROTEOMICS OR PROTEOGENOMICS

N. C. VerBerkmoes; New England Biolabs, Ipswich, MA.

n S5:5MATERNAL PRObIOTIC INTERVENTION PROTECTS AGAINST IMMuNE ALTERATIONS ANd GuT dYSfuNCTIONS IN THE MATERNALLY SEPARATEd RAT MOdEL Of IRRITAbLE bOWEL SYNdROME

J. Barouei, M. Moussavi, D. Hodgson; The University of Newcastle, Newcastle, AUSTRA-LIA.

This study aimed to examine whether maternal probiotic intervention can act to protect against immune and intestinal dysfunctions induced by early life and subsequent adult stress in Wistar rats. Dams had free access to drinking water supplemented with 3×109 CFU/mL of Bifidobacterium animalis subsp lactis BB-12® and 8.0×108 CFU/mL of Propionibacterium jensenii 702 from 10 days before conception until postnatal day (PND) 22 (weaning day), or control ad lib water. Pups were subjected to neonatal maternal separation (MS) from PND 2 to 14 or left undisturbed. In adulthood, ani-mals were exposed to restraint stress (AS) for 30 min/day between PND 83 and 85, or were left undisturbed as controls. Immediately after restraint stress animals were euthanized and blood samples were collected to assess plasma haptoglobin (an inflammation-inducible plasma protein) and immunoglobulin A (IgA) levels. Faecal pellets were also analysed to assess the composition of gut microflora. Ileal tissue samples were also collected to determine mRNA expression of corticotropin releasing hormone receptor 1 (CRH-R1). Neonatal stress was associated with significantly decreased plasma IgA levels compared to non-stressed controls (p ≤ 0.05). Neonatally stressed ani-mals also demonstrated significantly elevated haptoglobin levels and significantly declined faecal counts of total anaerobic bacteria and clostridia post adult stress compared to no-stress controls (p ≤ 0.05 in all cases). Counts of bifidobacteria were significantly decreased (p ≤ 0.05) in animals exposed to only adult stress compared with the controls. E. coli levels were significantly increased in animals exposed to

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ASM Conferences26

neonatal or adult stress (p ≤ 0.05). Exposure to neonatal and/or adult stress significantly increased ileal mRNA expression of CRH-R1 in adult females (p ≤ 0.05). Maternal probiotic intervention appeared to reverse the decline in plasma IgA levels and clostridia counts induced by neonatal stress. It was also associ-ated with significant reductions in haptoglobin levels and increased luminal IgA levels in stressed and non-stressed animals, and restored anaerobes, bifidobacteria and E. coli to normal levels in stressed animals. Finally maternal probiotic intervention induced significant re-ductions in ileal CRH-R1 mRNA expression in neonatally stressed females compared with their respective treatment groups in the vehicle subset. Our findings suggest that maternal probiotic intake can notably improve immune system and gut functions altered by early and/or later life stress. Underlying mechanisms however need to be further investigated.

n S5:6MICRObIOTA: dO YOu HAVE THE bEST TEAM WORkING fOR YOu?

B. P. Willing; University of British Columbia, Vancouver, BC, CANADA.

Microbiota: Do you have the best team work-ing for you? A unique population of microbes inhabits the gastrointestinal tract of each indi-vidual, yet we know relatively little about the consequences these variations have on host physiology. To further our understanding of the role of microbial composition on innate im-mune function and resistance to infection we use antibiotics and microbial transplantation as means to manipulate the microbiota. We have found that not all microbial populations are equal in their ability to regulate host antimicro-bial defense and that some microbes seem to actively suppress innate defense. Furthermore, host responses to antibiotic treatment vary depending on the initial colonizing bacteria. Finally, we have established that transfer of resistance can be achieved through microbial

transplantation, providing an additional mecha-nism to alter host resistance.

n S5:7INVESTIGATING THE SYSTEMIC ANd GuT-SPECIfIC MOuSE METAbOLOME bY NON-TARGETEd METAbOLITE PROfILING

S. S. Heinzmann, P. Schmitt-Kopplin; Helm-holtzZentrum Muenchen, Munich, GERMANY.

Many analyses have recently been performed to investigate the influence of the composi-tion of the gut microbiome on health and disease status. However, information about the metabolic functionality of the whole gut microbiome and species-specific metabolic activity and interaction with the host is sparse. Metabolite profiling provides the opportunity to characterize and quantify metabolites from different chemical classes. The non-targeted manner of metabolic profiling allows discovery of novel metabolites and a combination of complementary analytical techniques such as nuclear magnetic resonance (NMR) spectros-copy (1), mass spectrometry (MS) and ultra-high resolution MS (FT-ICR-MS) (2) provides a holistic picture of a broad range of metabo-lites present in each sample. To systematically characterize the metabolic composition of the gastro-intestinal tract and bodily tissues we collected gastro-intestinal contents, body fluids and tissues from n=3 C57BL/6 mice. The gut content was divided into 7 sections namely stomach, jejunum, ileum, cecum, proximal and distal colon, and feces to allow an overview of the procession of nutrient breakdown and mi-crobial activity in the gut. Metabolites were ex-tracted using a step-wise extraction with aque-ous (ACN:H

2O) and methanol solvents and

submitted to NMR spectroscopic analysis to obtain an overview of the metabolites. Pattern recognition analysis was employed to illustrate similarities and differences among the gut section. We were able to localize, where food digestion is completed (e.g. disappearance of stachyose/raffinose and simple carbohydrates) and the main microbial activity (e.g. appear-

Speaker Abstracts


ance of short-chain fatty acids and metabolites of protein fermentation) starts. Furthermore, metabolic evolution of bile acids from jeju-num, ileum and distal gut was apparent. The chosen method strategy allowed an overview of a wealth of metabolic information within one analytical experiment and the non-targeted approach opens the possibility to discover nov-el or unexpected metabolic information. This preliminary analysis unveils promising insights in the metabolic functionality of the symbiotic activity of host digestion and microbial activ-ity and provides the opportunity to link details about the gut microbiome composition to its direct effects on the host. (1) Heinzmann SS, Merrifield CA, Rezzi S et al. Stability and robustness of human metabolic phenotypes in response to sequential food challenges. J Proteome Res. 2012 Feb 3;11(2):643-55.(2) Jansson J, Willing B, Lucio M et al. Metabolo-mics reveals metabolic biomarkers of Crohn’s disease. PLoS One. 2009 Jul 28;4(7):e6386.

n S6:1A COMMON POLYSACCHARIdE IMPACTS THE SquId-VIbRIO SYMbIOSIS AT MANY LEVELS

E. Ruby; University of Wisconsin, Madison, WI.

One goal of systems biology is to understand a life process across a number of investiga-tive levels. In the context of a symbiosis, that can mean examining the interaction between two species all the way from underlying mo-lecular mechanisms, through the organism’s physiological and behavioral activities, to their evolutionary history and consequences. Much is known about the association between the Hawaiian squid Euprymna scolopes and its bioluminescence bacterial symbiont Vibrio fischeri, and integrating this information into a coherent picture of the symbiosis has revealed several common themes. One of those themes centers on the multiple roles of the polysac-charide chitin, a ubiquitous compound in the marine environment, in the biology of the

squid-vibrio association. Specifically, chitin, and its breakdown products, chitobiose and N-acetyl glucosamine, impact the symbiosis at the levels of microbial/host behavior, develop-ment, nutrition, signaling, gene expression and population biology. Bringing these multiple roles together creates a more coherent picture of the interaction of these species as a biologi-cal system.

n S6:2dIffERENCES IN ENERGY ANd NITROGEN METAbOLISM AMONG SYMbIONTS Of HYdROTHERMAL VENT GASTROPOdS RELATES TO GEOCHEMICAL NICHE

R. A. Beinart1, J. G. Sanders1, F. J. Stewart2, E. F. Delong3, P. R. Girguis1; 1Harvard Uni-versity, Cambridge, MA, 2Georgia Institute of Technology, Atlanta, GA, 3Massachusetts Insti-tute of Technology, Cambridge, MA.

Classic studies of niche partitioning, the pro-cess wherein coexisting organisms occupy distinct niches, have focused on how intrinsic traits affect niche utilization. Organisms can access novel niche space via symbiosis with other organisms, through the addition of the symbiont’s physiological capabilities. With growing awareness of the prevalence of micro-bial-animal symbioses, the interaction between niche utilization and symbiont physiology may prove key to understanding the structure of many communities. To that end, we compared the physiological state of the endosymbionts of hydrothermal vent snails, via transcriptomics, that inhabit different geochemical niches. Sym-bioses between invertebrates and intracellular bacteria dominate metazoan communities at deep-sea hydrothermal vents. In these symbio-ses, the bacterial endosymbionts oxidize vent-derived compounds such as hydrogen sulfide and methane for the energy to fix carbon, pro-viding primary nutrition to their host. Recently, a survey of the symbiotic snail genus Alvini-concha at vents across a regional geochemical gradient showed a structured biogeographic distribution of host types (likely cryptic spe-

Speaker Abstracts

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cies) and their specific symbiont phylotypes. Alviniconcha host types with ε-proteobacterial symbionts dominated at vents where hydrogen and hydrogen sulfide concentrations were high, while host types with γ-proteobacterial symbi-onts predominated at vents with lower concen-trations of these chemicals. This distribution pattern suggests that the different symbionts of Alviniconcha employ different chemoautotro-phic metabolism across the regional gradient, influencing niche utilization by the symbioses Here, we present data that reveal the symbi-onts’ physiological poise in situ. Via a novel in situ RNA sampling and preservation device, we compared the metatranscriptomes of the symbionts of Alviniconcha collected across the regional chemical gradient. The endosymbiont phylotypes show key differences in energy and nitrogen metabolism, which relate to both en-vironmental chemistry (i.e., the relative expres-sion of genes across the geochemical gradient) and symbiont phylogeny (i.e., the specific pathways employed by each symbiont type). Unexpectedly, we found dramatic differences in expression of transposases and flagellar genes, suggesting that the symbiont phylotypes may also have distinct life histories. These data further our understanding of these symbionts’ metabolic capabilities and their expression in situ, and suggest an important role for symbi-onts in mediating their hosts’ interaction with their geochemical niche. With our increasing awareness of the ubiquity of microbe-eukary-ote associations at hydrothermal vents and other ecosystems, this data underscores that symbiont physiology may be imperative to the structure of biological communities.

n S6:3AS SIMPLE AS POSSIbLE, buT NO SIMPLER: THE MuLTI-ORGANISMAL bASIS Of ANIMAL NuTRITION

A. E. Douglas; Entomology, Cornell Univer-sity, Ithaca, NY.

High throughput sequencing technologies are generating extensive datasets on symbioses

that are commonly too complex to be com-prehended by visual inspection alone. This difficulty applies particularly to the study of nutrient exchange, the core interaction in most animal-microbial symbioses. Systematic meth-ods for the construction and analysis of meta-bolic models are essential to obtain precise, testable hypotheses of nutritional interactions. Equally, empirical tests of these models are crucial, simply because plausible computa-tional scenarios can be wrong. Symbioses be-tween animals with single or low diversity of microorganisms can be considered as a sand-box in which systems-level approaches can be develop and tested, for subsequent application to more complex associations. I will focus on an association that naturally comprises just two genomes, one insect and one bacterial, in a stable association that has persisted for over >100 million years. Analysis of the metage-nome, metatranscriptome and metaproteome, together with metabolic modeling, has led to the identification of shared metabolic pathways between host and symbiont, in relation to the biosynthesis of key metabolites and nitrogen recycling; and yielded testable hypotheses on the mechanisms by which the bacterial popula-tions may be regulated. I will also address how understanding of the two-genome system can be applied to investigate the function of sys-tems comprising taxonomically-diverse micro-bial communities and their animal hosts.

n S6:4CONSERVATION Of SMALL RNA ExPRESSION IN REduCEd GENOMES Of THE ObLIGATE SYMbIONT buCHNERA

A. Hansen, P. Degnan, N. Moran; Yale Uni-versity, West Haven, CT.

Genomic architecture drastically changes when bacteria transition from a free-living lifestyle to an obligate intracellular association. Bu-chnera aphidicola is an example of such an endosymbiont, and possesses altered genomic features such as reduced genome size, A+T bias, and an increase in molecular evolutionary

Speaker Abstracts


rate. Buchnera has lost most of the regulatory mechanisms present in free-living relatives, and thus regulatory processes in Buchnera, an uncultivable microbe, are largely unknown. Recently, a computational approach based on divergent Buchnera genomes identified conserved putative regulatory elements within shared intergenic spacers. In this study, we empirically test if these predicted regulatory elements as well as other small RNAs are expressed using high-throughput directional RNAseq. We compare small RNA expression of five Buchnera strains from four divergent aphid species (Acyrthosiphon pisum [strains LSR1 and 5A], Acyrthosiphon kondoi [strain Ak], Uroleucon ambrosiae [strain Ua], and Schizaphis graminum [Sg]). RNA fragments <200nt were directionally sequenced using Illumina 35 nt reads. For each sample, most reads (65-78%) map to the corresponding Buchnera genome. Biological replicate strains Ap-5A and Ap-LSR1 share very similar small RNA expression profiles, for which coverage per base pair mapped is highly correlated in both directions (Pearson correlation coef-ficient= 87-97%) Out of a total of 110 IGS with regulatory elements predicted, we found that 67.3% are significantly expressed in at least one Buchnera taxon. This included 33 expressed in at least three divergent Buchnera taxa, and 19 expressed in all divergent taxa. Most predicted regulatory small RNA elements that are significantly expressed in all diver-gent taxa are expressed in intergenic spacers encompassed by coding sequences (CDS) ori-ented in the same direction, and appear to be putative novel small RNAs, riboswitches, and regulatory binding sites. In addition to pre-dicted elements, we also found numerous cases of antisense RNAs expressed within Buchnera CDS, which are conserved in all taxa. These results demonstrate how high-throughput ge-nomics can provide insights into novel regula-tory mechanisms of highly reduced symbiont genomes.

n S6:5NEW TOOLS fOR ExPLORING THE HuMAN GuT MICRObIOME

A. Goodman; Yale University, New Haven, CT.

Culture-independent surveys of human gut mi-crobial community composition have revealed an unexpected variability between individuals. While these personal microbial signatures are increasingly associated with wide-ranging aspects of human health, the underlying mech-anisms of microbial competition and selection that shape these communities remain obscure. This lack of knowledge means that we cannot identify which microbial pathways represent appropriate targets for therapeutic intervention, how to shift the balance of species in the gut, or how to successfully introduce new species into these communities. Thus, understanding how metabolites are exchanged between hu-man gut microbes and which of these networks represent the fundamental mechanisms that determine community structure is an important goal. We describe how integrating microbial genetics, germfree animal studies, and bioin-formatics can produce new insights into the functional basis of microbial interactions in the gut environment.

n S6:6dETECTING COdIVERSIfICATION IN AN INSECT GuT MICRObIOME

J. G. Sanders1, S. Powell2, D. J. Kronauer3, H. L. Vasconcelos4, N. E. Pierce1; 1Harvard Uni-versity, Cambridge, MA, 2George Washington University, Washington, DC, 3Rockefeller Uni-versity, New York, NY, 4Universidade Federal de Uberlandia, Uberlandia, BRAZIL.

To what extent does coevolutionary history shape commensal microbial communities? Partner fidelity, linking fitness outcomes among partners, is thought to be an important stabilizer in mutualisms; over long enough timescales, it may result in codiversification, as is frequently observed in the vertically

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transmitted endosymbionts of insects. But can historical partner fidelity operate effectively on complex extracellular communities, in the face of competition from exogenous microbes and greater opportunity for horizontal trans-mission? Here, we explore the hypothesis that codiversification in the Cephalotes ant gut community is important in structuring distri-bution of bacterial taxa among species in the host genus. Cephalotes are arboreal and largely herbivorous, frequently occupying similar habitats to Camponotus ants, which are known to host endosymbionts. Cephalotes have no known endosymbionts, but do host an unusu-ally dense microbial community in an enlarged hindgut. Using 454 pyrosequencing of 102 individuals from 26 Cephalotine species, we show that this community is extraordinarily conserved within the genus. To detect signs of codiversification, we used novel implementa-tions of two techniques. First, we grouped samples by the similarity of their communities and compared this to the host phylogeny. By systematically exploring the distribution of information in the dataset, we demonstrate not

only that communities tend to group according to the host phylogeny, but that the data under-lying these groupings are consistent with a history of codiversification across much of the bacterial community. Second, we searched the bacterial community for lineages whose phy-logenetic histories were similar to the host’s. Consistent with the community-based analysis, we find that many of the endemic members of the Cephalotes gut microbiome have codiver-sified with their hosts. Our results indicate a high degree of partner fidelity in this ant mi-crobiome, suggesting that vertical transmission of the entire community -- perhaps mediated by the ants’ eusocial colony structure -- may play an important role in the evolution and maintenance of the association. Intriguingly, a similar analysis of published mammalian mi-crobiomes yielded strikingly different results. As additional comparative microbiome data become available, the techniques presented here will become a powerful tool for exploring broad trends in the evolution of host-associated microbial communities.

Speaker Abstracts


Poster Abstractsn 1APAPER CuRRENCY AS fOMITE fOR bACTERIA WITH HuMAN PATHOGENIC POTENTIAL

S. Estrada1, C. Betancur2, M. T. Ceballos1, E. Sanchez2, A. M. Abad2, C. Vanegas1, L. M. Salazar2; 1Laboratorio Clínico Congregación Mariana, Medellín, COLOMBIA, 2Universidad CES, Medellín, COLOMBIA.

Introduction: Paper currency is one of the articles of highest circulation worldwide. In Colombia, the raw material for its manufacture is 100% cotton fiber, which gives it a porous structure that allows bills to lodge different types of debris and enables microbial coloniza-tion of this paper. Aim: Determine the pres-ence of bacterial contamination and its type in bills circulating in the city of Medellin. Mate-rials and methods: Out of convenience,101 bills were selected for microbiological exami-nation from December 2009 to May 2010. This analysis included the isolation of the bacterial microbiota present on both surfaces of the notes, using the swab technique and perform-ing the taxonomic classification of the isolates obtained. Results: 91.1% of the evaluated bills presented microbial contamination for a total of 124 isolates. The genus most frequently iso-lated corresponded to Bacillus; it was isolated in 65 cultures (52.4%). 41.9% of the isolates corresponded to bacteria potentially patho-genic to humans, such as coagulase-negative staphylococci (20.2%) and Enterobacteriaceae gramnegative rods (15.3%), Pantoea cloacae being the most frequently isolated species (8.9%). 17.7% of the isolates corresponded to enteric bacteria. Conclusion: Colombian bills can act as fomite for environmental microbiota and bacteria which are potentially pathogenic to people.

n 2BANTIMICRObIAL PEPTIdE fROM A RHIzObACTERIAL ISOLATE ANTAGONISTIC TO CLAVIbACTER MICHIGANENSIS SuSbP. MICHIGNENSIS: ISOLATION ANd PARTIAL PuRIfICATION

F. Mabood, A. Souleimanov, D. Smith; McGill University, Montreal, Ste Anne de Bellevue, QC, CANADA.

Clavibacter michiganensis subsp. michiganen-sis (Cmm) causes bacterial canker of tomato. The aim of the study was to isolate and char-acterize anti-cmm compound(s) produced by a novel rhizobacterial isolate antagonistic to Cmm. A novel rhizobacterial strain showing strong antibacterial activity against Cmm was isolated from the rhizosphere. Using an agar well diffusion assay, activity of the cell free su-pernatant was studied. The compound(s) were isolated, from bacterial culture, with n-butanol and further fractionated with HPLC. Purified fractions were subjected to SDS-PAGE analy-sis. Our results demonstrate that the antibacte-rial compound was produced during the late growth phase of the culture. Initial studies have shown that the antimicrobial compound is pro-teinaceous in nature and thus is a bacteriocin. SDS-PAGE of the bacteriocin shows that the molecular weight of the compound is less than 4 KDa. Further work on characterization of the compound(s) is underway.

n 3AESCHERICHIA COLI, THE INfLAMMASOME ANd CROHN’S dISEASE.

A. Thompson, J. Catchpole, D. J. Clarke; Uni-versity College Cork, Cork, IRELAND.

Crohn’s disease (CD) is an inflammatory bowel disease that affects millions of people globally. Although the etiology of the disease is not fully understood it is clear that both host genetics and environmental factors (e.g.

ASM Conferences32

bacteria) are important. In particular a group of Escherichia coli, called adherent-invasive E. coli (AIEC), have recently been strongly as-sociated with CD. AIEC have no obvious viru-lence factors yet they are characterized by their ability to invade epithelial cells and replicate within macrophages. We have been using a strain of AIEC, called HM605, recovered from a colonic biopsy from a CD patient to study the molecular mechanisms that facilitate AIEC replication in the macrophage. In order to iden-tify the metabolic pathways used by the bacte-ria for replication in this niche we constructed mutations in major catabolic and anabolic pathways. In this way, we show that both gly-colysis (ΔpfkAB) and pyrimidine metabolism (ΔpyrF) are important for bacterial replication in the macrophage. We then used the ΔpfkAB mutant to address the issue of whether the macrophage can distinguish between replicat-ing and non-replicating bacteria (indicative of potential pathogens and commensals, respec-tively) inside the macrophage. We show, using microarrays, that the macrophage transcrip-tional response to infection with HM605, the ΔpfkAB mutant or a non-pathogenic strain of E. coli, MG1655, is identical and dominated by NF-kB regulated genes. This suggests that the dominant signaling pathway(s) used by macrophages to respond to infection with E. coli cannot distinguish between replicating and non-replicating bacteria. However, subsequent cytokine analysis revealed that there was a sig-nificantly increased level of IL-1β secreted by macrophages infected with HM605 compared to either the ΔpfkAB mutant or MG1655. The production of IL-1β is controlled by the in-flammasome, a protein complex that activates caspase-1 in response to a variety of signals, resulting in the processing of cytoplasmic pro-IL-1β to the mature, secreted cytokine. Immunoblotting confirmed that caspase-1 is activated following infection of macrophages with HM605, suggesting that macrophages can use the inflammasome to distinguish between bacteria that can replicate (potential patho-gens) and viable bacteria that cannot replicate (commensals) inside the immune cell. This

work also potentially links, for the first time, activation of the inflammasome with Crohn’s disease.

n 4BTHE REGuLATION Of SYMbIOSIS IN PHOTORHAbduS.

L. Lango, S. A. Joyce, D. J. Clarke; University College Cork, Cork, IRELAND.

Photorhabdus is a genus of bioluminescent, Gram-negative bacterium belonging to the family Enterobacteriaceae. Photorhabdus is highly virulent to insect larvae whilst, at the same time, maintaining a mutualistic relation-ship with entomophagous nematodes of the family Heterorhabditiae. How does Photorhab-dus control these different relationships and make the appropriate life-style decisions? Our observation that pathogenicity is correlated with Photorhabdus exponential growth inside the insect whilst mutualism (i.e. the support of nematode growth and reproduction) is associ-ated with the post-exponential phase of growth suggested that life-style decisions might be linked to bacterial growth and/or metabolism. Photorhabdus elaborates an extensive sec-ondary metabolism during post-exponential growth that is also associated with mutualism. Products of this secondary metabolism include an antibiotic called 3-5-dihydroxy-4-isopropyl-stilbene (ST), an anthraquinone pigment (AQ) and bioluminescence. We identified a mutant that was unable to produce ST, AQ and light and this mutation was found to be in the mdh gene, encoding malate dehydrogenase, a key enzyme in the TCA cycle. The mdh mutant was unaffected in virulence but, importantly, the mdh mutant was not able to support nema-tode growth and development in vivo or in vitro. Furthermore the construction of muta-tions in key genes in other central metabolic pathways confirmed the critical role for the TCA cycle in both secondary metabolism and mutualism, but not in virulence. Therefore, the TCA cycle is required for the transition of Photorhabdus from pathogen to mutualist sug-

Poster Abstracts


gesting that a metabolic switch plays a key role in the regulation of life-style decisions in this bacterium. Finally, recent array studies have revealed the full extent of the changes in gene expression that occur during this metabolic switch. These changes can be used to describe the molecular contributions made by the bacte-ria to the mutualistic association and they also hint at an unexpected potential role for sulfur metabolism and the activated methyl cycle in controlling the transition from pathogenicity to mutualism in Photorhabdus.

n 5ASTRESSOR INduCEd REduCTION IN MuRINE-ASSOCIATEd LACTObACILLuS REuTERI RESuLTS IN INCREASEd COLONIC INfLAMMATION WHEN CHALLENGEd WITH CITRObACTER ROdENTIuM

J. Galley1, A. Mackos1, N. Parry2, M. Nelson3, Z. Yu1, M. Lyte4, M. T. Bailey1; 1Ohio State University, Columbus, OH, 2Massachusetts Institute of Technology, Cambridge, MA, 3Uni-versity of Connecticut, Storrs, CT, 4Texas Tech University, Lubbock, TX.

The inflammatory bowel diseases, as well as irritable bowel syndrome, are often exacer-bated during periods of psychological stress. The mechanisms by which stress enhances colonic inflammation are not well understood, but could involve disrupted homeostatic inter-actions between the mucosal immune system and intestinal microbiota. In a previous study involving the use of 454 pyrosequencing as well as quantitative polymerase chain reac-tion (PCR), we found that exposing mice to a stressor shifted the community profile of the gut microbiota and significantly reduced the abundance of colonic tissue-associated bacteria in the genus Lactobacillus, and in particular, Lactobacillus reuteri. Because L. reuteri has been shown to mediate mucosal immunity and to protect the host against pathogenic invasion, we hypothesized that stressor exposure would enhance colonic inflammation during pathogen challenge, and that administering L. reuteri

immediately following exposure would abro-gate this enhancement. Mice were subjected to a repeated social defeat stressor, called social disruption, prior to and during infection with Citrobacter rodentium, a murine pathogen that is homologous to enteropathogenic Escherich-ia coli. Mice exposed to the stressor had higher levels of pathogen colonization, higher levels of colonic tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) mRNA, as well as an increased colitis score (an evaluation of cellular infiltration, edema, hyperplasia, dysplasia, and epithelial defects) on Day 12 post-C. rodentium challenge. When the mice were fed L. reuteri after every cycle of the stressor, the effects of the stressor on colonic inflammation and histopathologic coli-tis were abrogated. The reduction in colonic inflammation occurred even though the C. rodentium burden was unaffected, indicating that L. reuteri acts through an anti-inflam-matory means rather than an anti-bacterial one. These data support the hypothesis that stressor-induced disruptions of gut microbiota, specifically of lactobacilli, exacerbate colonic inflammation during pathogen exposure. Ad-ministering L. reuteri through oral gavage re-duced the stressor-induced increase in colonic inflammation, demonstrating the importance of L. reuteri in regulating the inflammatory response in the colon.

n 6BOMPA-MEdIATEd bIOfILM fORMATION IS ESSENTIAL fOR COMMENSAL SOdALIS TO COLONIzE THE TSETSE fLY GuT

M. A. Maltz, B. L. Weiss, S. Aksoy; Yale Uni-versity, New haven, CT.

Many bacteria successfully colonize animals by forming protective biofilms. Molecular processes that underlie the formation and function of biofilms in pathogenic bacteria are well characterized. In contrast, the relation-ship between biofilms and host colonization by symbiotic bacteria is less well understood. Here, we used the tsetse fly (Glossina morsi-

Poster Abstracts

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tans) as a novel and cost-effective model to further investigate these processes. Tsetse flies are vertebrate blood feeding Diptera (Glossina spp.) that house 3 maternally transmitted sym-bionts, one of which is a commensal (Sodalis glossinidius) found in several host tissues, including the gut. We determined that Sodalis forms biofilms in tsetse’s gut, and that this process is influenced by Sodalis’s Outer mem-brane protein A (OmpA) through activation of the CpxRA two-component signaling pathway. Mutant Sodalis that do not produce OmpA (SodalisΔOmpA) fail to form biofilms in vitro, and are unable to colonize tsetse’s gut unless en-dogenous symbiotic bacteria are present. Our data indicate that in the absence of biofilms, SodalisΔOmpA are exposed to and eliminated by tsetse’s innate immune system, suggesting that biofilms help Sodalis evade its host’s immune system. Tsetse is the sole vector of pathogenic African trypanosomes, which also reside in the fly’s gut. Acquiring a better understanding of the dynamics that promote Sodalis coloniza-tion of tsetse’s gut may enhance the devel-opment of novel disease control strategies. Furthermore, basic concepts obtained from this study will be applicable to understanding the dynamics of symbiosis in the mammalian gas-trointestinal tract.

n 7ATHE uSE Of PREdATORY PROkARYOTES TO CONTROL HuMAN PATHOGENS ANd bIOfILMS

D. E. Kadouri, D. Godboley; University of Medicine and Dentistry of New Jersey, New-ark, NJ.

Disease-causing microorganisms that have be-come resistant to drug therapy are an increas-ing cause of wound infections. Bdellovibrio and Micavibrio are Gram-negative obligatory parasites that feed on other Gram-negative bacteria. The focus of the study was to evalu-ate the potential use of predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus to control pathogens associ-

ated with human infection. By coculturing B. bacteriovorus 109J and M. aeruginosavorus ARL-13 with selected pathogens, we have demonstrated that predatory bacteria are able to attack bacteria from a variety of genera in-cluding: Acinetobacter, Aeromonas, Aggrega-tibacter, Bordetella, Burkholderia, Citrobacter, Eikenella, Enterobacter, Escherichia, Fuso-bacterium, Klebsiella, Listonella, Morganella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio and Yersinia. Positive preda-tion was measured in single and multi-species microbial cultures as well as on monolayer and multilayer pre-formed biofilms. The predators were also able to prey and remove metabolical-ly inactive biofilms. Although predatory bacte-ria are extremely lethal to some Gram-negative bacteria, no toxic effects were observed when high doses of the predators were injected into the hemocoel of Galleria mellonella worms, or when human cell lines were incubated with the predators. In conclusion, the work presented here highlights the potential use of predatory bacteria as biological based agent for eradicat-ing infection.

n 8BCHANGES IN THE INTESTINAL MICRObIOME fOLLOWING fECAL MICRObIOTA TRANSPLANTATION fOR SEVERE CLOSTRIdIuM dIffICILE INfECTION

M. J. Sadowsky1, A. R. Weingarden1, M. J. Hamilton1, T. Unno1, A. Khoruts2; 1Univ. of Minnesota, St. Paul, MN, 2Univ. of Minnesota, Minneapolis, MN.

Clostridium difficile infection (CDI) is an antibiotic-associated diarrheal disease which continues to increase in incidence and severity worldwide. The most clinically severe forms of CDI can lead to fulminant colitis and megaco-lon, which is associated with a high mortality rate (~50%) and often requires surgical colec-tomy. Fecal microbial transplantation (FMT) is becoming a more accepted and widely used technology for treatment of recurrent Clostri-dum difficile infection (CDI), and may repre-

Poster Abstracts


sent a non-surgical alternative for treatment of severe disease. We recently reported the de-velopment and successful use of standardized, partially purified, frozen, fecal microbiota to treat CDI and have used 16S sequence analysis to follow changes in fecal microbiotia over time in patients receiving FMT. Here we report that deep DNA sequence analysis, done using barcoded primers targeting the V6 region and Illumina sequencing technology, revealed the initial engraftment of gut microbiota follow-ing transplantation of previously frozen fecal bacteria from a healthy donor. Prior to therapy the intestines of severe CDI patients were dominated by proteobacteria and relatively low numbers of Firmcutes and Bacteroidetes. This trend was reversed by FMT and resulted in restoration of microbial community structure and function, prompt clinical improvement, and rescue of patients from the operating table. However, the improvement was generally short-lived and an additional FMT therapy was required to clear the infection. Our results in-dicate that FMT is a potential alternative treat-ment to colectomy in severe CDI, but require more than one application to achieve full clear-ance of infection, complete clinical recovery, and conversion of gut microbial community structure to resemble that of the donor. This work is supported by NIH grant R21AI091907.

n 9ALactobaciLLus brevis kb290 ENHANCES IL-8 SECRETION bY vibrio paraHaemoLyticus-INfECTEd CACO-2 CELLS.

T. Yakabe1, T. Shimohata2, A. Takahashi2; 1Re-search Institute, Kagome Company, Ltd., Na-sushiobara, JAPAN, 2Institute of Health Biosci-ences, The University of Tokushima Graduate School, Tokushima, JAPAN.

Background. Vibrio parahaemolyticus in uncooked seafood causes acute gastroenteritis. The microorganism has two sets of type III se-cretion systems (T3SSs) and two hemolysins. When it injects its effector proteins into a host

cell via T3SS1, one of the T3SSs induces se-cretion of interleukin (IL)-8, a proinflammato-ry chemokine, through the phosphorylation of both ERK 1/2 and p38 MAPK. Although pro-biotics have beneficial effects on hosts and can help control some infectious diseases, there is a little research on the efficacy of probiot-ics in V. parahaemolyticus infection. Here we treated V. parahaemolyticus-infected human intestinal epithelial cells with L .brevis KB290 (KB290) a probiotic isolated from fermented vegetables, and demonstrated its efficacy in enhancing IL-8 secretion. Results. Among the 3 lactic acid bacterial strains we tested, KB290 induced the highest level of IL-8 secre-tions in the infected cells. Relative to control cells (Caco-2 cells pretreated with phosphate buffered saline), Caco-2 cells pretreated with KB290 expressed IL-8 earlier, although con-centrations were similar 450 min after infec-tion. KB290 pretreatment also induced earlier ERK 1/2 phosphorylation, greater p38 MAPK phosphorylation, and earlier IL-8 mRNA expression. Conclusions. KB290 accelerated IL-8 secretion, a host cell immune response, in V. parahaemolyticus-infected cells. We con-sider this to be beneficial because IL-8 plays an important defensive role against infection and in the repair of injured epithelial cells.

n 10BfECAL TRANSPLANTATION Of PATIENTS WITH CLOSTRIdIuM dIffICILE ASSOCIATEd dISEASE STudIEd WITH PHYLOGENETIC MICROARRAYS, HIGH-THROuGHPuT SEquENCING, ANd fLuORESCENT IN SITu HYbRIdIzATION

O. Paliy1, V. Shankar1, M. J. Hamilton2, T. Unno2, A. Kilburn1, A. Khoruts2, M. J. Sad-owsky2; 1Wright State University, Dayton, OH, 2University of Minnesota, Minneapolis, MN.

While the majority of the microbes inhabiting human gut are commensal, some have proven to cause a number of diseases and afflictions. One of these microbes, Clostridium difficile, is an opportunistic pathogen that is known to

Poster Abstracts

ASM Conferences36

cause enterocolitis. C. difficile can often bloom following an antibiotic administration to a particular individual, and Clostridium difficile associated disease (CDAD) in one of the main causes of antibiotic-induced diarrhea and coli-tis. While C. difficile infection can be treated with metronidazole or vancomycin, the disease has a high recurrence rate due to the resistance and survival of clostridial spores. One alterna-tive successful approach to combat CDAD is through fecal transplantation of distal gut mi-crobiota from healthy donor to the C. difficile-infected patient. In this study we have utilized phylogenetic Microbiota Array, high-through-put Illumina sequencing, and fluorescent in situ hybridization to investigate the specific changes in the CDAD patients’ gut microbial communities before and after fecal transplanta-tion, and we compared these communities to the composition of the donor’s fecal micro-biota. Fresh stool samples were collected from three CDAD patients, healthy donors, and from each patient over a four-month period follow-ing fecal transplantation. The original patients’ microbiota had low diversity, was dominated by members of Gammaproteobacteria and Bacilli, and had low numbers of Clostridia and Bacteroidia. In comparison, donor community was dominated by Clostridia and had signifi-cantly higher diversity and evenness. Because Microbiota Array provides quantitative esti-mates of genus and species level abundances, we determined specific genera prevalent in each sample. At the genus level, fecal samples of CDAD patients were rich in genera Veillon-ella, Lactobacillus, Streptococcus, Enterobac-ter, and Escherichia, all known to be prevalent in the human small intestine. The donor fecal samples had many abundant genera that in-cluded Coprococcus, Blautia, Faecalibacte-rium, Roseburia, Papillibacter, Bacteroides, and Akkermansia. The patients’ communities were largely replaced by the donor fecal microbiota within three days following fecal transplantation, and these transplanted com-munities remained stable in the patient for at least four months. This effect was consistent among all three transplantations. The trans-

planted microbiota was indistinguishable from that of the donor. The microarray results were concordant with high-throughput sequencing data and were confirmed by fluorescent in situ hybridization of class-specific probes. In each case, the gut microbiota replacement led to full patient recovery and symptom alleviation. We conclude that fecal transplantation results in the replacement of patient microbiota commu-nity with that of the donor.

n 11ACAENORHAbdITIS ELEGANS IN SALMONELLA PATHOGEN CHALLENGE IS PROTECTEd bY YEAST fERMENTATION PROduCT ExTRACT.

J. J. Peloquin1, S. G. Reeves2, T. M. Wer-ner1; 1Diamond V, Cedar Rapids, IA, 2Embria, Ankeny, IA.

Diamond V produced innovative and effective natural fermentation products for animal health for more than 64 years using our complex proprietary processes. Caenorhabditis elegans is an excellent whole animal model for host-pathogen interactions (Moy et al. 2006) and to show the influence of natural products on aging (Wilson et al. 2005). Instructed by these publications, Diamond V has developed a screening technology using C. elegans to iden-tify promising new product prototypes and to evaluate biological activities of product proto-types. Using a pathogen challenge model rely-ing on worm survival as a marker of biological activity, we show that a BSL-1 Salmonella enterica Typhimurium strain is pathogenic to C. elegans and significantly shortens their life compared to their normal laboratory diet of Escherichia coli OP50.1. Importantly, we also demonstrate that exposing nematodes through-out their lives to 70 μg/mL of a phosphate buffer extract of yeast fermentation product- sometimes called “yeast culture”- doubles the average survivorship of C. elegans exposed to said product when challenged with Salmonella enterica Typhimurium, whereas 7 μg/mL was not significantly protective. These yeast fer-

Poster Abstracts


mentation products don’t have significant en-dogenous anti-microbial properties, and there were no gut flora in these nematodes other than the bacteria they were fed. Thus the observed enhanced worm survival is likely mediated through enhanced innate immune response to the pathogen challenge.

n 12BCHARACTERIzING THE MICRObIOME Of THE kLEPTOPLASTIC MOLLuSC eLysia cHLorotica

S. P. Devine, K. N. Pelletreau, M. E. Rumpho; University of Connecticut, Storrs, CT.

The mollusc (sea slug) Elysia chlorotica forms an obligate, endosymbiotic relationship with chloroplasts (kleptoplasty) obtained from its algal prey Vaucheria litorea, allowing the sea slug to sustain itself photoautotrophically for several months. The diversity of other symbi-onts associated with this photosynthetic sea slug, and their potential role in contributing to the long-term success of the symbiosis through the provision of nutrients as well as develop-mental and protective benefits, has never been investigated. 16S rDNA-based metagenomic analysis was used to characterize the microbial diversity associated with two populations of E. chlorotica from Halifax, Nova Scotia and Martha’s Vineyard, MA. Whole animals were examined immediately after collection, after being starved of their algal prey for several months in the laboratory, and after being bred in the laboratory. The microbiome of labo-ratory-cultured V. litorea was also analyzed. Alpha-, beta-, and gamma-proteobacteria dominated all of the samples along with Acti-nobacteria, Bacilli, Flavobacteria, and Sphin-gobacteria. However, abundances and taxo-nomic diversity at the order and family levels varied between the two populations and among all samples except for the lab-bred sea slugs which had very similar bacterial profiles to one another, but dissimilar to the algal prey mi-crobiome. Bacteria capable of polysaccharide digestion and photosynthesis, as well as puta-

tive nitrogen fixation, vitamin B12

production, and natural product biosynthesis were associ-ated with the sea slug and algal samples. Mi-crobial diversity associated with the digestive gut and secreted mucus of E. chlorotica from Halifax was also characterized. A comparison of wild-captured vs. lab-starved (1 month) sea slugs showed that a change in habitat and diet affected gut microflora. Likewise, the gut and mucus of wild-captured E. chlorotica revealed different selection pressures for microbes. The most prevalent classes included α-, β-, and γ-proteobacteria as well as Actinobacteria, Bacilli, and Mollicutes. All samples had a high number of bacterial families in common, sug-gesting that these families represent transient environmental microflora acquired from the water of the native salt marsh. Most of the bacteria were chemoorganotrophs, although a number of phototrophs were identified. Puta-tive metabolic functions of identified bacteria included carbon cycling, denitrification, nitro-gen fixation, and vitamin B

12 production. This

study is the first to identify the bacteria living in association with E. chlorotica and V. litorea. Further localization and functional studies (metatranscriptomics and metabolomics) are needed to confirm the presence of specific symbionts and define their metabolic function in the Elysia-Vaucheria system.

n 13AA MuLTI-OMIC VIEW Of HOST-PATHOGEN-COMMENSAL INTERPLAY IN saLmoneLLa-INduCEd GASTROENTERITIS

B. L. Kaiser1, J. Li2, J. A. Sanford3, Y. Kim1, S. R. Kronewitter1, M. B. Jones4, C. T. Peterson4, S. N. Peterson4, B. Frank4, T. O. Metz1, R. D. Smith1, F. Heffron2, J. N. Adkins1; 1Pacific Northwest National Laboratory, Richland, WA, 2Oregon Health and Sciences University, Portland, OR, 3University of California San Diego, La Jolla, CA, 4J. Craig Venter Institute, Rockville, MD.

