the microbiome and its therapeutic potential in ... · fecal transplantation from a healthy donor...
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The microbiome and its therapeutic potential in inflammatory bowel diseases
Rossen, N.G.M.
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Citation for published version (APA):Rossen, N. G. M. (2016). The microbiome and its therapeutic potential in inflammatory bowel diseases.
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Download date: 19 Nov 2020
7fecal transplantation in ulcerative colitis: microbiota shifts and siGnatures that determine lonG-term success of therapy
Susana Fuentes and Noortje G. Rossen, Mirjam J. van der Spek, Jorn H.A. Hartman, Laura Huuskonen, Willem M. de Vos, Geert R. D’Haens, Erwin G. Zoetendal and Cyriel Y. Ponsioensubmitted
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ABSTRACT
Background and aimsFecal transplantation from a healthy donor can induce remission in ulcerative colitis. In this study, we aimed to identify bacterial signatures that are associated with sustained remission. To this end, we analysed the fecal and mucosal microbiota composition during and up to 3 years post transplantation.
MethodsFecal and rectal biopsy samples from UC patients and healthy donors who participated in the TURN trial (ClinicalTrials.gov Number: NCT01650038) were analysed. This trial compared the efficacy of two duodenal infusions of fecal homogenates after bowel preparation either from a healthy donor or autologous feces. Microbiota profiles were determined by 16S rRNA-gene based phylogenetic microarray and the number of Butyryl-coenzyme A (CoA)-CoA transferase genes (ButCoA) was determined by qPCR. To assess the effect of the therapy in the intestinal microbiota, patients were grouped into responders and non-responders (assessed at week 12 after FMT) and further stratified by sustained remission and relapse (assessed at ≥1 year follow-up).
ResultsAt baseline, non-responders showed reduced amounts of groups from the Clos-tridium cluster XIVa, and significantly higher levels of groups belonging to the Bacteroidetes as compared to donors. At twelve weeks these differences were retained. Redundancy analysis showed an almost complete separation of patients that although initially included in the responder group, relapsed at the ≥1-year FU. Sustained remission was highly associated with shift at twelve week to a Clostridium IV and XIVa enriched signature, including many potential butyrate producers, while relapse was associated with the phyla Proteobacteria and Bacteroidetes. ButCoA was shown to be enriched in responders, most prominently in patients who would achieve sustained remission. Remarkably, when sustained remission patients and relapsers were subdivided in placebo and donor feces recipients, only those placebo patients who at baseline had a microbiota close to a Clostridium IV and XIVa like signature were able to retain their response, while in donor recipients this was deter-mined by a Clostridium cluster IV and XIVa rich signature of the donor in all cases. No differences were observed in microbiota composition of mucosal biopsies between responders and non-responders.
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FMT in UC: microbiota shifts and signatures that determine success of therapy
ConclusionsIn UC patients a microbiota signature low in Clostridium clusters IV and XIVa and rich in Bacteroidetes and Proteobacteria is predictive of poor sustained response to FMT, unless they receive a successful engraftment from a Clostridium clusters IV and XIVa rich donor. This favorable signature is mainly dominated by butyrate producers and durable response is associated with a restoration of the significantly reduced butyrate production capacity at baseline compared to healthy donors.