The potential for commensal microorgan-isms indigenous to a host (the ‘microbiome’

Poster Abstracts

ASM Conferences38

or ‘microbiota’) to alter infection outcome by influencing host-pathogen interplay is largely unknown. We used a pan-omics “systems” approach, incorporating proteomics, metabolo-mics, glycomics, and metagenomics, to explore the molecular interplay between the murine host, the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), and commen-sal gut microorganisms during gastroenteritis. We find proteomic evidence that S. Typhimuri-um thrives within the infected 129/SvJ mouse gut without antibiotic pre-treatment, inducing inflammation and disrupting the intestinal mi-crobiome (e.g., suppressing Bacteroidetes and Firmicutes while promoting growth of Salmo-nella and Enterococcus). Alteration of the host microbiome population structure was highly correlated with gut environmental changes, including the accumulation of metabolites normally consumed by commensal microbiota. Finally, both proteomic and glycomic evidence suggest S. Typhimurium may take advantage of increased fucose moieties to metabolize fucose while growing in the gut. The application of multiple omics measurements to Salmonella-induced gastroenteritis provides insights into complex molecular strategies employed during pathogenesis between host, pathogen, and the microbiome.

n 14BYEAST COMMuNITIES Of dIVERSE dROSOPHILA SPECIES: COMPARISON Of TWO SYMbIONT GROuPS IN THE SAME HOSTS

J. A. Chandler, J. A. Eisen, A. Kopp; Univer-sity of California, Davis, Davis, CA.

The combination of ecological diversity with genetic and experimental tractability makes Drosophila a powerful model for the study of animal-associated microbial communities. De-spite the known importance of yeasts in Dro-sophila physiology, behavior, and fitness, most recent work has focused on Drosophila-bac-terial interactions. In order to get a more com-plete understanding of the Drosophila microbi-

ome, we characterized the yeast communities associated with fifteen different Drosophila populations collected around the world. These populations were chosen to sample the breadth of Drosophila phylogenetic, ecological and geographic diversity. Data was generated us-ing “fungal-specific” primers NL1 and NL4, which amplify the ribosomal large subunit, and the Roche 454 JR platform. For a dozen host populations, bacterial 16S data has previously been generated (Chandler et al., 2011, PLoS Genetics), and this strategy allowed us to com-pare the distribution and structure of the yeast and bacterial communities in the same host populations. We show that yeast communities are dominated by a small number of abundant taxa, that the same yeast lineages are associat-ed with different host species and populations, and that host diet has a greater effect than host species on the yeast community composition. These patterns closely parallel those observed in the Drosophila bacterial communities. However, Procrustes analysis implemented in QIIME does not detect a significant correlation between the yeast and bacterial communities of the same host populations. This suggests that knowledge of the community composition of one symbiont group is not sufficient to pre-dict the composition of a second. This research simultaneously looks at two symbiont groups associated with an animal host, future Dro-sophila-microbe work, whether it is focused on genetics, metabolism, or evolution, and animal-microbe research in general, should take a similarly holistic approach.

n 15ACOMPARATIVE TRANSCRIPTOMIC ANALYSIS Of ruminococcus aLbus STRAINS 7 ANd 8 GROWN ON CELLuLOSE ANd HEMICELLuLOSE

I. Kwon, I. Cann, R. Mackie; Univ. of Illinois at Urbana-Champaign, Urbana, IL.

Ruminococcus albus is known as a specialist bacterium for the degradation of both cellulose and hemicellulose in the rumen and colon.

Poster Abstracts


However, depending on strain, their pheno-typic and fibrolytic abilities are variable and no information is available on a comparison of genomic and transcriptional differences between strains of R. albus that underly this phenotypic variation. The regulation of genes involved in polysaccharide degradation in both strains grown on either cellobiose, phos-phoric acid-swollen cellulose (PASC) or wheat arabinoxylan (WAX) were compared using RNA sequencing approaches. Both strains of R. albus exhibited similar ability to degrade PASC as shown by growth rate, substrate deg-radation, and accumulation of fermentation end-products. When R. albus 7 was grown on PASC relative to cellobiose, 241 genes were expressed greater than 2-fold, and 289 genes were repressed greater than 2-fold (p < 0.001). Based on annotation using NCBI, RAST server and Pfam databases, 26 of 241 highly induced genes were predicted to code for glycoside hydrolase (GH) family domains. Although half (101 genes) of total induced genes were classified as hypothetical proteins, the other genes had functions predicted as carbohydrate binding proteins, membrane transporters and oxidoreductases. Similarly, during growth of R. albus 8 with PASC compared with cellobiose, 246 genes were expressed greater than 2-fold, and 218 genes were repressed greater than 2-fold (p < 0.001). Of the 246 induced genes, 23 genes were predicted to code for GH family domains. In contrast, during growth with WAX compared to cellobiose, both strains had dis-tinct and different physiological and transcrip-tional profiles. For R. albus 7, 151 genes were up-regulated greater than 2-fold, and 27 genes were down-regulated greater than 2-fold (p < 0.001). Of 151 up-regulated genes, 15 genes were predicted as GH’s. However, R. albus 8 had 649 up-regulated genes, and 46 genes of the total up-regulated genes were predicted to code for GH’s. It was also shown that R. albus 8 had more highly induced genes than R. albus 7 when comparing transcription profiles on WAX with PASC. During growth with WAX relative to PASC, 398 genes in R. albus 7 were induced greater than 2-fold (p < 0.001), and

13 of these genes were predicted as GH’s. In contrast, 541 genes in R. albus 8 were induced greater than 2-fold (p < 0.001), and 32 pre-dicted GH’s were induced greater than 2-fold. These transcriptional patterns were consistent with physiological data showing that R. albus 8 had higher maximum cell yield and level of fermentation end-products than R. albus 7 during growth with WAX. This study provides insight into the degradation of cellulose and hemicellulose based on analysis of genetic and transcriptional differences between the two R. albus strains and demonstrates that R. albus 8 has higher catalytic potential to degrade hemi-cellulose than R. albus 7.

n 16BCOMPARISON Of CRYPTOCOCCAL ANTIGENEMIA bETWEEN ANTIRETROVIRAL-NAïVE ANd ANTIRETROVIRAL-ExPERIENCEd HIV POSITIVE PATIENTS IN TWO HOSPITALS IN ETHIOPIA.

T. B. Tufa, Y. Woldeamanuel, D. Asrat; De-partment of Microbiology, Immunology, and Parasitology, College of Health Science, Addis Ababa University, Addis Ababa, ETHIOPIA.

Background: Cryptococcal meningitis is a major fungal opportunistic infection among HIV/AIDS patients and one of the most com-mon causes of deaths in stages of AIDS. It could be prevented by early screening and treating of patients. Objective: The purpose of the present study was to compare the preva-lence of cryptococcal antigenemia among HIV positive antiretroviral-naïve and experienced patients and to compare with their CD4 cell counts. Methods: A cross sectional compara-tive study was carried out in two hospitals (Asella teaching Hospital and Adama Hospital, Ethiopia). A total of 254 HIV positive consent-ing patients were enrolled in the study during the period of December, 2011 to May, 2012. Out of these, 127 were antiretroviral-naïve, 121 ARV experienced patients and 6 were discontinued treatment. The cryptococcal an-tigen was detected from the serum of patients

Poster Abstracts

ASM Conferences40

by latex agglutination tests. All positive sera were subjected for culture and identification using standard laboratory procedure. Results: Out 254 HIV positive patients screened, 26 (10.2%) were positive for cryptococcal anti-genemia. The rate for cryptococcal antigen-emia in antiretroviral-naïve, ARV experienced and those discontinued treatment were 14.2%, 4.1% and 50%, respectively. Out of the 26 positive for cryptococcal antigenemia, 4 (15.3%) were culture positive for Cryptococ-cus. Among the 26 positive for cryptococcal antigenemia, 13(22.0%), 10 (14.7%), 3(5.8%) and 0(0.0%) had CD4 count ≤100, 101-200, 201-350 and >350 /mm3, respectively. Con-clusion: The result of this study indicated that the target population for early cryptococcal antigenemia screening to prevent the develop-ment of cryptococcal meningitis are patients who ARV naive and have low cells CD4 count. Key words: HIV, Antiretroviral-experienced, Antiretroviral-naïve, CD4counts, Cryptococcal antigenemia, Ethiopia.

n 17ASIMuLTANEOuS SACCHARIfICATION ANd fERMENTATION Of CORN CObS TO bIO-ETHANOL bY CO-CuLTuRE Of aspergiLLus niger ANd saccHaromyces cerevisiae

J. U. Itelima, A. O. Salami; University of Jos, Nigeria, Jos, NIGERIA.

The production of bio-ethanol from corn is a mature technology that is not likely to see significant reduction in the production costs. Substantial cost reductions may be possible if cellulose based agricultural wastes such as corn cobs are used instead of corn. In this study, corn cobs which are in abundance and do not interfere with food security was sub-jected to simultaneous saccharification and fermentation process by co-culture of Aspergil-lus niger and Saccharomyces cerevisiae for 7 days. The corn cobs were sundried, milled into powder using hammer milling and stored at room temperature (25OC) before use. The

growth media used for culturing Aspergilus niger and Saccharomyces cerevisiae inocula were prepared respectively. Parameters such as biomass yield, cell dry weight, reducing sugar concentration, pH of the fermentation medium and the ethanol yield were determined at 24 hours intervals. The results of the study re-vealed that the yeast and mould biomass yield obtained from the corn cobs on the 7th day was 0.59 (OD), while the microbial cell dry weight obtained on the same day was 0.88mg/cm3. The substrate was hydrolyzed to produce 0.63mg/cm3 reducing sugar concentrations. The pH values of the fermentation medium varied between 3.05 and 7.58. Optimal ethanol yield of 10.08v/v was obtained after 7 days of fermentation. The results of this study suggest that agricultural wastes that contain ferment-able sugars can no longer be discarded into our environment, but should be converted to useful products like bio-ethanol.

n 18BMOLECuLAR bASEd STudY ON ROTAVIRuS CAuSING ACuTE dIARRHEA IN CHILdREN uNdER fIVE YEARS Of AGE VISITING TERTIARY CARE GOVERNMENT CHILdREN’S HOSPITAL Of kATHMANdu, NEPAL

S. Ansari, J. B. Sherchand; Institute of Medicine, Tribhuvan University., Kathmandu, NEPAL.

Background: Diarrheal diseases are major problem of developing countries. Though precise data on childhood mortality associ-ated with diarrheal diseases in Nepal is not available, it has been estimated that approxi-mately 25% of child death are associated with diarrheal disease, particularly acute diarrhea. Objective: The purpose of this study was to assess the incidence of rotavirus and specific genotype causing acute diarrhea in children under 5 years of age. Study design: A total of 525 children with acute diarrhea in a children’s hospital of Kathmandu, Nepal were enrolled between April 2011 to September 2011. Re-sult: The incidence of acute diarrhea due to

Poster Abstracts


rotavirus was 25.9% (136/525) by ELISA. The percentage of rotavirus infected male were higher 64.5% than female 35.5%. The frequency of rotavirus cases were higher in the children below 2 years of age among which the highest case 80.2% were found in age between 6-24 months (P<0.01). The genotypic charac-terization by reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that the serotype G12 represented 55.9% in this study associated with P-types either P[6], P[4] or P[8]. Further a total of eight G/P combination were identified, with G12P[6] 46.4% being the most common strain type of rotavirus in Nepal. Conclusion: The study indicates that the rota-virus diarrhea was 25.9% and the genotyping study revealed that G 12 and P [6] were found major genotypes causing rotavirus diarrhea in Nepal. Key word: Acute diarrhea, rotavirus, genotype, molecular, Nepal.

n 19AOxYGEN RELIEVES THE CO2 ANd ACETATE dEPENdENCY Of LACTObACILLuS jOHNSONII NCC 533.

R. Y. Hertzberger1, R. D. Pridmore2, C. Gysler2, M. Kleerebezem1, M. J. Teixeira de Mattos3; 1NIZO Food Research, Ede, NETH-ERLANDS, 2Nestlé Research Center, Vers-chez-les-Blanc, SWITZERLAND, 3University of Amsterdam, Amsterdam, NETHERLANDS.

The members of the Lactobacillus acidophi-lus group are of great importance to humans. Apart from their role in common food fermen-tations, they are also prevalent residents of the different human microbiota. Metabolic interde-pendencies between the species that constitute these complex microbial communities play a major role in the dynamics of the microbiota. The L. acidophilus group has shown to require a broad variety of nutrients for growth, rang-ing from nucleotides, fatty acids, and cofactors to CO2 and acetate, and is therefore strongly dependent on other species in the microbiome. We have studied the growth requirements and metabolism of one member of this group, Lac-

tobacillus johnsonii NCC 533, which has been recognized for its probiotic properties and is commercially available as a probiotic ingredi-ent in dairy products. Both on agar- and liquid-media, anaerobic growth of L. johnsonii NCC 533 requires CO2 supplementation of the gas phase. Switching off the CO2 supply induces growth arrest and cell death. The presence of molecular oxygen overcomes the CO2 depen-dency. Analogously, L. johnsonii NCC 533 strictly requires media with acetate to sustain anaerobic growth, although supplementation at a level that is 100-fold lower (120 microM) than the concentration in regular growth me-dium for lactobacilli already allows for regular growth. Analogous to the CO2 requirement, oxygen supply relieves the acetate-dependency for growth. The L. johnsonii NCC 533 genome indicates that this organism lacks genes coding for pyruvate formate lyase (PFL) and pyruvate dehydrogenase (PDH), which implies that endogenous acetyl-CoA production -a key precursor in numerous biosynthetic pathways- largely depends on pyruvate oxidase activity (POX). The proposed role of POX in CO2 generation is corroborated by the observation that in a POX deficient mutant of L. johnsonii NCC 533, oxygen is not able to overcome the CO2 dependency. Also, in contrast to the wild type, oxygen does not relief the acetate dependency of the pox-gene deletion mutant which confirms the role of pyruvate oxidase in the generation of C2-compounds. These results are unexpected, seen that aerobic environments generally do not enhance growth but cause severe oxidative stress. L. johnsonii NCC 533 produces up to 1 mM hydrogen peroxide in the presence of oxygen, which causes growth stagnation and cell death. These opposing con-sequences of oxygen presence contribute to the hypothesis that a micro-aerobic environment may be optimal for growth of L. johnsonii NCC 533.

Poster Abstracts

ASM Conferences42

n 20BPROfERTILITY EffECT Of LACTObACILLuS PLANTARuM 2621

P. Bhandari, V. Prabha; Panjab University, Chandigarh, INDIA.

Urogenital infections of bacterial origin have a high incidence among the world female population at reproductive age. Strains of Escherichia coli can colonize the vagina and replace the natural microflora. Lactobacillus, the predominant vaginal microorganism in healthy women maintains the acidic vaginal pH which inhibits the pathogenic microorgan-isms. Studies on Lactobacillus have shown that they can inhibit E. coli growth and vaginal colonization. As the efficacy of antimicrobial treatment is diminishing, an alternative thera-peutic approach to antimicrobial therapy is to reestablish Lactobacillus in this microbiome through probiotic administration. In the present work, we determined in a mouse animal model the capability of Lactobacillus plantarum 2621 strain with probiotic properties, to prevent the vaginal colonization of a pathogenic strain causing agglutination and immobilization of sperms leading to infertility. The preventive and curative effects produced by the probi-otic L. plantarum 2621 administered locally against E. coli in a murine vaginal tract model were studied. Screened mice were divided into three groups i.e. control, preventive and therapeutic. In preventive group, mice vagina were colonized with ten consecutive doses of Lactobacillus (108c.f.u./20 µl). After 48h, it was followed by 10 day intravaginal infection with E. coli (106 c.f.u/20µl) whereas, for the therapeutic group, vagina was colonized with (106c.f.u/20µl) E. coli for 10 consecutive days, followed by 10 day intravaginal administration with Lactobacillus. Controls each with Lacto-bacillus (108c.f.u/20µl)/E. coli (106c.f.u/20µl) / PBS for ten days were also set up. Upon mating and completion of gestation period, therapeutic, Lactobacillus and PBS group showed litter in contrast to preventive and E. coli group (control) which showed no litter.

The results indicated Lactobacillus intermitted colonization of pathogenic strains that resulted in reinforcement of natural microflora and resurge fertility.

n 21APRObIOTICS AS VACCINE AdjuVANTS: dOSE EffECTS Of L. rHamnosus GG ANd L. acidopHiLus ON MOduLATION Of PROTECTIVE IMMuNITY AGAINST HuMAN ROTAVIRuS IN GNOTObIOTIC PIGS

L. Yuan, K. Wen, F. Liu, G. Li, T. Bui, X. Yang, J. Kocher; Virginia Polytechnic Institute and State University, Blacksburg, VA.

Adjuvant effects of specific probiotic Lactoba-cillus strains have been reported for influenza, polio, rotavirus and cholera vaccines. Although strain-specific effects of probiotics have been well recognized, dose effects of probiotics on modulating innate and adaptive immune responses are not clearly understood. To ad-dress this question, we studied the dose effects of Lactobacillus rhamnosus GG (LGG) and L. acidophilus (LA) at a low, intermediate and high dose on antigen presenting cell (APC), IFN-γ producing T cell, regulatory T (Treg) cell, antibody-secreting cell and memory B cell responses in the intestinal (ileum) and systemic (spleen) lymphoid tissues and in the blood of gnotobiotic pigs vaccinated with an attenuated human rotavirus (HRV) vaccine. The protective efficacy of the vaccine against rotavirus shedding and diarrhea was assessed upon challenge with a virulent HRV. Low and intermediate dose LGG and intermediate dose LA increased frequencies and the matura-tion marker (CD80/86 or MHC II) and TLR9 expression, but decreased TLR3 and TNF-α expression of blood APC compared to the vaccine alone. Corresponding to the increased APC frequencies and function, the low and intermediate dose LGG and intermediate dose LA significantly increased rotavirus-specific effector/memory T cell, B cell and antibody responses and down-regulated the Treg cell responses, leading to significantly increased

Poster Abstracts


protection rate against challenge. In contrast, high dose LA induced strong Treg cell re-sponses and promoted IL-10 and TGF-β pro-duction by tissue-residing Treg cells, resulting in higher rate and longer duration of diarrhea compared to vaccine alone. Although low dose LA significantly enhanced IFN-γ producing T cell and decreased Treg cell responses, it did not enhance virus-specific B cell and antibody responses hence did not improve the vaccine efficacy. In conclusion, probiotics can skew the balance of Th1 and Treg cell responses in both intestinal and systemic lymphoid tissues in the context of vaccination. Different lactobacilli strains and the same strain at different doses can exert qualitatively different immunomodu-lating effects. Probiotics can be ineffective or even detrimental if not used at the optimal dos-age for the appropriate purposes, highlighting the importance of not only strain but also dose selection. Selected lactobacilli strains at appro-priate doses can significantly enhance protec-tive efficacy of rotavirus (and other) vaccines.

n 22BTEMPORAL ANd SPATIAL VARIAbILITY IN THE COLLEGE STudENT MICRObIOME

G. E. Flores1, G. Caporaso2, D. Domogala2, M. J. Gebert1, J. B. Henley1, G. Humphrey1, C. L. Lauber1, R. Dunn3, R. Knight1, N. Fi-erer1; 1University of Colorado, Boulder, CO, 2Northern Arizona University, Flagstaff, AZ, 3North Carolina State University, Raleigh, NC.

The increasing recognition that commensal and mutualistic microorganisms are necessary for many aspects of normal human physiology has altered the traditional pathogen-dominated view of human-associated bacteria. Despite this new perspective on human-microbe in-teractions, our understanding of the normal temporal and spatial variability of the human microbiome is limited because most previous studies have focused on single body habitats at one point in time or on disease states like obesity, dental caries and bacterial vaginosis. Here, using high-throughput sequencing of the

V4 region of the 16S rRNA gene, we charac-terized the temporal variability of the human microbiome across four body habitats (fore-head, gut, palm and tongue) of over 80 college students from three American universities with each individual sampled once per week over an 8 week period. Preliminary results show that different body habitats exhibit different degrees of variability, but intra-individual vari-ability was always less than inter-individual variability. Furthermore, we detected syn-chronicity in changes in microbial community composition across different body habitats. We used weekly surveys of health status to identify relationships between certain factors and behaviors that influenced community vari-ability. For example, we were able to quantify the affect of gender and antibiotic usage on the temporal variability of different body habitat communities. Overall, this study provides new insight into the temporal variability of the healthy human microbiome across multiple body habitats and provides clues to factors and behaviors that influence this variability.

n 23AdEEP dNA SEquENCE ANALYSIS REVEALS STAbLE ENGRAfTMENT Of GuT MICRObIOTA fOLLOWING TRANSPLANTATION Of fROzEN fECAL bACTERIA

M. J. Hamilton, A. R. Weingarden, T. Unno, A. Khoruts, M. J. Sadowsky; University of Min-nesota, Saint Paul, MN.

Fecal microbial transplantation (FMT) is becoming a more accepted and widely used technology for treatment of recurrent Clostri-dum difficile¬ infection (CDI). While previous treatments used freshly prepared fecal slurries as a source of fecal microbiotia for FMT, we recently reported the development and suc-cessful use of frozen fecal material to treat CDI. Here we report that deep DNA sequence analysis revealed the stable engraftment of gut microbiota following transplantation of previously frozen fecal bacteria from a healthy

Poster Abstracts

ASM Conferences44

donor. Similar bacterial taxa were found in post-transplantation samples obtained from the recipients and donor samples, but the relative abundance varied considerably be-tween patients and time points. All post FMT samples from patients showed an increase in the number of Firmicutes and Bacteroidetes, averaging about 75-80% of the total sequence reads. Proteobacteria and Actinobacteria were present in lower abundance (<5%) than found in patients prior to FMT. Post FMT samples from two patients were very similar to donor samples. One patient subsequently received antibiotics for an unrelated infection resulting in an increase in the number of intestinal Pro-teobacteria, primarily Enterobacteriaceae. Our sequence results demonstrate that frozen fecal microbiota from a healthy donor can be used to stably engraft patients treated for recurrent CDI resulting in restoration of a functioning gut microbiota, clearing of Clostridum difficile infection, and curing of patients.

n 24BCAN THE IMPACTS Of MICRObIOTA ON ANIMAL HEALTH bE ASSIGNEd TO INdIVIduAL MICRObIAL TAxA?

P. D. Newell, C. Wong, A. E. Douglas; Cornell University, Ithaca, NY.

It is well-established that the gut microbiota influences multiple aspects of host phenotype, including nutrition, immunity and behavior. The next challenge is to identify the microbial taxa and activities that mediate these effects. Our strategy is founded on a pair of alterna-tive hypotheses and an experimental system in which the association can be disassembled and reassembled with ease. We define mi-crobiota function as the difference in a host trait between untreated animals containing the microbiota and germ-free animals. Our hypotheses are that microbiota function (1) is highly interactive, i.e. microbiota impact on host traits requires multiple, interacting mi-crobial taxa, or (2) is mediated by single taxa, such that function can be coupled precisely to

microbial taxon. These hypotheses are likely to be extremes of a continuum, and different microbiota functions may vary in the identity and number of contributing taxa. Understand-ing patterns in the relationship between micro-bial taxa and function is crucial to explain and predict microbial impacts on animal health and fitness. Our experimental system is Drosophila, which has a gut microbiota of low taxonomic diversity, dominated by five bacterial species that are readily cultured. We have developed procedures for generating germ-free (axenic) D. melanogaster, and for re-associating these axenic hosts with cultured bacteria to generate gnotobiotic flies. For this study, we character-ized the impact of eliminating the microbiota on a range of host traits. Then, by generating gnotobiotic flies with five, single-species mi-crobiota, we tested the hypothesis that some functions of the microbiota can be mediated by a single taxon. Our comparison of germ-free and conventional flies identified multiple microbiota functions: promotion of devel-opmental rate and survival to adulthood on low-nutrient diets, regulation of body glucose and lipid levels, and up-regulation of immune genes, especially anti-microbial peptides. The pattern of traits in gnotobiotic flies colonized by each of the five dominant bacteria derived from the gut microbiota of untreated hosts demonstrated that some functions can be at-tributed to specific individual taxa. For exam-ple, flies from all gnotobiotic treatments with Acetobacter species developed at a normal rate while single-species treatments with Lac-tobacilli delay development compared to both conventional and axenic rearing. Other traits have an interactive basis, being dependent on multiple bacteria. Our analysis provides an ex-perimental framework for testing how changes in the microbiota composition, induced by diet, disease, age etc., can influence animal health and fitness. Furthermore, in combination with the bacterial genomic data and transcriptomic studies, these data generate specific, testable hypotheses on the mechanisms underlying microbe-dependent impacts on host traits.

Poster Abstracts


n 25AMICRObIAL ECOLOGY Of EARLY dEVELOPMENT ANd LARVAL SETTLEMENT IN TROPICAL CORALS

K. Sharp1, K. B. Ritchie2, J. Sneed3, V. J. Paul3; 1Eckerd College, St. Petersburg, FL, 2Mote Marine Laboratory, Sarasota, FL, 3Smithsonian Marine Station, Fort Pierce, FL.

Coral reefs represent significant marine eco-systems, home to a majority of the biodiversity in the world oceans. Corals have long been regarded as complex, multispecies associations consisting of the coral animal host, the photo-synthetic dinoflagellate Symbiodinium, bacte-ria, archaea, fungi, and viruses. Although the functional dynamics of coral-Symbiodinium associations have been well-studied, there is still very little known about the function of bacteria in tropical corals, and even less is known about the nature of Symbiodinium-bacterial associations. We have previously shown the Roseobacter clade of bacteria to be consistently present in early life stages of several tropical coral genera across the world, but to date, very little is known about how they may affect host fitness or larval ecology. In this study, we test whether the bacteria play a role in the development and fitness of the coral host. Here we describe a new putative function for Roseivivax sp. 46E8, a Symbiodinium-associated bacterial isolate representing the Roseobacter clade. Addition of Roseivivax sp. 46E8 liquid cultures, both in log and in station-ary phase, increases larval settlement rates in two Caribbean corals, Porites astreoides and Montastraea faveolata. Spent medium (0.22um filtered cultures) from log phase cultures also enhances larval settlement. Monospecific biofilms of 46E8 formed in the laboratory result in increased larval settlement rates. These results are in contrast to treatments with exposure to other Symbiodinium-associated bacteria, which do not elevate larval settle-ment rates. The mechanism by which 46E8 induces the developmental changes in corals is not yet elucidated; however, our conventional

organic extraction suggests that the induc-tion is not due to release of organic molecules by the cells. Our results demonstrate that Roseivivax sp. 46E8 plays an important role in coral fitness in the environment by direct-ing developmental changes in the host coral larvae, though the mechanism by which they confer this change is unclear. We are using a combination of PCR-based techniques to detect and characterize diversity of bacteria from the Roseobacter clade in adult and larval stages of the corals P. astreoides and M. faveo-lata. In addition, sequence-specific probes and fluorescence in situ hybridization (FISH) are being used to characterize potential shifts in microbial biofilms caused by the addition of Roseivivax cultures. The results of this study characterize a new mechanism through which coral-associated bacteria have evolved to influ-ence larval ecology.

n 26BPRObIOTIC POTENTIALS Of VAGINAL LACTIC ACId bACTERIA ON GENITAL PATHOGENS

F. M. Agboola; University of Ibadan, Ibadan, NIGERIA.

Oral probiotics has been found useful in treat-ment or prevention of urogenital infections. The ability of probiotic organisms to serve as biotherapeutic agents can be attributed to vari-ous properties they possess, one of which is the capacity to inhibit disease causing organisms by producing substances such as hydrogen peroxide and lactic acid. This study employed human vaginal strains of lactic acid bacteria (LAB); reports their potential probiotic proper-ties and antagonistic activities against some genital pathogens. Thirty two LAB strains were randomly picked from the culture of vaginal swab samples of healthy females in Nigeria. The isolates were stored at 4ºC in MRS broth supplemented with glycerol 12% (v/v). Ten clinical strains of genital pathogens (Staphylococcus aureus, Escherichia coli, Klebsiella spp., and Candida albicans) were

Poster Abstracts

ASM Conferences46

used. Antibiotic susceptibility of the vagina LAB isolates was done using antibiotic multi-discs. The in-vitro antimicrobial activity of LAB against pathogens was determined by well diffusion method. 50µl of LAB superna-tant from centrifuged culture broth were placed in wells (8mm diameter) cut on cooled agar already seeded with pathogens. Quantitative determination of Lactic acid and hydrogen peroxide was done by growing LAB isolates in MRS broth for 96 hours; samples were col-lected 24-hourly and centrifuged, followed by titration method of A.O.A.C in 1990. The selected LAB were characterized as Lactoba-cillus fermentum, L. jensenii, L. delbrueckii, L. plantarum, L. acidophilus and L. lactis. All isolates demonstrated antimicrobial activ-ity against the genital pathogens in varying degrees except in some tests on Candida albi-cans. L. fermentum had high antagonistic effect with zones of inhibition of 24mm & 25mm against Klebsiella sp. & E. coli respectively. The inhibitory capability of these vaginal isolates indicates a good probiotic potential. Percentage antibiotic resistance of the isolates reflects that 86% of the isolates exhibited re-sistance to both amoxacillin and gentamicin; 73% to ampiclox and 64% to rocephine while low percentage resistance from 0% to 5% was observed for the fluoroquinolones and augu-mentin. The knowledge of the antimicrobial resistance of exogenously applied probiotic is of interest to predict their behaviour in pa-tients subject to any type of chemotherapy, as well as to consider the concomitant use of the probiotic and antibiotics for the restoration of the normal urogenital flora. The concomitant application of probiotic LAB with the floro-quinolones, augumentin or streptomycin may not be advised. Production of both hydrogen peroxide and lactic acid were detected in all the LAB isolates. L. fermentum produces more hydrogen peroxide when compared with the other isolates and also had the highest zones of inhibition against genital pathogens, hence, can be considered as an isolate with outstand-ing probiotic potential.

n 27AMOduLATING THE GuT MICRObIOTA WITH A PRObIOTIC MIxTuRE: PROTECTIVE EffECTS IN AN ANIMAL MOdEL Of ANTIbIOTIC-ASSOCIATEd COLITIS.

S. E. Martz1, C. Noordhof1, S. He1, D. Hurl-but1, E. Allen-Vercoe2, E. O. Petrof1; 1Queen’s University, Kingston, ON, CANADA, 2Univer-sity of Guelph, Guelph, ON, CANADA.

Background: Antibiotics disrupt the normal balance of the gut microbiota and may subse-quently reduce colonization resistance, allow-ing pathogens such as Clostridium difficile to colonize the intestine and cause disease. By re-introducing normal microbiota from a donor, colonization resistance against pathogenic bac-teria can be restored. Based on this premise, a complex “probiotic” mixture (ME) of intestinal non-pathogenic bacteria containing 33 strains of commensal bacteria was derived from the stool of a single healthy human donor. This gut microbial ecosystem has been success-fully used to treat two patients with C. difficile infection, but mechanisms of action remain to be elucidated. We hypothesize that ME can be used to protect the host against disease by restricting the growth of other opportunistic pathogens; we tested our hypothesis using a Salmonella mouse model of colitis. Experi-mental Methods: An antibiotic-associated colitis mouse model was employed to investi-gate the potential mechanisms underlying the protective effects of ME. C57bl/6 female mice were pretreated with oral streptomycin prior to receiving one dose of ME or vehicle control by oral gavage. Mice were then gavaged with Salmonella enterica serovar Typhimurium to induce enterocolitis. Serum cytokine levels were measured by ELISA. Bacterial loads in the colon and spleen were measured by plat-ing serial dilutions on MacConkey agar plates containing 100 µg/ml streptomycin. Results: Infected mice that were given the ME mixture lost less weight than mice not given ME. The Salmonella bacterial loads in the colon were depleted in those mice that received ME as a

Poster Abstracts


pretreatment, and there was reduced bacterial translocation to the spleen. Serum levels of the proinflammatory chemokine MCP-1 were reduced in mice that received ME. Conclu-sions: The reduction in bacterial loads in both the colon and spleen show ME may protect during a Salmonella infection by restricting the growth of the pathogen, thus leading to a re-duced infection. A less severe infection would lead to reduced weight loss and decreased MCP-1 serum levels. ME may exert its protec-tive effect by depleting the pathogenic bacteria involved in the infection.

n 28BMETAbOLIC ANd COLONIC MICRObIAL RESPONSES TO INCREASING dIETARY LEVELS Of RESISTANT STARCH IN ObESITY-PRONE ANd ObESITY-RESISTANT RATS

C. T. Christophersen, A. R. Bird, D. P. Belo-brajdic; Commonwealth Scientific & Industri-al Research Organisation (CSIRO) Food Fu-tures National Research Flagship and CSIRO Animal, Food and Health Sciences, Adelaide, SA, AUSTRALIA.

Resistant starch (RS) reduces live weight gain and adiposity in rats (Belobrajdic et al., unpublished) but the responses differ between lean and obese rats. Although it is understood that the end products of RS fermentation in the colon are involved in mediating these benefits little is known about the impact of the compo-sition of the gut microbiota. We used a targeted QPCR approach to determine the colonic abundances of short chain fatty acid (SCFA) producing bacteria, and other microbes consid-ered important to gut health, in male obesity-prone (OP) and obesity-resistant (OR) rats (Sprague-Dawley, n=60). The rats were fed a diet containing 0, 4 or 8% RS (as high amylose maize starch) for 4 wk. In the month preced-ing the dietary intervention they were fed a moderate-fat diet to classify their phenotype. Those that gained the most weight (40%) were classified as OP and the remaining 60% as OR.

RS stimulated fermentation dose-dependently as shown by a lowering of colonic pH and in-creased concentrations of total and individual SCFA, including butyrate. The response in SCFA to feeding RS was greater in OP rats. Phenotype also influenced the composition of the microbiota. Bifidobacteria spp. and Para-bacteroides distasonis were more abundant in OR than OP rats but were not affected by RS. RS (at 4%) increased Lactobacillus spp. abundances in OP rats, and Clostridium leptum group and Akkermansia muciniphila abundanc-es in OR animals. The Clostridium coccoides group was smaller on 4% RS diet compared to 0% (but not 8% RS), and the response was independent of phenotype. Multivariate analy-sis showed that phenotypic- and RS-induced changes in microbial community structure were explained in part by fermentation-related biochemical variables, especially butyrate, propionate and pH.

n 29AIN VITRO EVALuATION Of AN ALTERNATIVE TREATMENT STRATEGY fOR ERAdICATION Of GRAM NEGATIVE OPPORTuNISTIC PATHOGENS WITH NISIN- A LANTIbIOTIC Of PRObIOTIC ORIGIN.

D. Begde1, P. Mahatme2, A. Upadhyay3; 1Dr. Ambedkar College,, Nagpur, INDIA, 2PGTD Biochemistry, RTM Nagpur University, LIT Campus,, Nagpur, INDIA, 3Hislop School of Biotechnology, Hislop College,, Nagpur, IN-DIA.

The classical approach of treating bacterial infections involves several challenges, includ-ing unwanted selection of resistant bacterium in the infected host during antibiotic therapy. Therefore treatment strategies that challenge the virulence rather than the growth of the pathogen might represent a better alterna-tive therapeutic model. Critical evaluation of pathogenesis within the host has demonstrated the crucial role of quorum sensing (QS) in disease manifestation. QS is a small molecule based signaling mechanism in bacteria that

Poster Abstracts

ASM Conferences48

collectively regulate and trigger the expression of certain genes in the bacterial population, including those associated with bacterial viru-lence. The present study is an attempt to test whether host symbiotic or probiotic bacteria’s extracellular molecules could be utilized to reduce pathogen colonization and thereby disease manifestation through their QS attenu-ation. An autoinducer, pheromone peptide, Nisin, naturally produced by a probiotic bacte-rium, Lactococcus lactis, was selected for the said analysis. Nisin - a lantibiotic - does not challenge the growth of Gram negative bacte-ria but this does not necessarily prove its inef-ficiency against this class of microorganisms. In our study design, as a supplement in the growth medium, nisin appeared to attenuate the QS mechanisms of three common Gram negative opportunistic pathogens viz. Proteus mirabilis, Pseudomonas aeruginosa and Esch-erichia coli, when tested in vitro. Nisin effec-tively downregulated the QS controlled mecha-nisms such as swarmer cell metamorphosis, biofilm formation and virulence factor expres-sion (viz. hemolysin in P. mirabilis, curli fiber and hemolysin expression in pathogenic E. coli and rhamnolipid expression in P. aeruginosa), that are closely associated with disease patho-genesis. Although swarmer cell differentiation and biofilm formation are considered mutually exclusive events, nisin seemed to effectively inhibit both at similar concentrations. Fur-thermore, nisin treatment leading to complete dislodgement of the adherent P. mirabilis cells from their well-established preformed biofilms seemed to provide a proof of its utility not only in preventive but also in remedial therapy. Moreover, nisin alone could stimulate isolated human neutrophils to form very effective, anti-bacterial, neutrophil extracellular traps (NETs) in vitro. Therefore from the overall results it could be proposed that, the probiotic, lantibi-otic, nisin, with its immunostimulatory as well as QS attenuating ability, perhaps carries a great anti-infective therapeutic potential.Ad-ditionally, our results also hint towards further investigation of plausible roles of extracellular

molecules from beneficial microbes in preser-vation and restoration of healthy state of host.

n 30BAN IN-VITRO STudY Of PRObIOTIC bACTERIA ANd PREbIOTIC SubSTRATES fOR APPLICATION TO MILITARY RATIONS

W. S. Muller, A. Sikes, D. Anderson; US Army, Natick Soldier Research, Development and Engineering Center, Natick, MA.

Stress of combat operations may degrade im-mune function; and sudden change in dietary intake (from fresh food sources to operational rations) may negatively impact gastrointestinal function. Pre- and probiotics may be helpful for attenuating immune function decrements and gastrointestinal problems. Additionally, military rations must meet storage require-ments (3 years at 80°F or 6 months at 100°F), which are more stringent than commercial products. Thus, there is interest in strategies to incorporate probiotic/prebiotics in military rations. The objective of this in-vitro study was to determine the most suitable commer-cial combination of pro- and prebiotics for inclusion into a low water activity ration. The growth of four probiotic bacteria were evalu-ated in the study: Lactobacillus acidophilus La-5, Lactobacillus reuteri DSM17938, Lacto-bacillus rhamnosus GG, Bifidobacteria anima-lis BB-12 and three commercial prebiotic sub-strates were studied: sc FOS, inulin and GOS (GTC Nutrition). A MRS-like medium incor-porated 1% of the prebiotic into the medium, with 1% glucose used as positive control, and no additional substrate as negative control. The growth of bacteria was determined by measur-ing OD

600 over 48 hrs under anaerobic condi-

tions. In all cases, the glucose control exhibited the highest OD, and the no substrate control the lowest OD. La-5 utilized both scFOS (OD

max0.97) and GOS (OD

max0.86) very well,

while inulin was utilized at a lesser rate (OD-

max0.45). L. reuteri utilized GOS (OD

max1.65)

the best, while both scFOS (ODmax

0.47) and inulin (OD

max0.47) exhibited much lower rates

Poster Abstracts


of growth. LGG utilized all three prebiotics at lower rates with inulin (OD

max0.81) and scFOS

(ODmax

0.80) being the best. BB-12 utilized both scFOS (OD

max1.14) and GOS (OD

max0.95)

very well exhibiting a shorter lag period than glucose. Persistence of the probiotic is espe-cially important for military application with the Soldier’s irregular dietary schedule in the field. A human feeding study is planned to examine persistence of a probiotic with and without the selected prebiotic incorporated into a military ration. In conclusion, based on the growth data the combination of L. reuteri and GOS would be the best choice for inclusion in a military ration.

n 31AuNCORkING THE dIVERSITY Of WOLbACHIA ANd dROSOPHILA SPECIES AT INdIANA WINERIES

R. Hicks, K. Sheehan, I. Newton; Indiana University, Bloomington, IN.

Wolbachia is an obligately intracellular bac-terium known for manipulating the biology of their hosts in favor of continued widespread infection. Belonging to the Rickettsiaceae fam-ily, Wolbachia infection is a global epidemic amongst insects, with roughly 40% of spe-cies carrying the parasite. To survey diversity and extent of Wolbachia infection within the Brown County, Indiana area, we collected Dro-sophila species at local wineries using traps and net sweeping over the course of the sum-mer (2012). In addition to visual identification, the fruit fly hosts were also sorted via sequence analysis of the mitochondrial cytochrome oxi-dase I gene. Primers designed to amplify the Wolbachia surface protein wsp were used to test for infection and acquire strain information for these bacteria. These data revealed that the Southern Indiana wineries are home to a vari-ety of Drosophila host species. Of the samples collected, we found a large amount of diversity in Wolbachia strain within and between host species across both time and space. Under-standing the extent of Wolbachia diversity is

important for building a larger knowledge base concerning this important organism. Possess-ing a greater ecological perspective can lead to more efficient and thorough approaches to researching Wolbachia vertical and horizontal transmission dynamics. This could also result in an improved model for studying the effects Wolbachia may have on host species evolution as a result of its prolonged intracellular pres-ence.

n 32BTOWARd uNdERSTANdING THE ROLE Of bACTERIAL CONSORTIA IN CORAL’S HEALTH: ECOLOGICAL INfERENCES fROM GENOMIC-bASEd COMMuNITY ANALYSIS

M. Rodriguez-Lanetty1, C. A. Granados-Cifuentes2, A. Bellantuono1, C. Basti-das3; 1Florida International University, Mi-ami, FL, 2University of Louisiana at Lafayette, Lafayette, LA, 3Universidad Simon Bolivar, Caracas, VENEZUELA, BOLIVARIAN RE-PUBLIC OF.

The coral animal forms associations both intra-cellularly and extracellularly with a large mi-crobiota. This multipartite symbiosis between coral host and its intracellular eukaryotic algae symbionts and the diverse microorganisms (bacteria, archaea, fungi, and viruses) found in association with coral tissue has been termed the holobiont. To date, it is well-known that the microbial biota, specifically bacteria, living on the surface (coral mucus) and in the tissue of many coral species is complex and dynamic, which supports the notion that this bacterial consortium is likely to play a key role in the holobiont’s function and health. However, at the moment, there is a lack of understanding of the principles underlying the assembly and structure of these complex microbial commu-nities associated with scleractinian corals. The comprehension of these processes is essential if we are to dissect the functional role of the microbial diversity associated with corals. In this study, we applied well-accepted ecologi-cal models to examine and detect processes

Poster Abstracts

ASM Conferences50

underlying structuring patterns of bacterial communities associated with the Caribbean scleractinian coral, Porites astreoides. Five samples per population were collected in a total of six coral populations within a range of 30 Km on the south reef of Curacao Island. From these, the microbial communities were examined using Illumina HiSeq sequencing of the V5-16S rRNA region. The most abundant classes among the coral samples were Gamma- and Alpha-proctobacteria, and Cyanobacteria; however, the proportion of these groups dif-fered among populations. UniFrac, ANOSYM and non-metric multidimensional scaling analyses of OTUs also showed clearly geo-graphical patterns in the bacterial community assembly. With the use of genomic-based mea-sures of community diversity and abundance, we further tested stochastic (neutral) and deterministic (niche) ecological models, and learned that both processes contribute to the shaping of coral-associated microbial commu-nity assembly. We also discussed that the kind of approaches used in this study will contribute to establish baselines from which to detect differences associated with diseases and with the response to environmental stressors, such as global warming and ocean acidification that threaten one of the most diverse ecosystems on the planet: coral reefs.

n 33AbACILLuS THuRINGIENSIS IN THE SERVICE Of AGRICuLTuRE ANd HEALTH: COMbATING INSECT PESTS bY PYRAMIdING CRY ANd CYT GENES

A. Zaritsky1, Z. Mendel2, G. Gindin2, D. Wein-thal1, T. Tzfira3, E. Ben-Dov4; 1Ben-Gurion University of the Negev, Be’er-Sheva, ISRAEL, 2Agricultural Research Organization, Bet Dagan, ISRAEL, 3Be’er-Sheva, Be’er-Sheva, ISRAEL, 4Achva Academic College, Shikmim, ISRAEL.

Each ssp. of Bacillus thuringiensis (Bt) pro-duces during sporulation a proteinacious crystal [[Unsupported Character - Symbol

Font ]]-endotoxin specific against a narrow range of insects, pests of crops and disease vectors, some serve as biological con-trol agents [1]. Over 70 Cry toxin families (224 holotypes) and 3 Cyt families (11 holotypes) have been found [2], all included in crystals of at least 82 serovars [3]. Many transgenic crops are resilient to the pests by expressing cry genes [4] thus reduce use of synthetic pesticides that damage the environment. Pest resistance to Cry following extensive use of such crops or the bacteria [4] can be avoided by pyramiding genes coding for at least two toxins operating in different modes of action [5], reflected in synergy. A combination of 2 cry genes with cyt1Aa from the mosquito lar-vicidal Bt israelensis expressed in the cyano-bacterium Anabaena PCC 7120 demonstrated synergy in killing Aedes aegypti larvae [6], and a combination of cry1Ac with cry1Ca highly synergized larval mortality of the Lepidopteran pest Spodoptera littorallis [7]. To combat larvae of the Coleopteran flat-headed borer Capnodis tenebrionis developing in the roots of stone-fruit trees, we bioassayed 28 field-isolates of Bt [8] using a recently developed artificial diet [9], and found from one of the toxic isolates a new, 640th cry gene (accession # JX282317 in NCBI; [2]) designated cry8Ta1. The gene will be expressed in acrystalliferous Bt [10] and its product assayed for toxicity to C. tenebrionis larvae. If positive, it will be modified according to a codon usage com-mon to several plant species [11], including Arabidopsis thaliana to serve as a fast bioas-say model system, and cloned into the binary vector pPZP-RCS2 [12] as the first in a series of pyramided toxin genes, aiming to devise a rootstock Prunus that will control Coleopteran pests. [1] Crickmore N, 2006. J Appl Microbiol 101:616 [2] http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ [3] Xu D, Côté J-C, 2008. Appl Environ Microbiol 74:5524 [4] Sanahuja G et al, 2011. Plant Biotechnol J 9:283 [5] Sanchis V, Bourguet D, 2008. Agron Sust Dev 28:11 [6] Khasdan V et al, 2003. FEMS Microbiol Lett 227:189 [7] Khasdan V et al, 2007. Arch Microbiol 188:643 [8] Ben-

Poster Abstracts


Dov E et al, 1997. Appl Environ Microbiol 63:4883 [9] Gindin G et al, 2009. Eur J Ento-mol 106:573 [10] Cohen S et al, 2007. J Biol Chem 282:28301 [11] Veksler I et al, 2012, in prep [12] Zeevi V et al, 2012. Plant Physiol 158:132

n 34BdIVERSITY-GENERATING RETROELEMENTS IN THE HuMAN GuT COMMENSAL bACTEROIdES fRAGILIS

Y. Wang, M. Gingery, L. Clark, J. F. Mill-er; University of California-Los angeles, Los angeles, CA.

The human gastrointestinal tract houses ap-proximately 1014 microorganisms of more than 1500 species, and represents one of the most diverse and dynamic microbial ecosystems. Bacteroides fragilis, a common member of the normal gut microbiota, confers potent immunomodulatory effects against chronic inflammation but little is known about the mechanisms by which B. fragilis colonizes the host and is maintained in the densely populated intestinal niche. With knowledge gained from studies of the prototype diversity-generating retroelement (DGR) in Bordetella phage BPP-1, bioinformatic analyses revealed that DGRs are widely distributed in the ge-nomes of diverse bacterial species, including those that inhabit the human gut. DGRs are a newly discovered family of retroelements that function through a template-dependent, reverse transcriptase-mediated mechanism to introduce nucleotide substitutions at specified sites in protein-coding sequences. Diversified residues are displayed in ligand-binding pockets, creat-ing vast repertories of receptors for ligand in-teractions. We have discovered that B. fragilis 638R contains a complete diversity-generating retroelement (DGR) within its genome. Our work aims to test the hypothesis that B. fra-gilis has evolved a DGR-based mechanism to generate diversified surface proteins for novel binding specificities, and this diversity contrib-utes to bacterial colonization and long-term

maintenance in the host. Our analysis estimates that repertoires of 1025 unique polypeptide sequences in the target protein can potentially be generated by the B. fragilis 638R DGR, far exceeding the capabilities of any naturally occurring protein diversification system de-scribed to date. Further examination of the locus and its flanking regions suggests that the DGR is carried by a conjugative transposable element. We are currently evaluating cis- and trans-acting requirements for DGR function using a PCR-based mutagenic retrohoming assay. Since conjugative transposition is a major driver of the dissemination of antibiotic resistance in the gut microbiota, we are also evaluating intra- and inter-species transfer of the B. fragilis DGR.

n 35AEPOxIdE-MEdIATEd dIffERENTIAL PACkAGING Of OMV

A. E. Ballok, G. A. O’Toole; Geisel School of Medicine at Dartmouth, Hanover, NH.

Background: Host-association often requires a dynamic interplay of sensing and response to the environment. One understudied mechanism of host-association used by both pathogens and symbionts is production of outer membrane vesicles (OMV). These vesicles can act as long-distance communication systems with the host and other bacteria during associa-tion. Pseudomonas aeruginosa OMV have been shown to contain a number of factors important for survival within the host including quorum sensing molecules, phospholipases, alkaline phosphatases, and Cif (CFTR in-hibitory factor); a protein capable of altering host protein recycling. In fact, Cif efficacy is 17,000-fold greater when delivered by OMV to host cells. Cif is regulated transcription-ally by CifR which binds to the cif promoter to prevent transcription. In the presence of an inducer ligand, such as a host-derived epoxide, CifR will bind the ligand and release repres-sion of Cif. We sought to better understand the OMV packaging of this and other proteins

Poster Abstracts

ASM Conferences52

to gain a clearer understanding of host-asso-ciation for Pseudomonas. Methods: For this study we purified vesicles from P. aeruginosa cultures grown with an without epoxides, via filtration and ultracentrifugation. Cif, OprF and other proteins were visualized by western blot or silver stain from fractionated cells and ves-icles. Phospholipase and alkaline phosphatase activity of vesicles were measured by enzy-matic assay. Results: We found that incubation with epoxides resulted in a dramatic change in the protein profile of the OMV, including an increase in Cif protein in vesicles which was independent of CifR. Additionally, increases were seen in other proteins including OprF, a porin previously shown to increase over time in the CF lung. Enzymatic analysis showed no change in alkaline phosphatase activity of these vesicles and phospholipase activity decreased. Finally silver stained SDS-PAGE of purified vesicle contents revealed a second population of vesicles with a unique protein profile in the epoxide-treated samples. Conclu-sion: From these studies we can conclude that Pseudomonas is capable of altering vesicle packaging in response to stimuli, such as ep-oxides. This manipulation of OMV may be important in the transition from early to long-term infection. We anticipate further study of this phenomenon will yield insight into the mechanisms involved in OMV packaging and host-associtaion in general.

n 36BTHE ObLIGATE MuTuALIST Of TSETSE (dIPTERA: GLOSSINIdAE), WiggLesWortHia, dEMONSTRATES SPECIES-SPECIfIC NuTRIENT PROVISIONING, CONTRIbuTING TOWARdS HOST PHENOTYPIC VARIATION

A. K. Snyder1, A. F. Savage2, B. A. Clark1, R. E. Hewitt1, R. V. Rio1; 1West Virginia Univer-sity, Morgantown, WV, 2Yale University, New Haven, CT.

Microbial symbionts contribute towards host evolutionary diversification. The tsetse fly

(Diptera: Glossinidae), the vector of African trypanosomes causing human sleeping sick-ness and the veterinary wasting disease Na-gana, maintains an obligate mutualism with the Gammaproteobacterium Wigglesworthia glossinidia. The ancient historical symbiosis of Wigglesworthia with tsetse has led to es-sential contributions by this symbiont towards host immune development and the nutritional supplementation complementing the strict tsetse blood diet, particularly with B vitamins. Comparative analysis of Wigglesworthia genomes isolated from two tsetse species, Glossina brevipalpis (Wgb) and Glossina morsitans (Wgm), reveals significant chromo-somal synteny despite an estimated separation of eighty million years. A unique feature of Wgm is the complete retention of loci involved in chorismate biosynthesis, an essential inter-mediate in aromatic amino acids and vitamin production, as well as the capability to convert chorismate to folate (Vitamin B9). Interest-ingly, G. morsitans, relative to G. brevipalpis, has enhanced vector competence, that is the ability to acquire and transmit the parasite Trypanosoma brucei ssp.. Trypanosomes are unable to produce neither chorismate nor folate, yet encode transporters for their exogenous uptake. While vector competence distinctions between tsetse species have been described, contributions by symbionts towards this phenotype remain largely unknown. In this study, we hypothesize that the retention of the chorismate and folate biosynthetic capabilities by Wgm are integral to G. morsitans biology and contribute to vector competence. Tran-scriptional analyses of multiple unique Wgm loci involved in chorismate biosynthesis and its conversion into folate show enhanced expres-sion at times of increased nutrient demand by the host, specifically during pregnancy and trypanosome infection. The quantitative PCR analyses of loci expression at different stages of trypanosome infection (midgut versus sali-vary gland) are described. Administration of a chorismate biosynthesis pathway inhibitor in host blood meals results in decreased diges-tion and survival in a concentration dependent

Poster Abstracts


manner. The unique contribution of nutrients by this symbiont species aids in our under-standing of phenotypic variation among tsetse host species, including distinctions in parasite transmission, and may provide novel avenues for vector control.

n 37AINfLuENCE Of INTERACTIONS bETWEEN MATERNAL TRANSMITTEd RESIdENT MICRObES ANd PGRP-Lb ON SYMbIOSIS ANd PARASITISM IN TSETSE’S OffSPRING

J. Wang, Y. Wu, S. Aksoy; Yale School of Epi-demiology and Public Health, New Haven, CT.

Beneficial microbe functions range from host dietary supplementation to development and maintenance of host immune system. In mam-mals, newborn progeny are quickly colonized with a symbiotic fauna that is provisioned in mother’s milk and that closely resembles that of the parent. Tsetse fly (Diptera: Glossini-dae) also depends on the obligate symbiont Wigglesworthia for nutritional supplementa-tion, optimal fecundity, and immune system development. Tsetse progeny develop one at a time in an intrauterine environment and receive nourishment and symbionts in mother’s milk. We show that the host Peptidoglycan Recogni-tion Protein (PGRP-LB) is expressed only in adults and is a major component of the milk that nourishes the developing progeny. The amidase activity associated with PGRP-LB may scavenge the symbiotic peptidoglycan and prevent the induction of tsetse’s Immune Deficiency pathway that otherwise can damage the symbionts. Reduction of PGRP-LB experi-mentally damages Wigglesworthia in the milk through induction of antimicrobial peptides, including Attacin, which in turn diminishes fe-male fecundity. Larvae that receive less mater-nal PGRP-LB and fewer Wigglesworthia give rise to adults with lower Wigglesworthia level and hyperimmune responses. Such adults also suffer dysregulated immunity, as indicated by the presence of higher trypanosome densities in parasitized adults. We show that recPGRP-

LB has antimicrobial and antitrypanosomal activities that may regulate symbiosis and impact immunity. Thus, mutualistic interac-tions between vertical transmitted PGRP-LB and symbionts play a pivotal role in shaping offspring’s symbiosis and immunity.

n 38BExPLORATION Of HOST-PRObIOTIC INTERACTIONS IN e. coLi NISSLE 1917 bIOfILM fORMATION PHENOTYPE MuTANTS

J. Nzakizwanayo, W. M. Macfarlane, B. V. Jones; University of Brighton, Brighton, UNITED KINGDOM.

The probiotic Escherichia coli Nissle 1917 (EcN) has been demonstrated to effectively protect against enteropathogens, and maintain remission of inflammatory bowel diseases by virtue of its anti-inflammatory properties. However, little is known regarding the mecha-nisms underlying these beneficial effects and strategies which facilitate the genetic manipu-lation of this organism in order to elucidate host-probiotic interactions are required. Our aim was to establish random transposon mu-tagenesis of EcN and use the mutant bank to explore host-probiotic interactions using in vitro co-culture models of the human intestinal Caco-2 cells, in conjunction with mini-Tn5bio-film mutants of EcN. Amini-Tn5 mutagenesis system pRL27 was conjugally transferred to EcN by mating with donor E. coli strain BW29427. Loss of the suicide plasmid vector in EcN was determined by plasmid prepara-tion and restriction digestion, while mini-Tn5 chromosomal insertion was verified by South-ern blot analysis. An EcN mutant bank was generated and subsequently screened in a high-throughput crystal violet (CV) staining-based assay for quantification of biofilm formed in 96-well polystyrene plate format. Mutants were defined as biofilm deficient or enhanced (BFD/BFE) only if their ability to form bio-films was significantly different to EcN wild-type (p = <0.05) but growth in LB broth was

Poster Abstracts

ASM Conferences54

unaffected. Disrupted genes were identified using a “cloning free” PCR-based approach. A Caco-2-EcN co-culture model was optimized to determine EcN:Caco-2 ratios and exposure time affecting the intestinal cell viability using the tryptan blue exclusion assay. A sub-set of mutants were selected to study internaliza-tion, invasion, and effect of EcN on Caco-2 cell lifecycle using gentamicin protection and Hoechst-propidium iodide staining assays. Apoptosis was analyzed by measuring caspase 3/7 activity using the Caspase-Glo® 3/7 As-say. The pRL27 system derived mutants were confirmed to be free from the suicide plasmid delivery vector and to possess mini-Tn5 single random chromosomal insertions. A bank of 11,328 mutants has been generated, of which 4,116 were screened in a high-throughput CV assay. 25 mutants were identified and nucleo-tide sequence data for Tn flanking regions ob-tained. Four BFD and three BFE mutants were studied for adhesion and invasion, effect of EcN on Caco-2 cell lifecycle. While adhesion onto and internalization into Caco-2 cells was found to be independent of biofilm formation ability in selected mutants, one mutant dis-rupted in KfiB gene was demonstrated to el-evate induction of apoptosis in Caco-2 cells, as compared to EcN wild-type. These preliminary results indicate potential novel functions of the EcN genes in modulating apoptosis in intes-tinal cells. Further study of these interactions will provide insight into mechanisms through which probiotics can influence epithelial integ-rity in the human gut.

n 39APRObIOTIC-MEdIATEd CHANGES IN THE MuCOSALLY-AdHERENT MICRObIOTA PROMOTE COLITIS-ASSOCIATEd COLORECTAL CANCER IN IL10-/- MICE

J. C. Arthur1, J. M. Uronis1, E. Perez-Cha-nona1, A. A. Fodor2, C. Jobin1; 1University of North Carolina at Chapel Hill, Chapel Hill, NC, 2University of North Carolina at Char-lotte, Charlotte, NC.

Patients with inflammatory bowel diseases (IBD) have a high risk of developing colorectal cancer (CRC). Furthermore, IBD and CRC patients generally have a dysbiotic microbiota. Millions of individuals worldwide consume probiotic bacteria with the goal of improving gastrointestinal health by restoring microbial homeostasis. We hypothesized that inter-ventional treatment with the FDA-approved VSL#3 probiotic would alter microbial com-munity composition and cancer development in a mouse model of IBD-associated CRC. Germ-free Il10-/- mice were conventionalized in specific pathogen free (SPF) housing for seven weeks to establish intestinal inflamma-tion, after which tumorigenesis was initiated with azoxymethane (AOM). VSL#3 was orally administered at this time and daily for the remainder of the experiment (1 billion CFU / animal / day). Fifteen weeks later, minia-ture endoscopy was performed to visualize tumor development. Stool and colon tissue biopsies were collected to assess luminal and mucosally-adherent microbial communities by Illumina HiSeq2000 sequencing (V6 16S region). Inflammation and tumorigenesis were evaluated by histology. Colon tissue cytokines were measured using an inflammation PCR array (SABiosciences). Surprisingly, VSL#3 intervention enhanced tumor penetrance (p=0.011), tumor multiplicity (p=0.002), histologic neoplasia (p=0001), and adenocar-cinoma invasion (p=0.001), relative to VSL#3-untreated AOM/Il10-/- mice. Histologic inflam-mation and inflammatory cytokines were not affected by VSL#3 treatment, suggesting that tumorigenesis was enhanced independently of inflammation. High-throughput sequencing revealed that VSL#3 altered community com-position of both the luminal/stool (Analysis of Similarity (ANOSIM) R=0.306, p=0.032) and mucosally-adherent microbiota (ANOSIM R=0.525, p=0.008). In the mucosally-adherent microbiota, the abundance of several bacterial groups was altered by VSL#3 treatment. Cory-nebacteriaceae were more abundant in VSL#3-fed animals (p=0.004), and this positively

Poster Abstracts


correlated with dysplasia (Spearman R=0.776, p=0.014), suggesting a cancer-promoting role for this bacterial family. Verrucomicrobiaceae/Akkermansia and Porphyromonadaceae were less abundant in VSL#3-fed animals (p=0.013, p=0.018), and this correlated negatively with the extent of dysplasia (Spearman R=-0.852, p=0.004, and R=-0.785, p=0.012), suggesting a protective role for these bacterial groups. Our results indicate that intervention with VSL#3 alters microbial community composition, most notably in the mucosally-adherent microbiota, and these changes correlate with enhanced CRC development.

n 40BfRIENd VS fOE: fuNCTIONAL AMYLOId IN COMMENSALISM?

M. Bois, S. Singh, P. N. Lipke, M. C. Gar-cia; Brooklyn College, Brooklyn, NY.

Candida albicans, a dimorphic yeast and an opportunistic pathogen, possesses an array of adherence factors including members of the agglutinin-like sequence (Als) family of man-noproteins. The adhesin Als5p mediates adhe-sion to many substrates, and is upregulated during the commensal stage, but is downregu-lated during active C. albicans infections [1]. An amyloid forming core sequence at residues 325-331 is important for function, because a single amino acid substitution at position 326 (V326N) greatly reduces Als5p-mediated adherence [2]. We evaluated the role of Als5p in host-microbe interactions, using Caenorhab-ditis elegans as a host model and feeding them Saccharomyces cerevisiae expressing Als5p on the surface. Als5p-expressing yeast had 3.5 and 8.5-fold increased intestinal accumulation rates when compared to amyloid- deficient Als5pV326N or non-expressing S. cerevisiae accordingly. Surprisingly, this accumulation delayed S. cerevisiae-induced killing of C. elegans. The median survival time was nearly twice as long as nematodes fed non-express-ing- or non-amyloid forming Als5pV326N-expressing S. cerevisiae. Treatment with the

amyloid inhibiting dye Congo red or repres-sion of Als5p expression abrogated the protec-tive effect of Als5p. This study is the first, to our knowledge, to show that expression of amyloid-forming Als5p in S. cerevisiae can attenuate S. cerevisiae pathogenicity in C. el-egans. Supported by NIGMS SC1 GM083756

n 41AISOLATION ANd CARACTERIzATION Of SOME bACTERIAL STRAINS fROM ALGERIAN SOILS WITH PGP CARACTERS, ANTIfuNGAL ACTIVITY AGAINST PHYTOPATHOGENS fuNGI ANd POTENTIAL PROMOTING WHEAT ANd bARLEY GROWTH uNdER GREENHOuSE CONdITIONS.

N. Mokrane1, D. Dahel1, N. Tabli1, E. Nabti1, M. Dary2, M. Megias2; 1Laboratoire de Biomasses et des Energies Renouvellables. Facutés des sciences de la nature et de la vie.University of A MIRA. Bejaia. Algeria, Bejaia, ALGERIA, 2LaboratoireLaboratoire RESBIOG. Facuté de Pharmacie.University of Sevilla., Sevilla, SPAIN.

In this work, we proposed to study the plant growth promoting activity of certain local strains isolated from different soils (black soil farming, red soil, rhizospheric soil of potato phyllosphere and rhizospheric soil of the prickly) of the East Coast of the Wilaya of Bejaia (Algeria). Twenty strains were isolated based on their morphological aspects on dif-ferent isolation media: PCA, YMA, JENSEN, M2, Nfb. Morphological and biochemical tests are performed: Gram stain, cell morphology, mobility, catalase, citrate utilization, nitrate reductase, VP, RM, urease, tryptophanase, TDA, use of certain carbon sources (glucose, lactose and mannitol), H2S production and growth at 3% of NaCl. The presence of char-acters plant growth promoters (production of indole acetic acid, solubilization of inorganic phosphorus, siderophore and several enzymes production: lipase, protease, chitinase, amylase and urease) and antagonistic activity against of some phytopathogenic fungi are searched.

Poster Abstracts

ASM Conferences56

The effect of these strains on growth of wheat and barley is performed in pots under green-house conditions. The different biochemical and identification tests show high biodiversity of our strains belonging to different bacterial genera. These strains have a good potential for promoting plant growth with different PGP characters and variables antagonistic activities vis-à-vis of the different tested fungi. Some of these strains show good growth promoting of wheat and barley under greenhouse conditions. Keywords: PGPB, biodiversity, AIA, solubili-zation of inorganic phosphorus, siderophores, antagonism, plant pathogens, wheat, barley.

n 42BTHE MICRObIOME’S ROLE IN COLONIzATION RESISTANCE AGAINST C. dIffICILE

A. M. Schubert, P. D. Schloss; University of Michigan, Ann Arbor, MI.

Colonization resistance (CR) is the preven-tion of invading microbes from colonizing a given body site. The gut microbiome, a complex bacterial community, plays a critical role in establishing CR and limiting infec-tions. Antibiotic treatments alter the structure of the microbiome and in turn alter CR to populations that might attempt colonization, although the exact mechanism of this disrup-tion is unknown. Specifically, epidemiological studies demonstrate that antibiotic use is a major risk factor for acquiring a C. difficile infection (CDI), which has become the leading nosocomial infection in the United States. We hypothesize that disruption of key features of the microbiome by antibiotics decreases CR. To test this hypothesis we are investigating the role of the microbiome in CR against C. diffi-cile in both mice and humans. We differential-ly manipulated the murine microbiome using clindamycin and cefoperazone. Using varying titrations of spores, we tested the ability of C. difficile strain 630 to colonize and persist in the mouse gut over the course of 10 days. Groups of mice treated with either antibiotic

were challenged with spores ranging from 20 to 106 spores/ml. Treatment with clindamycin, a lincosamide antibiotic, is expected to deplete the bacterial community of anaerobes. Spore titrations for clindamycin treated mice ranged from 102 to 106 spores/ml and resulted in 106 to 107 C. difficile CFU/g feces respectively. Colonization in this model was transient, and C. difficile was not detected in culture by 6 days post challenge regardless of spore dose. In contrast, treatment with cefoperazone, a cephalosporin antibiotic, is expected to broadly deplete the microbiome of bacterial diversity. Spore titrations for cefoperazone treated mice ranged from 20 to 105 spores/ml and resulted in 107 to 108 CFU/g feces respectively. Unlike the clindamycin treated animals, this level of colonization persisted in the mice throughout the 10 day observation period following C. difficile challenge. These two antibiotics alter the community in differing ways. Clindamycin treatment results in a gut microbiome struc-ture that has greater colonization resistance compared with the community structure of cefoperazone treated mice. This is clear given that clindamycin treated mice were colonized to initially lower levels than cefoperazone treated mice, even given a greater spore dose. Additionally the level persistence of C. difficile in these mice is lower, suggesting that these microbiomes have greater CR. 16S rRNA gene sequencing of these microbiomes with diverse levels of colonization resistance will elucidate the specific populations necessary for CR.

n 43AIMMuNOMOduLATORY PROPERTIES Of bACILLuS SubTILIS R0179 SPORES ANd VEGETATIVE CELLS ON HuMAN INTESTINAL EPITHELIAL CELLS

C. MacPherson, T. A. Tompkins, P. Bur-guière; Lallemand Inc., Montreal, QC, CANADA.

Presently the probiotic market is strongly dom-inated by Lactobacillus and Bifidobacterium strains. Alternatively, new research is advanc-

Poster Abstracts


ing into other potentially beneficial effects of other microbial species. One such microbe is Bacillus subtilis R0179 (CNCM I-3471) a commercially available probiotic strain manu-factured by Lallemand Inc., which is resistant to different physico-chemical stress, and higly stable over time in food matrices or dietary supplements. In this study, the immunomodu-latory properties of B. subtilis R0179 were examined using a custom-designed two-color expression immune-array consisting of 1354 human immune genes targeting 17 pathways associated with innate immunity and barrier function. For that purpose, human intestinal epithelial cells (HT-29) were co-incubated for 3 h with either spores or vegetative cells of B. subtilis R0179 prior to RNA isolation. LOW-ESS normalization and statistical analysis were conducted using the Limma Package from BioConductor in R software (version 2.8.1). Gene expression differences were considered statistically significant with a p-value <0.05 and a cut-off of 1.4-fold change in transcript abundance. B. subtilis R0179 immunomodu-latory properties are both quantitatively and qualitatively highly dependent on the cell physiological state. Indeed, vegetative cells had a greater impact on immune genes ex-pression compared to spores with 59 and 20 genes modulated, respectively. Moreover, immune response modulation by spores re-sulted in genes associated with the MAPK signalling pathway (e.g. DUSP1, DUSP6 and MAP3K14). Interestingly, genes connected to a pro-inflammatory (TH1) response were all down-regulated (e.g. IL8, IL17C, CCL20, CXCL1, CXCL2 and CXCL3) inferring an anti-inflammatory effect. Conversely, the veg-etative cells had a quite different effect by in-ducing a moderate pro-inflammatory oriented response (e.g. TNF, IL8, IL17C, IL32, CCL20, CXCL1, CXCL2, CXCL3 and NFκB1); in addition, down-regulating the expression of key anti-inflammatory (TH2 response) genes, IL-10 and TGF-β. We speculate that the switch from spore to vegetative form in the intestine may be an important mechanism of action for this beneficial microbe.

n 44BGENETIC TOOLS fOR PROPIONIbACTERIuM ACNES

M. S. Wollenberg, K. P. Lemon; Forsyth Insti-tute, Cambridge, MA.

Propionibacterium and Staphylococcus are two of the most common bacterial genera detected in the microbiota of the healthy adult human nostrils (anterior nares). One central hypoth-esis of our lab is that natural isolates of the anterior nares produce diffusible substances that both inhibit and stimulate the in vitro growth of Staphylococcus aureus. As confir-mation of this hypothesis, our lab has demon-strated the existence of P. acnes-produced S. aureus-inhibiting small-molecules using an in vitro co-culture assay. In this poster, we out-line both targeted gene disruption and random mutagenesis approaches as part of a further effort to identify and understand the function of these diffusible substances. We describe the adaptation of methods for creating marked and markerless deletion mutants in primary nostril isolates of P. acnes. In addition, the develop-ment of a 96-well-based screen to detect P. acnes mutants defective in the ability to influ-ence S. aureus growth in vitro is presented.

n 45AA MATTER Of LIfE ANd dEATH?: dETECTION Of C. ALbICANS CELL WALL AMYLOIdS.

M. C. Garcia1, N. Lysak1, S. Singh1, R. So-bonya2, S. A. Klotz2, P. N. Lipke1; 1CUNY Brooklyn College, Brooklyn, NY, 2University of Arizona, Tuscon, AZ.

Als5p, a member of C. albicans cell wall ad-hesins, contains amyloid forming sequences which are conserved in other Als family mem-bers. These sequences are critical for soluble peptide and protein amyloid fibril formation. The Als5p amyloid sequence is critical for cell adhesion and biofilm formation (Garcia et al, PLoS ONE e17632). Assays thus far have not addressed the existence of C. albicans

Poster Abstracts

ASM Conferences58

cell wall amyloids in the human host. Here we used autopsy sections from patients with invasive candidiasis to determine the pres-ence of yeast amyloid interactions in the host. Autopsy sections from the gut of infected patients stained for both yeast and hyphae with amyloid specific dyes thioflavin T, Congo red and thioflavin S. Histological sections obtained from the spleen served as a negative control and exhibited minimal or no fluorescence. Autopsy sections from the gut, stained with Congo red exhibited birefringence in areas where there were large abscesses. We also de-veloped fluorescent peptide probes containing the conserved Als and Hwp2 amyloid forming sequence. We found that these peptides bind to yeast and hyphal forms of C. albicans. Ad-ditionally, the Als peptide was used to detect fungal cells in autopsy sections. These tissues were counterstained with calcofluor white to show colocalization with the FITC-labeled amyloid peptide. We observed that yeast in the gut tissues stained with colocalization of thioflavin T and the fluorescent peptide. Col-lectively, our data demonstrates that fungal amyloid-like interactions exist in the host. Sup-ported by NIGMS SC1 GM083756 and R01 GM098616

n 46BdISENTANGLING THE ROLE Of CORRINOIdS IN STRuCTuRING HuMAN GuT MICRObIAL COMMuNITIES

P. H. Degnan1, N. Barry1, K. Mok2, M. Taga2, A. L. Goodman1; 1Yale University, New Haven, CT, 2University of California Berkeley, Berke-ley, CA.

Genome sequencing efforts have revealed the complete nucleotide sequences of hundreds of human gut symbionts however, the mecha-nisms required for microbial competition and cooperation in the gut are largely undefined. Human gut microbes depend on proteins with distant homology to vitamin B

12 transporters

to colonize the mammalian gut in gnotobiotic mouse models. Notably, gut microbes possess

elaborate systems for uptake of vitamin B12

and related compounds (corrinoids) and gener-ally encode multiple paralogous transporters with conserved accessory genes. To understand how these systems determine the fitness of hu-man gut microbes in vivo, we combined genet-ic, gnotobiotic, and computational approaches to characterize corrinoid transport in the prominent human gut symbiont Bacteroides thetaiotaomicron. We demonstrate that each of the three outer membrane corrinoid transport-ers encoded in the B. thetaiotaomicron genome can facilitate B

12-dependent growth in vitro

and that conserved accessory proteins missing from E. coli and other standard model organ-isms are also required. Competition assays further reveal patterns of corrinoid specificity and possible tradeoffs between binding affinity and transport effectiveness in these systems. Complementary studies in gnotobiotic mice indicate that the role of corrinoid exchange in shaping community assembly can be modu-lated through control of host diet. Moreover, analysis of over 400 sequenced human gut microbial genomes indicates that the human gut microbiome is replete with complex, pre-viously unappreciated systems for corrinoid transport. In sum these results demonstrate how genetic and gnotobiotic approaches can complement genome sequencing to shed light on underlying mechanisms that shape human gut microbial community composition.

n 47ASTudY THE SuRVIVAL Of LACTObACILLuS ACIdOPHILuS AS A PRObIOTIC ANd ITS EffECT ON SOME fACTORS Of quALITY Of WHITE IRANIAN CHEESE IN bRINE

T. Moosavi, L. Mohammadyar; Islamic Azad University, Tehran, IRAN, ISLAMIC REPUB-LIC OF.

The survival of the probiotic strains Lactoba-cillus acidophilus (PTCC 1643) was inves-tigated in the Iranian white cheese in brine. Four treatments of Iranian white cheese were produced: control I (C1), with a commercial

Poster Abstracts


starter culture, control II (C2),with commercial starter culture and the probiotic L. acidophilus, LC with a commercial starter culture and pro-biotic L. acidophilus (uncapsulated) and LCC with commercial starter culture and probiotic L. acidophilus capsulated with Sodium Algi-nate by extrusion method. The cheese samples were ripened at 8-12 oC for 60 days and the viability of cultures and also moisture, salt, protein, fat in solids, pH were determined on days 1,15,45 and 60. At day 1, the numbers of L. acidophilus in the samples C2, LC and LCC were 1.74×106, 2.5×106 and 2.7×106 cfu g-1 respectively. These figures reached to 9×105,9.4×105. and1.48×106 cfu g-1 after 60 days of storage, respectively. The reduction was significant (P<0.001). The final numbers of L. acidophilus, in all of the cheese samples were lower than the minimum numbers of the recommended therapeutic products (107 cfu /g). Moisture content and pH of all cheeses were decreased and salt and protein and fat in dry matter content increased during ripping. The maximum and minimum range of loss of moisture at the end of ripening period was39.2 % and 37.2%which belonged to LC and C1 respectively. The maximum and minimum range of loss of pH at the end of ripening pe-riod belonged to C1,C2 and LCC respectively.Therefore probiotic Lb. acidophilus both form free and microencapsulated must be used with higher doses in Iranian white cheese in brine. Keywords: Probiotic, Iranian white cheese in brine, Capsulated , Lactobacillus acidophilus (PTCC 1643)

n 48BCONTRIbuTION Of VIbRIO fISCHERI TCP PILuS IN THE COLONIzATION Of THE SquId EuPRYMNA SCOLOPES.