INTRODUCTION
The prevailing hypothesis on the pathogenesis of ulcerative colitis (UC) involves either (I) an excessive immune response to the normal microbiota colonizing the gastrointestinal (GI) tract, or (II) an altered composition of the microbiota that induces a disturbed epithelial barrier function leading to a pathological mucosal immune re-sponse.1,2 Fecal Microbiota Transplantation (FMT) has been used as a clinical therapy in UC to restore the abnormal microbiota associated with the disease resulting in varying outcomes.3–6 FMT can change the fecal microbiota in UC patients, and has been shown to have beneficial effects on disease activity in several case series as well as two randomized controlled trials (RCT).6–9 How and why a “hard reset” of the GI microbiota can have beneficial effects on disease activity in UC is still elusive. Furthermore, the impact of a flare of UC itself on the microbiota composition is also poorly understood. The effects of FMT have mainly been studied for Clostridium dif-ficile infections, where patients display a largely reduced microbiota diversity, which invariably recovers towards a healthy profile after cure by FMT.10
The microbiota of patients with IBD has been characterised by a lower abundance of Firmicutes, more specifically Clostridia, Ruminococcaceae, and Lactobacillus, as well as Actinobacteria such as Bifidobacterium.11–13 While findings related to microbiota composition in Crohn’s disease (CD) seem to be more consistent, for patients suffer-ing from UC conflicting data have been reported. In UC, the aberrant GI microbiota has been linked to a decrease in short chain fatty acids (SCFAs) production, such as butyrate and propionate.14–16 These SCFAs, notably butyrate, mainly produced by species within the Firmicutes phylum, are known to downregulate proinflammatory responses in intestinal epithelial cells and are found to modulate activity of relevant sentinel cell types.17 Specifically in UC, Firmicutes species such as Roseburia hominis and Faecalibacterium prausnitzii were found in lower abundance in feces from patients as compared to healthy controls by quantitative PCR (qPCR) analysis.18 Bacteroidetes are anaerobic, bile-resistant, non-spore forming negative rods cover-ing approximately 25% of all species in the human colon.19 These bacteria ferment complex carbohydrates resulting in production of fatty acids utilized as energy by the
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host. There is a wide inter-individual variation in relative abundance and composition of Bacteroidetes, and outcomes from studies comparing the abundance in fecal samples from UC patients and healthy individuals are conflicting. Nevertheless, strains of Bacteroides vulgatus and enterogenic Bacteroides fragilis (ETBF, causing peritonitis or appendicitis) have been implicated in IBD but the mechanism of action is not yet fully understood.19,20
Although signature groups in the GI microbiota have been reported in many studies for a wide variety of diseases, consistent observations are largely lacking which is in part due to the factors such as diet, antibiotics, other life style influences and host-specificity of the microbiota. Consequently, to date, there is no reference standard of a healthy microbiota.21–23 Additionally, the mucosal adherent microbiota is also unknown to play an important role, with direct crosstalk with the mucosal immune system. In healthy individuals, the mucosal microbiota composition differed from the composition in fecal samples, and in patients with Irritable Bowel Syndrome (IBS) the microbiota diversity was also found to be different at both levels.24,25 Further-more, differences between IBD patients and healthy subjects were found not only at microbiota composition but also in their metabolic profiles of the luminal content, which illustrates the complexity of the etiology of IBD.26
We recently conducted a randomized trial of FMT in mild to moderately active UC patients, which provides a unique human model to study the impact of changes in the microbiota on disease activity.9 A number of patients experienced a com-plete remission of their disease activity, which was sustained for at least 1 year. Responders were observed both in patients treated with donor feces and in patients receiving autologous infusion, which indicates that signatures need to be identified for responders and non-responders in both groups.The microbial signatures and changes thereof of this subgroup of patients that sustained remission, as compared to those that did not respond initially or relapsed within one year, is the focus of this study. Hence, we aim to unravel how patients can benefit from specific bacterial groups, as well as whether specific microbial signatures can predict sustained response to therapy.
MATERIAL AND METHODS
Study design, subjects and samplingPatients with mild to moderately active UC and healthy donors participated in a randomized placebo-controlled study, the TURN trial, comparing the efficacy of two duodenal infusions at a three week interval of fecal homogenates after bowel preparation from a healthy donor (FMT-D) or autologous feces (FMT-A) as placebo.9 The complete procedure for both FMT-A and FMT-D preparation and infusion was
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FMT in UC: microbiota shifts and signatures that determine success of therapy
described previously.9 Patients who achieved the primary endpoint of the trial (clini-cal remission; defined by a Simple Clinical Colitis Activity Index (SCCAI) ≤2 together with a decrement of ≥ 1 point in Mayo Endoscopic Score from baseline on repeat sigmoidoscopy at week 12) were called ‘responders’ and patients who did not achieve the primary endpoint were called ‘non-responders’. Patients were followed up to three years after inclusion and were requested to fill out an SCCAI and a questionnaire on medication use every two months. Sustained remission at one year (or more) after inclusion was defined as clinical remission (SCCAI ≤4) without the need for rescue therapy.27 Relapse was defined as a SCCAI >5 and or need for rescue therapy during follow-up. Patients who did not achieve the primary endpoint maintained the ‘non-responders’ classification during follow-up. Healthy subjects who participated in the trial as donors underwent extensive screening on bacterial, viral and parasitic stool pathogens and were classified as ‘donors’.Fecal samples were collected as described previously at baseline (t0), week 6 (t6w), week 12 (t12w) and 1 year or more (t≥1y) follow-up (FU) after FMT from all patients and were subsequently used for microbiota profiling9. Donor samples were collected at baseline (at screening of the donor) or on the day of fecal donation. If a donor donated twice for the same patient, only one fecal sample was collected for assess-ment of the microbiota composition.9 To investigate whether the impact of FMT on the mucosal adherent microbiota, biopsies from the rectum were collected from UC patients during the baseline, week 6 and week 12 by flexible sigmoidoscopy after preparation with a sodium phosphate enema. Biopsies were placed in sterile tubes immediately on the endoscopy room, snap frozen in liquid nitrogen and stored at -80°C.