C. Bongrand; University of Wisconsin, Madi-son, WI.

Vibrio fischeri is a marine luminous gram-neg-ative bacterium that can live in symbiosis with the squid Euprymna scolopes. The host pro-vides nutrients to the bacteria which, in return,

produce bioluminescence during the night to help the squid hide from predators. This sym-biosis is a good model to study host-microbe interactions in a natural system. To establish the symbiosis, the bacterium aggregates on the ciliated appendages of the nascent light organ, travels to and through surface pores, migrates down ducts, and colonizes six crypts of the light organ. The different steps of colonization involve many squid-bacteria and bacteria-bacteria interactions that still remain to be determined. V. fischeri possesses ten separate pilus gene clusters, which may be involved in the process of colonization. One of these is the Tcp pilus, which has been determined as essential for the V. cholerae infection. Because the Tcp pilus has been involved in micro-colonies formation, we hypothesized that this pilus could be involved in the establishment of the symbiosis. To address this possibility, we constructed a deletion in tcpA, the major pilin subunit of the Tcp pilus and performed single-strain and competitive colonization ex-periments of the juvenile squid. Interestingly, we observed that wild type V. fischeri is able to outcompete the tcpA mutant under certain conditions, suggesting that the pilus may be involved in successful aggregation. We are currently characterizing the impact of the tcpA deletion in both culture and symbiosis.

n 49AANTIbACTERIAL MEMbRANE ATTACk bY A PORE-fORMING INTESTINAL C-TYPE LECTIN

S. Mukherjee, H. Zheng, Q. Jiang, L. V. Hooper; UT Southwestern Medical Center at Dallas, Dallas, TX.

Mammalian epithelial surfaces are associated with resident bacterial communities that per-form beneficial metabolic functions but can threaten health if they invade host tissues. C-type lectins of the RegIII family are bactericid-al proteins that protect the intestinal epithelial surface and are essential for maintaining mutu-alistic relationships with the intestinal micro-

Poster Abstracts

ASM Conferences60

biota. Here we elucidate the mechanistic and structural basis for RegIII bactericidal activity. We show that human RegIIIα binds membrane phospholipids and kills bacteria by forming a membrane permeabilizing oligomeric pore. Using cryo-electron microscopy (cryoEM), we have determined the three-dimensional structure of the RegIIIα pore, finding that it is a unique hexameric assembly resembling two stacked three-winged propellers. Our cryoEM model, which is supported by structure-based mutagenesis results, indicates that the indi-vidual RegIIIα subunits are oriented with the carbohydrate binding loop pointed towards the central pore and with the N-terminus, which regulates RegIIIα lipid binding activity, oriented toward the lipid bilayer. Lipopolysac-charide inhibited RegIIIα pore-forming activ-ity, explaining why RegIIIα is bactericidal for Gram-positive bacteria but not Gram-negative bacteria. Our findings identify C-type lectins as mediators of membrane attack in the mucosal immune system, and provide structural insight into an antibacterial mechanism that promotes mutualism with the resident microbiota.

n 50BbACTEROIdES THETAIOTAOMICRON uSES MuLTIPLE CARbOHYdRATE bINdING dOMAINS TO TARGET dIffERENT STRuCTuRAL fEATuRES Of STARCH

E. A. Cameron, N. M. Koropatkin, E. C. Mar-tens; University of Michigan, Ann Arbor, MI.

A prominent function of the human gut micro-biota is to break down complex carbohydrates that our digestive enzymes are unable to de-grade. Bacterial fermentation of sugars liber-ated from these polysaccharides produces short chain fatty acids, providing the host with a sig-nificant source of nutrients and increasing the health of colonic tissue. The Bacteroidetes, one of the dominant phyla in the gut, are capable of degrading a wide variety of diet- and host-de-rived carbohydrates. This task is accomplished via the expression of multi-component systems

composed largely of outer membrane and peri-plasmic proteins that bind, degrade and import polysaccharides. The starch utilization system (Sus) of Bacteroides thetaiotaomicron (Bt) was the first such system described and is essential for growth on various forms of starch. Two proteins in this system, SusE and SusF, belong to a highly abundant family of proteins that is enriched in the gut metagenome, yet their function(s) remains unknown. We solved the crystal structures of SusE and SusF, revealing that they each contain multiple carbohydrate binding modules (CBMs), with SusE having two tandem CBMs and SusF having three. Based on the architecture of the CBM binding sites, we hypothesized that they possess unique affinities for different forms of starch. Thus, we used two different assays to investigate the individual and cooperative contributions of these CBMs to binding starch or its oligosac-charides. Using an adsorption/depletion assay, we determined that individual CBMs varied in their affinity for insoluble cornstarch, with one of the SusF CBMs being unable to bind this substrate at all. Additionally, using isothermal titration calorimetry we show that the SusE and SusF CBMs vary in their preference for linear versus cyclic starch oligosaccharides as well as their overall binding strength for these shorter substrates. Expression of several starch binding domains with varying specific-ity may endow species like Bt with the ability to deploy a single protein system to degrade a series of structurally related but heterogeneous molecules (e.g., different forms of amylose, amylopectin, glycogen), while economizing on the total numbers of genes that it must possess to attack these important nutrients. These stud-ies provide insight into the mechanisms that have evolved to allow human gut symbionts to cope with structural variability in the myriad of carbohydrates that they encounter in the human gut.

Poster Abstracts


n 51AAdAPTIVE EVOLuTION Of E. COLI AuxOTROPH CO-CuLTuRES

X. Zhang, J. L. Reed; University of Wisconsin Madison, Madison, WI.

Microbial mutualism is a widespread phenom-enon and is important in maintaining natural microbial communities and host-microbe inter-actions. Although mutualistic interactions are prevalent in nature, little is known about the processes which allow their initial establish-ment, govern population dynamics and affect evolutionary processes. To explore these ques-tions, we constructed an artificial mutualistic co-culture using two E. coli auxotrophs, a ΔlysA and a ΔleuA mutant, which cannot grow individually in minimal medium unless lysine or leucine is supplied, respectively. However, when co-cultured together both strains can grow in minimal medium, implying an ex-change of leucine and lysine takes place. We have performed independent parallel adaptive evolutionary experiments of ΔlysA and ΔleuA co-cultures in glucose minimal medium for ~40 days. Exponentially growing cells were repeatedly passed into fresh medium before reaching stationary phase, and the growth rates, biomass yields, and ratio of the two strains were measured over evolution of the co-culture. Results showed that the co-culture growth rates increased by ~3 fold within the first 10 days for all independently evolved co-cultures. The biomass yields of the co-cultures also increased during adaptive evolution. The ratios of the two mutants in three indepen-dently evolved co-cultures stayed close to 1:1, indicating that one particular strain does not become dominant over the other. While these community-level trends were conserved, the individual trajectories of the parallel evolved co-cultures did show some differences. In-dividual ΔlysA and ΔleuA isolates from the evolved co-culture populations were character-ized in monoculture and co-culture. Surpris-ingly, in monoculture with exogenous lysine or leucine, the evolved isolates had lower growth

rates compared to the un-evolved strains (6~40% for ΔleuA and 40~65% for ΔlysA). However, co-cultures of evolved isolates (ΔlysA or ΔleuA) with the un-evolved partner strain were able to grow 1.4~ 1.7 times faster than the un-evolved co-culture. Therefore, both ΔlysA and ΔleuA strains evolve to improve the fitness of the co-culture. A genome-scale meta-bolic co-culture model was also built and used to investigate the impacts of amino acid (leu-cine or lysine) production and consumption on growth and composition of the co-culture, and to suggest mechanisms for improving com-munity fitness.

n 52BGuT HOST-MICRObIAL INTERACTIONS fROM A PROTOCHORdATE PERSPECTIVE

L. Dishaw1, J. Flores-Torres1, B. Leigh1, C. Karrer1, G. Mueller1, A. Liberti2, D. Melillo2, I. Zucchetti2, R. De Santis2, M. Pinto2, G. W. Lit-man1; 1University of South Florida, St. Peters-burg, FL, 2Stazione Zoologica Anton Dohrn, Naples, ITALY.

Recent studies of gut microbial communities have demonstrated essential roles for bacteria in normal host physiology. Defining the rules of gut-microbial homeostasis has significant prognostic value in understanding disease eti-ologies and overall health. However, details of how the immune system tolerates millions of microbial inhabitants at the epithelial surfaces of the gut are not fully understood. In order to help define this dialog, we are using the protochrodate, Ciona intestinalis, as a model for studies of host gut-microbial interactions. As a filter feeder, Ciona is exposed to a literal sea of microbes from which it selectively discriminates indicating that complex mecha-nisms supporting gut-microbial homeostasis likely has evolved for several hundred million years. Ciona may aid in determining how a host adapts to seasonal and dietary encounters with microbiota. In recent studies we have observed that, 1) the Ciona gut possesses what appears to be a complex, immune-associated

Poster Abstracts

ASM Conferences62

lamina propria whose development appears to be tightly regulated; 2) like in vertebrates, Ciona secretes a soluble immunoglobulin-domain containing protein into the gut that interacts with luminal bacteria and can be specifically recognized by phagocytic amoe-bocytes; 3) inflammatory responses in the gut can be experimentally manipulated and gauged with common markers of innate immunity, and 4) starvation, antibiotics, and semi-sterile conditions can induce microbial dysbiosis. As a model system, Ciona lacks vertebrate-type adaptive immune tissues and responses. Stud-ies of host gut and microbial interactions at the epithelial surface in this species will provide unique insight into the mechanisms that govern intestinal immune homeostasis.

n 53AquANTITATIVE INTESTINAL IMAGE ANALYSIS REVEALS dIETARY IMPACT ON bACTEROIdES LOCALIzATION

K. Earle, K. Huang, J. Sonnenburg; Stanford University, Stanford, CA.

The gut microbiota impacts many facets of host biology. Investigations into microbiota membership have revealed compositional shifts in a variety of situations related to host health and disease, dietary change, and a number of other factors. Such enumeration studies typi-cally focus on the composition of bulk fecal material. How the numerous relevant variables (e.g., diet) impact the localization of bacteria in the gut, and how changes in localization, in turn impact the host and microbial ecosystem is poorly understood. Here we have combined fluorescence in situ hybridization with quan-titative high-throughput image analysis to analyze the spatial distribution of thousands of bacterial cells per sample in the proximal and distal colon of gnotobiotic mice. In this study, we have applied the new technique to determine how dietary change influences the localization of the human gut symbiont B. thetaiotaomicron relative to the host colonic epithelium. Previous work suggests that in a

healthy mouse model, bacteria are largely ex-cluded from the gut epithelium by a complex interplay of host immune effectors and the mucosal barrier. Consistent with this previ-ous work, we found bacteria to be excluded from the tight inner mucus layer in the distal colon. However, in mice fed a polysaccharide-deficient diet, we found a significant increase in bacteria invading the mucus layer and contacting the epithelium. The dependence of polysaccharide-deprived B. thetaiotaomicron on host mucus as a carbon source is a likely contributor to this change in localization. These findings and technique set the stage for quantitatively assessing how numerous factors impact the proximity of resident microbes to one another and to host tissue.

n 54BEVER-CHANGING fRIENdS? POSITIVE MICRObE-MICRObE INTERACTIONS IN A VARIAbLE GuT MICRObIOTA Of dROSOPHILA.

C. Wong, J. M. Chaston, A. E. Douglas; Cor-nell University, Ithaca, NY.

The gut microbiota is a critical determinant of animal health. Although changes in microbiota composition have been linked to different health states, the factors that shape the gut microbiota are not well-understood. To bet-ter understand the patterns and underlying processes in the composition of the gut micro-biota, we examined the contribution of host phylogeny, bacteria in the environment/food, and interactions among the microbiota, using 18 Drosophila species as our experimental sys-tem. The bacteria in dissected guts were identi-fied by 454 pyrosequencing of 16S rRNA gene amplicons. We have demonstrated: (1) The composition of the gut microbiota was not con-gruent with Drosophila phylogeny, indicating that the gut microbiota in Drosophila has nei-ther co-evolved with the host over evolutionary time, nor tracked evolutionary changes in gut physiology that vary in parallel with phyloge-netic relatedness between different host taxa.

Poster Abstracts


(2) The community structure is likely driven by stochastic processes, as well as factors intrinsic to the bacteria and host, since the microbiota composition varied greatly within and among Drosophila cultures over generations. (3) The gut bacteria may be propagated by fecal-oral transmission. Specific members may cooperate synergistically to promote colonization and retention of each other, as co-occurrence of certain bacterial species is significantly greater than predicted by chance. We noted that no an-tagonistic interactions among the bacteria were evident. Our conclusions that the composition of the gut microbiota is determined by the conditions and resources in the Drosophila gut and diet, the vagaries of fecal-oral transmis-sion, and mutually beneficial among-bacterial interactions have been made possible by the low bacterial diversity in Drosophila guts, which permits near-comprehensive inventories of the gut microbiota. Our results yield specific hypotheses about the ecological determinants of gut bacterial community assembly that can be tested in more complex associations, includ-ing mammals.

n 55AA NOVEL GENE CLuSTER bYPASSES quORuM SENSING IN vibrio fiscHeri duRING SYMbIOSIS

T. Miyashiro1, D. Oehlert1, Z. Liu2, J. Zhu2, E. G. Ruby1; 1University of Wisconsin-Madison, Madison, WI, 2University of Pennsylvania, Philadelphia, PA.

Quorum sensing describes the general signal-ing mechanism used by bacteria to regulate group behaviors. The marine bacterium Vibrio fischeri integrates multiple quorum-sensing systems via a phosphorelay to regulate biolu-minescence, motility, and colonization of its natural squid host, Euprymna scolopes. This phosphorelay is conserved among Vibriona-ceae members, and controls transcription of the small regulatory RNA Qrr1, which post-transcriptionally regulates the global regulator LitR. From a mutagenesis screen, we have

identified a plasmid-linked gene cluster that regulates qrr1 expression. A point mutation in VF_B0006 results in the elevated expression of the five-gene operon containing VF_B0006, which in turn leads to an increase in qrr1 ex-pression. This regulatory effect depends on the response regulator LuxO, but is independent of the phosphorelay. We show that overexpression of the plasmid-associated genes also affects quorum sensing in other species of Vibriona-ceae. Colonization and luminescence levels of squid light organs are reduced when the symbi-onts overexpress the plasmid-associated genes. Further, we observe cell-to-cell heterogeneity in expression levels of the plasmid-associated genes in an isogenic bacterial population within individual crypts of the light organ. Our results demonstrate a mechanism by which individual cells can bypass quorum sensing and introduce phenotypic heterogeneity within a clonal population of bacteria.

n 56BCHARACTERIzATION Of LACTObACILLI WITH PRObIOTIC POTENTIAL IN THE PRAIRIE VOLE microtus ocHrogaster

S. Assefa1, A. Achterhof2, V. Reynolds3, Y. Chen1, J. T. Curtis1, G. A. Koehler1; 1Okla-homa State University Center for Health Sci-ences, Tulsa, OK, 2Tulsa Community College, Tulsa, OK, 3Oral Roberts University, Tulsa, OK.

Increasing evidence is pointing towards an in-tricate involvement of the intestinal microbiota in the bidirectional communication between the CNS and the gut. We are particularly inter-ested in the potential role of intestinal probiot-ics in ameliorating neurological disorders that are associated with social withdrawal symp-toms. Probiotics are live organisms that confer health benefits to their host when administered in adequate amounts. Most probiotic bacteria belong to the genus Lactobacillus. For in-depth studies using prairie voles (Microtus ochrogas-ter) as model system for the behavioral impact of environmental toxins and potential remedia-

Poster Abstracts

ASM Conferences64

tion by probiotics, lactobacilli were isolated from the vole gastrointestinal tract and tested for probiotic properties. Using anaerobic cul-ture techniques and selective media, 30 bacte-rial strains were identified from the vole ce-cum, colon and small intestine and classified to the species level by 16S ribosomal RNA gene sequencing. Random Amplification of Poly-morphic DNA (RAPD) PCR was employed for strain fingerprinting. Both approaches revealed a close relationship of the 30 strains to Lacto-bacillus johnsonii, a species known to include probiotic strains with beneficial effects in humans. RAPD analysis allowed for categori-zation into nine different RAPD clusters. Since orally administered probiotics must survive passage through the highly acidic stomach and withstand adverse intestinal conditions, the strains’ acid tolerances as well as resistances to bile and bile salts were determined. Six strains demonstrated tolerance to low pH levels in addition to strong resistance to bile and the bile acid taurocholate. The lactobacilli and their culture supernatants were also evaluated for antimicrobial effects towards fungi and bacteria. While few of the isolates were able to inhibit growth of the fungus Candida albicans, potent antibacterial properties were detected in at least eleven of the strains. For future in vivo studies, the strains’ resistance to toxic metals and adhesion to intestinal epithelial cell lines were also examined. Five L. johnsonii strains have been selected as probiotic candidate strains based on their performance in the afore-mentioned assays. In conclusion, this study demonstrates that lactobacilli with probiotic potential are present in the vole intestine. The L. johnsonii isolates identified in this study will provide a basis for the investigation of probiotic effects in the vole behavioral model system.

n 57ATRANSCRIPTIONAL ANd PHYSIOLOGICAL RESPONSES Of THE SOYbEAN SYMbIONT bradyrHizobium japonicum TO OxIdATIVE STRESS

W. Chang, A. J. Donati, H. Lee, J. Jeon; Uni-versity of Texas-Arlington, Arlington, TX.

Bradyrhizobium japonicum is a soil bacterium capable of establishing a symbiotic relation-ship with the leguminous soybean plant (Gly-cine max). During the nodulation process, in which the bacterium forms nitrogen-fixing nodules on the soybean roots, B. japonicum must resist the oxidative burst produced by the host plant as a defense mechanism against invasive bacterial cells. We investigated the ef-fect of oxidative stress on the gene expression and physiological properties of B. japonicum. Hydrogen peroxide (H

2O

2) and paraquat were

used as sources of the oxidative stress in this study. Genes encoding F

0F

1-ATP synthase,

chemotaxis, transport, and ribosomal proteins were mainly upregulated during prolonged exposure (PE) to paraquat, while the expres-sion of a large number of ABC transporter genes was detected during PE to H

2O

2. Inter-

estingly, FixK2-related genes, which are known

to be induced in bacteroid-like microaerobic environments, were upregulated by fulminant shock (FS) in both paraquat and H

2O

2 condi-

tions. However, genes encoding catalase and superoxide dismutase were not detected in either paraquat or H

2O

2 conditions presumably

because those genes are expressed constitutive-ly to detoxify endogenous reactive oxygen spe-cies. Instead, organic hydroperoxide resistance genes, which are known to typically respond to organic peroxide but not H

2O

2 in other bac-

teria, were significantly upregulated by FS of H

2O

2. Interestingly, the paraquat-mediated oxi-

dative stress enhanced the exopolysaccharide (EPS) production of B. japonicum; however, EPS production was not induced by H

2O

2 at

the given experimental conditions.

Poster Abstracts


n 58BdISRuPTION Of PNEuMOCOCCAL COLONISATION bY THE PRObIOTIC STREPTOCOCCuS SALIVARIuS.

J. Manning, E. Dunne, C. Satzke; Murdoch Childrens Research Institute, Melbourne, AUS-TRALIA.

There is mounting evidence that probiotic bac-teria could play a role in reducing disease in the respiratory tract. Streptococcus pneumoni-ae (the pneumococcus) is an important paedi-atric pathogen, causing pneumonia, meningitis, septicaemia and otitis media. Pneumococci often colonise the nasopharynx of children, and this carriage is considered a prerequisite for pneumococcal disease. Previous studies have shown the probiotic bacterial species Streptococcus salivarius can disrupt pneumo-coccal adhesion to HEp-2 pharyngeal epithelial cells when added before, during, but not after pneumococcal colonisation is established. S. salivarius strain K12 is known to inhibit patho-genic bacteria such as Streptococcus pyogenes through production of megaplasmid-encoded bacteriocin-like inhibitory substances (BLISs) in vitro and has been used as commercial probiotic for more than a decade. In order to further investigate the role of the megaplas-mid in S. salivarius-mediated inhibition of S. pneumoniae colonisation, we are developing a duplex quantitative PCR assay for the simul-taneous detection of both chromosomal and megaplasmid-encoded S. salivarius genes.

n 59AMICRON-SCALE STRuCTuRE IN THE HuMAN ORAL MICRObIOME ANALYzEd uSING CLASI-fISH

J. L. Mark Welch1, A. M. Valm1, B. Rossetti1, C. G. McKinney1, F. E. Dewhirst2, G. G. Bori-sy1; 1Marine Biological Laboratory, Woods Hole, MA, 2The Forsyth Institute, Cambridge, MA.

Many cooperative and competitive interactions among microbial cells are critically dependent on distance between the cells, particularly in

habitats characterized by salivary flow in the human mouth. Analysis methods based on DNA sequencing have identified dozens of bacterial genera and several hundred species-level taxa in dental plaque, but the process of nucleic acid extraction destroys fine-scale spa-tial information. Fluorescence in situ hybrid-ization (FISH) targeting ribosomal RNA has been used to identify microbes and investigate their spatial arrangement, but is generally em-ployed to differentiate only two or three taxa simultaneously. We have developed a method, Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH), that can be used to in-crease dramatically the number of fluorescent labels that can be differentiated simultane-ously, and that thus is capable of identifying and visualizing sufficient taxa to begin to describe the structural complexity of oral bio-films. We employ probes targeting genus-level and family-level groups, followed by applica-tion of probes targeting species and groups of species to define more precisely the taxonomy of cells that form structures of interest. Ap-plication of CLASI-FISH with genus- and family-level probes to methacrylate-embedded sections of human dental plaque reveals strik-ingly complex corncob-like structures made up of at least four distinct bacterial taxa identified as members of the genera Corynebacterium, Streptococcus, and Porphyromonas and the family Pasteurellaceae (genera Haemophilus and Aggregatibacter). In addition to the clas-sic corncob structure of Corynebacterium filaments surrounded by Streptococcus cells, more complex structures are found in which the Streptococcus cells are surrounded by an additional layer of Pasteurellaceae. Porphy-romonas cells are found both intermingled with Streptococcus and forming corncob structures of their own, immediately adjacent to Streptococcus-dominated corncobs. Applica-tion of probes targeting subsets of these genera and families suggests that individual species occupy distinct niches within the plaque struc-ture. These CLASI-FISH data call attention to taxa that may be involved in both cooperative and potentially competitive interactions in

Poster Abstracts

ASM Conferences66

dental plaque, and suggest avenues for further genomic, biochemical, and metabolic studies to elucidate the roles and functions of the taxa involved.

n 60BMICRObIAL COMMuNITIES SuCCESSION IN A COMPOSTING OPERATION AT THE SãO PAuLO zOO PARk, bRAzIL

L. Principal1, L. Farage1, J. C. de Oliveira2, R. C. Pascon2, D. Barbosa1, L. A. Digiampietri1, J. B. Cruz3, L. Juliano4, S. Verjovski-Almeida1, J. C. Setubal1, A. M. da Silva1; 1Universidade de São Paulo, São Paulo, BRAZIL, 2Universi-dade Federal de São Paulo, Diadema, BRAZIL, 3Fundação Parque Zoológico de São Paulo, São Paulo, BRAZIL, 4Universidade Federal de São Paulo, São Paulo, BRAZIL.

Biodegradation and conversion processes dur-ing composting are carried out by complex microbial communities whose structure chang-es depending on temperature, pH, aeration, humidity and type of organic solids. Previous analyses of different composting environments by either cultivation-dependent or DNA-based approaches have identified major bacterial orders and fungal groups in composting pro-cesses. However these studies were limited to the detection of abundant microbial groups and to biases imposed by rRNA gene-cloning or DNA probing approaches. In order to describe in a greater detail the microbial community composition dynamics in composting as well its gene and functional diversity related to biomass degradation, we performed direct sequencing of metagenomic DNA samples isolated from a composting facility constructed inside the São Paulo Zoo Park. This facility is designed to compost dropped tree leaves, plant debris and grass clippings collected from an Atlantic Rain Forest fragment and gardens located inside the park, water recycling slurry from its artificial lake, wastewater treatment sludge, bedding materials and animal feed wastes plus animal excrements from ~400 spe-cies. The end compost humus-rich material

obtained after 80-100 days (600 tons compost per year) is used as fertilizer and soil amend-ment in the São Paulo Zoo farm, thus complet-ing the full cycle of recycling. Here we report results of analyses of a 1.8 Gbp data set gener-ated by Roche 454 GS FLX Titanium shotgun sequencing of metagenomic DNA extracted from five samples collected 1, 30, 64, 78 and 99 days after the beginning of the composting process in a 8m3 open concrete chamber. The average temperature of the compost on the day of sample collection was, respectively, 60oC, 58oC, 53oC, 57oC and 49oC, and the pH, was 5.0 and 7.9 for the first two samples and 7.0 for the three other samples. rRNA-based and protein-based community profiles revealed that the microbial community structure changed significantly along the composting process. While the sample from day 1 appears to be en-riched in Pseudomonadales and Bacteriodales, sample from day 64 was enriched in Bacillales, Actinomycetales and Rhizobiales. Moreover, despite their relatively low sequence abun-dance, Clostridiales were detected throughout the composting process. Our initial analyses indicate that along the process, the Zoo com-posting presents the same taxonomic groups but their relative proportions vary significantly. Future work will focus on the transcripts and metabolites generated from these samples in order to determine the metabolic activity of the microbes found in composting. Financial Sup-port: FAPESP, CNPq and CAPES.

n 61ATRAdITIONAL fOOd MICRObIAL GENOMES, “VIVE LA dIVERSITé”

M. Almeida1, A. Hebert2, S. Rasmussen3, C. Delbes4, C. Monnet2, C. Farrokh5, M. Mon-tel4, F. Irlinger2, P. Renault1; 1INRA, Jouy en Josas, FRANCE, 2INRA, Thiverval-Grignon, FRANCE, 3CBS, Copenhaagen, DENMARK, 4INRA, Aurillac, FRANCE, 5CNIEL, Paris, FRANCE.

In fermented foods such as cheeses, microbial consortia may be rather complex and > 260

Poster Abstracts


microorganism species have been isolated from cheeses, while only a small part of them are actually used in starter cultures and characterized at the genomic level. The envi-ronmental origin of most species is still uncer-tain, but may include in raw milk or different components of the cheese production system. Many of these species display environmental counterparts (water, grass, animal and human microbiota) rising the question of microbial fluxes in the food chain. However, these still poorly characterized species play also an important role in cheese process and confer their typical taste and aroma. Lastly, foods displaying high microbial diversity may also play a beneficial role for health by interact-ing with our gut as suggested by the “hygiene hypothesis”. We aimed characterizing these bacteria in order to better define their meta-bolic abilities in processes, their origin and specific adaptation to milk ecosystem in order promote their use as food starters or probiot-ics. We collected 147 strains encompassing 140 different species from 65 genera which 18 of them did not display reference genomes. They are taxonomically related to gram nega-tive bacteria (38%), low (33%) and high GC (29%) gram-positive bacteria, mainly isolated in France and EU, from different products made from goat, ewe, camel or cow milks. The genomic sequences were generated with an innovative method allowing decreasing dra-matically the cost of library construction based on recent advance in metagenomic assembly and clustering. Draft quality was assessed by different procedures, including HMP criteria, indicating that 78 passed “high quality” and 28 “standard quality” draft criteria. Draft genome analysis provided answer to questions such as the origin of food strains (i.e. Escherichia coli living on the surface of smeared soft cheeses, Pseudohalteromonas species present on the surface of over 50% cheeses), or specific ad-aptative features of food strains compared to their environmental corresponding strains (i.e. dairy Streptococcus infantarius versus human

commensals). These data provide a dedicated food microbial resource that will allow better exploiting metagenomic data in food ecosys-tem the potential of these bacteria in food pro-cesses or as probiotics.

n 62BMICRObIAL COMMuNITY COMPLExITY IN A PROTOCHORdATE MOdEL fOR INTESTINAL IMMuNE HOMEOSTASIS

B. A. Leigh1, J. Flores-Torres1, K. Gemayel1, C. Karrer1, M. Mueller1, A. Liberti2, D. Mel-illo2, I. Zucchetti2, R. De Santis2, M. Rosaria Pinto2, G. W. Litman1, L. J. Dishaw1; 1Univer-sity of South Florida, St Petersburg, FL, 2Stazi-one Zoologica Anton Dohrn, Napoli, ITALY.

Ciona intestinalis is a new model being de-veloped for studies of gut microbial immune interactions. Ciona is a filter-feeding sessile marine invertebrate which is exposed to a nearly infinite, seasonally dynamic, assort-ment of marine microbes; however, we have found that its gut is inhabited by distinct com-munities of microbes. Compositional changes in gut microbiota are being monitored using various molecular approaches that include: rapid screening by restriction fragment length polymorphisms (RFLP) analysis of 16s PCR products, random sequencing of small 16s product libraries, 454 pyrosequencing, and real-time PCR monitoring of distinct bacterial communities. Preliminary analyses reveals evi-dence for a core microbial community in wild populations of Ciona, which are dynamically influenced by seasonal changes and environ-ment. The timing and colonization of microbes in Ciona juveniles is being disrupted in various ways (i.e., dysbiosis) to monitor effects on the development of gut associated immune tis-sues. Gut immune tissue development is tightly regulated and may be influenced by more than resident microbial communities.

Poster Abstracts

ASM Conferences68

n 63AELuCIdATING kEY fEATuRES REquIREd fOR OPTIMIzEd RECOMbINEERING IN LACTObACILLuS REuTERI ATCC PTA 6475

J. van Pijkeren, K. Neoh, R. A. Britton; Mich-igan State University, East Lansing, MI.

Single-stranded DNA (ssDNA) recombineer-ing is a technology which is used to generate subtle changes in the chromosome of several bacterial genera. We recently adapted ssDNA recombineering to a range of lactic acid bacte-ria, and the goal of this work was to determine parameters contributing to the efficiency of ssDNA recombineering in Lactobacillus re-uteri ATCC PTA 6475. The ability to avoid the mismatch repair (MMR) system has been shown to be critical to improving recombineer-ing efficiency in Escherichia coli. To address this in L. reuteri we determined recombineer-ing efficacy with oligonucleotides yielding individual mismatches and showed that the hierarchy of mismatch repair in L. reuteri is G•G, T•C > A•A, T•G, C•A > A•G, C•C; this is comparable to E. coli. However, the differ-ence in efficacy of mismatch repair between a G•G (2x103 recombinants per 109 bacteria) and a C•C mismatch (~8x106 recombinants per 109 bacteria) is ~4,000-fold in L. reuteri versus 370-fold in E. coli. Also, evasion of MMR in L. reuteri with an oligonucleotide yielding a C•C mismatch and a second or third mismatch is strongly dependent on sequence context; however, four and five consecutive non-complementary bases evade the MMR system regardless of the presence of a C•C mismatch. Optimum oligonucleotide length for recombineering in L. reuteri is 60-90 bases, and a single non-complementary base in a 80mer oligonucleotide may be located between base 9-76 without affecting recombineer-ing efficiency. Chromosomal encoded RecT contributes to recombineering efficiency in L. reuteri wild type bacteria; however, it does not impact recombineering efficiency when RecT is expressed from a plasmid. Taken together, these data suggest that different rules apply to

evade the MMR system of L. reuteri compared to E. coli, and this work provides insight on how to adapt high efficiency recombineering to other lactic acid bacteria.

n 64BEubACTERIuM RECTALE dISTINGuISHES SOLubLE VERSuS RESISTANT dIETARY STARCH ANd INduCES SEPARATE ENzYME RESPONSES

N. M. Koropatkin, M. A. Maynard; University of Michigan Medical School, Ann Arbor, MI.

A primary metabolic function of the human gut microbiota is the degradation of other-wise inaccessible dietary polysaccharides and fermentation to host-absorbable short-chain fatty acids (SCFA). The SCFA butyrate is the preferred food source of colonocytes, and may prevent and/or reduce tissue inflammation as-sociated with inflammatory bowel diseases, as well as inhibit the onset and progression of colorectal cancer. Butyrate is produced by bacteria in the microbiota of the phyla Fir-micutes, including Eubacterium rectale. This Gram-positive organism is adept at degrading a variety of dietary polysaccharides, although its abundance increases consistently in human volunteers consuming resistant starch type 2 (RS2). Our hypothesis is that E. rectale directly degrades (RS2), capturing this polymer at the cell surface via the expression of one or more α-amylases tethered to the cell membrane or peptidoglycan layer. We demonstrate that E. rectale (DSM 17629) grows well on a variety of different starches, including RS2, soluble starch, and maltooligosaccharides. In compari-son to growth on glucose, the two predicted cell wall/membrane-localized α-amylases, Eur_21100 and Eur_01860 display different transcriptional responses depending on their starch exposure. Eur_21100 is a predicted 145 kDa α-amylase with an LPXTG motif for teth-ering to the peptidoglycan layer and contains several putative starch-binding carbohydrate-binding modules, while Eur_01860 is a 60 kDa α-amylase containing a putative N-terminal

Poster Abstracts


transmembrane helix. The transcription of Eur_21100 is upregulated ~150-fold on RS2 compared to growth on glucose, and upregu-lated 10-20 fold on soluble starch and maltose, suggesting that the inducing signal, presum-ably generated by starch hydrolysis, is a struc-ture unique to RS2. Conversely, Eur_01860 is uniformly upregulated ~10 fold on resistant starch, soluble starch and maltose compared to cells grown on glucose. Proteomic data on the membrane fraction of E. rectale grown on soluble starch, revealed that the most abundant protein is Eur_01830, a predicted solute-binding protein that is part of a genetic locus encoding an ABC transporter as well as Eur_01860. This is the first study identifying the enzymes and proteins responsible for resis-tant starch degradation in E. rectale.

n 65AMOLECuLAR ANd PHENOTYPIC CHARACTERIzATION Of INTERACTIONS bETWEEN A MOdEL HOST (CAENORHAbdITIS ELEGANS) ANd 46 bACTERIAL ISOLATES COLLECTEd fROM ITS NATuRAL ENVIRONMENT

A. C. Wollenberg1, M. Felix2, J. E. Irazo-qui1; 1Massachusetts General Hospital, Boston, MA, 2Institut de Biologie de L’Ecole Normale Supérieure, Paris, FRANCE.

The nematode Caenorhabditis elegans is best known as a genetic model for developmental biology, but as a natural bacterivore, it also serves as a tractable system in which to inves-tigate interactions between bacteria and the eukaryotic digestive tract. In the past decade, the use of C. elegans to study the invertebrate immune system has yielded considerable infor-mation about the molecular crosstalk between nematodes and medically relevant pathogens. However, until the recent and pioneering work of Dr. MA Felix demonstrated that wild C. elegans populations are most often found on rotting fruit [1,2], it was unclear what types of microbial communities would be naturally

encountered by C. elegans and what sorts of beneficial and detrimental interactions might occur between C. elegans and bacteria in the wild. We are beginning to address these ques-tions using a panel of 46 bacterial isolates that were collected by Dr. MA Felix at seven mi-crosites in France (e.g. a rotting apple) where C. elegans populations were also found [2]. The isolates have been assigned to 20 genera on the basis of 16S rDNA sequencing and rep-resent 2 Gram-positive phyla (Actinobacteria, Firmicutes) and 4 Gram-negative phyla (Alp-haproteobacteria, Betaproteobacteria, Gamma-proteobacteria, Bacteroidetes). Our hypothesis is that this collection of bacterial isolates will include not only potential pathogens, but also potentially beneficial microbes able to protect nematodes from more pathogenic bacteria, perhaps via colonizing the intestine or by priming the immune system. We will present data showing that the 46 isolates vary in (a) their ability to promote nematode growth and reproduction, (b) their effect on the lifespan of adult animals, and (c) their capacity to elicit immune responses such as induction of puta-tive antimicrobial genes. Importantly, of the isolates that elicit an immune response, not all are pathogenic, and we are investigating the possibility that some of these non-pathogenic bacterial isolates can prime the nematode im-mune system for future pathogen exposure. Additionally, we are investigating how com-binations of isolates influence the nematode response. Because nothing is currently known about the natural microbiota that inhabit the C. elegans intestine, our work is likely to generate novel insights. Furthermore, because nematodes are the most abundant animals on the plant, our findings will shed light on a broadly relevant bacteria/host interaction. 1. Felix, M-A and Braendle, C (2010) The natural history of Caenorhabditis elegans. Curr Biol 20:R965. 2. Felix, M-A and Duveaur, F (2012) Population dynamics and habitat sharing of natural populations of Caenorhabditis elegans and C. briggsae. BMC Biol 10:59.

Poster Abstracts

ASM Conferences70

n 66BLATITudINAL VARIATION Of dEfENSIVE SYMbIOSIS IN THE MARINE bRYOzOAN buGuLA NERITINA

N. B. Lopanik1, G. E. Lim-Fong2, J. Linneman1, D. Paulus2; 1Georgia State Uni-versity, Atlanta, GA, 2Randolph-Macon Col-lege, Ashland, VA.

Larvae of the temperate marine bryozoan, Bu-gula neritina, are defended from predators by high concentrations of unpalatable bryostatins, polyketide molecules that have been shown to be produced by the uncultured proteobacterial symbiont, “Candidatus Endobugula sertula.” Previous genetic investigations into differ-ent populations of B. neritina in the Western Atlantic revealed two cryptic species: a Shal-low genotype with “Ca. Endobugula sertula” and defensive bryostatins found south of Cape Hatteras, and a Northern genotype that does not contain “Ca. Endobugula sertula” or defen-sive bryostatins found in Delaware and Con-necticut. It has been hypothesized that higher predation levels at lower latitudes prevented establishment of the undefended Northern sibling species in locations with the defended Shallow sibling species. Furthermore, the lack of symbiotic Shallow colonies that co-occur with Northern sibling species north of Cape Hatteras could be due to cost associated with having the symbiont and consequent competi-tive disadvantage of the symbiotic Shallow. In this study, we performed a survey of host genotypes along the Western Atlantic by re-striction analysis of host cytochrome oxidase I (COI) mitochondrial genes. We detected the presence of the Northern sibling species at lower latitudes (North Carolina) where sym-biotic Shallow colonies traditionally inhabit, and Shallow at higher latitudes (Virginia) with Northern colonies. Interestingly, symbiont-spe-cific PCR of the 16S rDNA gene and of a por-tion of the bryostatin biosynthetic gene cluster revealed that Northern colonies found in NC appear to possess “Ca. Endobugula sertula” and HPLC analysis demonstrated the pres-

ence of bryostatins in their larvae. Conversely, Shallow colonies collected from Virginia were shown not to possess the symbiont. As “Ca. Endobugula sertula” is thought to be vertically transferred from one generation to the next and to date, has not been found in surrounding seawater, there are significant questions as to the transmission of the symbiont and the sta-bility of the relationship. The absence of “Ca. Endobugula sertula” at higher latitudes, and the presence at lower latitudes, independent of host genotype, suggests that predation pres-sure may be responsible for maintenance of the symbiosis in both sibling species.

n 67ATHE INfLuENCE Of fOOd MATRICES ON SuRVIVAL Of A PRObIOTIC duRING TRANSIT THROuGH THE IVIdIS MOdEL SIMuLATING THE HuMAN uPPER GASTRO-INTESTINAL TRACT

I. Mainville1, Y. Arcand1, P. Burguière2, T. A. Tompkins2; 1Agriculture and Agri-Food Can-ada, St-Hyacinthe, QC, CANADA, 2Lallemand Inc., Montreal, QC, CANADA.

The heat-stable probiotic strain Bacillus sub-tilis R0179, possessing the GRAS (Generally Recognized As Safe) status, has been used for more than 15 years to support gut health. This strain, shown to resist to industrial produc-tion processes such as baking, freezing and extrusion, thus it is an excellent candidate to be incorporated into a variety probiotic food products. The aim of the study was to measure the viability of B. subtilis R0179 incorporated in 3 products, 2% milk fat (w/v) pasteurized milk (Québon), shelf-stable orange juice “Pure breakfast” (Oasis), and a cranberry-blueberry muffin (Nutri-France) during an in vitro diges-tion using the IViDiS model simulating the human upper gastro-intestinal tract. The IVi-DiS model is a dynamic computer-controlled system that simulates mastication, the stomach conditions and the duodenum conditions. Sur-vival of the bacteria was measured at the start of digestion and every 30min after ingestion.

Poster Abstracts


Sampling of the duodenum started 90 min after ingestion. Samples were plated on Tryptic Soy Agar (TSA) and incubated at 37°C for 48h. Repeated measures ANOVA’s were performed on geometric average of duplicate plating for 4 different digestion assays of each food ma-trix. The overall survival of B. subtilis R0179 through the simulated human upper gastro-intestinal tract was very good (especially when compared to that of other non-sporulated probiotic microorganisms like lactobacilli). However, the survival rate was linked to the food matrix. The in vitro digestion has no significant impact on B. subtilis survival in both the milk and the orange juice. Conversely, there is a significant reduction of the bacterial population in the muffin in the stomach com-partment (1.5-log decrease after 150 minutes) whereas a trend for recovery is observed in the duodenum. These results highlight that con-sistency of the matrix is probably determinant for survival. A solid food will undergo longer exposure to stomach conditions whereas a quicker gastric emptying rate will be observed for liquid foods.

n 68BTHE ROLE Of GuT MICRObIOTA IN MAINTAINING CANdIdA ALbICANS GI COLONIzATION RESISTANCE

D. Fan, L. A. Coughlin, M. R. Savani, J. S. Hamilton, M. M. Neubauer, A. Y. Koh; Uni-versity of Texas Southwestern Medical Center, Dallas, TX.

Background: Candida spp are the most com-mon human fungal pathogen and the 4th lead-ing cause of bloodstream infections in the U.S. C. albicans harmlessly colonizes the gastroin-testinal (GI) tract but can disseminate after GI microbial dysbiosis and immune suppression. While 50-60% of humans are colonized with C. albicans, adult mice are resistant to Candida colonization. Materials: C3H/HeN, C57BL/6, and Balb/c mice from Harlan, Taconic, and Jackson were given penicillin G (PCN), strep-tomycin (STR), PCN/STR, or no treatment in

their drinking water. Mice were then gavaged C. albicans strain SC5314 (107 cfu). C. albi-cans GI colonization levels were assessed on days 1, 2, 3, 4, 7, 21, 28 days after gavage. Bacterial gDNA was isolated from fecal pel-lets. 16S rRNA amplicons (V4 variable region) were generated and sequenced on Roche 454 GS-FLX sequencer. The abundance of specific intestinal bacterial groups was determined by qPCR using group-specific 16S rRNA gene primers. qPCR for quantifying gene expression of antimicrobial peptides (AMP) and cytokines from distal small intestine was done using SsoAdvanced SYBR Green Master Mix (Bio-rad) and specific primers. Results: Neonatal, germ-free and antibiotic-treated mice can be colonized with C. albicans. Wild-type mice of all three strains from all vendors were resis-tant to Candida colonization. Only antibiotics that markedly depleted the bacterial phylum Firmicutes and more specifically, reduced clos-tridial clusters IV and XIVa allowed for sus-tained C. albicans GI colonization. The same mouse strain from different vendors, receiving the same antibiotics, displayed widely vari-able Candida colonization phenotypes. In mice colonized with C. albicans, the introduction of Blautia producta (clostridial cluster XIVa) resulted in complete elimination of C. albicans from the murine GI tract, whereas B. fragilis, L. acidophilus, E. coli resulted in slightly di-minished C. albicans colonization and P. aeru-ginosa had no effect. AMP (notably CRAMP and defensins) and cytokine (esp IL17A, IL22) gene expression was significantly decreased in colonization susceptible mice compared to colonization resistant mice. Butyrate, the primary SCFA produced by cluster XIVa and IV bacteria, inhibits C. albicans’ germination, hyphal formation, and growth; enhances the AMP LL-37 killing of C. albicans; and signifi-cantly decreases C. albicans murine GI coloni-zation and dissemination in mice. Conclusion: The composition of the murine gut microbiota is a critical determinant of whether C. albicans can colonize the murine GI tract. Firmicutes, particularly butyrate-producing anaerobes,

Poster Abstracts

ASM Conferences72

appear to be essential for maintaining Candida colonization resistance. Gaining insight into the mechanisms by which the murine GI tract is C. albicans colonization resistant may lead to novel therapeutic modalities for preventing C. albicans infections in humans.

n 69AHAbITAT SELECTION bY bACTERIAL TAxA INHAbITING THE HuMAN MOuTH

S. Huse1, A. M. Eren2, J. Mark Welch2; 1Brown University, Providence, RI, 2Marine Biological Laboratory, Woods Hole, MA.

Using the Human Microbiome Project (HMP) 16S rRNA sequence data, we demonstrate that subtaxa within bacterial genera show separate patterns of niche selection between the differ-ent habitats within the human mouth. To date, most analyses of HMP data have employed relatively coarse taxonomic classifications, cal-culating summary indices measuring the over-all degree of difference between communities, or using genus-level taxonomy or clustering algorithms that create operational taxonomic units (OTUs) by grouping sequences differ-ing by up to 3%. A single 3% OTU frequently encompasses several named species, particu-larly in intensively-studied habitats such as the human mouth. Here we employ a method known as oligotyping to identify key sites of nucleotide variation, allowing the identification of lineages that differ by as little as a single nucleotide within the sequenced region but show differential distribution across samples. Applying oligotyping to trimmed HMP 16S ribosomal RNA sequences from hypervariable regions 4 through 5 (V4-V5) from 8 differ-ent genera of particular interest for human oral health (Actinomyces, Corynebacterium, Fusobacterium, Porphyromonas, Prevotella, Streptococcus, Treponema, Veillonella), 3 families (Lachnospiraceae, Neisseriaceae, Pasteurellaceae), and one phylum (TM7), we separated each of these taxa into subtypes and examined the subtype distribution in over 200 patients across 9 different oral sites (buccal

mucosa, tongue dorsum, hard palate, keratin-ized gingiva, supragingival plaque, subgingival plaque, palatine tonsils, throat, and saliva). The oligotypes show distinct preferences for certain of the oral habitats and many of these preferences differ between subtypes of the same taxa, implying variable functional roles or habitat adaptations for species of the same genus. These subtype variations are not dis-cernable using the Ribosomal Database Project (RDP) Classifier and do not correspond to 3% OTUs, but most of them are 98.5% to 100% identical to sequences in the Human Oral Microbiome Database (HOMD) or the CORE Oral Database. Although oligotype selection is not consistently correlated between most habi-tats, oligotype selections for the subgingival and supragingival plaque habitats are similar to each other and substantially different from those of the other oral sites.

n 70BHuMAN NOSTRIL/SkIN-ASSOCIATEd ACTINObACTERIA PROduCE SMALL MOLECuLES THAT IMPACT STAPHYLOCOCCuS AuREuS

B. E. Jacobson1, M. S. Wollenberg1, J. Clae-sen2, E. E. Pagano1, M. Zimmermann3, K. L. Goguen1, K. L. Aldridge4, T. C. Scharschmidt2, M. A. Fischbach2, K. P. Lemon4; 1Forsyth In-stitute, Cambridge, MA, 2University of Califor-nia, San Francisco, San Francisco, CA, 3In-stitute of Molecular Systems Biology, Zurich, SWITZERLAND, 4Forsyth Institute and Boston Children’s Hospital, Cambridge, MA.

Although the majority of bacteria that colonize human epithelial surfaces behave as com-mensals/mutualists, humans are host to a small number of bacterial species that can behave both as pathogens and as commensals/mu-tualists. One such species is Staphylococcus aureus. S. aureus colonizes the anterior nares (nostrils) of ~30% of the U.S. population and the majority S. aureus carriage is asymptom-atic. However, colonized individuals are at an increased risk for serious S. aureus infections,

Poster Abstracts


most of which appear to be due to endogenous S. aureus strains. It is well established that S. aureus strains are not alone in colonizing and persisting on epithelial surfaces of the human body. In addition to S. aureus, other commen-sal/mutualistic bacteria that colonize the same body sites as S. aureus make up what is likely to be a long-standing and co-evolving microbi-al community. We hypothesize that individual members of these communities produce small molecules that impact the growth and behavior of S aureus. As confirmation of this hypothesis we have used co-culture assays with primary isolates from healthy adult nostrils to demon-strate that some strains of the common nostril and skin genera Propionibacterium and Cory-nebacterium produce diffusible substances that impact S. aureus growth. Using both extracts of culture medium and a candidate-molecule approach, we have begun to identify some of the molecules responsible for these interspe-cies interactions. Understanding the influence of small-molecule-mediated interactions on the bacterial community structure in the hu-man nostril will both provide insights into the chemical ecology of this host-associated microbial community, and facilitate the devel-opment of new strategies for preventing and/or eliminating S. aureus colonization, drastically decreasing the risk for human infection.

n 71AGLObAL dISCOVERY Of COLONIzATION dETERMINANTS IN VIbRIO fISCHERI

J. F. Brooks1, M. C. Gyllborg1, D. C. Cronin1, A. L. Goodman2, M. J. Mandel1; 1Northwest-ern University Feinberg School of Medicine, Chicago, IL, 2Yale University School of Medi-cine, New Haven, CT.

Animals form intimate, beneficial relation-ships with bacteria. To study the processes by which animal hosts acquire their specific symbiotic bacteria from the environment, we take advantage of a natural model host system

that is colonized by a single symbiont. The light organ of the Hawaiian bobtail squid, Euprymna scolopes, is colonized exclusively by the luminous Gram-negative bacterium, Vibrio fischeri. This mutualistic relationship is characterized by the protection each spe-cies confers to the other: the bioluminescence produced by V. fischeri masks E. scolopes from predators at night, and E. scolopes provides V. fischeri with a privileged nutrient-rich environ-ment. The goal of this project is to define novel gene products, molecules, and behaviors that mediate specific host colonization. Insertional Sequencing (INSeq) is a high-throughput ge-netic screening approach that combines high-density transposon mutagenesis with Illumina deep sequencing technology to enumerate pool composition. We have adapted INSeq to identi-fy genes encoding factors that are required for optimal fitness under specific selective regimes (e.g., culture-based enrichments or squid colo-nization). By applying this technology during squid colonization, we identified a large num-ber of founder symbionts per host, which sup-ports the suitability of this approach for sym-biotic V. fischeri. We expanded our analysis to 500 hosts (i.e., 40,000 colonizing symbionts) and identified colonization-defective mutants in genes that code for stress-responsive factors. These included factors that regulate protein folding and degradation, oxidative and nitrosa-tive stress responses, and envelope integrity. We found that the protein folding chaperone DnaJ is required for normal host colonization, and in culture exhibits severe defects in biofilm formation and luminescence, two processes known to be required for robust colonization. The squid environment is known to be replete with toxic compounds (including nitric oxide, hydrogen peroxide, and hypochlorous acid), and these mutant candidates provide tools to examine how the bacterial symbionts signal and survive during symbiotic development. We are now expanding this approach to screen the symbiont genome to saturation and to examine our preliminary candidates in further detail.

Poster Abstracts

ASM Conferences74

n 72BdISCOVERING INHIbITORS AGAINST THE CANdIdA ALbICANS MET6P PROTEIN uSING x-RAY CRYSTALLOGRAPHY, dIffERENTIAL SCANNING fLuORIMETRY ANd ENzYME kINETICS

D. Ubhi; University of Texas at Austin, Austin, TX.

Background: Candidemia is a serious health concern which afflicts millions and results in considerable mortality. The Candida albi-cans species is the most prevalent cause of candidemia and has been the focus of previ-ous studies which identified an essential C. albicans gene, Met6. This gene encodes the zinc-dependent fungal enzyme cobalamin-in-dependent methionine synthase (Met6p) which synthesizes methionine by directly transferring a methyl group from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (L-Hcy). This 88 kDa protein is structurally and mecha-nistically different from the modular 140 kDa cobalamin-dependent methionine synthase (MetH) found in mammals. Hypothesis: Since the Met6p active site has the potential to be a potent antifungal target based on its pre-dicted structure and essential gene function, we should be able to find Met6p inhibitors which retard pathogen growth with minimal toxicity to the host. Methods: Met6p variants susceptible to protein crystallization were cre-ated by identifying a high entropy cluster on a surface exposed loop of wild-type Met6p using a web-based SER prediction (SERp) server. Charged residues in the 103LysLysAl-aThrGlu107 cluster were mutated to Ala, Thr or Tyr. The differential scanning fluorimetry (DSF) method was used in 96-well format to identify compounds that bind Met6p and increase its melting temperature (Tm). DSF was used to screen the entire NIH clinical collection (NCC) consisting of 731 drug-like compounds and the first 2,000 compounds from the Chembridge Fragment and KinaSet libraries. Ligands which shifted the Tm by ≥ 0.3 °C were considered hits. These were

validated using an established enzyme activity assay at competing substrate (5-methyl-THF and L-Hcy) and 500 uM inhibitor concentra-tions. Hits which reduced the enzyme activity by ≥ 30% were tested for their IC50 values in the presence of competing 5-methyl-THF and saturating L-Hcy concentrations. Results: All 3 surface engineered Met6p proteins crystal-lized in an “open” conformation, retained full enzymatic activity, and were used to map the active site. The DSF assay identified 26 hits from NCC, 13 from the Fragment and 20 from the KINASet library. 6 NCC, 4 Fragment and 14 KINASet compounds sufficiently reduced the enzyme activity. We found one inhibitor (N-(4-aminophenyl)benzamide) from the Frag-ment library with an IC50 of ~175 uM. Three potential inhibitors, 3-({[4-(benzoylamino)phenyl]amino}sulfonyl)-N-(4-bromophenyl)benzamide, N-[(2-hydroxy-1-naphthyl)(3-nitrophenyl)methyl]benzamide, and N-[(2-hydroxy-1-naphthyl)(4-nitrophenyl)methyl]-3-methoxybenzamide were discovered from the KINASet with IC50 values of ~60 uM, ~75 uM, and ~100 uM, respectively. Conclu-sion: A chemically diverse set of compounds was found to inhibit the fungal Met6p enzyme activity and are worthy of further study as po-tential drugs against candidemia.

n 73APREVENTION Of MENOPAuSE RELATEd OSTEOPOROSIS IN MICE bY TREATMENT WITH PRObIOTIC LACTObACILLuS REuTERI

R. A. Britton, R. Irwin, J. Zhang, L. Schaefer, S. Raehtz, D. Quach, N. Parameswaran, L. McCabe; Michigan State University, East Lansing, MI.

Osteoporosis and osteopenia are estimated to affect over 200 million people worldwide. While men, women, and children can be af-flicted with various maladies that contribute to bone loss, the largest population affected by osteoporosis is post-menopausal women. Current therapies that prevent bone loss have undesirable side effects, thus novel treatments

Poster Abstracts


need to be developed. Evidence in animal stud-ies suggests that post-menopausal bone loss is partly due to pro-inflammatory cytokine production in the bone. Therefore, we tested if the anti-inflammatory probiotic strain Lactoba-cillus reuteri ATCC PTA 6475 would be able to prevent bone loss in ovarectomized (ovx) mice. Surprisingly, L. reuteri 6475 was able to completely suppress bone loss associated with ovariectomy. Four weeks after ovariectomy, mice displayed a nearly 50% loss in trabecular bone volume fraction (femur and vertebrae). Daily supplementation of L. reuteri 6475 pre-vented this bone loss; L. reuteri treated mice displayed bone volume fractions similar to control mice. Not all probiotic strains tested were equally successful in suppressing osteo-porosis, suggesting that L. reuteri 6475 was having a specific effect on bone health. Analy-sis of microbial communities demonstrated that estrogen depletion altered the intestinal microbiota of mice while supplementation of L. reuteri 6475 partially restored these commu-nities towards those observed in wild-type ani-mals. Currently we are attempting to elucidate the mechanism(s) by which L. reuteri 6475 and the intestinal microbiota influence bone metab-olism and to identify if the anti-inflammatory activity of L. reuteri 6475 is required for the amelioration of osteoporosis.

n 74BSTRAIN-LEVEL GENETIC MAPPING IN VIbRIO fISCHERI WITH TfOx-INduCEd TRANSfORMATION

M. C. Gyllborg, M. J. Mandel; Northwestern University, Chicago, IL.

The bioluminescent Gram-negative bacterium Vibrio fischeri forms a natural monospecific light-organ association with Euprymna scol-opes squid. During each host generation, the relationship is recapitulated: E. scolopes hatch lacking symbionts and secrete mucus to col-lect environmental bacteria. This specificity is exquisite as only V. fischeri, and only certain strains of V. fischeri, colonize E. scolopes

squid. As such this mutualism is a valuable model in which to define the molecular basis for specific acquisition of symbionts, and we seek to define the molecular factors that me-diate host colonization specificity. Here we present a genetic method to directly map speci-ficity factors. It has recently been shown in the Vibrionaceae in general, and in V. fischeri spe-cifically, that chitin induces DNA competence facilitating transformation of exogenous DNA. Evidence suggests that large DNA fragments (>20 kb) are readily transferred by this mecha-nism in V. cholerae and competence can be in-duced in V. fischeri in the absence of chitin by overexpression of the TfoX regulatory protein. As V. fischeri lack other robust mechanisms for transformation (or transduction) we hypoth-esized that tfoX-induced transformation may provide a tool by which relevant phenotypes could be mapped by both classical and next-generation genetic tools. To test this approach, we analyzed V. fischeri strain ES114-L that was highly similar to its wild-type presumed parent ES114 but exhibited an inability to grow on glycerol and failed to export siderophore. We introduced ES114 genomic DNA into ES114-L to map fragments that conferred the ability to utilize glycerol as a carbon source; all 26 transformants restored siderophore export and mapped to a 31 kb region of chromosome II, which upon Illumina sequencing revealed a 10 kb deletion in ES114-L relative to ES114. Ad-ditional protocol improvements have allowed for quantitative linkage mapping, and in the future we will apply this technology to identify host specificity factors in symbiotic V. fischeri.

n 75AAGE dEfINES THE bENEfICIAL GuT MICRObIOME COMPOSITION ANd fuNCTION

Z. E. Ilhan, D. Kang, R. Krajmalnik-Brown; Biodesign Institute, Arizona State University, Tempe, AZ.

The gut microbiome benefits its host by regu-lating the immune response, maintaining an

Poster Abstracts

ASM Conferences76

energy balance and promoting health by pro-ducing valuable metabolites. Its composition and function may change with age affecting the interactions with its host. We hypothesized that adults have different and more diverse ben-eficial microbiome than children. In order to identify the beneficial gut microbiome changes by age; we compared fecal microbiome com-position of 20 healthy children and 10 healthy adults by pyrosequencing the V2-V3 region of the 16S rDNA. We analyzed the data using QIIME 1.4.0 suite, and binned the operational taxonomic units into metabolic groups that can potentially have a beneficial function, such as fatty acid producers (lactate, propionate, butyr-ate), and hydrogen consumers. This metabolic grouping approach helped us recover rare but functionally important genus-level phylotypes that were overshadowed by highly abundant ones. The alpha diversity within all metabolic groups was higher in adults than children, indi-cating that the children microbiomes have not reached their full metabolic potential. Within each metabolic group, the abundance of phy-lotypes significantly varied between adults and children suggesting an interesting functional redundancy and that by aging, specific gut microbiome functions might be accomplished by different microbiota. Butyrate producers, which maintain the integrity and health of the colon by feeding the colonocytes were mainly composed by Subdoligranulum and Faceli-bacterium (60-80%) in adults and children, and Bifidobacterium was significantly more abundant in children. Propionate is often as-sociated with a reduction in cholesterol and the relative abundance of propionate producers was significantly higher in adults suggesting a possible contribution from the gut microbiome to battle rising cholesterol levels as the human ages. The composition of propionate producers showed a higher abundance of Akkermansia in children and Prevotella in adults. Lactate is a precursor of fatty acids and benefits the host by stimulating the immune system and protect-ing against colorectal cancer. We observed that possible dominant lactate producers were Bifi-dobacterium in children and Dialister in adults.

Hydrogen consumers allow fermentation to proceed favorably in the gut, and they were more abundant in adults than children probably due to a larger fermentation capacity and need in the adults. Dominant hydrogen consumers included: homoacetogenic Blautia in adults and Eubacterium in children. With real time PCR, we observed another group of hydrogen consumers, methanogens, were more prevalent in adults however; children that exhibit them had higher copy numbers than adults. Our re-sults suggest that the gut microbiome evolves by aging to respond to the needs of the host and different microbial associations maintain the gut homeostasis.

n 76BGACA TRANSLATIONAL REGuLATION Of LuMINESCENCE fuNCTIONS dOWNSTREAM Of THE LuxO quORuM SENSING CASCAdE

A. E. Ballok1, A. Septer2, R. A. Foxall3, E. V. Stabb4, C. A. Whistler3; 1Dartmouth College, Hanover, NH, 2Harvard University, Cambridge, MA, 3University of New Hampshire, Durham, NH, 4University of Georgia, Athens, GA.

Bioluminescence of Vibrio fischeri is essential for mutualism with its squid host Euprymna scolopes. Luminescence is induced by two AHL quorum sensing systems, and by GacA which also coordinates other phenotypes im-portant in symbiosis. In other bacteria, GacA activates several small RNAs including CsrB which bind and sequester the translational repressor CsrA. If the CsrA regulatory cascade functions as it does in Vibrio cholerae, the GacA luminescence defect would be due to an inability to inhibit CsrA, leading to activation of Qrr1 via LuxO, resulting in low levels of the quorum sensing regulators LitR and LuxR. This would reduce transcription of the entire lux operon. V. fischeri possesses two CsrB RNAs and we hypothesize that they func-tion in symbiosis as intermediates of GacA regultion. We confirmed that GacA regulates both CsrBs using transcriptional fusions.

Poster Abstracts


When these genes were expressed in trans they complemeted all known defects of the GacA mutant in culture and during squid colonza-tion. Furthermore, a CsrB1/2 double mutant mirrored the GacA mutant for all phenotypes. Spontaneous CsrA mutations also suppress the GacA mutant. However, CsrA mutants are also less efficient than wild-type at squid coloniza-tion which indicates the presence of CsrA and its proper regulation by GacA is important for early stages of symbiosis. We further investi-gated how GacA, the CsrBs and CsrA interface with quorum sensing. The inability of litR and luxR or AHL to complement the GacA-lumi-nescence defect, and the dark phenotype of a GacA/LuxO mutant suggest a unique regula-tory hierarchy. Moreover, expression of lux-ICDABEG from a GacA-independent promoter failed to complement lumiescence, suggesting a post-transcriptional mechanism. We identi-fied a putative CsrA binding site within the coding region of luxI whose alteration witha silent mutation (GGAAGA) increases lumines-cence 5-fold. In conclusion, GacA regulates luminescence and other host-association phe-notypes through the established CsrA/CsrB paradigm; however, luminescence regulation is predominatly through translational repression of the lux operon. Thus, V. fischeri exhibits novel circuitry among the published Vibrios.

n 77ASTAbILITY Of HuMAN MICRObIOTA IN THE CONTExT Of TRACkEd HOST HEALTH ANd bEHAVIOR

L. A. David1, A. Materna2, J. Friedman3, I. Baptista4, M. Blackburn5, A. Perrotta3, S. Preheim3, E. Alm3; 1Harvard University, Cambridge, MA, 2CLC bio, Boston, MA, 3Mas-sachusetts Institute of Technology, Cambridge, MA, 4Rockefeller University, New York, NY, 5École Polytechnique Fédérale de Lausanne, Lausanne, SWITZERLAND.

Daily microbiome changes may be caused by minor alterations in human health or routine but few studies have linked microbiome dy-

namics to longitudinal studies of human well-ness and lifestyle. Here, we integrated high-throughput sequencing of bacterial 16S ribo-somal RNA in 751 fecal and saliva samples collected from two subjects over the course of one year with more than 10,000 self-reported measurements of host health and behavior. We identified two host factors -- prolonged travel abroad and acute enteric infection -- capable of causing major microbiota disturbances. Travel was associated with a near doubling of the Bacteroidetes to Firmicutes ratio. En-teric infection resulted in sharp and persistent abundance declines for over half of formerly common bacterial taxa. Yet aside from these two disturbances, metrics of overall gut and salivary microbiota state remained stable for months. Nearly all tracked host factors did not appear to affect the dynamics of individual bacterial taxa within these stable periods, with the notable exception of fiber-consumption. Our data support the hypothesis that changes to human health or behavior can reveal alterna-tive ecological stable states, but suggest that most human actions do not influence microbial dynamics within those stable periods.

n 78BGASTROINTESTINAL MICRObIOTA ANd SOCIAL bEHAVIOR: THE PRAIRIE VOLE MOdEL

S. Assefa1, Y. Chen2, J. T. Curtis1, G. Koe-hler1; 1Oklahoma State University Center for Health Sciences, Tulsa, OK, 2Oklahoma State University Center for Health Sciences, Tulsa, OK.

Prairie voles, Microtus ochrogaster, have been suggested as an excellent animal model to study the neurobiology of social behavior. Un-like many other mammal species, prairie voles exhibit socially monogamous and biparental behavior. Our long-term objective is the expan-sion of this model system towards elucidating the interactions of gastrointestinal microbiota with the social brain. Recent behavioral studies have shown that male voles exhibit social defi-

Poster Abstracts

ASM Conferences78

cits after toxic metal (e.g. mercury) ingestion while female voles appeared to be unaffected. To study the effect of toxic metal exposure on the vole gut microbiota and detect potential correlations with behavioral outcomes, we de-termined the composition of the gut microbiota in experimental and control animals of both sexes. To this end, DNA was extracted from various areas of the vole GI tract or from fe-cal pellets and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analyses were used to search for major differences in microbial populations. For detailed composi-tional characterization of the microbiota, pyro-sequencing of 16S rRNA-derived PCR ampli-cons was employed. Analysis of classified se-quencing results revealed strong dominance of Firmicutes in cecal samples of all voles. Com-parison of the composition of the microbiota in experimental and control groups revealed sex-specific and treatment specific differences. While toxic metal ingestion led to specific changes in the overall abundance of some bac-terial groups, female-specific compositional variants were identified that might confer pro-tective effects against metal toxicity. Real-time PCR provided quantitative insights into how selected bacterial groups with potentially exac-erbating or ameliorating roles in metal toxicity were affected by mercury treatment. To our knowledge, this study presents the first charac-terization of gut microbiota in prairie voles. In addition, a basis for further studies on the inte-gration of the intestinal microbiota in gut-brain communication is provided in a new model system for social behavior.

n 79AdROSOPHILA ENdOSYMbIONTS: THE EffECTS Of INfECTION ON d. MELANOGASTER uPON ATTACk bY TWO PARASITOId WASPS

J. Xie, G. Sanchez, S. Butler, M. Mateos; Tex-as A&M University, College Station, TX.

Maternally-transmitted associations between endosymbiotic bacteria and their insect hosts

are common in nature. Heritable microbes with imperfect vertical transmission are expected to be lost in the absence of compensational mechanisms to increase their persistence in their host lineages (e.g. host reproductive ma-nipulation and fitness benefits to host). Indeed, many inherited facultative endosymbionts are reproductive manipulators, and several others defend their hosts against viruses, nematodes and parasitoids. Two closely related strains of the heritable endosymbiont Spiroplasma infecting distantly related Drosophila hosts, protect their respective hosts against a para-sitic wasp (Leptopilina heterotoma; Lh) and a nematode. Although these Spiroplasma defen-sive strains are not reproductive manipulators, several closely related strains manipulate host reproduction by killing the sons of infected females. One such male-killing Spiroplasma strain is MSRO, which naturally infects D. melanogaster. Goal 1: Examine whether strain MSRO also enhances host survival against wasp parasitism by Lh. Goal 2: Determine whether MSRO protects its host against an-other species of Leptopilina (L. boulardi; Lb), which differs from Lh in host evasion/suppres-sion strategies. Goal 3: Evaluate the potential interaction of Wolbachia (another common heritable endosymbiont of D. melanogaster), with the protective mechanism of Spiroplasma. To address this, we examined whether larval to adult survival of D. melanogaster subjected to oviposition attacks by the two different species of wasps differed among four endo-symbiont infection treatments: uninfected flies; Spiroplasma MSRO-infected flies; Wolbachia-infected flies; and doubly-infected flies. Goal 4: Compare the growth of wasp larvae in the above four endosymbiont infection treatments to better understand the protective mechanism. We found that: (1) Spiroplasma MSRO caused high mortality of the two species of wasps (~96% in Lh and ~97% in Lb); (2) MSRO sig-nificantly enhanced fly larva-to-adult survival against Lh and Lb (~3.28 and ~3.13%, respec-tively; Spiroplasma infection only), compared to their endosymbiont-free counterparts (~0.86 and 1.26%, respectively); (3) Wolbachia had

Poster Abstracts


a weaker, but additive positive effect on fly survival against Lh and Lb (~7.78 and ~6.72%, respectively, Spiroplasma+Wolbachia double infection), which was only significant against Lh; and (4) Spiroplasma slowed down larval growth of both wasp species at different rate. The observation that the male-killer MSRO provides weaker protection to its host than the non-male-killer of D. hydei against para-sitoid wasps, suggests that the male-killing and defensive mechanisms are complimentary strategies of Spiroplasma MSRO to achieve persistence in its host population.

n 80BTHE uSE Of GNOTObIOTIC zEbRAfISH fOR STudYING INTESTINAL MICRObIOTA ASSEMbLY

W. Z. Stephens1, A. R. Burns1, S. Wong2, M. J. Taormina1, M. Jemielita1, K. Stagaman1, J. S. Hampton1, R. Parthasarathy1, J. F. Rawls2, B. J. Bohannan1, K. Guillemin1; 1University of Oregon, Eugene, OR, 2University of North Carolina, Chapel Hill, NC.

Microbial colonization of the digestive tract is a crucial event in vertebrate development, required for maturation of host immunity and establishment of normal digestive physiology. We have developed the zebrafish as a model system to study how intestinal microbial com-munities are established in time and space. We have profiled the gut microbiota of replicate populations of zebrafish across developmen-tal time. We find that bacterial communities change dramatically between larval, juvenile, and adult stages. Neutral models of commu-nity assembly explain a substantial amount of interindividual variation, suggesting that sto-chastic assembly may be important. We have assembled a collection of zebrafish-associated bacteria that represent all genera detected in multiple populations of laboratory-reared and wild-caught fish. This collection was used to inoculate germ-free zebrafish larvae with sim-ple communities of defined bacterial species. The relative abundances of members of these

simple communities mimicked those of natu-rally occurring communities. Furthermore, as-sembly of these simple gut microbiota revealed ecological interactions between community members. We have further explored intestinal bacterial community assembly in the intestines of optically transparent larval zebrafish using light sheet microscopy. Our data are consistent with a model of colonization by a relatively small founder population that expands inside the intestine and that can exclude subsequent invasion. We are currently exploring the bacte-rial genetic requirements for colonization and the role of bacterial competition in determining gut microbiota composition.

n 81A16S RRNA PROfILING REVEALS TEMPORAL STAbILITY Of THE HINdGuT MICRObIOME IN THE TERMITE RETICuLITERMES fLAVIPES

J. Benjamino, M. C. Nelson, J. Graf; Univer-sity of Connecticut, Storrs, CT.

The eastern subterranean termite, Reticuli-termes flavipes is dependent upon its hindgut symbionts for the digestion of lignocellulose and the provision of essential nutrients. A complex community of Archaea, Bacteria, and Eukarya comprise the hindgut microbiome, totaling more than 107 organisms. While the protozoal community is limited to a few spe-cies, the prokaryotic community is more di-verse and not well understood. Colonies of R. flavipes were established in the laboratory, and the stability of the microbiome was monitored. Termites were collected from the field in card-board traps, maintained for 56 days on spruce, 14 days on cardboard, followed by a final 56 days on spruce. In addition to the time course of workers, the microbiome from workers from two sites and soldiers was characterized. The microbiome was examined by paired-end, 2x150 bp sequencing of the V4 region of the 16S rRNA gene using universal primers on an Illumina MiSeq, generating >15,000 sequences

Poster Abstracts

ASM Conferences80

per sample. Read pairs were merged to cre-ate a single read comprising the entire V4 amplicon and processed using Qiime. Alpha diversity was similar across all samples, with Shannon (H’) indices ranging from 4.6-5.9. The microbial community of each sample was dominated by Spirochaetaceae (32-67%) and Endomicrobia (24-37%). Spirochaetaceae play an important role in host nutrition by produc-ing acetate and nitrogen compounds, with acetate being the primary energy source for the host. The Endomicrobia are endosymbionts of certain hindgut Protozoa and provide amino acids to both its protozoal host and the termite. The next most abundant taxa were Rickettsi-ales, Spingomonadaceae, Desulfovibrionales, and Pseudomonadaceae, which combined account for ~4% of the reads. Despite using universal V4 primers, less than 1% of total reads were assigned to the Archaea. Beta di-versity analyses using phylogenetic (UniFrac) and OTU (Bray-Curtis) based metrics revealed temporal stability of the hindgut microbiome over the 126-day period, despite changes in diet. This could be explained by the process of proctodeal feeding, wherein worker termites contribute their gut microbiota to other mem-bers of the colony. While proctodeal feeding is not the primary method of feeding in termites, it occurs after molting when the gut microbiota is shed. This infrequent sharing of symbionts could create a homogenous microbiome within the hindguts of a single colony. The temporal stability of the R. flavipes hindgut microbiome reinforces the synergistic symbiosis between the host and its symbionts in the degradation of lignocellulose, resulting in the provision of nutrients for each partner.

n 82BPARTNER SPECIfICITY IN AN ENVIRONMENTALLY-ACquIREd INSECT-buRkHOLdERIA SYMbIOSIS ANd THE EffECT Of HOST HAbITAT

J. R. Garcia, E. Chung, N. M. Gerardo; Emo-ry University, Atlanta, GA.

Many organisms acquire beneficial microbes from the environment. Although these associa-tions can be quite specific and evolutionarily stable, they are also more prone to lower part-ner specificity and partner switching. Here, we investigated the partner specificity in an insect-Burkholderia symbiosis and tested the hypothesis that habitat location can affect the symbiont diversity within a host. We screened four sympatric broad-headed bug species and their habitat, including soil and food plants, for the diversity of Burkholderia symbionts using 16S rRNA gene sequences of cultivated and non-cultivated symbiont strains. We found a diversity of Burkholderia in the insects’ midgut crypts, including strains that grouped with a clade of insect symbionts and strains that grouped with free-living and rhizosphere-associated Burkholderia. One Burkholderia strain (JRG1) was highly abundant in all four species. This strain groups within a clade of insect symbionts and has also been found to be the dominant symbiont in two Asian broad-headed bug species. Consequently, there was no significant difference in symbiont strains by host species. However, some individual insects of all four species were infected with a mono-culture of a different symbiont strain or were co-infected with two or more symbiont strains. There was also a significant difference in sym-biont strains in hosts by host habitat location. Finally, we investigated the bacterial diversity in the nitrogen-fixing root nodules of the in-sects’ food plant, Lespedeza spp., and found a number of Burkholderia strains. Strikingly, all of these strains grouped with the clade of insect symbionts and some strains were nearly identical to strain JRG1, the most common symbiont strain in the insects. This finding indicates sympatric plant and insect hosts may harbor the same or similar symbionts. We conclude that there is likely partner choice for strain JRG1 in multiple broad-headed bug species, but stochasticity in the environmental presence or abundance of Burkholderia may partially determine which strains establish in a host.

Poster Abstracts


n 83AbIOLOGICAL CHARACTERISTICS Of crinum asiaticum L. ANd ITS APPLICATION IN MICRObES: fROM PRECLINICAL INVESTIGATES TO INTERNATIONAL CLINICAL TRIALS

M. Mollik; Peoples Integrated Alliance, Dha-ka, BANGLADESH.

Crinum asiaticum L. is a tropical plant and belongs to the plant family Amaryllidaceae Jaume St.-Hil.. It is traditionally used for the remedy of killer diseases as well as debilitat-ing diseases. Although there is no report on physicochemical and antimicrobial potencies of leaves, skin of leaves, fillet of leaves to the plant, it is an attempt to investigate. The parameters investigated are minerals profile, moisture content, solid content, ash content, fatty acid content in the Crinum asiaticum L. leaves, skin of leaves, fillet of leaves and also the antimicrobial potencies of Crinum asiati-cum L. leaves skin of different ages of plants. Mineral profile was estimated by the atomic absorption spectroscopy (AAS). Outcomes showed that Crinum asiaticum L. leaves are good sources of Iron (Fe), Potassium (K), Sodium (Na), Calcium (Ca), Magnesium (Mg) and also few minerals were obtained such as Manganese (Mn), Phosphorus (P), Arsenic (As), Zinc (Zn), Chromium (Cr), Copper (Cu). It was seen that mineral content ranges in the order 6th months>9th months>3rd months ages of plants. Methylated fatty acids of Crinum asiaticum L. leaves skin of different ages of plants were analyzed by the gas chromatog-raphy-mass spectrometry (GC/MS) technique and good amount of methyl laurate (C

13H

26O

2),

methyl myristate (C15

H30

O2), methyl palmitate

(C17

H34

O2), methyl margarate (C

18H

36O

2) and

so on were obtained. On considering composi-tions can arrange in the order 3rd months<6th months<9th months ages of plants. Antimicro-bial potencies investigation shows that Crinum asiaticum L. leaves skin 80% ethanol (C

2H

6O)

extract produced zone of inhibition against bacteria (Staphylococcus aureus Rosen-

bach, and Salmonella species) and potencies obtained in case of Staphylococcus aureus Rosenbach, and Salmonella species i.e. resis-tant 66.60% and 80.95% for 3rd months, resis-tant 57.14% and 71.43% for 6th months, and resistant 43.32% and 59.61% for 9th months ages of plants. The indigenously available medicines and technologies can prove an asset in the tropical and developing countries of the world. At the same time developed countries also can be benefited because of safety profile of the plant extracts and microbial strains agents.

n 84BA ETHANOL-dEPENdENT PATHWAY Of METHANOL REduCTION TO METHANE IN A NEWLY ISOLATEd METHANOSPHAERA SP. fROM THE WESTERN GREY kANGAROO.

M. Morrison1, E. Hoedt2, P. Evans1, W. Smith1, P. Ó Cuív1; 1CSIRO Animal, Food and Health Sciences, St Lucia, AUSTRALIA, 2School of Chemistry and Molecular Biosciences, Univer-sity of Queensland, St Lucia, AUSTRALIA.

The Australian macropodids (kangaroos and wallabies) have been shown by metagenom-ics and other microbial methods to possess a distinctive microbiota that contributes to its reduced methane emissions, compared to ruminants. However, methanogenic archaea do colonize and persist in the macropodid fore-gut, but there is scant understanding of these microbes. An isolate taxonomically assigned to the Methanosphaera genus (WGK6) was isolated from the anterior sacciform forestom-ach of a Western Grey kangaroo (Macropus fuliginosus). Like the isolate from the human gut, Methanosphaera stadtmanae, WGK6 was demonstrated to be a hydrogen-dependent methylotroph, with no capacity for autotrophic growth; nor was it capable of growth with formate and hydrogen. Interestingly, our isola-tion work also suggested some capacity for the isolate to utilize ethanol to support growth. Subsequent studies showed WGK6 indeed

Poster Abstracts

ASM Conferences82

utilizes ethanol to support growth, but princi-pally as a source of reducing power. Growth of WGK6 was still dependent on the provision of methanol, but ethanol could be used in place of hydrogen gas. A draft sequence of the WGK6 genome was produced and is comprised of 1,731,982 base pairs with a G+C content of 27.7%, not unlike the characteristics noted for M. stadtmanae. Genome alignments using MAUVE revealed that WGK6 possesses con-tiguous genes encoding a putative alcohol- and an aldehyde- dehydrogenase, which are lack-ing in the human isolate. These genomic differ-ences provide a plausible explanation for the ability of WGK6 to utilize ethanol for metha-nol reduction to methane; and furthermore, support a greater cell yield per mol of methane formed, compared to when hydrogen is avail-able. Such findings also suggest how methano-genic archaea can persist in the herbivore gut even with relatively low methane emissions.

n 85ATRI-TROPHIC uNdERGROuNd SYMbIOSIS bETWEEN, bACTERIA, A bEETLE ANd A dESERT PLANT

Y. Helman1, O. Shelef2, A. L. Friedman3, A. Be-har1, S. Rachmilevitch2; 1The Hebrew Univer-sity of Jerusalem, Rehovot, ISRAEL, 2Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, ISRAEL, 3Tel-Aviv University, Tel-Aviv, ISRAEL.