Microbiota profiling and Butyryl-coenzyme A (CoA)-CoA transferase assessmentDNA was isolated from samples as previously described.28,29 Isolated DNA was used for profiling of the microbiota using the Human Intestinal Tract Chip (HITChip), a phylogenetic microarray containing a duplicated set of over 5000 probes based on 16S rRNA gene sequences of over 1140 intestinal bacterial phylotypes.30
The total bacterial number in samples was quantified by qPCR using 0.5ng of fecal DNA and universal bacterial primers targeting the 16S rRNA gene.28,31 Primer concen-tration in each reaction was 0.2μM. The thermal cycling conditions included an initial DNA denaturation step at 95°C for 5min followed by 40 cycles of denaturation at 95°C for 20s, annealing at 50°C for 20s, extension at 72°C for 30s, and an additional incubation step at 82°C for 30s. The fraction of butyrate producers was determined by qPCR targeting the Butyryl-coenzyme A (CoA)-CoA transferase (ButCoA) gene using 25ng of template DNA and 0.5μM primer concentration in each reaction as
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previously described.32 qPCR amplifications were done in triplicate using MX3005P Real-Time PCR System (Stratagene, La Jolla, CA, USA) in a volume of 25μl. Each reaction was amplified by using 5μl of HOT FIREPol EvaGreen qPCR Mix Plus, no ROX (Solis BioDyne, Tartu, Estonia).
Data analysis and statisticsDemographic data were collected for all subjects included in the study, and expressed as the mean and 95% confidence interval (CI) of the mean or when they were not normally distributed, the median and interquartile range (IQR) was given. Differences between study groups (in both fecal and biopsy samples) were calculated by T-test (for parametric data) and Mann-Whitney test (for non-parametric unpaired data). Three or more group-wise comparisons were performed by Kruskal-Wallis testing at different time points. For selected comparisons patients considered as responders at 12 weeks were subdivided into those with sustained remission or relapse at 1 (or more) year FU, and donors who donated for responders were selected as “healthy controls”. P-values were corrected for false discovery rate (FDR) to accommodate for multiple testing where necessary.33 Statistical analyses were performed using SPSS v. 22.0 software (Chicago, IL), R version 3.2.0 and Canoco5.34
Redundancy analysis (RDA) was used to determine associations between the mi-crobiota composition of samples (130 genus-like groups included in the HITChip) and host variables (or explanatory variables) including age, gender, disease duration, site of disease, use of medication, randomization (FMT-D or FMT-A), time, subject (donor or patient), response and sustained remission (or relapse) at ≥1 year FU. Significance of the explanatory variables was assessed by Monte Carlo permutation testing (MCPT) as implemented in the Canoco 5 software.Pearson product-moment correlations were calculated to establish similarity be-tween samples over time (moving window), or to their donors and baseline samples. Diversity of the microbiota was calculated by the Shannon-index using the oligo-nucleotide level of the HITChip, and compared between groups by T-test.
RESULTS
From UC patients, a total of 98 fecal samples collected at baseline, week 6 and week 12 were used for microbiota profiling and ButCoA gene quantification. Another 27 fecal samples were collected at 1-3 years after inclusion. Ten fecal samples from donors who donated for patients who responded at week 12, were selected as healthy control samples.
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FMT in UC: microbiota shifts and signatures that determine success of therapy
Forty-eight rectal biopsies were available from UC patients collected at baseline, week 6 and week 12 and were used for microbiota profiling. Study subjects charac-teristics are shown in Table 1.
Table 1. Baseline characteristics of Donors, Non-responders and Responders.