Inhabitants of arid ecosystems face severe nitrogen and water limitations. Inventive adap-tations by organisms occupying such habitats are essential for survival. Here, we describe a unique tri-trophic symbiotic interaction be-tween a plant (Salsola inermis), a beetle (wee-vil -Conorhynchus pistor), and bacteria (Kleb-siella pneumonia). In many desert ecosystems, plant communities are dominated by annual species, which typically exhibit rapid wet-season growth followed by dormancy or decay during the dry summer. An exception to this general rule of thumb is S. inermis, an annual shrub that flourishes in saline soils during the

dry summer. The unusual dry-season vitality of this plant is exploited by the beetle, which survives by living within a unique mud struc-ture it affixes to the plant’s roots. We found that active nitrogen-fixing bacteria, harbored within the beetle’s gut, support this interaction by supplying nitrogen to the system, which eventually promotes seed development. These microbial symbionts were shown to fix nitro-gen only during the underground larval phase, which is nourished by root carbon exudates, and not in the imago gut. Out of over 60,000 known weevils, this is the first documentation of weevils living most of their life underground without harming plants. The unique tri-trophic symbiotic interaction described here signifies the important ecological role of desert plant roots and provides an example for a sustain-able consortium of living organisms coping with the challenging desert environment.

n 86BTHE EffECTIVE ENTEROCOCCuS HIRAE bACTERIA ON THE STRIPEd CATfISH (PANGASIANOdON HYPOPHTHALMuS) fINGERLING Of SuRVIVAL RATE ANd GROWTH RATE

N. T. Tam; Faculty of Applied Biology - Taydo University, Cantho, VIET NAM.

The studying aim to evaluate the effective supplement Enterococcus hirae in fish feed on striped catfish fingerling of the growth and survival rate. The experiment was disposed comepletely random with 3 different treat-ments each triplicated. The control treatment (un-supplement Encterococcus hirae in fish feed), the second treatment (supplement 30 ml Enterococcus hirae broths Kg- fsh feed) and the third treatment (supplement 50 ml Entero-coccus hirae broth Kg- fish feed). The feeding pellets (Quaxcel-40N) were used to make feed for fish in this study. It took four weeks to con-duct this study. The results showed the highest survival rate of fish (76.6%) in third treatment, the lowest survival rate of fish (55.5%) in control treatment and significant difference (p

Poster Abstracts


< 0.05) betwwen them. Besides, the highest final mean weight and length of fish (39.0g and 16.4 cm) in the third treatment, then the second treatment (37.2g và 16.1 cm) and significant difference (p <0.05) between this two treat-ments and control treatment. To sum up, could be used to supply Enterococcus hirae in fish feed to obtain the higher effect. Key words: Striped catfish fingerling, Enterococcus hirae, survival rate, growth rate.

n 87AENVIRONMENTAL RESERVOIRS fOR fuTuRE CORAL GENERATIONS Of THE MICRObIAL dINOfLAGELLATE SYMbIOdINIuM: THE bENEfICIAL SYMbIONT Of CORAL REEfS

C. Granados-Cifuentes1, P. Leberg1, M. Ro-driguez-Lanetty2; 1University of Louisiana at Lafayette, Lafayette, LA, 2Florida International University, Miami, FL.

Coral reefs are among the most diverse eco-systems of the world, which provides goods and services to humans estimated as billions of dollars each year. Scleractinian corals provide the framework of this ecosystem, which in turn depend on an obligate endosymbiosis with microbial dinoflagellates of the genus Sym-biodinium. The basis of this beneficial sym-biosis is nutritional; where up to 90% of the photosynthetic products of Symbiodinium are released to the coral host. This allows corals to thrive in the oligotrophic, tropical waters they inhabit. As important as these symbionts are, nearly 80% of the corals need to acquire their symbionts a new each generation. While Sym-biodinium should be available in the environ-ment for the coral larvae, very little is known about the distribution and dynamics of Sym-biodinium populations during the free-living stage. In this study, we sampled different habi-tats across various reef sites of the Caribbean island Curacao during three sampling periods to identify the environmental reservoirs used by the symbiotic Symbiodinium. These habi-tats included the water column, coral rubble,

sediment, macroalgae of the genera Halimeda spp., Dictyota spp. and Lobophora spp, and for the first time, leaves of the seagrass Thalas-sia testudinum. We recovered Symbiodinium from genetic clades A, B, C, F, G, and H us-ing the chloroplastic 23S hypervariable DNA region. Further phylogenetic analyses resolved Symbiodinium types detected here with previ-ously detected symbiotic types. This makes the present study the first one to find a direct correspondence between the free-living Sym-biodinium types and those found in symbiosis with scleractinian corals, octocorals, zoanthids, sponges, and foraminiferans. We also recov-ered new Symbiodinium types that probably represent true, non-symbiotic types. Log-linear modeling showed that the main effects (sym-biont clade, sampling time, and habitat) and the interactions symbiont clade × habitat and symbiont clade × sampling time are important to explain the observed dynamics. Overall, we detected habitats that could be playing a role of environmental reservoir of symbiotic Symbio-dinium that could potentially engage in sym-biosis with future generations of corals. The identification of these habitats are crucial if we are to better understand and conserve coral reefs, especially with the current and predicted threats of global warming and ocean acidifica-tion that this ecosystem faces.

n 88BVALORIzATION Of AGRO-RESIduES fOR PROduCTION Of xYLANASE fROM baciLLus LicHeniformis

B. Kumar, K. Manhas; University of Jammu, Jammu, INDIA.

Investigations of microbial xylanases from novel sources which display high activity and stability under hostile industrial processes are still in progress. In the present study, bacterial isolate Bacillus licheniformis P11(C) efficient-ly utilized agro-residues as carbon and nitro-gen sources to generate substantial amount of xylanase which exhibited activity and stability over broad pH range (5-11) and over elevated

Poster Abstracts

ASM Conferences84

temperatures (40-100°C), and even in presence of potential inhibitors (triton, SDS, EDTA). Purification of xylanase (4.24-fold) by am-monium sulphate precipitation and DEAE-sepharose chromatography, and analysis by SDS-PAGE and zymography showed that B. licheniformis P11(C) produced two xyanases (17.5 kDa and 23 kDa). Furthermore, xylanase displayed exciting potential for application in fruit juice processing and bakery processes; enzyme was found to be highly effective in getting enhanced sugar extraction from fruit juices, clarification of fruit juices, and substan-tial dough-raising in bakery.

n 89AuTILIzATION Of SuGAR INduSTRY WASTE WATER fOR PHb PROduCTION fROM bACILLuS SubTILIS NG05

N. K. Aggarwal, G. Singh, A. Mittal, A. Ya-dav; Kurukshetra University,Kurukshetra, Kurukshetra, INDIA.

Abstract: The fossil derived plastic material is now causing serious environmental problems. The best available alternative is the use of Biodegradable plastic. Among Biodegradable plastic Poly β hydroxybutyrate is drawing much attention due to its resemblance with petrochemical based plastic. But high pro-duction costs restrict its wider applications. Considering the industrial interest in poly - β -hydroxy butyrate (PHB), its high production cost, and pollution caused by sugar industry waste water, work has been undertaken for the production of PHB by Bacillus subtilis NG05 using sugar industry waste as substrate to mini-mize the production cost as well as to control the pollution by sugar industry waste water. Accumulation of PHB granules as food reserve materials in the organism was analyzed by Su-dan Black method. The PHB was qualitatively analyzed by Nile Blue A staining and Nuclear Magnetic Resonance (NMR) methods, Fourier Transform Infrared (FTIR), and Differential Scanning Calorimetry. The maximum yield of 5.132g/L was obtained using sugar industry

waste water as carbon source. This ongoing study suggested that sugar industry waste water could be effectively used for the produc-tion of Poly β-hydroxybutyrate. Keywords: Poly β-hydroxybutyrate, Nile Blue A, Nuclear magnetic resonance, Fourier Transform Fourier transform infrared spectroscopy, Differential Scanning Calorimetry etc.

n 90BENTEROTYPES ARE SHAREd bY HuMANS ANd CHIMPANzEES

A. Moeller1, P. Degnan1, M. Wilson2, A. Pusey3, B. Hahn4, H. Ochman1; 1Yale University, New Haven, CT, 2University of Minnesota, Minneapolis, MN, 3Duke University, Raleigh-Durham, NC, 4University of Pennsylvania, Philadelphia, PA.

The communities of microbes inhabiting the gastrointestinal tracts of humans have been divided into multiple classes, termed ‘entero-types’, which are each overrepresented by distinct sets of bacterial genera. To test for the presence of enterotypes in a non-human ape, we have assayed the gut microbial communi-ties present within fecal samples of chimpan-zees from Gombe, Tanzania. We report that the gut microbiotae of chimpanzees assort into enterotypes that are compositionally analogous to those of humans. Through the analysis of longitudinal samples, we show that the micro-bial signatures of the enterotypes are stable over time, but that individual hosts switch between enterotypes over periods longer than a year. These results support the hypothesis that enterotypic variation was present in popula-tions of great apes before the divergence of humans and chimpanzees.

n 91ACHARACTERIzATION ANd IdENTIfICATION Of PROduCTIVITY-ASSOCIATEd RHIzObACTERIA IN WHEAT

M. Anderson, J. Habiger; Oklahoma State University, Stillwater, OK.

Poster Abstracts


The rhizosphere is populated by a numer-ous and diverse array of rhizobacteria, many impact productivity in largely unknown ways. Here we characterize the rhizobacteria com-munity in a wheat variety categorized accord-ing to shoot biomass using 16S rRNA pyrose-quencing abundance data. Plants were grown in homogenized field soil under greenhouse conditions, DNA extracted and pyrosequenced resulting in 29,007 quality sequences. Opera-tional taxonomic units (OTUs) that were sig-nificantly associated with biomass productivity were identified using an exact test adjusted for false discovery rate. The productivity deviation expressed as a percentage of the total mean square for regression (PMSR) was determined for each OTU. Out of 719 OTUs, 42 showed significant positive and 39 significant negative associations (q-value < 0.05). OTUs with the greatest net positive associations were: Du-ganella #43 and #3, Janthinobacterium #278, Pseudomonas #588, and Cellvibrio #1847, and negative associations: Bacteria #273, Chrys-eobacterium #508, Proteobacteria #249, and Enterobacter #357. Shoot biomass productiv-ity was strongly correlated with the balance between the overall abundance of positive to negative productivity associated OTUs. High productivity rhizospheres contained 9.2 sig-nificant positives to every negative, while low productivity showed 2.3 significant negatives for every positive associated rhizobacteria. Three phylogenetic groups showed the greatest relative positive contribution to wheat shoot productivity, including: Duganella-Janthino-bacterium (10.3%), Rhizobium (2.6%) and Acidobacteria Gp6 (2.6%); and negative con-tribution: unknown Proteobacteria (-10.7%), unknown Bacteria (-6.5%) and Chryseobacte-rium (-5.1%). These may represent evolution-ary determined phylogenetic hotspots in the plant-microbe productivity association. Root plastids as a whole showed a strong negative relationship to productivity due their dramatic increase in low productivity plants. This may reflect a redirection of plastid based metabolic machinery to serve deleterious or pathogenic rhizobacteria. Overall rhizobacteria com-

munity diversity as measured by the Chao1, Shannon and Simpson indexes was nonlinearly related to productivity, closely fitting a wave like cubic equation. We conclude that shoot biomass productivity is strongly related to the ratio of positive and negative associated rhizo-bacteria in the rhizosphere.

n 92BfRuCTAN uTILISATION bY bACTEROIdES OVATuS

S. Shapiro, A. Basle, D. Bolam; Newcastle University, Newcastle, UNITED KINGDOM.

Dietary polysaccharides resistant to degrada-tion in the early digestive tract provide the main sources of energy and carbon for our resident colonic microbiota. Fructans are plant derived beta-linked fructose polymers widely utilised by our intestinal microbiota. Despite prevalence in the diet and use as prebiotic supplements there is a paucity of data regard-ing the mechanisms by which fructans are ac-cessed and degraded by members of this com-plex community. Bacteroides, prominent mem-bers of the normal gut flora, are able to access a range of polysaccharides by selective ex-pression of discrete polysaccharide utilisation loci (PULs) encoding a suite of cell envelope located proteins, collectively known as a Sus-like system, each targeting a specific glycan. The core component of all Sus-like systems are homologs of SusD and C, outer membrane located oligosaccharide binding and import proteins. The prototypic Sus also comprises a surface located polysaccharide binding pro-tein known as SusE, but sequence divergence makes it hard to distinguish SusE-like proteins in many PULS and thus their distribution and importance in glycan use is unclear. Bacteroi-des ovatus upregulates a Sus-like system in response to inulin (β2-1 linked fructan) which contains two putative cell surface proteins belonging to the CAZy Glycoside Hydro-lase (GH) family 91, but no obvious SusE candidate. The majority of fructan degrading enzymes belong to GH family 32, making this

Poster Abstracts

ASM Conferences86

system unusual. Here we show that while one of these GH91s is an endo acting inulinase, the other, BACOVA_04502, displays no enzymatic activity, but binds tightly to inulin. Studies conducted using isothermal titration calorim-etry reveal that a discrete C-terminal domain in BACOVA_04502 mediates fructan binding. A crystal structure of the C-terminal domain was obtained with an inulin-derived oligosac-charide bound and the molecular determinants of specificity and affinity probed using alanine scanning mutagenesis. These data suggest that BACOVA_04502 is fulfilling the role of the surface polysaccharide binding protein in the B. ovatus fructan utilisation system and provide insight into the distribution and impor-tance of SusE-like proteins in gut Bacteroides.

n 93AfROM GOOd TO bAd: uNREGuLATEd bENEfICIAL MICRObES AS A SOuRCE fOR HYbRId MORTALITY

R. M. Brucker, R. Pauly, S. R. Borden-stein; Vanderbilt University, Nashville, TN.

It is well understood that multiple factors con-tribute to the dynamics of the gut microbiota that benefit an animal host. However, little is known about how these microbiotas change during and after recent speciation events in their hosts. The focus of this research is to de-termine (i) if variation in microbiotas between closely related animal species recapitulates host evolutionary relationships, (ii) how inter-specific hybridization disrupts the beneficial microbiotas observed within species resulting in hybrid mortality, and (iii) how interspecific hybridization in germ-free conditions rescues hybrid mortality. For the proposed studies, we utilize three sister species of Nasonia parasit-oid wasps: N. vitripennis (Nv), N. longicornis (Nl), and N. giraulti (Ng). Nv diverged one million years ago from the ancestor of Nl and Ng, which themselves diverged ~400K years ago. The recent period of speciation permits hybridization between the species with varying levels of hybrid incompatibility depending on

the cross. We used barcoded pyrosequenc-ing of 16S rRNA gene amplicons to measure microbial communities during development of the three species and their hybrids. Finally, using two divergent Nasonia and their hybrids (Nv and Ng) we reared the developing wasps under germ-free conditions to determine if the microbiota are implicated in hybrid mortality. We then reintroduced the native microbiota into the germ-free individuals to reinstate hybrid mortality. We report four key results. First, all three Nasonia species share simple larval microbiotas that are dominated by the γ-proteobacteria, Providencia rettgeri; as lar-vae develop into pupae and adults, microbial successions occur in which the taxonomic diversity markedly increases. Second, the evolutionary relationships of the three Nasonia sibling species are congruent with those of their microbial communities. Third, the micro-biotas of hybrid offspring of the three host spe-cies differ significantly from the microbiotas of their parental species. Fourth, the absence of microbiotas in Nasonia causes a significant rescue of hybrid mortality and is reduced when native microbiotas are reintroduced. These results demonstrate that the evolution of micro-bial communities is closely allied with that of their hosts’ evolutionary history. Thus, micro-biotas can be as important as nuclear genes in the evolution of new host species.

n 94BCHARACTERIzATION Of THE SOdALIS GLOSSINIdIuS ACHROMObACTIN IRON ACquISITION SYSTEM

C. L. Smith1, B. Weiss2, L. J. Runyen-Janecky1, S. Aksoy2; 1University of Richmond, Rich-mond, VA, 2Yale University, New Haven, CT.

Sodalis glossinidius is a facultative intra-cellular midgut symbiont of the tsetse fly, which also harbors Trypanosoma brucei (the causative agent of African trypanosomiasis). Sodalis has a putative achromobactin sidero-phore iron acquisition system on the pSG1 plasmid. RT-PCR analysis showed that there

Poster Abstracts


was one polycistronic mRNA containing twelve transcribed achromobactin genes and that the operon was expressed when Sodalis was within the tsetse fly. Expression of the Sodalis achromobactin operon was repressed in iron-replete conditions; in a Fur mutant, ex-pression was aberrantly de-repressed in these iron-replete conditions, indicating that the Fur protein repressed achromobactin gene expres-sion when iron is plentiful. Three putative Fur binding sites within the Sodalis achromobactin promoter were identified using bioinformat-ics; however, only one of these three sites was found to actively bind Fur in Fur titration as-says. Sodalis strains with insertion mutations in the acsD putative achromobactin biosynthet-ic gene and the acr putative outer membrane ferri-achromobactin receptor were constructed; these strains grew poorly in Brain-Heart Infu-sion media, relative to the parental Sodalis strain. The reduced growth of the acsD mu-tant, but not the acr mutant, was rescued with spent media containing achromobactin from wildtype Sodalis. Unlike wildtype Sodalis, the acsD mutant did not produce detectable sidero-phore in Chrome Azurol-S assays. Preliminary data also showed that, relative to the parental strain, the acsD mutant had reduced growth in tsetse flies, suggesting that this Fur-regulated, functional siderophore system has a role in Sodalis iron acquisition in the tsetse host.

n 95APLANT MICRObIOMICS: uNIquE SOuRCE Of bENEfICIAL MICROORGANISMS.

A. Chaurasia, D. Ahrivar; NBAIM,ICAR, Mau Nath Bhanjan, INDIA.

Hypothesis: In disease infected agricultural field, few plants remain comparatively healthy & unaffected by the pathogens. One of the rea-sons behind it may be the presence of unique endophytic microorganisms (actinomycetes) in such plant which produces specific biomol-ecules inhibiting or killing the microbial patho-gen responsible for the disease occurrence. Other plants of the same species, lacking such

endophytes become disease susceptible. Such useful endophytic actinomycetes can be used as biocontrol & PGPR agent. Objectives: 1. Isolation of culturable endophytic actinomy-cetes from the healthy & pathogen infected wheat crop & their comparative analysis. 2. PGPR and biocontrol (against disease causing microbial pathogen) potential evaluation. 3. Fractionation of bioactive metabolites isolated from endophytic actinomycetes. 4. Pot/Field evaluation of the potent actinomycetes strains. Materials and methods: 1. Endophytic acti-nomycetes were isolated from the wheat root, stem & leaf using actinomycetes isolation agar, starch casein agar & various other media used for the isolation of rare endophytic actinomy-cetes by surface sterilization protocol & steril-ity check as control. 2. PGPR & biocontrol potential of the isolates were evaluated against Dreschlera sp. Alternaria sp. Curvularia sp. Rhizopus sp. using dual plate technique. These fungal pathogens were isolated from the infected wheat plant using Potato Dextrose agar media. 3. Pre pot & pot experiment of the isolates were performed as per the protocols. Active metabolites were fractionated. Poten-tial isolates were identified using polyphasic approach. Results: Diversity of culturable endophytic actinomycetes was different in healthy & disease infected wheat plant. Unique actinomycetes could be identified in the root of healthy wheat plant. Out of thirty five isolates seven showed better biocontrol potential. Iso-late NBAIM-WR-20 showed best result. Three isolates NBAIM-WR-20, 32, 06 also showed better PGPR activity. Bioactive ingredients were extracellular in nature, soluble in organic solvent chloroform and identified using HPLC and other spectroscopic techniques. Isolate NBAIM-WR- 20 was identified as Streptomy-cetes sp. Conclusions: Roots of healthy wheat plant were found to have unique endophytic actinomycetes having PGPR & biocontrol potential. Certain wheat plants were healthy in disease infected field because of the presence of these endophytic actinomycetes. Isolate NBAIM-WR-20 can be used as biocontrol agent against wheat Phytopathogens and for

Poster Abstracts

ASM Conferences88

enhancing crop productivity. Plant Micro-biomics is going to be a very important area for the future research. References: [1]Franco, C.M.M., Coombs, J.T. 2003.Visualization of an Endophytic Streptomyces species in wheat seed. Applied and Environmental Microbiol-ogy 69:4260-4262.

n 96BdOES THE PRESENCE Of NORMAL bENEfICIAL MICRObES INTERfERE IN GuT COLONIzATION ANd IMMuNO-MOduLATION Of PRObIOTICS!

R. Chaudhry, N. Sharma, A. Gahlowt, P. Sharma, V. Bamola, M. Chaudhary; AIIMS, Delhi, INDIA.

Background: Probiotic have shown to have significant clinical beneficial effects in the prevention and management of gastrointestinal complication and other conditions. A double blind randomized control study was designed, to study the in-vivo colonization and immuno-modulation by intervention of selective pro-biotics (P1 and P2) in pediatric population. Methods: Fecal samples of enrolled cases from pediatric wards (5mth to 5yrs age) were collected before and after probiotic interven-tion (5days- twice a day). All fecal samples were processed for anaerobic flora. The isola-tion of flora were carried out by culture on BHIA, MRS Agar and identified by API 50 CH system. For immuno-modulation viable colonocytes were isolated from sample (~1g) by somatic cell sampling and recovery (SCSR) process and immunoglobulin receptors (IgA and IgG) were studied by Flowcytometry. Re-sults: A total of seventy five cases comprising 20 breast fed and 55 Non-breast fed babies were enrolled. Twenty-five cases received pro-biotic P1(group A), 24 cases received probiotic P2( group B) and 26 cases received placebo (Group C). In 42 cases beneficial microbes (56%) were grown before intervention includ-ing breast and non-breastfed babies. Before in-tervention 55% (11/20) of breast fed and 53% (29/55) of non-breast fed babies grew Lac-

tobacillus spp. We observed presence of 134 Lacbacillus sp. in 75 cases enrolled. Predomi-nant Lactobacillus strains were L.plantarum, L.fermentum, L.salivarius, L.acidophilus, and L.brevis. Probiotic colonization occurred with P1 in 27% and none with P2. The concentra-tion of IgA receptor were higher than IgG in all the samples. Immunoglobulin receptors did not show any statistically significant difference between probiotic group and control group. Conclusion: We found that the Indian Pediat-ric gut flora is rich in indigenous Lactobacillus spp. which may interfere with colonization of administered probiotics. Further studies based on clinical application are required to know the therapeutic potential and immunomodulatory effects of probiotics in Indian population.

n 97AbENEfICIAL HINdGuT MICRObIOTA IN TERMITES: ROLES bEYONd NuTRITION?

R. B. Rosengaus1, K. Schultheis1, M. Bul-mer2; 1Northeastern University, Boston, MA, 2Towson University, Towson, MD.

Termites have had a long co-evolutionary his-tory with their hindgut microbes. Historically, the role of these anaerobic obligate symbionts has been attributed to the nutritional welfare of the host. We hypothesized that the nature of this mutualistic interaction may extend beyond the nutritional benefits and that gut symbionts may enhance the host’s defenses against patho-gens at both the individual and colony levels. Through a series of in vitro and ex vivo experi-ments combined with the use of chromogenic gels, we report that the primitive dampwood termite Zootermopsis angusticollis harbors hindgut microorganisms that synthesize mul-tiple active β-1,3-glucanases, enzymes known for their fungistatic activity. The elimination of the microbial consortia via termite oxygenation was accompanied by the loss of all but two endogenous β-1,3-glucanases in the hindgut. Fractionation of hindgut microbiota indicated that the β-1,3-glucanases are synthesized by the protozoa and their associated bacteria and

Poster Abstracts


not by the free living bacteria colonizing the gut walls or gut lumen. Furthermore, 12 day ex vivo anaerobic cultures of the protozoa and associated bacteria exhibited β-1,3-glucanase activity. The multiple β-1,3-glucanases are also excreted along with the termite’s fecal material, which is then incorporated into the termites’ nesting structure. The deposition of feces on the nest galleries likely diminishes the risk of fungal overgrowth within the decayed wood as termite feces are known to possess antifungal properties. Currently we are test-ing if β-1,3-glucanase are responsible for the fungistatic effect of feces. The externalization of microbial-derived antifungal enzymes via termite defecation represents an extension from individual-level immunocompetence to colony-wide protection (i.e. social immunity). This research establishes a novel role for the mutualistic association between termites and their hindgut microbial consortia and provides new insights into factors, other than lingo-cellulolytic activity, that could have influenced the origin and/or maintenance of this complex mutualism, the evolution of termite sociality and the host’s individual and social immune responses.

n 98BbIOLOGICAL CONTROL Of POSTHARVEST CROWN ROT ON bANANA VAR. LATuNdAN(MuSA x PARAdISIACA L.)

R. S. Flamiano, M. B. Solidarios, H. T. Arab; Mindanao State University, General Santos City, PHILIPPINES.

Biological control of postharvest banana crown rot by antagonistic microorganisms is a potential alternative to the use of chemical treatments to improve the shelf life of commer-cial bananas. Biological control experiments were conducted to test the effects of the dif-ferent populations of the two bacterial isolates Bacillus subtilis and Psedomonas sp against Colletotrichum musae infecting on Musa x paradisiaca L. The antagonistic activity was assessed by recording the occurrence of necro-

sis in terms of internal necrotic surface area, INS (mm) 14 days after inoculation of the pathogen. Results of this study revealed that Bacillus subtilis and Pseudomonas sp at 108

CFU/ml applied singly and the combination of these bacteria at 106 CFU/ ml had signifi-cantly higher antagonistic activity with INS of 84.45, 117.78 and 115.55 respectively than the chemical treatment (0.025% Potassium alum and 0.025% chlorine solution with higher INS value of 308.89. The highest percentage of protection at 91.84 was observed at 108 CFU/ml Bacillus subtilis followed by the 106 CFU/ml Bacillus subtilis and Pseudomonas sp. com-bination(83.67%) and Pseudomonas sp at 108

CFU/ml (79.59%). This study further revealed that Bacillus subtilis and Pseudomonas sp. had further increased the shelf life of bananas by 1-2 days more than chemically-treated bananas.

n 99AbACILLuS bACTERIA AS PROSPECTIVE PRObIOTICS

I. Sorokulova; Auburn University, Auburn, AL.

Probiotic prophylaxes and therapies are gain-ing wider acceptance as more scientific data emerge regarding the interaction between pathogen and beneficial microbes in the hu-man intestinal tract and molecular mechanisms of probiotics’ action. Bacteria of the Bacillus genus possess an enormous potential as probi-otic cultures. We isolated Bacillus strain and showed its safety in vitro and in animal mod-els. No evidence of pathogenicity was found in vitro testing. The median lethal dose for this strain was >1010 CFU/mouse, which is equal to >10 therapeutic human doses. Bacillus strain was resistant to bile and acid. High specific activity of tested Bacillus strain against a wide spectrum of pathogens (including multiresis-tant cultures) in vitro studies was confirmed in animal models of infections. Thus, protective effect against Campylobacter pathogens was 80-100% after oral pretreatment of mice with

Poster Abstracts

ASM Conferences90

probiotic culture. We isolated and character-ized new antibiotic, produced by Bacillus strain, with the original spectrum of specific activity. We demonstrated high efficacy of this probiotic in treatment of acute diarrhea in randomized double-blind controlled clinical trials. In clinical trials, 66 adults with acute diarrhea were treated with Bacillus probiotic strain. Patients received bacterial suspension two times a day during a 4 to 10 day period, and patients in the control group were treated with commercial prescribed Lactobacillus probiotic. A pronounced effect was shown by using Bacillus probiotic in comparison to Lactobacillus probiotic in the reduction of the diarrheal phase (3.0 ± 0.22 days vs. 5.2 ± 0.16 days, P< 0.05), length of abdominal pain (2.6 ± 0.12 days vs. 4.1 ± 0.27 days, P<0.05) and pathogen elimination time from the intestine (3.1 ± 0.31 days vs. 5.5 ± 0.24 days, P<0.05). Following treatment with Bacillus probiotic, there were no pathogens cultured from patients (100% elimination). In the control group, pathogens could still be cultured from 20% of those treated with a commercial Lactobacillus probiotic. This was the first evidence of the clinical efficacy of probiotics in treatment of acute diarrhea and demonstrated that Bacillus probiotics were more effective for treatment of these infections than prescribed therapy with Lactobacillus, especially in the elimination of pathogens from intestine. We conclude that Bacillus cultures have high efficacy as probi-otic bacteria.

n 100Buraria picta (jACq.) dC.: NOVEL STRATEGY fOR dRuG dISCOVERY fROM THE ROOTS ExTRACTS fOR ANTIMICRObIAL CHARACTERISTICS IN CLINICALLY IMPORTANT PATHOGENIC MICRObIAL STRAINS

M. Mollik; Prescience Trust Funds, Phoenix-ville, PA.

Uraria picta (Jacq.) DC. belongs to the plant family Fabaceae Lindl.. There is no report on

antibacterial characteristics of roots on this plant, the attempt to studies. The studies were undertaken to evaluate in vitro antimicrobial characteristics of methanol, acetone, and N, N-dimethylformamide extracts from roots of Uraria picta (Jacq.) DC.. In vitro antimicrobial characteristics of all the extracts were done by agar disc diffusion assay. 100 clinically important strains were used for the studies, which were both clinical isolates as well as identified strains. Piperacillin and gentamicin were used as standards for antibacterial assay, while nystatin and flucanazole were used as standards for antifungal assay. Antimicrobial characteristics were determined by measure-ment of inhibition zone around each paper disc. For each extract four replicate trials were conducted against each organism. The antibac-terial characteristics were more pronounced against bacteria than fungal strains. The gram positive bacteria were more susceptible than gram negative bacteria. The methanol extract showed best antibacterial characteristics. Ura-ria picta (Jacq.) DC. roots extracts showed better antibacterial characteristics than com-mercially used antibiotics. Demonstration of antimicrobial characteristics of Uraria picta (Jacq.) DC. provides the scientific basis for the use of this plant in the traditional treat-ment of diseases and may help to discover new chemical classes of antibiotic substances that could serve as selective agents for infectious disease chemotherapy and control. The studies have opened up the possibility of the use of this plant in drug development for human con-sumption possibly for the treatment of various infections caused by microbes.

Poster Abstracts


n 101AuSING quANTITATIVE PROTEOMICS TO IdENTIfY CHANGES IN THE AduLT HEMOCYTE PROTEOME duE TO COLONIzATION STATE IN THE ASSOCIATION bETWEEN THE SquId, euprymna scoLopes, ANd THE bACTERIuM, vibrio fiscHeri

T. R. Schleicher, S. V. Nyholm; University of Connecticut, Storrs, CT.

The Hawaiian bobtail squid, Euprymna scol-opes, and the luminescent bacterium, Vibrio fischeri, form a highly specific beneficial light organ symbiosis. Not only does the host have to select V. fischeri from the environment, but it must also prevent subsequent colonization by non-symbiotic microorganisms. Macrophage-like hemocytes are the main component of the host’s cellular innate immune system and are believed to play a role in mediating the sym-biosis with V. fischeri. Previous data suggest that the host hemocyte response changes spe-cifically towards V. fischeri depending on the colonization state of the light organ. To further understand the molecular mechanisms behind this process, we used isobaric tag for relative and absolute quantification (iTRAQ) label-ing and liquid chromatography tandem mass spectrometry (LC MS/MS) to compare and quantify the adult hemocyte proteomes from colonized and uncolonized (antibiotic-treated/cured) hosts. Overall, iTRAQ analyses allowed for the quantification of 1,534 hemocyte pro-teins. Nine hundred and thirty of these proteins had 2 or more peptides and a ProteinPilot score of greater than or equal to 2.0 (>99% confi-dence). Proteins significantly less abundant in hemocytes from cured hosts included two ca-thepsins, a cell adhesion molecule, histone H4, a ganglioside GM2 activator precursor, and a sodium/calcium exchanger (p < 0.05). Proteins significantly more abundant in hemocytes from cured hosts included two putative calcium-binding proteins, an annexin A7-like protein and a calumenin-like protein (p < 0.05). These results suggest that colonization state may

influence hemocyte calcium signaling and cell adhesion. Current work is focused on deter-mining whether these and other proteins are involved in mediating hemocyte binding and recognition of V. fischeri. LC MS/MS analyses of circulating hemocytes also identified a puta-tive carbohydrate binding protein similar to molluscan galectins, a protein group that can act as host pattern recognition receptors of mi-croorganisms. The full open reading frame of the E. scolopes galectin (EsGalectin) transcript predicted four carbohydrate recognition do-mains. Western blot analysis using a polyclonal antibody generated to EsGalectin revealed a 64.9 kD protein and immunocytochemistry of circulating hemocytes localized galectin to the pseudopodia of attached cells. RT-PCR analyses showed that the EsGalectin transcript is expressed in various tissues, including adult hemocytes, the symbiont-containing light or-gan central core, gills, mantle tissue and diges-tive tract. Future work will focus on determin-ing whether EsGalectin can bind V. fischeri and whether expression/production of this and other hemocyte proteins is altered by challenge with symbiotic and non-symbiotic bacteria.

n 102BROLE Of TROPOdITHIETIC ACId PROduCTION ANd bIOfILM fORMATION IN THE PRObIOTIC ACTIVITY Of PHAEObACTER SPECIES S4

W. Zhao, M. Karim, C. Dao, M. Gomez-Chiar-ri, D. C. Rowley, D. R. Nelson; University of Rhode Island, Kingston, RI.

Recent reports demonstrate that some Pha-eobacter species exhibit probiotic activity, protecting fish or shellfish from infection by various pathogenic Vibrio species. We isolated a putative probiotic bacterium, Phaeobacter species S4, from the inner surface of an oyster shell. A growth inhibition assay and HPLC were used to demonstrate that S4 produces the antibiotic tropodithietic acid (TDA), a small sulfur-containing compound active against many marine and human pathogens, includ-

Poster Abstracts

ASM Conferences92

ing Vibrio species. Additionally, Phaeobacter species are typically excellent biofilm formers and dominant colonizers of surfaces in marine environments. Biofilm formation is thought to be essential for probiotic activity by a variety of mechanisms including competition for adhesion sites, oxygen, nutrients, and by pre-venting contact between pathogens and hosts. Initial studies showed that when present prior to or concurrently with the oyster pathogen Vibrio tubiashii, S4 protected oyster larvae from infection and death. We hypothesized that the probiotic activity of S4 may be the result of one or more factors, including TDA production and biofilm formation. In order to examine the specific roles of TDA produc-tion and biofilm formation in the probiotic activity of Phaeobacter S4, mutations in TDA biosynthesis genes, clpX, and an exopolysac-charide biosynthesis gene (exoP) were created by insertional mutagenesis using homologous recombination. Mutations in TDA biosynthesis genes, constructed by single crossover ho-mologous recombination, resulted in the loss of TDA production, decreased biofilm forma-tion, and decreased larval protection. Mutation of clpX resulted in the loss of TDA production, no decline in biofilm formation, and decreased larval protection. Mutation of exoP resulted in a ~50% decline in biofilm formation, no decline in TDA production, and decreased larval protection. Complementation of the clpX mutation partially restored the wild type phenotype. Examination of biofilm formation by GFP-tagged V. anguillarum in the presence of orange fluorescent protein (OFP)-tagged S4 wild type or S4 mutant strains revealed that wild type S4 suppressed V. anguillarum biofilm formation. In contrast, both the clpX mutant and the exoP mutant did not block V. anguil-larum biofilm formation. Our observations suggest that probiotic activity by Phaeobacter species S4 involves both biofilm formation and the production of the antibiotic TDA. It is interesting to note that these two activities appear to be linked, as mutations to several TDA biosynthesis genes result in both the loss

of TDA production and a decline in biofilm formation.

n 103AA MIxEd CuLTuRE Of SELECTEd OSMOTOLERANT YEAST STRAINS SHOW STRONG ANTAGONISM Of bACTERIAL PATHOGENS IN VITRO, ANd IMPROVEMENT IN GASTROINTESTINAL fuNCTION IN PRE-CLINICAL TESTS

M. Islas-Romero1, E. M. Loza-Valerdi2, S. R. Trejo-Estrada1; 1IPN, Puebla, MEXICO, 2METCO, DF, MEXICO.

Among the probiotic microorganisms, both bacterial (mostly lactic acid bacteria and bifi-dobacteria), and less frequently yeast cultures (namely Saccharomyces boulardii, a well established commercial probiotic), have been developed for their application in food or nutraceutic products. The in vitro antibacte-rial activity is one of the main characteristics of potential probiotics. In a recent study, both LAB and osmotolerant yeast (OY) cultures, were screened for their ability to antagonize bacterial pathogens. Osmotolerant yeast strains showed a consistent wide spectrum antibacte-rial activity both in agar media and in liquid cultures. In the present study, we report the use of previously selected three strains of LAB and two osmotolerant yeast isolates, for their abil-ity to grow in natural agave juices, their surviv-al as viable cultures after different stabilizing treatments, and their effect in pre-clinical tests, as oriented towards gastrointestinal health and function. Static fermentation methods were used for the production of LAB and OY strains, and several culture media formulations, based on agave sap and yeast extract, were evaluated. Preservation methods in high sugar substrates at low temperature, integration with carrier agents, as well as lyophilization, were evaluated. A higher survival rate was obtained for OY cultures, than for LAB strains. A group of 90 patients were daily treated with different mixtures of microbial LAB and OY selected strains, for a 4-week period. A very evident

Poster Abstracts


increase in gastrointestinal health and regular-ity was registered for at least two formulations. Evidence of a positive effect in the control of irritable bowel syndrome (IBS) was also de-tected. The osmotolerant yeast mixed culture provided a more active, clinically significant culture, than the LAB mixed culture. Extensive work is under way, to determine the biochemi-cal determinants of this activity.

n 104BMEdICINAL PROPERTIES Of OCIMuM SANCTuM: ROLE Of ENdOPHYTIC ACTINOMYCETES.

A. chaurasia; NBAIM,ICAR, Mau Nath Bhan-jan, INDIA.

Hypothesis: Endophytic microorganism’s spe-cially endophytic actinomycetes present in the medicinal plant produces bio-active molecules effective against various pathogens and are ac-tually responsible for the medicinal properties of the medicinal plants. Objectives: 1. Isola-tion of culturable endophytic actinomycetes from the leaf, stem & leaf of the medicinal plant Ocimum sanctum. 2. Polyphasic identi-fication of the potent endophytes 3. Biocontrol potential evaluation of the endophytes. 3. Screening for bioactive metabolites responsible for biocontrol potential & its identification using various spectroscopic means. Materials and methods: Endophytic actinomycetes were isolated from the leaf/stem/root of the Ocimum sanctum plant using sterility check. Rare endo-phytic actinomycetes were isolated using vari-ous specific media. Isolates were evaluated for biocontrol potential evaluation using dual plate technique. Potent isolates were identified using polyphasic approaches. Bioactive metabolites were isolated using solvent extraction & were fractionated using HPLC & being identified using various spectroscopic means. Results: Rare endophytic actinomycetes were found to have excellent biocontrol potential against various pathogens. Bioactive metabolites were soluble in organic solvent & were of aromatic in nature. Conclusion: Rare endophytic ac-

tinomycetes present in Ocimum sanctum root have biocontrol potential against most of the pathogens tested & produced bioactive aromatic molecules soluble in the organic sol-vent. We conclude that these rare endophytic actinomycetes contribute substantially toward medicinal properties of the medicinal plant like Ocimum sanctum.

n 105ACOMPARATIVE SYSTEMS bIOLOGY Of LACTIC ACId bACTERIA; LactobaciLLus pLantarum METAbOLISM ANd dIVERSITY

A. McLeod1, F. Santos2, D. Bellomo2, B. Teusink2, I. Rud1, L. Axelsson1; 1Nofima, Nor-wegian Institute of Food, Fisheries and Aqua-culture Research, Ås, NORWAY, 2Center for Integrative Bioinformatics, Vrije Universiteit Amsterdam, Amsterdam, NETHERLANDS.

By using a comparative systems biology ap-proach, our aim is to understand similarities and differences in fermentation behavior of four Lactic Acid Bacteria (LAB): Lactococcus lactis, used in dairy food applications; Entero-coccus faecalis, a major (fecal) contaminant of food and water as well as a contributor to food fermentation, and Streptococcus pyogenes, an important human pathogen. The fourth organism is the highly versatile and industri-ally important species Lactobacillus (Lb.) plantarum, found in many different niches, and associated with human health (probiotic). The major regulatory events at the genetic level (adaptive mutations), the transcription, translation, enzymatic and metabolic level up to the final output (functional) level, is quanti-fied and then integrated in iterations between experimentation (all~omics) and modeling (network structure, flux balances, dynamics, control, regulation). Mathematical, compre-hensive, models are developed that quantita-tively describe the catabolic (product forma-tion) response of the organisms under different conditions. The effect of growth rate in four genome sequenced strains of Lb. plantarum is

Poster Abstracts

ASM Conferences94

currently being investigated. The strains origi-nate from various environments (human saliva, grass silage, pickled cabbage) and one is mar-keted as a probiotic. Samples at steady-state from the strains grown in anaerobic glucose limited chemostats at high compared with low growth rates are analyzed by transcriptomic, proteomic, metabolic and enzymatic analyses. Preliminary results show only small differenc-es in end-product formation between the two growth rates and between the different strains, reflecting the robust metabolism of the bacte-rium. However, there is some strain variation in the production rates of D- and L-lactate, and in the metabolism of some amino acids. Still, the similarities are striking given the different ecological niches. Proteins involved in protein synthesis, amino acid biosynthesis, fatty acid and phospholipid metabolism, as well as en-ergy metabolism, are differentially expressed. Scanning electron microscopy images show morphological differences between the fast and slow growing cells. The obtained basic knowl-edge on the metabolism and strain diversity of Lb. plantarum will add to the understanding of this beneficial microbe and its behavior in dif-ferent habitats.

n 106BRHIzObACTERIA fROM NATuRAL PLANT SETTINGS: in vitro AuxIN PROduCTION ANd GROWTH STIMuLATION Of triticum aestivum.

B. Ali; University of the Punjab, Lahore, Paki-stan, Lahore, PAKISTAN.

Phytohormones especially indole-3-acetic acid (IAA) is considered very critical in plant growth and development. IAA production potential of plant associated bacteria offer good opportunity to use these microbes as biofertilizers to enhance the growth and yield and agronomically important crops. The main objective of this study was to investigate the potential of bacterial strains associated with natural plant settings to enhance endogenous IAA content and growth of Triticum aestivum

var. Inqalab-91 under axenic and natural envi-ronmental conditions. Fifteen bacterial strains belonging to genera Bacillus, Pseudomonas, Escherichia, Micrococcus and Staphylococcus from wild herbaceous plants were identified by 16S rDNA sequencing. Strains were evaluated for in vitro auxin production in the presence of 1mg ml-I L-tryptophan. GC-MS analysis revealed that bacterial strains produce 0.6-8.22 µg ml-I IAA in liquid culture medium. In vitro experiments were conducted in the presence of 500 mg L-tryptophan to evaluate the potential of bacterial strains to enhance endogenous IAA content of wheat and this strategy used as a basic criterion for the selection of effective plant growth promoting rhizobacteria (PGPR). Strains of Bacillus were more effective to increase IAA content as compared to other inoculations. For instance, Bacillus sp. EhH-5, Bacillus sp. TpP1, Bacillus sp. NpR-1, Bacil-lus sp. AaH-1 and Bacillus sp. BP-1 increased 208%, 139.70%, 108%, 104.60% and 104% endogenous IAA, respectively, over control. Plant growth under axenic conditions recorded 11.30% and 40% increase in shoot length and shoots fresh weight with Bacillus sp. NpR-1 and Pseudomonas sp. AvH-4, respectively, over control. At final harvest the highest in-crease in shoot length and number of tillers was 29.16% and 97.35% with Pseudomonas sp. AvH-4 and Bacillus sp. NpR-1, respec-tively, whereas maximum increase in grain yield was 13.70% with Bacillus sp. MiR-4. In the end it was concluded that bacterial strains especially of genus Bacillus and Pseudomonas have the ability to increase the endogenous IAA content and growth of T. aestivum var. Inqalab-91. Bacterial strains used in this study mostly belong to genus Bacillus that may have agricultural importance because they can be applied as spores on seeds and show signifi-cant protective effects against phytopathogens as well as growth promoting activities. Thus, microbial strains isolated from natural plant settings can be effectively used on the basis of their auxin production potential to enhance the growth and yield of agronomically important crops.

Poster Abstracts


n 107AIMMuNOSTIMuLATORY EffECTS Of SELECTEd PRObIOTIC ANd POTENTIAL PRObIOTIC LACTIC ACId bACTERIA

H. Jensen1, M. Taraldrud2, L. Axelsson1, S. Grimmer1; 1Nofima, Norwegian Institute of Food, Fisheries and Aquaculture Research, Ås, NORWAY, 2Norwegian University of Life Sci-ences, Ås, NORWAY.

Probiotics are defined as live microorgan-isms which when administered in adequate amounts confer a health benefit on the host. Stimulation of immunomodulatory cells and secretion of cytokines are among the suggested mechanisms by which probiotic bacteria can interact with the human host and mediate their health benefits. In this study seven Lactobacil-lus strains (L. reuteri DSM 20016, L. reuteri DSM 17938, L. reuteri ATCC PTA 6475, L. plantarum MF1298, L. plantarum NC8, L. plantarum 299v and L. rhamnosus GG), of which three are commercially marketed as probiotcis (DSM 17938, 299v and GG), were tested for their ability to induce secretion of cytokines from the monocytic THP-1 cell line and THP-1 derived macrophages. The secretion of IL-6, IL-8, IL-10 and TNF-α was investigated following 6 h co-culture with cell lines and Lactobacillus strains. Both live and UV-inactivated bacteria were investigated. All three L. reuteri strains stimulated to an overall high secretion of cytokines, whereas L. plan-tarum NC8 and L. rhamnosus GG induced low secretion of cytokines compared to the other strains. The ability to induce cytokine secretion was similar for live bacteria as for the UV-inactivated bacteria, except for L. rhamnosus GG where UV-inactivated cells induced higher levels of cytokines than the corresponding liv-ing cells. In conclusion, all seven Lactobacillus strains tested had a stimulatory effect on the monocytic cell line THP-1 and THP-1 derived macrophages. Interestingly, the three L. reuteri strains induced higher levels of cytokine secre-tion compared to the other strains investigated. As there were no major differences in the

cytokine secretion induced by live and UV-inactivated bacteria, the bacteria itself, and not its metabolites, is responsible for the observed cytokine secretion.

n 108BMONITORING TEMPORAL dYNAMICS Of THE GuT MICRObIOTA ASSOCIATEd WITH INfLAMMATORY bOWEL dISEASE

K. Li1, R. Lamendella2, M. Dunklebarger2, J. Halfvarson3, C. Tysk3, J. Jansson1; 1Lawrence Berkeley National Lab, Berkeley, CA, 2Juniata College, Huntingdon, PA, 3Örebro University Hospital, Örebro, SWEDEN.

Inflammatory bowel disease (IBD) consists of the subtypes Crohn’s disease and ulcerative colitis, both of which are characterized by gas-trointestinal bleeding, diarrhea, and abdominal discomfort. As a chronic and increasingly prevalent condition, it is imperative that we introduce new methods for streamlined diag-nosis and personalized treatment plans. There is strong evidence that the etiology of IBD involves an intricate relationship between host immune response and the heterogeneity of the gut microbiota. In this regard, the correlations between disease status and microbial com-munities of the gut represent a critical aspect of disease management. This study aims not only to establish specific microbial signatures associated with each IBD subtype, but also to chronicle the evolution of gut consortia over a given time course. Additionally, patient meta-data will highlight any correlations between the changes of the gut microbiota and lifestyle factors. We hypothesize that there will be a trend of persistent dysbiosis that correlates with disease presentation, such as inflam-mation . Fecal samples were collected from 119 IBD patients every three months over a two year time. In a high throughput format, genomic DNA was extracted and sequenced using the Illumina-tag (itag)16S rRNA gene targeting strategy. We have retrieved more than 250 million rRNA gene sequences from nearly 800 samples. These sequence data were

Poster Abstracts

ASM Conferences96

analyzed using Qiime pipeline on the Amazon Web Services Elastic Compute Cloud. Subse-quent analysis of 16S rRNA gene profiles re-vealed a wide distribution of species diversity along with relative bacterial abundance. The results show that there were significant shifts in gut microbial composition over the various patient time points with high intra-patient dif-ferences. This suggests a relationship among the distinct bacterial signatures within a patient and IBD progression. In due course, a more holistic characterization of the gastrointestinal microflora will allow us to discern the possible constituents of its pathology and could be the source of future potential biomarkers for the disease.

n 109AdIffERENTIAL CELLuLAR RESPONSE Of juVENILE euprymna scoLopes TO SYMbIOTIC ANd NON-SYMbIOTIC bACTERIA

B. A. Rader, K. Putman, H. Olmstead, S. V. Nyholm; University of Connecticut, Storrs, CT.

The binary association between the squid Eu-prymna scolopes and its symbiont Vibrio fisch-eri is an experimentally tractable system that offers a powerful tool for the investigation of symbiont-dependent activation of the immune system through manipulation of colonization state in juvenile and adult hosts. Previous evi-dence of symbiotic modulation of host immune activation comes from an in vitro binding assay in which adult hemocytes, the macrophage-like blood cell of the squid, differentially bound closely related species within the Vibrio group. V. fischeri bound poorly to hemocytes isolated from colonized host, but significantly increased in binding to hemocytes from hosts where the symbiont had been removed, while binding of non-symbiotic bacteria remained unchanged. These data suggest that adult he-mocytes may become conditioned to the pres-ence of the symbiont. To better characterize this hemocyte response in juvenile squid dur-

ing the first 96 hours of the symbiosis, we used end-point PCR to amplify immune transcripts such as members of the NF-κB pathway, from hemocytes isolated from colonized and uncolonized animals. To characterize juvenile hemocyte behavior we assayed bacterial bind-ing and phagocytosis using either V. fischeri or non-symbiotic Vibrio harveyi. Colonization state had no significant affect on the number of V. fischeri or V harveyi bound, although the number of either bacteria bound per hemocyte increased over time from roughly 1 to 4. Time-lapse photography indicated that while isolated hemocytes bound to either bacterium, V. har-veyi was preferentially internalized. V. fischeri was never observed inside juvenile hemocytes. Colonization state had no significant affect on the number of hemocytes containing internal-ized V. harveyi (26 – 43%), or the number of V. harveyi phagocytosed (1.25 to 1.80 bacte-rial cells/hemocyte). Finally, internalized V. harveyi co-localized with acidic compartments stained with Lysotracker Red (Invitrogen). These results show that during initiation, ju-venile hemocytes express canonical immune genes and that even at an early stage of the symbiosis, squid hemocytes can distinguish between V. fischeri and other closely related bacteria. These data, along with the previ-ous adult hemocyte study, suggest hemocytes undergo a maturation process, perhaps first safeguarding the initiation of the symbiosis, and later allowing for immune “tolerance” of V. fischeri.

n 110BINSIGHTS INTO HOST RANGE AdAPTAbILITY THROuGH COMPARATIVE GENOMIC ANALYSIS Of AEROMONAS VERONII

S. M. Colston, J. Graf; University of Con-necticut, Storrs, CT.

Aeromonas veronii is capable of associating with different hosts with outcomes ranging from beneficial to pathogenic symbioses. While also abundant in the aquatic environ-

Poster Abstracts


ment, A. veronii forms a beneficial digestive-tract symbiosis with the medicinal leech, Hirudo verbana, the zebrafish, Danio rerio, and is found to reside in the digestive tract of the Dengue fever mosquito, Aedes aegypti. A. veronii strains exhibit variable degrees of virulence in the animal models, Galleria mel-lonella and Mus musculus, and is implicated in diseases such as gastroenteritis in humans and hemorrhagic septicemia in fish. The spectrum of animal hosts in which A. veronii can propa-gate in nature is a manifestation of its genetic breadth and capability. The study of A. veronii provides a unique opportunity to investigate a group of bacteria that plays dichotomous roles depending on the host or location within the host. One complete and 17 A. veronii draft genomes of strains obtained from leeches (7), clinical (9) or environmental sources (1) were analyzed for loci that correlate with source. One conserved locus that was present in all leech and clinical isolates but absent in en-vironmental isolates was a type III secretion system (T3SS) and its cognate effectors. This T3SS has been previously shown to be impor-tant in both the beneficial association with the medicinal leech as well as virulence in mice. Further investigation of the genomes revealed the presence of a second T3SS (T3SS-2) in at least four out of seven leech isolates that was absent in the clinical isolates and environmen-tal isolate. Sequence comparison of the T3SS-2 shows the highest degree of similarity to that of Edwardsiella species, which are known fish pathogens, and to an insecticidal toxin, tccC, from Photorhabdus nematophila. Other genes of interest encode toxins, antibiotic re-sistance, outer membrane proteins, and sialic acid utilization proteins. Single and double mutants for T3SS-1 and T3SS-2 were gener-ated using a gene deletion approach. Currently, new virulence models using Drosophila me-lanogaster and D. rerio are being established to examine the role of these loci in beneficial and pathogenic associations. By combining genome comparisons, mutational analyses, and multiple animal models, we may uncover the genetic requirements for the host range and

elucidate the molecular mechanisms underly-ing multi-host colonization and gain insight into the evolution of pathogenic and beneficial associations.

n 111ALONG LASTING REMISSION INduCTION Of dIAbETES bY ORAL L.Lactis SECRETING HuMAN PRO-INSuLIN PLuS IL-10 COMbINEd WITH SHORT-TERM LOW-dOSE ANTI-Cd3

L. Steidler; ActoGeniX, Zwijnaarde, BEL-GIUM.

Type 1 Diabetes (T1D) and other autoim-mune diseases arise from a breakdown of self-tolerance. Re-educating the immune system to establish antigen-specific-tolerance is an important treatment goal. According the concept of oral tolerance induction, adminis-tration of autoantigenic peptides via the gut provides an interesting strategy to selectively target autoreactive T-cells. The use of geneti-cally modified Lactococcus lactis (L.lactis) is an innovative method of protein delivery to the gut. The goal of the present study was to investigate combination therapy (CT) of daily oral administered L.lactis secreting human pro-insulin and interleukin 10 (IL-10) and short term daily subtherapeutic doses of anti-CD3 for diabetes remission in new-onset diabetic (NOD) mice. Several combinations of L. lactis +/- IL-10 +/- proinsulin +/- anti-CD3 were tested. Interestingly, our most effective treat-ment was the combination of anti-CD3 and L.lactis secreting pro-insulin plus IL-10 (CT) which induced long-term diabetes remission (>100 days) in 59% of the new-onset diabetic mice (n=61). Untreated mice did not spontane-ously revert and had to be sacrificed due to disease progression, monotherapy of anti-CD3 alone (n=19) or L.lactis secreting pro-insulin plus IL-10 alone (n=13) yielded 27% and 15% of diabetes reversal, respectively. This yield was increased by combining anti-CD3 together with L.lactis secreting IL-10 or L.lactis secret-ing pro-insulin, which induced 40% and 48%

Poster Abstracts

ASM Conferences98

of diabetes remission, respectively, while mice which received anti-CD3 together with con-trol L.lactis with an empty vector had similar results to the anti-CD3 alone with 23% of dia-betes reversal (n=22). CT cured mice presented a significant increase of CD4+CD25+FOXP3+ T-cells (regulatory/tolerogenic phenotype) in pancreatic draining lymph nodes at stop of L.lactis treatment (42d), lasting up to 100 days post-start of treatment. In addition to that, isolated CD4+CD25+ cells from cured CT-treated mice demonstrated suppressive func-tion in vitro and in vivo as well as higher IL-10 production when relevant antigen is present in culture, showing the long-term installation of a highly functional, antigen specific regulatory compartment. The L.lactis delivery platform has been successfully used in human clinical experimentation before, so we conclude that CT of low-dose anti-CD3 and L.lactis secret-ing pro-insulin plus IL-10 is a promising tool for the treatment of T1D. Takiisih et al. J Clin Invest. 2012;122(5):1717-1725

n 112BTHE bIOCHEMICAL fuNCTION Of WOLbACHIA PIPIENTIS TYPE IV EffECTORS ExPLOREd THROuGH A GENOMIC SCREEN

I. L. Newton1, C. F. Lesser2, R. R. Isberg3; 1In-diana University, Bloomington, IN, 2Harvard University, Cambridge, MA, 3Tufts University, Boston, MA.

Regardless of the effect on the host, all invad-ing intracellular microbes face similar chal-lenges: adherence to host, induction of phago-cytosis, escaping lysosome fusion, and sub-sequent proliferation. We know from genetic and genomic studies that both mutualists and pathogens use similar strategies to overcome these challenges, including the use of secretion systems. Wolbachia pipientis wMel encodes a type IV secretion system that directly injects into host cells proteins that manipulate eukary-otic cellular processes. Identification of these translocated proteins would allow us to better understand how Wolbachia promotes infection

and causes reproductive effects. Here we pres-ent a large-scale functional genomic screen of putative Wolbachia translocated substrates us-ing yeast as a model system. Substrates of bac-terial protein translocation systems can often confer toxic phenotypes when expressed in the yeast Sacchromyces cerevisiae. Identification of this subset of effectors only requires that the bacterial orfs be expressed in yeast, making this approach perfect for the study of Wolba-chia, which cannot be genetically manipulated. Over 300 putative translocated substrates were identified in the wMel genome using bioinfor-matics methods and we present the results of phenotypic and biochemical characterization of a subset. The methods used have led to the first successful identification of Wolbachia factors interacting with core elements of eu-karyote cell biology, providing clues as to how Wolbachia may manipulate the host cell during infection. We focus on Wolbachia proteins that target the host cell cytoskeleton and what these results in yeast might mean for successful colonization of the insect host.

n 113APREdICTING HOST-MICRObE NuTRITIONAL INTERACTIONS fROM HOST GENOTYPE: A SYSTEMS-LEVEL ANALYSIS

J. M. Chaston, A. J. Dobson, P. D. Newell, C. N. Wong, D. R. Sannino, S. L. Hermann, A. E. Douglas; Cornell University, Ithaca, NY.

The impact of the resident microbiota on animal nutrition can be substantial. Gastroin-testinal microbes can modify ingested food, and exchange metabolites with their animal hosts. Differences in host nutritional status across individuals may result from differ-ences in host genotype, bacterial complement and function, or both. Our hypothesis is that host nutritional status varies predictably with both host genotype and the composition and activities of the gut microbiota. Our goal is to identify host genetic predictors of host- and microbiota-dependent host nutritional status. To address how genetically-distinct hosts

Poster Abstracts


respond to their microbiota, we studied Dro-sophila melanogaster and its gastrointestinal tract bacterial community, which is dominated by just five bacterial taxa (>99% numerical representation). We measured fly nutritional indices in multiple, genetically diverse D. melanogaster fly lines to assess the impact of host genotype on host nutritional indices. We assessed bacterial contributions to fly nutrition by quantifying the same nutritional indices in experimentally-generated germ-free flies. Our results demonstrate that host genotype and microbial colonization interact to shape host nutritional status. Specifically, host nutritional status varies with microbial treatment and the magnitude and direction of microbe-dependent differences in nutritional status vary across lines. Genome-wide association of fly line allelic variation with nutritional index and trait values is revealing individual-level microbiota-responsive gene networks that affect host nutritional status. The distribution of these putative host factors across different fly lines demonstrates how similar biological processes are mediated by different host-microbe interac-tions in genetically-distinct individuals. This analysis enables us to test the capacity of host genotype to predict microbial impacts on host nutritional health. It is a test-bed for assessing the feasibility of incorporating microbe-depen-dent effects on host health into genome-based personalized medicine.

n 114BMuCuS AS A POTENTIAL VEHICLE fOR THE HORIzONTAL TRANSMISSION Of A GuT SYMbIONT, aeromonas veronii bIOVAR SObRIA, TO THE EuROPEAN MEdICINAL LEECH, Hirudo verbana

B. M. Ott, G. Altimus, T. K. Maxwell, R. V. Rio; West Virginia University, Morgantown, WV.

Transmission mode can impact symbiont partner specificity, with strict vertical trans-mission leading to highly specialized associa-tions. However, knowledge of the evolution

of vertical transmission remains elusive. The European medicinal leech, Hirudo verbana, provides an ideal symbiosis model system, as the crop, the largest leech digestive tract organ, consistently houses only two partners: the Gammaproteobacterium Aeromonas veronii and a Bacteroidetes Rikenella-like bacterium. These symbionts are believed to provide nu-trients to the host, prevent putrefaction of the host blood meal diet, and hinder the establish-ment of unwanted microbes. Although these bacteria offer significant fitness benefits to the leech host, imperfect vertical transmission has been observed. Additionally, through phyloge-netic studies, a lack of co-evolution has been reported between A. veronii and its host, de-spite the species-specificity of this symbiosis. In this study, we aim to identify how host mu-cosal secretions, released from mucosal glands found within the leech epidermal layer, may complement imperfect vertical transmission. Interestingly, leeches appear to be attracted to mucus of conspecifics, thereby providing a potential mechanism for partner adherence of the symbiosis. We hypothesize that these secretions serve as potential vehicles for sym-biont horizontal transmission to aposymbiotic juveniles following cocoon emergence. To investigate this, mucus samples were obtained at known time points and examined for A. veronii presence using diagnostic PCR. Real-time quantitative PCR was used to measure the temporal proliferation of the symbiont in the mucus, while transmission was tested by both contact and no-contact dependent means. To gain additional insight on the microbial com-munity composition of mucus, a 16S rRNA clone library was created using isolated total mucus DNA, followed by RFLP typing. Our results indicate that mucus houses a wide range of bacterial species, many of which have been previously described within the leech. In addition, A. veronii proliferation within the mucus was observed to be synchronous to host shedding cycles. These results support a work-ing hypothesis where horizontal transmission of A. veronii via mucus may serve to comple-ment imperfect vertical transmission, thereby

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ASM Conferences100

ensuring the establishment and cohesiveness of critical relations.

n 115AALTEREd METAbOLIC RESPONSES IN bACTEROIdES THETAIOTAOMICRON CELLS ExPOSEd TO GLYCAN MIxTuRES

E. C. Martens, T. E. Rogers, N. A. Pudlo, K. Urs; University of Michigan Medical School, Ann Arbor, MI.

The Gram-negative Bacteroidetes are among the most numerous bacteria present in the hu-man distal gut. Many species in this phylum can degrade a broad range of different glycans that are found either in our diet or in protec-tive intestinal mucus. The metabolic breadth of these glycan generalists is exemplified by the prototypic symbiont Bacteroides thetaio-taomicron (Bt), which is capable of growing on at least a dozen different glycans. Since Bt is likely to routinely encounter multiple gly-cans at varying concentrations in the gut, we wanted to determine how exposure to various glycan mixtures influences expression of this species’ numerous polysaccharide utilization loci (PULs) that are involved in processing glycan substrates. Using antibodies that are specific for cell surface proteins involved in degrading different glycans, we show for the first time that single cells are capable of simul-taneously targeting multiple nutrients. At the transcriptional level, Bt populations that were pre-grown in medium containing only glucose responded extremely rapidly when exposed to a new glycan source, reaching maximum expression of glycan utilization genes within minutes after exposure. Pre-exposure to a mix-ture of other glycans did not alter response ki-netics, but substantially limited the maximum expression level of some PULs and reduced their sensitivity to low abundance glycans up to several orders of magnitude. Growth of Bt in a mixture containing all twelve glycans that it degrades revealed that it uses some glycans prior to others. This metabolic hierarchy is not influenced by previous glycan exposure, as

re-introduction of high priority substrates after they were depleted re-initiated repression of genes involved in degrading the lower prior-ity glycans. O-linked glycans derived from intestinal mucus are consistently given low-est priority, either when present in a complex mixture or in pairwise combinations with other glycans. This latter finding suggests that Bt is metabolically programmed to avoid foraging these protective host molecules when alterna-tive glycans are present in sufficient quantity. These results provide insight into how a broadly saccharolytic gut bacterium modifies its behavior in response to the dynamic glycan environment in the gut and provides a base on which to build experiments to examine com-plex metabolic behavior in vivo.

n 116BOPTIMIzATION Of TERPENOId PRECuRSORS IN THERMObIfIdA fuSCA uSING IN SILICO MOdELING

N. Vanee, S. S. Fong; Virginia Commonwealth University, Richmond, VA.

Natural products play an important role in manufacturing of several active pharmaceutical ingredients (APIs). APIs or precursors of APIs can be produced in living organisms with the major challenge of designing and optimizing metabolic pathways to obtain the compounds of interest. In this capacity, living organisms can act as renewable catalysts with high prod-uct specificity to produce APIs with potential cost savings over purely synthetic chemistry synthesis routes. Thus, efforts are being made to understand and design industrially usable microorganisms that can act as a host system for the purpose of production of these com-pounds. Cellulolytic organisms are being heav-ily studied for the production of biofuels, given that lignocellulosic biomass would be a cheap, abundant, and renewable starting material for chemical production. A challenge with cel-lulolytic microorganisms is that they are typi-cally poorly characterized and often difficult to genetically manipulate. Here we propose

Poster Abstracts


that the aerobic, cellulolytic actinobacterium, Thermobifida fusca, may be a good candidate for cellulolytic production of APIs. Our group has recently developed a genetic engineering technique for T. fusca and has constructed a genome-scale metabolic model to study its metabolic capabilities. In the current study, in silico analysis of the non-mevalonate pathway (or DXP pathway) will be performed using a genome-scale metabolic model of T.fusca. The DXP pathway leads to the production of terpe-noid precursors that have applications in nutra-ceutics and pharmaceutics. Modeling analysis illustrates the presence of the genes required to form: 1) Gerenyl pyrophosphate GPP, which is a substrate for producing monoterpenoids, 2) Farnesyl pyrophosphate or FPP, which is sesquiterpenoids precursor, and 3) Geranyl geranyl pyrophosphate GGPP, which is a diterpenoids precursor. The constructed meta-bolic model provides a basis for conducting in silico strain design predictions to optimize the production of terpenoid precursors through metabolic flux analysis to provide a rational basis for determining the distinct possible tar-get gene sets for increased product synthesis. In parallel with these computational predic-tions, experimental work is being conducted to verify specific biochemical pathways and to implement novel pathways in T. fusca. Overall, this work will provide a computational basis for terpenoid production from cellulose and experimental testing and implementation of genetically modified strain designs.

n 117AMICRObIOTA ANd dIET REGuLATE fATTY ACId AbSORPTION IN THE zEbRAfISH INTESTINE

I. Semova1, J. D. Carten2, J. Stombaugh3, L. C. Mackey1, R. Knight4, S. A. Farber2, J. F. Rawls1; 1University of North Carolina, Chapel Hill, NC, 2Carnegie Institution for Science, Baltimore, MD, 3University of Colorado, Boul-der, CO, 4Howard Hughes Medical Institute, University of Colorado, Boulder, CO.

The intestinal microbiota is known to impact host nutrition and energy balance, and has been implicated as an environmental factor contrib-uting to obesity and malnutrition. However, the role of the microbiota in intestinal absorption and subsequent extra-intestinal metabolism of dietary fat has remained unclear. We used in vivo imaging of fluorescent fatty acid (FA) analogs delivered into gnotobiotic zebrafish to reveal that the microbiota stimulates FA uptake and lipid droplet (LD) formation within absorptive enterocytes in the intestinal epithe-lium. Comparison of animals that were unfed or fed a sterile diet revealed that the microbiota promotes epithelial LD number, but not size, in a diet-dependent manner. The presence of food resulted in enrichment of Firmicutes bacteria in the microbiota of the zebrafish intestine, but not in the surrounding water. Monoassociation studies revealed that epithe-lial LD number is increased by diet-enriched Firmicutes and LD size is increased by other bacterial types. Epithelial LD number was also increased in germ-free zebrafish exposed to Firmicutes-conditioned media, indicating that bacterial products are sufficient to stimulate intestinal FA absorption. These results show that members of the microbiota regulate intes-tinal FA absorption via distinct mechanisms, and identify the gut microbiota as a new target for controlling dietary fat absorption. This work also shows that diet-induced alterations in gut microbiota membership might indirectly influence FA absorption, thereby providing an ecological explanation for the diet-dependent nature of this host-microbe interaction.

n 118BCHEESE: A MOdEL SYSTEM fOR PRObING MICRObIAL COMMuNITY dYNAMICS

J. E. Button, R. J. Dutton; Harvard Univer-sity, Cambridge, MA.

Background: Microbes typically live in com-plex, multi-species communities in the natural world. Advances in DNA sequencing technol-ogy have made it possible to characterize mi-

Poster Abstracts

ASM Conferences102

crobes present in diverse environments, includ-ing in and on the human body, and studies have demonstrated that these communities greatly impact our health. Defining the principles that govern microbial community development and persistence will help us to manipulate these communities to our advantage. Detailed, mechanistic analyses of microbial behavior in multi-species communities will benefit from an experimentally tractable model system that is culturable, simple, and reproducible. We have previously conducted a culture-independent survey of 160 cheeses in order to catalog the microbes present on the rinds of cheese (R. Dutton and B. Wolfe, unpublished results). These data show that cheese communities consist of no more than 17 bacterial taxa in any given community. Furthermore, triplicate wheels of cheese from the same batch house nearly identical bacterial communities. Thus, cheese communities exhibit two of the proper-ties of an ideal model system: simplicity and reproducibility. We propose that cheese com-munities are also highly culturable, and a com-plete collection of cultured cheese microbes can be used to reconstruct whole microbial communities in the lab. Methods and Results: In order to show that cheese communities are culturable, the composition of bacterial com-munities on triplicate wheels of cheese was determined by both culture-independent and culture-dependent methods: after extraction of DNA from rind samples, cheese com-munities were analyzed by 16S amplicon sequencing using the Illumina HiSeq platform, and samples were also plated onto standard microbiological media. Single colonies were isolated, and species were identified by Sanger sequencing of the bacterial 16S gene. Com-munity composition as determined by both culture-independent and -dependent methods were highly similar. Species that were isolated from a single cheese were then inoculated onto a cheese-like medium in order to reconstruct a simple cheese community. Development of the lab community over time was compared to microbial succession observed on an actual cheese rind, as determined by bacterial 16S

amplicon sequencing. In the lab, we were able to reproduce patterns of microbial succession highly similar to those observed on cheese. Conclusions: The bacterial communities pres-ent on the rinds of cheese are highly culturable, and all members of a rind community can be isolated in the lab. Using a collection of iso-lates from the rinds of cheese, we can recon-struct whole microbial communities. Because of the simplicity of the system, systematic manipulation of these communities is possible, and thus, they can be used to rigorously test hypotheses about microbial community devel-opment and response to perturbation.

n 119ACHARACTERIzING THE ROLE Of A bACTERIAL CONSORTIuM IN THE REPROduCTION Of THE MOdEL SYMbIOTIC SquId, EuPRYMNA SCOLOPES

A. H. Kerwin1, S. Frasca1, K. O’Donnell2, D. A. Sutton3, S. V. Nyholm1; 1University of Con-necticut, Storrs, CT, 2Bacterial Foodborne Pathogens and Mycology Research Unit, Peo-ria, IA, 3University of Texas, San Antonio, TX.

The Euprymna scolopes - Vibrio fischeri as-sociation has been a model for the elucidation of numerous beneficial host/microbe interac-tions, including quorum sensing, host immune response to beneficial and environmental microbes, and symbiont specificity. Recently, the bacterial consortium contained within the accessory nidamental gland (ANG), part of the reproductive system of female E. scolopes, has come under scrutiny. Ribosomal fluores-cence in situ hybridization of freshly laid eggs showed that the jelly coat contains populations of bacteria similar to that found in the ANG, suggesting that bacteria are deposited directly into the eggs. The role of these bacteria in the reproduction of this and other cephalopod spe-cies is unknown but has been hypothesized to be involved with providing protection for the developing embryos, possibly by the preven-tion of biofilm fouling. To begin to explore the function of the ANG bacterial consortium, we

Poster Abstracts


conducted a series of experiments whereby development of egg clutches (with and with-out antibiotic treatment) was monitored over a four-week period. Treatment of squid egg clutches with chloramphenicol led to the devel-opment of a thick biofilm and the death of the embryos (0% viability; n = 7 clutches). Clutch-es treated with ethanol (the chloramphenicol vehicle; n = 5 clutches) or those left untreated (n = 7 clutches) had no observed biofilms and developed and hatched at normal levels (>62% viability). Numerous fungal hyphae were observed in DAPI-stained biofilms by fluorescence microscopy. The fungal biofilm generally appeared 11-19 days after the antibi-otic-treated clutches were deposited. A fungal culture was isolated on inhibitory mold agar with gentamicin. Morphological characteriza-tion and preliminary multi-locus sequence typing using four genes (TEF1-α, RPB2, ITS rDNA, and the 5’ end of LSU rDNA) suggest that this isolate is a haplotype of an unnamed phylogenetically distinct species (designated FSSC 2), located within the Fusarium solani species complex (FSSC), a species that in-cludes pathogens of marine animals. Taken together, these data suggest ANG bacteria may protect developing squid embryos from fungal biofilms, similar to what has been described for bacterial egg protection of other marine in-vertebrates. Current work is focused on testing whether cultured ANG bacterial strains inhibit fungal and other microbial biofilms, character-izing bacterial gene expression in the eggs and ANG, and monitoring the bacterial egg popula-tion during embryogenesis.

n 120BISOLATION ANd CHARACTERIzATION Of dENITRIfYING ALkALIPHILIC bACTERIA fROM ALkALINE SOdA LAkE Of ETHIOPIA, LAkE CHITu.

R. A. Ibrahim; Addis Ababa University, Addis Ababa, ETHIOPIA.

Lake Chitu is one of the productive alkaline soda lakes in the Ethiopian Rift Valley system.

It consists of high algal bloom and feathers of the lesser flamingo with protein and other ni-trogenous compounds. As a result high amount of partially degraded organic nitrogenous and carbon compounds are accumulated in the lake. It also receives different nitrogen source from external environment like animal dung from inhabitants near by the lake. In the pres-ence of such large amount of waste in the lake, it is not deteriorated with characteristic of foul odor and color change (Tesfaye Minuta, 2006). The above points indicate the presence of ac-tive nitrogen cycle and denitrification in that environment. The objective of the study is to isolate and characterize denitrifying and alka-liphilic bacteria from Lake Chitu. Water and mud sample were collected from Lake Chitu using standard microbiological technique. The samples were inoculated to enrichment media and potential denitrifiers were selected by creating anoxic environment. For Organism that show relatively better rate of denitrifica-tion, growth rate determination, denitirfication ability and morphological characterizations were evaluated. BACC118 and BACC280 were the fastest in denitrification rate. They are fast grower reaching their stationary phase at around 24th hours. They can grow at high concentration of NaCl and Na

2CO

3. They have

ability to denitrify Nitrate in the presence of up to 5 percent NaCl concentration. Between 0.5 to 5% concentration of Na

2CO

3 there was

denitrification. These two organisms can also denitrify from 8.5 to 12 pH. Lake Chitu has microorganisms that are very important for nitrogen recycling in the lake. There are also active denitrification that reduce the nitrogen compounds in to gaseous nitrogen both aero-bically and anaerobically. The two isolates have the potential to treat wastes that contain high amount of nitrogen compounds, high salt concentration and high pH values like tannery waste water.

Poster Abstracts

ASM Conferences104

n 121AMYCObIOTA, MYCOTOxINS, ANd HEAVY METALS fROM INSTANT NOOdLES IN SAudI ARAbIA

L. A. Nasser; Princess Norah Bint Abdulrah-man, Riyadh, SAUDI ARABIA.

Forty-two samples of instant noodles (fast food meals) were collected from several markets at El-Riyadh, Saudi Arabia and examined for the occurrence of fungi, mycotoxins and pathogen-ic bacteria in addition to cadmium, lead and aluminum, to stand on their safety for human consumption. Twenty-five fungal species, in addition to one species variety belonging to 9 genera were isolated on both glucose-Czapek’s agar and PDA (Potato Dextrose Agar) media at 28oC, before and after cooking process of in-stant noodle products used. Three fungal gen-era, isolated with different frequencies on both media of cooked and uncooked noodle samples used in this study, were Aspergillus (31 - 50%), Penicillium (9.5 - 33.3%), and Cladosporium (9.5 - 19%). Chromatographic analysis re-corded the presence of citrinin which detected in only one out of 42 uncooked samples. The bacteriological analysis of instant noodle sam-ples according to NMKL, indicated that noodle samples used were free from pathogenic bac-teria. The cadmium, lead & aluminum concen-trations in each instant noodle samples were determined using atomic absorption spectro-photometer (AAS). The highest concentrations were recorded for cadmium (0.076 ppm), lead (1.143 ppm) and aluminum (11.250 ppm).

n 122BCuLTuRAbLE MICROORGANISMS ASSOCIATEd WITH SISHEN IRON ORE ANd THEIR POTENTIAL ROLES IN bIObENEfICIATION

R. A. Adeleke, M. Maila; Agricultural Re-search Council, Pretoria, SOUTH AFRICA.

Increasing demand and consumption of iron ore has triggered the search for processing technologies that can be utilised to “purify” the

low-grade iron ore. Our study investigated in-digenous bacteria associated with Sishen iron ore minerals and their potential to reduce high levels of unwanted potassium and phosphorus contents of the minerals. Twenty-three bacte-rial strains that belong to Proteobacteria, Fir-micutes, Bacteroidetes and Actinobateria were isolated and identified with molecular and phy-logenetic methods. Abilities of these isolates to lower the pH of the growth medium and solu-bilisation of tricalcium phosphate were used to screen them as potential mineral solubilisers. Eight isolates were successfully screened and utilised in shake-flask experiment. All the eight isolates produced high quantity of gluconic acid but lower quantities of acetic, citric and propanoic acid. Scanning Electron Microscopy and Fourier transform infrared analyses also helped to uncover the role that biofilm and extracellular polymeric substances could play in mineral solubilisation. Aims:(1) To isolate and characterise culturable bacterial population inhabiting the iron ore surfaces, (2) To screen the isolates that are potential organic acids-pro-ducing bacteria through the use of microbial features-characteristics such as ability to lower the pH of the growth medium and dissolution of insoluble phosphorus were utilised, and (3) To investigate the biobeneficiation (K and P reduction) potential of the selected isolates Methods and results: Two types of iron ore samples were collected from the Sishen mine located in the Nothern Cape Province of South Africa. These samples were originally charac-terised by the company as KGT (conglomer-ates) and SK (shale). The iron ore materials were milled and sieved into different sizes. Methods used to achieve the aim of this study included isolation of bacteria from iron ore samples, screening of P-solubilising and low pH-driven isolates, molecular identification of the isolates, leaching experiments, microscopy, fourier transform infrared (FTIR) spectroscopy as well as statistical analyses. The abilities of the isolates to lower the pH of the growth medium and solubilisation of tricalcium phos-phate were used to screen them as potential mineral solubilisers. Eight isolates were suc-

Poster Abstracts


cessfully screened and utilised in shake flask experiments using iron ore minerals as sources of K and P. The shake flask experiments re-vealed that all eight isolates have potentials to produce organic acids that aided the solubilisa-tion of the iron ore minerals. In addition, all eight isolates produced high concentrations of gluconic acid followed by relatively lower concentrations of acetic, citric and propanoic acid. Scanning electron microscopy and FTIR analyses also indicated extracellular polymeric substances could play a role in mineral solu-bilisation.

n 123ASuRVEY ON ANTIbIOTIC RESISTANCE Of LACTObACILLuS SPP. ISOLATEd fROM GARMSAR TRAdITIONAL dAIRY PROduCTS

T. Moosavi1, L. Mohammadyar2, N. Davar-zani2; 1Garmsar Branch, Islamic Azad Uni-versity, Garmsar, IRAN, ISLAMIC REPUBLIC OF, 2Garmsar branch,Islamic Azad University, Garmsar, IRAN, ISLAMIC REPUBLIC OF.