Donors*n= 6
Non-respondersn= 22
Respondersn= 12
p-value
Median age, years (IQR) 26.7 (25.5- 36.2) 42.0 (28.5- 48.5) 44 (36.0- 58.5) 0.03^
Male sex, n (%) 5 (83.3) 11 (45.8) 5 (41.6) 0.26
Median disease duration, years (range) - 8.0 (1-27) 11.5 (0-27) 0.25
Extent of disease, n (%)
-
1.0
E1, proctitis 0 (0) 0 (0)
E2, left-sided 12 (50.0) 6 (50.0)
E3, pancolitis 12 (50.0) 6 (50.0)
Concomitant drug treatment n, (%)
-
Mesalamine oral 16 (66.7) 8 (66.7) 1.0
Mesalamine/ corticosteroid rectal 5 (20.8) 4 (33.3) 0.69
Immunosuppressants 7 (29.2) 4 (33.3) 1.0
Systemic corticosteroids <10 mg/day 4 (16.7) 3 (25.0) 0.66
Median SCCAI score at inclusion (range)
- 8 (4-11) 9 (4-11) 0.77
Mayo endoscopic score at inclusion, n (%)
Mayo 1 3 (12.5) 2 (16.7) 0.32
Mayo 2 13 (54.2) 9 (75.0)
Mayo 3 8 (33.3) 1 (8.3)
Site of disease at inclusion, n (%)
-Rectum only 3 (12.5) 1 (8.3) 0.03
Left side of colon 15 (62.5) 10 (83.3)
Proximal to the splenic flexure 16 (66.7) 1 (8.3)
Allocated to FMT-D, n (%) - 10 (41.7) 7 (58.3) 0.72
Clinical outcome at 1 year, n (%)
-
- -
sustained remission 6 (50.0)
relapse 6 (50.0)
NOTE. *Donors from responders were selected as healthy controls for comparisons. FMT: Fecal Microbiota Transplantation. FTM-D: FMT with donor feces. SCCAI: Simple Clinical Colitis Activity Index. Extent of disease was scored according to the Montreal classification. The Mayo endoscopic score was assessed at the baseline sigmoidoscopy. FU: Follow-up. ^Donors were significantly younger than non-responders (p= 0.049) and responders (p=0.009). There was no significant difference in age between non-responders and responders (p=0.23).
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Shifts in microbiota composition after FMTProfiling of the intestinal microbiota at the genus-like level (130 groups) revealed sig-nificant differences between healthy donors, non-responders and responders, both at baseline (t0) and 12 weeks after treatment (Fig. 1, Suppl. Table 1). At baseline, non-responders showed reduced amounts of groups from the Clostridium cluster XIVa, and significantly higher levels of groups belonging to the Bacteroidetes as compared to donors, with differences ranging from 1.2 to over 11 fold higher in the
Non Responders Rel SustRem Donors Responders -4 -2 0 2 4
Row Z-Score
Color Key
Coprobacillus catenaformis et rel. Ruminococcus obeum et rel. Ruminococcus gnavus et rel. Outgrouping clostridium cluster XIVa Lachnospira pectinoschiza et rel. Eubacterium ventriosum et rel. Eubacterium rectale et rel. Eubacterium hallii et rel. Dorea formicigenerans et rel. Coprococcus eutactus et rel. Clostridium sphenoides et rel. Clostridium nexile et rel. Bryantella formatexigens et rel. Anaerostipes caccae et rel. Peptostreptococcus micros et rel. Peptostreptococcus anaerobius et rel. Veillonella Uncultured Selenomonadaceae Enterococcus Sutterella wadsworthia et rel. Serratia Escherichia coli et rel. Burkholderia Tannerella et rel. Prevotella tannerae et rel. Prevotella ruminicola et rel. Parabacteroides distasonis et rel. Bacteroides vulgatus et rel. Bacteroides stercoris et rel. Bacteroides splachnicus et rel. Bacteroides ovatus et rel. Bacteroides fragilis et rel.
*
* * * *
*
* * *
Coprobacillus catenaformis et rel. Outgrouping Clostridium cluster XIVa Clostridium sphenoides et rel. Clostridium nexile et rel. Veillonella Uncultured Selenomonadaceae Sutterella wadsworthia et rel. Tannerella et rel. Prevotella tannerae et rel. Prevotella ruminicola et rel. Bacteroides vulgatus et rel. Bacteroides stercoris et rel. Bacteroides splachnicus et rel. Bacteroides ovatus et rel. Bacteroides intestinalis et rel. Bacteroides fragilis et rel.
*
Non Responders Rel SustRem Donors Responders
A
B
Figure 1. Kruskal-Wallis comparison of the 130 genus-like groups included in the HITChip of donors, non-responders and responders, the latter subdivided into patients that relapse (Rel) or sustain re-mission (SustRem) at 1 year follow up. Shown significant different groups with patients at A: time t0 (p<0.05; FDR<0.3) and B: t12 weeks (p<0.05; FDR<0.2). Representation of abundance of the differ-ent bacterial groups range from less (blue) to more (red) abundant. Red and blue squares highlight genus-like groups in patients samples either enriched or at lower levels respectively. *Marks genus-like groups containing known butyrate producing bacteria.
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FMT in UC: microbiota shifts and signatures that determine success of therapy
case of Bacteroides vulgatus and related species. Remarkably, this pattern was also observed when 12-week responders were stratified into patients that at ≥1 year FU relapsed or those with sustained remission, with some marked differences between these. Patients that relapsed showed similar profiles to non-responders at baseline, with high levels of Bacteroidetes showing fold differences when compared to donors ranging from 1.2 to 17 times higher, being the highest for Bacteroides vulgatus and related species. Patients that sustained remission showed profiles more similar to those of healthy donors.After treatment (t12w), differences within the Bacteroidetes remain with all patients showing high levels as compared to donors (Fig.1). Enterococcus species, which were found 3-fold higher compared to donors in patients’ samples at t0, were de-creased in responders (levels similar to donors) and increased in non-responders (7.5-fold higher) after therapy. Furthermore, and opposite to profiles of responders, non-responders showed significant lower levels of bacteria belonging to the Clostridium cluster XIVa, including many known butyrate producers. Responders showed levels similar to those in donors. Proteobacteria, including potential pathogenic bacteria, are also increased in patients’ samples, being relatively higher in non-responders as compared to responders. Separation of responders into future relapse and sustained remission revealed significant differences mostly in the Prevotella melaninogenica et rel. and Prevotella oralis and related species (p<0.05; FDR<0.15), found at over 10-fold higher in responders with sustained remission but also in healthy donors.
The mucosal microbiota composition after FMTSimilar comparisons were performed at the mucosal biopsy level at baseline and t12 weeks between responder and non-responder samples, but no significant differ-ences between the study groups were found (FDR>0.8) (Suppl. Table 2).The microbiota composition of feces and biopsies was also compared, and differ-ences between samples were highly reduced after therapy, which could indicate that FMT also had an effect on the mucosal recolonization (Suppl. Table 3).
Microbial signatures associated with short- and long-term responseRedundancy analysis showed an almost complete separation of patients with sus-tained remission and those that relapsed at the ≥1-year FU (Fig. 2A, FDR<0.05). Samples at baseline (t0) of most patients, as well as those from donors from patients that relapsed (lower right quadrant) were positively associated with the phylum Proteobacteria (including Escherichia coli or Aeromonas among other potentially pathogenic and pro-inflammatory bacteria). Furthermore, these samples were highly associated with Ruminococcus gnavus (Fig. 2B). Of note, Ruminococcus gnavus in donors of patients that relapse (Fig. 2D.) was a 3.8 fold higher than in those of pa-
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tients that sustained remission (2.34%±2.53 and 0.61%±0.25 respectively, p=0.08) (Fig. 2C), indicative of a signature donor bacterial group prone to failure of FMT.Samples from patients that relapse were also positively associated with groups from the Bacteroidetes phylum (including B.vulgatus and B.fragilis among others). Patients with sustained remission at ≥1-year FU as well as healthy donors were found mostly associated to the Clostridium clusters IV (including Faecalibacterium prausnitzii and related species among others) and cluster XIVa (including E.hallii, Roseburia intestinalis or Butyrivibrio crossotus and related species among others), all known to contain butyrate producing bacteria.These bacterial associations are of importance also when looking at the effect of the treatment (FMT-D and FMT-A, Fig. 2A). For the FMT-A treatment to be successful
B A
C D
Donor
t0
t12w
t≥1y
FMT-D
t0
t12w
t≥1y
FMT-A -1.0 1.0
9.33%
-1.0 1.0
-1.0 1.0
-1.0 1.0
9.33%
10.26% 10.26%
p6
p6
p10
p10
p6
p10
Bacilli Clostridium clusters IV & XIVa
p3
p3
p8
p8
p12
p12
p3
p8*
p8
p12
Bacteroidetes
Proteobacteria
Dp4
p4 p4
p7
p7
p9
p9
p11
p11
Dp11-I
Dp11-II
Dp7-II Dp7-I
Dp9-I
p4
p7 p9 p11 Dp9-II
Dp2 Dp1
p1
p1
p2
p2
p5**
p5 Dp5
p1
p2
Sustained remission Relapse FM
T-A
FM
T-D
I III
II
IV Bacilli Clostridium clusters IV & XIVa
Bacteroidetes
Proteobacteria
Figure 2A. Redundancy analysis (RDA) of samples at t0, t12 weeks and t1 year of FMT-A with sus-tained remission (A) and relapse (B) and FMT-D with sustained remission (C) and relapse (D) and their corresponding donors (of first or second FMT, shown as –I or –II). Grey arrows represent the 65 best fitting genus-like bacterial groups, shown grouped into phyla (detailed information in Fig. 2B). 20% of the variation in the dataset is explained in the first two axes. *Patient 8 includes 2 samples after 1 year. **≥1 year sample from Patient 5 did not fulfil quality control criteria of the HITChip microarray.