From old times ,Lactic acid bacteria have played an important role in food technology .There is a long history of consumption of them by human for producing and storage of food. Many studies have been done concern-ing about the good effects of different strains of lactic acid bacteria in the gastrointestinal tract in humans and animals and describe their mechanism of action. Some of these benefits include improved lactose digestion and treat-ment of diarrheal disease.Recently, many stud-ies concerning the clinical potential of lactic acid bacteria, especially those that are known as probiotics has been made. In this study, 40 subjects from the local Dairy of Garmsar city (including cheese, yoghurt, Kashk and Doogh) with sanitation in the container lid was steril-ized before you collect the necessary tests on them were in the least possible time. Lactoba-cillus average counts in our samples,yoghurt 5×107 cfu/ml,cheese 5/6×106 cfu/gr , Kashk 1×108 cfu/ml and Doogh 9×106 cfu/ml were reported. From 40 subjects 24 lactobacilli

were isolated, 7 of which belonged to the strain L.gasseri, 4 samples strains belong-ing to L.fermentum & L.animalis , 5 samples strains belonging to L.farciminis, 1 samples belonging to the strain L.reuteri & L.buchneri and 2 strains belonging to L.brevis.All strains was suspectible to amoxicillin[25mg] , ampicillin[10mg] , erythromycin[15mg] , clindamycin[2mg] , penicillin [10mg] , tetracycline[30mg] , oxytetracycline[30mg] , nitrofurantoin [300mg] and cephazolin[30mg] . All strains was resistant to gentamicin[10mg] , trimethoprimsulphametoxazol[1.25mg] and vancomycin[30mg].There was also percentage of moderately suspectible of penicillin. Survey on antibiotic resistance of isolated from a local traditional dairy, can prevented of many rare and unexpected of anti biotic resistant. Key words: Lactobacillus,Probiotics,Traditional dairy products,Garmsar city

n 124BMICRObIAL dIVERSITY IN MINE HEAPS: dIffERENT bIOGEOCHEMICAL NICHES

J. Garcia Meza1, S. J. Casas-Flores2; 1Geo-microbiología-UASLP, San Luis Potosi, MEX-ICO, 2División de Biología Molecular-IPICyT, San Luis Potosi, MEXICO.

Due to their huge and spatiotemporal hetero-geneity of heaps, in terms of mineral reactiv-ity, irrigation efficiency, temperature, pH, pO2, pCO2, ORP, dissolved solutes, available nutrients, etc., a considerable diversity of re-siding acidophilic Archaea and Bacteria may inhabited it, with diverse metabolic capaci-ties, not only chemolitotrophics, which play a directly or indirect role in biomining. In this Molecular Geomicrobiology study, microor-ganisms directly obtained from a sample of a mine-heap were identified. Total DNA extrac-tion, PCR amplification of rDNA 16S, cloning, sequencing and analysis of the genomic rDNA 16S genes were done. The results show the presence of chemolithoauto- and chemolitho-heterotrophics, aerobics and microaerophilics, acidophiles and circumneutral bacterias. Thus,

Poster Abstracts

ASM Conferences106

in the bioleaching columns different microen-vironments were established. Three groups of microorganism were recognized: the bioleach-ers sensu stricto as Sulfobacillus, Acidithioba-cillus and Thiobacillus; chemoorganotrophs as Sphingomonas, Micrococcus, Ochrobactrum and Rhodopseudomonas (fix N2), and an-aerobics heterotrophs. At least, microorgan-isms of these three groups must be common inhabitants of mine heaps. Mine-heap is a real man-made environment but governed by basic ecological rules, as the symbiotic interaction through chemolithotrophic and other metabolic groups

n 125AISOLATION Of NOVEL ANTIbACTERIAL COMPOuNd WITH HIGH IN-VITRO ANTIMICRObIAL ACTIVITY AGAINST vibrio cHoLerae O1 ANd O139 PROduCEd bY pseudomonas aeruginosa

N. Bhuiyan1, K. U. Ahmed1, G. B. Nair2, H. P. Endtz3, M. Alam1; 1International Centre For Diarrheal Disease Research, Bangladesh, Dhaka, BANGLADESH, 2Translational Health Science Technology Institute, Gurgaon, Hary-ana, INDIA, 3Department of Medical Microbi-ology and Infectious Diseases,, Erasmus MC, Rotterdam, NETHERLANDS.

Background: Pseudomonas aeruginosa is common inhabitant of soil and water. A strain of P. aeruginosa was isolated, which produced blue pigments in broth culture having vibrio-cidal activity. Objective: Characterize a new bactericidal substance produced by P. aerugi-nosa. Methodology: The strain was inoculated in 50-ml, Luria bertani broth and incubated at 37 degree Centigrade for 24 hours. Centri-fuged and filtered through 0.22 micrometer membrane. The filtrate was lyophilized and re-suspended in 2.5 ml distilled water and sub-jected to sephadex column. Antibacterial activ-ity was examined by the agar diffusion method against V. cholerae O1, V. cholerae O139 and also other pathogenic enteric bacteria. Results:

Five different fragments were found in sep-hadex column. Vibriocidal activity was pres-ent only in blue fragment. The antibacterial substance produced by P. aeruginosa shown inhibitory effect on the growth of V. cholerae O1, O139, non O1/non-O139, Aeromonas spp., V. parahaemolyticus, E. faecales, Cam-pylobacter spp., S. aureus, and V. mimicus but not on Shigella spp., Salmonella spp., and E. coli. This substance was very heat-stable, non-evaporating and not digested by proteinase K. Conclusion: Unknown substance produce by P. aeruginosa showed significant bactericidal activity against multidrug resistant V. cholerae. Further chemical characterization of the com-pound is under way. Acknowledgement: This research was funded by core donors which provide unrestricted support to icddr,b for its operations and research. Current donors pro-viding unrestricted support include: Australian Agency for International Development (Aus-AID), Government of the People’s Republic of Bangladesh; Canadian International Develop-ment Agency (CIDA), Swedish International Development Cooperation Agency (Sida), and the Department for International Development, UK (DFID). We gratefully acknowledge these donors for their support and commitment to icddr,b’s research efforts.

n 126BMICRObIAL COMMuNITIES Of TWO dIffERENT ExTREME INdOOR ENVIRONMENTS

A. Mahnert1, L. Oberauner2, C. Högenauer3, K. Smolle3, A. Probst4, C. Moissl-Eichinger4, G. Berg1; 1Institute of Environmental Biotech-nology of the Graz University of Technology, Graz, AUSTRIA, 2Institute of Environmental Biotechnology of the Graz University of Tech-nology, Austrian Centre of Industrial Biotech-nology GmbH, Graz, AUSTRIA, 3Department of Internal Medicine, Medical University of Graz, Graz, AUSTRIA, 4Department for Mi-crobiology and Archaea Centre, University of Regensburg, Regensburg, GERMANY.

Poster Abstracts


People in developed countries spend most of their lifetime in indoor environments, which are often dominated by human-associated mi-croorganisms. While potential pathogens are quite well-studied, less is known about harm-less or beneficial indoor organisms. Hence, a detailed characterization of indoor microbi-omes is an important step for understanding parameters possibly influencing their diversity and for developing strategies how human-beneficial microbes could help to control these artificial environments. For this study, two different indoor habitats that can be con-sidered extreme for microbes were selected: an intensive care unit (ICU) of the University hospital (Graz, Austria) and a clean room facility (Friedrichshafen, Germany), which is regularly controlled to maintain a low-particle and -biomass environment for modern industry processes. Deep sequencing of 16S rRNA gene PCR amplicons (454 GS-FLX+ TitaniumTM) derived from metagenomic DNA from several sites showed great variations in microbial rich-ness for each sample. In both environments Proteobacteria and Firmicutes were the domi-nant phyla. While Pseudomonas and Propioni-bacterium were found as the most abundant genera in the ICU, the clean room environment was dominated by Gram-positive genera such as Staphylococcus and Anaerococcus. The ICU analyses revealed differences between microbial communities present on floors (Aci-netobacter, Streptococcus and Staphylococcus) and those found on devices and workplaces (Propionibacterium, Pseudomonas, Burkhold-eria and Bradyrhizobium). However, the clean room environment seemed to selectively enrich Firmicutes over Proteobacteria and Actinobac-teria, which were primarily found in the office and changing room located in close vicinity. We assume that the composition of the micro-bial communities is highly dependent on the surrounding environment, maintenance proce-dure and human activity. In sum, the detected communities reflect a combination of the microbial diversity present in the surrounding natural environment and a specifically adapted

microbiome, including human pathogens and potentially beneficial microbes. The presented data is a very useful prerequisite for further analyses with respect to microbial community monitoring, management and -if necessary- confinement. Finally, these results should also help to develop strategies for identification and support of beneficial microorganisms in artifi-cial indoor environments. Key words: indoor microbiology, artificial environments, intensive care unit, clean room, microbial diversity, microbial management, 16S rRNA gene deep sequencing

n 127AISOLATION ANd MOLECuLAR CHARACTERIzATION Of fuNGAL STRAINS CAPAbLE Of dEGRAdING POLYVINYL CHLORIdE (PVC)PLASTICS

M. I. Ali1, G. Robson2, S. Ahmed1, N. Atiq1, N. Ali1, A. Hameed1; 1Quaid-i-Azam University Islamabad, Pakistan, Islamabad, PAKISTAN, 2University of Manchester, Manchester, UNIT-ED KINGDOM.

Polyvinyl chloride (PVC) has been one of the most widely used synthetic polymers world-wide due to its versatile properties. Though, its recalcitrant nature creates serious environ-mental concerns during manufacturing and disposal. The present study screened out dif-ferent soil fungi involved in the biodegradation of PVC films. Low molecular weight thin films of PVC were treated in soil burial and shake flask experiments. After 10 months of incuba-tion in soil, it was observed that a number of fungal strains were flourishing on PVC films. On morphological as well as on 18rRNA gene sequence basis they were identified as Pha-nerochaete chrysosporium PV1, Lentinus tigri-nus PV2, Aspergillus niger PV3 and Aspergil-lus sydowii PV4. The biodegradation ability of these fungal isolates was further checked in shake flask experiments by taking thin films of PVC (C source) in mineral salt medium. In both the experimental setups the PVC films showed a significant change in color and sur-

Poster Abstracts

ASM Conferences108

face deterioration which was further confirmed through SEM. During shake flask experiments, Phanerochaete chrysosporium PV1 produced maximum dry biomass of about 2.57 mg/ml followed by Aspergillus niger PV3. The rela-tive growth of the fungi proved to be strongly correlated with production of CO2 in strum test. Phanerochaete chrysosporium PV1 showed significant reduction (178, 000 Da) in Molecular weight of the PVC film then control ( 20,2530 Da) observed by gel permeation chromatography (GPC) . Appearance of new peak at 2370-2350 cm -1 region indicating the change in the structure of the PVC by Fou-rier transform infrared spectroscopy (FTIR). Nuclear Magnetic resonance (cNMR) also indicated attribution of new signal range 29-47 ppm in treated film of PVC, confirming the degradation pattern and structural changes in PVC films by fungal treatment.

n 128BfuNGI; AS NObLE METAL NANO-fACTORY ANd ECO-REMEdIATOR, AN EMERGING GREEN TECHNOLOGY APPROACH.

S. Z. NAQVI, S. Zainab, A. H. Naeem, A. Ha-meed, S. Ahmad, N. Ali; Quaid-i-Azam Uni-versity, Islamabad, PAKISTAN.

There are both beneficial and harmful as-pects of microbes.Among all microorgan-isms’ fungi found to be most dynamic in nature involving in multiple procedures of environmental concern. The synthesis of in-organic nanoparticles(NPs) is a vibrant field of interest for academia and applications in nanotechnology.A diversity of physico-chemical processes available for the synthesis of metallic NPs. However,these procedures accompanying different issues like;use of toxic solvents,generation of perilous by-products,and high energy and cost expendi-tures.So,there is a vital requirement to build up environmental friendly protocols for the syn-thesis of inorganic NPs.Biological synthesis of NPs is a growing innovative approach,which is relatively cheaper and environmental friendly.

Among various microbes,fungi found to have promising role in synthesis of nanomaterials.In this study, extracellular mycosynthesis of silver NPs using four different Aspergillus species;A.niger,A.fumigatus, A.flavus&A.terreus was investigated.The UV-visible spectra recorded for the aqueous medium containing silver ions at different times of reaction have shown the strong surface plasmon resonance centered between 400-420 nm,which clearly increased in intensity with time.X-ray diffraction(XRD) spectra have shown peaks of various intensi-ties with respect to angle of diffraction(2θ) corresponding to the silver NPs. Transmission electron microscopy(TEM) images further confirmed formation of silver NPs of 3-80 nm in size by the four Aspergillus species.At the ambient experimental conditions the fungus A.fumigatus synthesized NPs faster than the others while the A.flavus showed more drift towards monodispersity than others.After the optimization with silver NPs,the mycogenesis of gold and platinum NPs were also studied through Aspergillus niger and A.fumigatus.The formation of gold and platinum NPs was confirmed by TEM.The process of extracel-lular reduction proved to be considerably fast and could help in the downstream processing and scaling up of the process.Furthermore,the comparison of dye degradation rates of two azo dyes i.e. AR151 & Orange II were mea-sured between fungus A.niger,myco-genized silver NPs and commercially available silver NPs.Optimization of reaction condition was done for better control on degradation rate and adsorption equilibrium.Afterwards, best optimized conditions were applied to study the biodegradation of both the azo dyes by A.niger and by silver NPs.The decolorization of both the azo dyes was routinely monitored by vi-sual observations,UV-Visible spectroscopy and FTIR.Dye degradation rate found to be catalyzed by NPs.Along with this preliminary study,phytotoxicity and cytotoxicity assays of biodegradation byproducts were also car-ried out.Thus, we can deduce that microbes especially fungi can be employed in the huge valuable features.

Poster Abstracts


n 129AREMOVAL EffICACY Of HARMfuL MICRObES ANd CONTAMINANTS IN bIOSANd fILTER MICROCOSMS

N. M. Horne, B. Biddanda, R. Rediske, J. O’Keefe; Grand Valley State University, Mus-kegon, MI.

Freshwater is a shrinking resource in large parts of the Earth, with overuse and contami-nation in underdeveloped countries further rendering the available supply of water unsafe. An inexpensive remedy is the use of Biosand filters of 60-100 L volume (BSFs) in every household, classroom, and hospital, delivering safe potable water - a UN Millennium goal. Whereas physical filtration of particulate im-purities through graded sand is well known, there is poor understanding of the effectiveness of the microbial biofilm within BSFs in ridding water of harmful contaminants. To address this void, we investigated the efficacy of BSFs in filtering harmful contaminants: total coliforms, E. coli, arsenic, and microcystin-LR in a 3-phase study, using self-designed microcosm syringes of 60 mL volume - micro scale ana-logs of BSFs. Muskegon Lake, a mesotrophic urban lake in Michigan, served as the source water. In Phase I, filters exhibited significant pathogen removal from source water, upon bio-film maturity. Over a 3-day period, a reduction of total coliforms from 12.2 - 6.3 MPN/100mL in input water to 1.0 - <1 MPN/100mL in output water was observed - a 92 - 86% re-moval efficiency (RE). Concurrently, E. coli was reduced from 7.5 - 5.2 MPN/100mL in input water to 1.0 - <1 MPN/100mL in output water - an 87 - 83% RE. In a repeated experi-ment, over a 6-day period, a reduction in total coliforms from 42.0 - 8.5 MPN/100mL in input water to 2.0 - <1 MPN/100mL in output water was observed, a 95 - 89% RE. Scale-up into the full-sized Hydraid® BSF (Cascade Engineering) showed a reduction in total co-liforms ranging from 104 - 21.6 MPN/100mL in input water to 3.0 - 1.0 MPN/100mL in output water over a 3-day sampling period, a 99 - 86% RE. In Phase II, addition of rusted

Fe filings to microcosms resulted in reduction of arsenic (a common groundwater contami-nant in many parts of Asia) from 100μg As in input water to 0μg As in output water, a 100% mean RE (n=4) in two separate experiments. Without rusted Fe fillings, mean RE was 65-75%. Phase III involved the investigation of microcystin-LR (a hepatotoxin produced by bloom-forming cyanobacteria in eutrophic lakes worldwide) removal in the absence and presence of a mature biofilm. Before biofilm maturity, mean removal efficiency was 17-30% (n=4) over a 3-day period. Following biofilm maturation, mean removal efficiency ranged from 82-95% over a 6-day period. Upon scale-up into the full-sized Hydraid® BSF, a reduc-tion in microcystin-LR ranged from 94 - 93% mean RE (n=3) over a period of 3 days. Our study demonstrates that mature BSFs effec-tively remove common pathogenic microbes and harmful cyanobacteria toxins and chemical contaminants, making them a viable option for delivering safe point-of-use drinking water in undeveloped countries.

n 130BuNdERSTANdING HOW NITRILE HYdROLYzING ENzYMES HELP r. rHodocHrous STRAIN dAP 96253 CELLS dELAY THE RIPENING Of SEVERAL fRuIT SPECIES.

G. Perry, F. Du, A. Keller, G. Pierce; Georgia State University, Atlanta, GA.

Americans are developing a desire for a healthier diet; a trend that has led to an in-crease in fresh vegetable and fruit consump-tion. Currently chemicals are used to retard fruit ripening and produce spoilage. The agricultural industry wants organic ways to delay and naturally ripen fruit. Rhodococcus rhodochrous is a common rhizobacterium. R. rhodochrous strain DAP 96253 cells cultured on induced YEMEA media (supplemented with 16mM cobalt and 125mM urea) displayed the ability to delay the ripening of several fruit species. Propylene was used to simulate phyto

Poster Abstracts

ASM Conferences110

hormones released by plants. Induced YEMEA media increased NHase activity to 18 times (units/mg cdw) greater than non-induced cells, while induced YEMEA and propylene media increased activity to 37 times (units/mg cdw) higher than non-induced cells. SDS analysis of cells cultured on induced YEMEA and pro-pylene media displayed increased expression of NHase, amidase, nitrilase,alkene monoxy-genase (AMO) and epoxide carboxylase (EC) proteins, 24kDa, 27kDa, 35kDa, 40kDa, 45kDa, 53 kDa, and 57kDa. The results from this study suggest that R. rhodochrous strain DAP 96253 cells over express nitrile hydratase (NHase), amidase, and/or nitrilase to convert indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). Large concentrations of IAA then cause increased production of ethylene and cyanide by plants cells. The R. rhodochrous strain DAP 96253 cells use AMO and EC to degrade and metabolize ethylene and/or cya-nide. The high IAA concentration prevents ethylene from rebinding to ETR complexes on plant cell surface. The potential process is ca-pable of deterring the fruit from ripening.

n 131AANTAGONISTIC POTENTIAL Of LACTObACILLuS STRAINS AGAINST PATHOGENIC MICROORGANISMS COMMONLY fOuNd IN PACkEd SAuSAGES IN MExICO

O. Lazo1, S. Trejo2, C. Ramirez1; 1IPN, Puebla, MEXICO, 2IPN, Pueb;a, MEXICO.

An increasingly important market exists for packed sausages and franks in Mexico. Several health risks are posed by deficient sanitary and microbiological controls. In order to man-age this situation companies have invested in research to have alternatives of quality control methods that not necessary involve the abuse of chemical preservatives. Therefore as a result of this problematic the current investigation was developed. 50 strains of lactobacillus were isolated and purified from different samples of agave sap (aguamiel), pulque and processed

meat such as sausages among other samples that came from rural areas of the Mexican highlands . All the strains were tested to evalu-ate their antagonistic capacity against patho-genic microorganisms such as Salmonella typhi, Salmonella cholerae, Staphylococcus aureus, Escherichia coli and Listeria monocy-togenes. From the 50 strains tested, 8 of them selectively proved their antagonistic capacity in different scale against some of the pathogen-ic strains.This tests were made using selective media for the lactobacillus strains and for the pathogenic strains. The antagonistic capacity was measured by inhibition halos after both bacteria were cultivated.

n 132BAGGREGATION ANd NATuRAL TRANSfORMATION MEdIATEd bY TYPE fOuR PILI IN THE EARTHWORM SYMbIONT VERMINEPHRObACTER EISENIAE

G. Dulla1, R. Go1, N. Pinel2, J. Barkley1, D. A. Stahl1, S. K. Davidson1; 1University of Wash-ington, Seattle, WA, 2Integrated Systems Biol-ogy, Seattle, WA.

The earthworm Eisenia fetida harbors bacteria in excretory organs called nephridia. The bac-terial symbiont Verminephrobacter eiseniae is deposited in egg capsules as part of a mixed microbial community prior to colonization of the embryo nephridia. Colonization of ne-phridia by V. eiseniae requires both flagella and type four pili (TFP) to reach the symbiotic organ. Observations of V. eiseniae in culture lead to the study presented here which tested the hypotheses that TFP were also involved in DNA uptake and aggregate formation of V. eiseniae. Experiments that compared V. eiseni-ae TFP mutants to wild type demonstrated that V. eiseniae is naturally competent and employs the TFP to import DNA from the environment. V. eiseniae, both in culture and when resident in the egg capsule, was able to assimilate ex-tracellular DNA into the chromosome. This ability was greatly attenuated in nonpiliated pilBC- and hyperpiliated pilT- mutants with-

Poster Abstracts


out functional TFP. Growth phase and carbon availability influenced the frequency of NT suggesting an increase in DNA uptake from the environment under carbon limiting condi-tions. Increasing presence of nonhomologous DNA had minimal impact on NT suggesting sequence specificity in DNA uptake. The dy-namics of aggregate formation were also influ-enced by TFP. When grown as static culture in polystyrene wells, V. eiseniae initially settled as a smooth layer of cells then formed evenly spaced small aggregates which rearranged into larger aggregates with wider spacing in 2-3 days. The nonpiliated pilBC- mutant failed to form aggregates, and remained as a smooth layer of cells, where as the hyper-piliated

pilT- mutant formed an irregular rough mass, failing to form distinct aggregates. A flagellar, flgKL-, mutant produced only small aggregates and failed to rearrange to the larger structures, indicating swimming motility is also involved in the aggregate formation dynamics. Addition of DNase into cell culture results in deforma-tion and spreading of distinct aggregates. Conversely, excess nonhomolgous DNA slows formation of larger aggregates suggesting the presence of DNA as structural component in aggregates. Our data show the potential for horizontal gene transfer and cell-cell contact mediated by V. eiseniae TFP in the nephridia and egg capsules of earthworms.

Poster Abstracts

ASM Conferences112

IndexAbad, A. M. 1AAchterhof, A. 56BAdeleke, R. A. 122BAdkins, J. N. 13AAgboola, F. M. 26BAggarwal, N. K. 89AAhmad, S. 128BAhmed, K. U. 125AAhmed, S. 127AAhrivar, D. 95AAksoy, S. 37A, 6B,

94B, S3:6Alam, M. 125AAldridge, K. L. 70BAli, B. 106BAli, M. I. 127AAli, N. 127A,

128BAlicki, E. R. S1:2Allen-Vercoe, E. 27AAlm, E. 77AAlmeida, M. 61AAltimus, G. 114BAnderson, D. 30BAnderson, M. 91AAndrade, F. S1:2Ansari, S. 18BArab, H. T. 98BArcand, Y. 67AArthur, J. C. 39AAsrat, D. 16BAssefa, S. 56B, 78BAtiq, N. 127AAxelsson, L. 105A,

107ABailey, M. T. 5ABain, J. R. S5:2Ballok, A. E. 35ABallok, A. E. 76BBamola, V. 96BBanse, A. V. S4:6Baptista, I. 77ABarbosa, D. 60BBarkley, J. 132BBarouei, J. S5:5Barry, N. 46BBasle, A. 92B

Chaudhry, R. 96Bchaurasia, A. 104B, 95AChen, Y. 56B, 78BCheng, J. S5:2Christophersen, C. T. 28BChung, E. 82BCiche, T. A. S4:4Claesen, J. 70BClardy, J. S4:4Clark, B. A. 36BClark, L. 34BClarke, D. J. 3A, 4BClemente, J. C. S5:2Colston, S. M. 110BCoughlin, L. A. 68BCrawford, J. M. S4:4Cronin, D. C. 71ACruz, J. B. 60BCurtis, J. T. 56BCurtis, J. T. 78Bda Silva, A. M. 60BDahel, D. 41ADai, D. S1:6Dale, C. S2:6Dao, C. 102BDary, M. 41ADavarzani, N. 123ADavid, L. A. 77ADavidson, S. K. 132Bde Oliveira, J. C. 60BDe Santis, R. 52B, 62BDearing, M. S2:6Degnan, P. 90B, S6:4Degnan, P. H. 46BDelbes, C. 61ADelong, E. F. S6:2Devine, S. P. 12BDewhirst, F. E. 59ADigiampietri, L. A. 60BDishaw, L. 52BDishaw, L. J. 62BDobson, A. J. 113ADomogala, D. 22BDonati, A. J. 57ADouglas, A. E. 113A

Bastidas, C. 32BBauman, J. S2:2Begde, D. 29ABehar, A. 85ABeinart, R. A. S6:2Bellantuono, A. 32BBellomo, D. 105ABelobrajdic, D. P. 28BBen-Dov, E. 33ABenjamino, J. 81ABerg, G. 126BBetancur, C. 1ABhandari, P. 20BBhuiyan, N. 125ABiddanda, B. 129ABird, A. R. 28BBlackburn, M. 77ABohannan, B. J. 80BBois, M. 40BBolam, D. 92BBongrand, C. 48BBordenstein, S. R. 93ABorisy, G. G. 59ABosch, T. C. S2:7Britton, R. A. 63ABritton, R. A. 73ABrooks, J. F. 71ABrucker, R. M. 93ABryan, N. S. S1:2Bui, T. 21ABulmer, M. 97ABurguière, P. 43A, 67ABurns, A. R. 80BButler, S. 79AButton, J. E. 118BCameron, E. A. 50BCann, I. 15ACaporaso, G. 22BCarten, J. D. 117ACasas-Flores, S. J. 124BCatchpole, J. 3ACeballos, M. T. 1AChandler, J. A. 14BChang, E. B. S1:6Chang, W.-S. 57AChaston, J. M. 113A, 54BChaudhary, M. 96B


Douglas, A. E. 24B, 54B, S6:3

Du, F. 130BDulla, G. 132BDuncan, A. E. S5:2Dunklebarger, M. 108BDunn, R. 22BDunne, E. 58BDutton, R. J. 118BDzutsev, A. S3:4Earle, K. 53AEisen, J. A. 14BEndtz, H. P. 125AEren, A. M. 69AEstrada, S. 1AEvans, P. 84BFaith, J. J. S5:2Fan, D. 68BFarage, L. 60BFarber, S. A. 117AFarrokh, C. 61AFelix, M.-A. 65AFerreyra, J. A. S1:4Fierer, N. 22BFischbach, M. A. 70BFlamiano, R. S. 98BFlores, G. E. 22BFlores-Torres, J. 52B, 62BFodor, A. A. 39AFong, S. S. 116BFonseca, J. S4:2Foxall, R. A. 76BFrank, B. 13AFrasca, S. 119AFriedman, A. L. 85AFriedman, J. 77AFujimoto, M. S2:2Gahlowt, A. 96BGalley, J. 5AGarcia, J. R. 82BGarcia, M. C. 40BGarcia, M. C. 45AGarcia Meza, J. 124BGebert, M. J. 22BGemayel, K. 62BGerardo, N. M. 82BGindin, G. 33AGingery, M. 34BGirguis, P. R. S6:2Go, R. 132B

Godboley, D. 7AGoguen, K. L. 70BGomez-Chiarri, M. 102BGonzalez, A. S5:2Goodman, A. S6:5Goodman, A. L. 46B, 71AGordon, J. I. S5:2Graf, J. 110B, 81AGranados- Cifuentes, C. 87AGranados- Cifuentes, C. A. 32BGrimmer, S. 107AGuillemin, K. 80BGuillemin, K. J. S4:6Gyllborg, M. C. 71A, 74BGysler, C. 19AHabiger, J. 91AHahn, B. 90BHalfvarson, J. 108BHameed, A. 127A,

128BHamilton, J. S. 68BHamilton, M. J. 10B, 23AHamilton, M. J. 8BHampton, J. S. 80BHansen, A. S6:4Hartmann, A. A. S4:2He, S. 27AHeath, A. C. S5:2Hebert, A. 61AHeffron, F. 13AHeidt, A. J. S4:4Heinzmann, S. S. S5:7Helman, Y. 85AHenley, J. B. 22BHermann, S. L. 113AHertzberger, R. Y. 19AHewitt, R. E. 36BHicks, R. 31AHochwind, K. S4:2Hodgson, D. S5:5Hoedt, E. 84BHögenauer, C. 126BHooper, L. V. 49A, S4:1Horne, N. M. 129AHsiao, W. S3:4Huang, K. 53AHumeida, U. H. S5:1Humphrey, G. 22B

Hurlbut, D. 27AHuse, S. 69AHuttenhower, C. S1:5Ibrahim, R. A. 120BIlhan, Z. E. 75AIrazoqui, J. E. 65AIrlinger, F. 61AIrwin, R. 73AIsberg, R. R. 112BIslas-Romero, M. 103AItelima, J. U. 17AJacobson, B. E. 70BJansson, J. 108BJemielita, M. 80BJensen, H. 107AJeon, J.-M. 57AJiang, Q.-X. 49AJobin, C. 39AJones, B. V. 38BJones, M. B. 13AJones, S. E. S3:2Joyce, S. A. 4BJuliano, L. 60BKadouri, D. E. 7AKaiser, B. L. 13AKang, D.-W. 75AKaplan, H. B. S1:2Karim, M. 102BKarrer, C. 52B, 62BKeller, A. 130BKepert, I. S4:2Kerwin, A. H. 119AKhoruts, A. 10B, 23AKhoruts, A. 8BKilburn, A. 10BKim, K. S1:6Kim, K.-S. S4:4Kim, Y.-M. 13AKing, N. S2:5Kleerebezem, M. 19AKlotz, S. A. 45AKnight, K. L. S3:2Knight, R. 117A,

22B, S1:1, S5:2

Kocher, J. 21AKoehler, G. 78BKoehler, G. A. 56BKoh, A. Y. 68B

Index

ASM Conferences114

Index

Kohl, K. D. S2:6Kopp, A. 14BKoropatkin, N. M. 50B, 64BKrajmalnik- Brown, R. 75AKrauss- Etschmann, S. S4:2Kronauer, D. J. S6:6Kronewitter, S. R. 13AKumar, B. 88BKwon, I. 15ALamendella, R. 108BLango, L. 4BLauber, C. L. 22BLazo, O. 131ALeadbetter, J. R. S2:1Leberg, P. 87ALee, H.-I. 57ALeigh, B. 52BLeigh, B. A. 62BLemon, K. P. 44BLemon, K. P. 70BLesser, C. F. 112BLi, G. 21ALi, J. 13ALi, K. 108BLiberti, A. 52B, 62BLim-Fong, G. E. 66BLindow, S. E. S4:5Linneman, J. 66BLipke, P. N. 40B, 45ALitman, G. W. 52B, 62BLiu, F. 21ALiu, Z. 55ALopanik, N. B. 66BLovett, B. S2:2Loza-Valerdi, E. M. 103ALysak, N. 45ALyte, M. 5AMabood, F. 2BMacfarlane, W. M. 38BMackey, L. C. 117AMackie, R. 15AMackos, A. 5AMacPherson, C. 43AMahatme, P. 29AMahnert, A. 126BMaila, M. 122BMainville, I. 67AMaltz, M. S3:6

Maltz, M. A. 6BMandel, M. J. 71A, 74BManhas, K. 88BManning, J. 58BManter, D. K. S2:4Mark Welch, J. 69AMark Welch, J. L. 59AMarsh, T. L. S2:2Martens, E. C. 115A, 50BMartin, A. R. S4:4Martz, S.-L. E. 27AMateos, M. 79AMaterna, A. 77AMatzinger, P. S3:4Maxwell, T. K. 114BMaynard, M. A. 64BMcCabe, L. 73AMcKinney, C. G. 59AMcLeod, A. 105AMegias, M. 41AMelillo, D. 52B, 62BMendel, Z. 33AMetz, T. O. 13AMiller, J. F. 34BMittal, A. 89AMiyashiro, T. 55AMoeller, A. 90BMohammadyar, L. 123A, 47AMoissl- Eichinger, C. 126BMok, K. 46BMokrane, N. 41AMollik, M. 100B, 83AMonnet, C. 61AMontel, M.-C. 61AMoosavi, T. 123A, 47AMoran, N. S6:4Moran, N. A. S5:3Morgun, A. S3:4Morrison, M. 84BMoussavi, M. S5:5Muehlbauer, M. J. S5:2Mueller, G. 52BMueller, M. 62BMukherjee, S. 49AMuller, W. S. 30BNabti, E. 41ANaeem, A. H. 128BNair, G. B. 125ANAQVI, S. Z. 128B

Nasser, L. A. 121ANelson, D. R. 102BNelson, M. 5ANelson, M. C. 81ANeoh, K. 63ANeubauer, M. M. 68BNewell, P. D. 113A, 24BNewgard, C. B. S5:2Newton, I. 31ANewton, I. L. 112BNg, K. M. S1:4Nirenberg, P. S2:2Noordhof, C. 27ANyholm, S. V. 101A,

109A, 119A, S3:5

Nzakizwanayo, J. 38BÓ Cuív, P. 84BO’Donnell, K. 119AO’Keefe, J. 129AO’Toole, G. A. 35AOberauner, L. 126BOchman, H. 90BOehlert, D. 55AOlmstead, H. 109AOtt, B. M. 114BPagano, E. E. 70BPaliy, O. 10BParameswaran, N. 73AParry, N. 5AParthasarathy, R. 80BPascon, R. C. 60BPaul, V. J. 25APaulus, D. 66BPauly, R. 93APelletreau, K. N. 12BPeloquin, J. J. 11APerez-Chanona, E. 39APerrotta, A. 77APerry, G. 130BPeterson, C. T. 13APeterson, S. N. 13APetrof, E. O. 27APetrosino, J. F. S1:2Pierce, G. 130BPierce, N. E. S6:6Pinel, N. 132BPinto, M. 52BPowell, S. S6:6


Index

Prabha, V. 20BPreheim, S. 77APridmore, R. D. 19APrincipal, L. 60BProbst, A. 126BPudlo, N. A. 115APusey, A. 90BPutman, K. 109AQuach, D. 73ARachmilevitch, S. 85ARader, B. A. 109ARaehtz, S. 73ARaes, J. S1:3Ramirez, C. 131ARasmussen, S. 61ARavel, J. S3:4Rawls, J. F. 117A, 80BRediske, R. 129AReed, J. L. 51AReeves, S. G. 11ARenault, P. 61ARey, F. E. S5:2Reynolds, V. 56BRidaura, V. K. S5:2Rio, R. V. 114BRio, R. V. 36BRio, R. V. S4:3Ritchie, K. B. 25ARobson, G. 127ARodriguez, R. S2:3Rodriguez- Lanetty, M. 32B, 87ARogers, T. E. 115ARosaria Pinto, M. 62BRosengaus, R. B. 97ARossetti, B. 59ARowley, D. C. 102BRuby, E. S6:1Ruby, E. G. 55ARud, I. 105ARumpho, M. E. 12BRunyen-Janecky, L. J. 94BSadowsky, M. J. 10B, 23A,

8BSalami, A. O. 17ASalazar, L. M. 1ASanchez, E. 1ASanchez, G. 79ASanders, J. G. S6:2, S6:6

Sanford, J. A. 13ASannino, D. R. 113ASantos, F. 105ASatzke, C. 58BSavage, A. F. 36BSavani, M. R. 68BSchaefer, L. 73AScharschmidt, T. C. 70BSchleicher, T. R. 101ASchloss, P. D. 42BSchmid, M. S4:2Schmidt, T. M. S1:6Schmitt- Kopplin, P. S4:2, S5:7Schubert, A. M. 42BSchultheis, K. 97AScribner, K. S2:2Semova, I. 117ASepter, A. 76BSetubal, J. C. 60BShankar, V. 10BShapiro, S. 92BSharma, N. 96BSharma, P. 96BSharp, K. 25ASheehan, K. 31ASheflin, A. S2:4Shelef, O. 85ASherchand, J. B. 18BShimohata, T. 9AShirasu-Hiza, M. S3:3Shulzhenko, N. S3:4Sikes, A. 30BSingh, G. 89ASingh, S. 40B, 45ASloup, R. E. S4:4Smith, C. L. 94BSmith, D. 2BSmith, R. D. 13ASmith, W. 84BSmolle, K.-H. 126BSneed, J. 25ASnyder, A. K. 36BSobonya, R. 45ASogin, M. S1:6Solidarios, M. B. 98BSomvanshi, V. S. S4:4Sonnenburg, J. 53ASonnenburg, J. L. S1:4Sorokulova, I. 99A

Souleimanov, A. 2BStabb, E. V. 76BStagaman, K. 80BStahl, D. A. 132BSteidler, L. 111AStephens, W. Z. 80BStewart, F. J. S6:2Stombaugh, J. 117ASutton, D. A. 119ATabli, N. 41ATaga, M. 46BTakahashi, A. 9ATam, N. T. 86BTaormina, M. J. 80BTaraldrud, M. 107ATeixeira de Mattos, M. J. 19ATeusink, B. 105AThompson, A. 3ATompkins, T. A. 43A, 67ATrejo, S. 131ATrejo-Estrada, S. R. 103ATribble, G. S1:2Troll, J. V. S4:6Tufa, T. B. 16BTysk, C. 108BTzfira, T. 33AUbhi, D. 72BUnno, T. 10B, 23AUnno, T. 8BUpadhyay, A. 29AUronis, J. M. 39AUrs, K. 115AUrsell, L. K. S5:2Valm, A. M. 59Avan Hemert, S. S4:2van Pijkeren, J.-P. 63AVanee, N. 116BVanegas, C. 1AVasconcelos, H. L. S6:6VerBerkmoes, N. C. S5:4Verjovski- Almeida, S. 60BVivanco, J. S2:4Walters, W. A. S5:2Wang, J. 37AWang, Y. 34BWeingarden, A. R. 23A, 8BWeinthal, D. 33A

ASM Conferences116

Weiss, B. 94B, S3:6Weiss, B. L. 6BWeiss, R. B. S2:6Wen, K. 21AWerner, T. M. 11AWhistler, C. A. 76BWilling, B. P. S5:6Wilson, M. 90BWoldeamanuel, Y. 16BWollenberg, A. C. 65AWollenberg, M. S. 44B, 70B

Wong, C. 24BWong, C. 54BWong, C.-N. N. 113AWong, S. 80BWu, Y. 37AXie, J. 79AYadav, A. 89AYakabe, T. 9AYang, X. 21AYoung, V. B. S1:6Yu, Z. 5A

Yuan, L. 21AZainab, S. 128BZaritsky, A. 33AZhang, J. 73AZhang, X. 51AZhao, L. KS1Zhao, W. 102BZheng, H. 49AZhu, J. 55AZimmermann, M. 70BZucchetti, I. 52B, 62B

Index

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