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FMT in UC: microbiota shifts and signatures that determine success of therapy
at the ≥1-year FU, baseline samples need to be closer to the “sustained remission” section (e.g. patients 6 and 10). This is however not the case for the FMT-D treat-ment, where patients that show a more disturbed baseline microbiota (i.e. patients 7 and 11) can be recovered after treatment when transplanted with a healthy donor profile. However, patients that received FMT from a donor in the “relapse” section (i.e. patients 1, 2, or 5) failed to sustain remission. This same holds for all the FMT-A patients with baseline samples in the “relapse” section.Table 2 demonstrates the clinical outcome and use of concomittant medication for Non-responders and Responders at 1 year Follow-up. Explanatory variables such as time (more specifically ≥1 year samples), sustained remission and medications “category 1” (i.e. mesalamine, low dose steroids or thiopurines used from week 0 until week 12) and “category 2” (medication 1 including high dose of steroids or anti-TNF at ≥1 year FU) did impact the microbiota composition as calculated by
Associations
Donor
Relapse
SustRem
I III
II
IV
Bacilli Clostridium clusters IV & XIVa
Bacteroidetes Proteobacteria
I III Roseburia intestinalis et rel.* Bacteroides uniformis et rel. Clostridium colinum et rel. Parabacteroides distasonis et rel. Lachnospira pectinoschiza et rel.* Bacteroides ovatus et rel. Ruminococcus lactaris et rel. Bacteroides plebeius et rel. Wissella et rel. Tannerella et rel. Prevotella ruminicola et rel. Prevotella tannerae et rel. Papillibacter cinnamivorans et rel. Bacteroides vulgatus et rel. Allistipes et rel.* Bacteroides fragilis et rel. Eubacterium siraeum et rel. Bacteroides intestinalis et rel. Subdoligranulum variable et rel. Bacteroides stercoris et rel. Streptococcus intermedius et rel. Bacteroides splachnicus et rel. Aerococcus Clostridium stercorarium et rel. Eubacterium biforme et rel. Sutterella wadsworthia et rel. Veillonella Aeromonas Faecalibacterium prausnitzii et rel.* Anaerotruncus colihominis et rel.
Anaerostipes caccae et rel.* Haemophilus Clostridium difficile et rel.* Clostridium symbiosum et rel.* Streptococcus mitis et rel. Aneurinibacillus Butyrivibrio crossotus et rel.* Uncultured Selenomonadaceae Uncultured Clostridiales I Klebisiella pneumoniae et rel. Actinomycetaceae Bulleidia moorei et rel. Anaerofustis Dialister Lactobacillus plantarum et rel. Oceanospirillum Uncultured Mollicutes Bilophila et rel. Leminorella Peptostreptococcus micros et rel. Ruminococcus callidus et rel. Gemella Eubacterium hallii et rel.* Escherichia coli et rel.
Streptococcus bovis et rel. Peptostreptococcus anaerobius et rel.
Prevotella oralis et rel. Eggerthella lenta et rel. Prevotella melaninogenica et rel. Dorea formicigenerans et rel.
II IV Lactobacillus salivarius et rel. Clostridium ramosum et rel. Megamonas hypermegale et rel. Eubacterium ventriosum et rel.* Ruminococcus gnavus et rel.
Figure 2B. Grey arrows represent the 65 best fitting genus-like bacterial groups, shown grouped into phyla (detailed information of bacterial groups associated to quadrants I-IV in table). Crosses indicate association of explanatory variable “Sustained Remission” (which includes groups “relapse”, “sustained remission” and “donors”) with samples and bacterial groups. *Groups including known butyrate producing bacteria.
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MCPT (p<0.05, FDR<0.1). However, the impact of medication correlated differently as that of time, indicating they did not determine the success or failure of remission.
Stability of the fecal microbiota after FMTStability of the fecal microbiota was assessed by moving window correlation of sam-ples in time: from baseline to ≥1-year FU, both for responders and non-responders separated into the treatment groups (i.e. FMT-D and FMT-A) (Fig. 3). Stability over time in the FMT-A group was similar for responders and non-responders (Fig.3 A and B). This was not the case in the FMT-D group (Fig. 3, C and D), where response led to a significantly more stable microbiota in responders in the period after treatment (between 6 and 12 weeks, p=0.02) than non-responders, with average similarities of r2=0.91±0.06 and 0.81±0.09 respectively. When comparing treatments (FMT-D and FMT-A), the similarity of samples at 6 weeks after treatment (t6w) with the baseline sample (t0) was significantly lower (both in responders and non-responders) in patients treated with donor feces. This finding suggests a general engraftment of the donor microbiota in the FMT-D group, which led to a different composition than the one prior to the therapy.After therapy (t12 weeks), responders in the FMT-D group showed a significantly higher similarity to their donors than non-responders (r2=0.79±0.07 and 0.70±0.09 respectively, p=0.01). Furthermore, comparison of samples at 12 weeks and ≥1 year after treatment with baseline samples (at t0) revealed that non-responders returned to a profile more similar to their original microbiota after 1 year of treatment (Fig. 4).In addition, and as previously observed, in this study the diversity of the microbiota in UC patients was similar to that observed for healthy donors.9 Stratification of
Table 2. Clinical outcome and medication use of Non-responders and Responders at 1 year Follow-up
Non-respondersn= 22
Responders
n= 12
p-value
Allocated to FMT-D, n (%) 10 (41.7) 7 (58.3) 0.72
Clinical outcome at 1 year, n (%)
- -Sustained remission 6 (50.0)
Relapse 6 (50.0)
Concomitant drug treatment at 1 year FU, n (%)
No medication, Mesalamine oral, Mesalamine/ corticosteroid rectal
13 (54.2) 7 (58.3) 0.81
Immunosuppressants and/ or steroids <10mg/day 7 (29.2) 4 (33.3) 1.0
Systemic corticosteroids >10 mg/day, Anti-TNF 4 (16.7) 1 (8.3) 0.65
NOTE. FMT: Fecal Microbiota Transplantation. FTM-D: FMT with donor feces. FU: Follow-up. Anti-TNF: anti- Tumor necrosis factor.
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FMT in UC: microbiota shifts and signatures that determine success of therapy
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Chapter 7
148
Tab
le 3
. R
elat
ive
abun
danc
e (±
sta
ndar
d de
viat
ion,
SD
) of
sig
nific
ant
diffe
rent
gro
ups
(p<
0.05
, FD
R<
0.3)
bet
wee
n re
spon
ders
and
non
-res
pond
ers
at t
12
wee
ks. “
Fold
” sh
ows
rela
tive
diffe
renc
es in
abu
ndan
ce b
etw
een
resp
onde
rs/n
on-r
espo
nder
s. *
Gro
ups
incl
udin
g kn
own
buty
rate
pro
duci
ng b
acte
ria.
Rel
ativ
e ab
un
dan
ce ±
SD
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lum
/Cla
ssG
enu
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vel
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n-r
esp
on
der
sR
esp
on
der
sFo
ldp
.val
ue
FDR
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strid
ium
clu
ster
XI
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strid
ium
clu
ster
X
IVa
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ostr
epto
cocc
us a
naer
obiu
s et
rel
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081
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0.00
20.
090.
0078
0.11
33
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naer
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es c
acca
e et
rel
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387
±1.
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0±
1.46
11.
460.
0219
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36
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lost
ridiu
m n
exile
et
rel.
0.59
9±
0.42
71.
174
±0.
689
1.96
0.00
410.
0766
Clo
strid
ium
sph
enoi
des
et r
el.
0.66
8±
0.36
61.
391
±0.
875
2.08
0.00
270.
0710
*C
lost
ridiu
m s
ymbi
osum
et
rel.
1.47
7±
0.77
32.
910
±2.
595
1.97
0.02
990.
2775
*C
opro
cocc
us e
utac
tus
et r
el.
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9±
2.90
87.
954
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932
2.93
0.00
150.
0659
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ea fo
rmic
igen
eran
s et
rel
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761
±1.
044
3.03
1±
0.84
01.
720.
0005
0.03
19
*E
ubac
teriu
m h
allii
et
rel.
0.61
1±
0.55
40.
917
±0.
422
1.50
0.00
470.
0766
*E
ubac
teriu
m v
entr
iosu
m e
t re
l.0.
446
±0.
471
1.86
3±
2.96
74.
180.
0219
0.24
36
*La
chno
spira
pec
tinos
chiz
a et
rel
.1.
416
±1.
036
3.70
4±
2.60
02.
620.
0020
0.06
65
Out
grou
ping
Clo
strid
ium
clu
ster
XIV
a0.
658
±0.
652
2.48
3±
3.16
53.
770.
0003
0.03
19
Rum
inoc
occu
s gn
avus
et
rel.
0.54
8±
0.85
60.
669
±0.
525
1.22
0.02
440.
2436
Rum
inoc
occu
s la
ctar
is e
t re
l.0.
204
±0.
289
0.46
0±
0.53
32.
250.
0244
0.24
36
*R
umin
ococ
cus
obeu
m e
t re
l.7.
155
±7.
532
10.9
34±
4.32
31.
530.
0041
0.07
66
149
FMT in UC: microbiota shifts and signatures that determine success of therapy
t0 t12w
6.7 fold
0.00E+00
2.00E-05
4.00E-05
6.00E-05
8.00E-05
1.00E-04
1.20E-04
1.40E-04
1.60E-04
Bu
tCo
A/1
6S r
RN
A
* p<0.05
Figure 5.A. qPCR of Butyryl CoA (normalized to 16S rRNA gene copies) of donor samples and patient samples at baseline (t0) and 12 weeks after treatment (t12w). Patients are divided into non-responders (NR), and responders, subdivided into those that relapse (R-Rel) or sustain remission (R-SustRem) at 1 year follow up. Levels of Butyryl CoA were significantly lower in patients (non-responders and responders) at t0 and in non-responders at t12. Levels of ButCoA in responder samples with sustained remission at 1 year increase by 6.7 fold from t0 to t12 weeks.
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Chapter 7
150
samples from Responders into relapse or sustained remission calculated at the 1-3 year FU revealed no significant differences between these groups in time, between them or with the non-responders.
Association of butyrate with responseA variety of genus-like groups associated with butyrate production were significantly higher in responders as compared to non-responders (Table 3, p<0.05, FDR<0.3). Hence, the effect of the treatment on the butyrate metabolic capacity of the micro-biota was also assessed by qPCR of ButCoA. Samples from donors and patients before and after treatment revealed significantly lower levels of this gene in UC pa-tients as compared to healthy donors (Fig. 5A, p<0.05). These levels were restored after therapy, and increased in patients that responded to the treatment, particularly in those that stayed in remission at ≥1 year FU. In these patients, gene counts of ButCoA were found increased by 6.7-fold. When comparing the relative abundance (%) of genus-like groups that include known butyrate producing bacteria in feces, significantly higher levels were found in responders compared to non-responders at baseline in donors compared to non-responders at baseline and week 12 (Fig. 5B-I). For biopsy samples, responders were significantly higher abundant in butyrate producing bacteria as compared to non-responders (Fig. 5B-II).
DISCUSSION
Our previous findings showed that the fecal microbiota altered in composition after FMT treatment.9 Remarkably, these findings were observed in patients receiving the microbial fraction of donor feces used for FMT, but also in patients who got their own microbes infused, although microbiota shifts were different. While patients who responded to FMT-A shifted in the direction associated with an increase in Bacilli, Proteobacteria and Bacteroidetes, patients who responded to FMT-D showed an association with Clostridium clusters IV, XIVa, and XVIII, as well as a reduction in Bacteroidetes. In the current study, analyses of the baseline microbiota revealed differences in patients non responsive to FMT as compared to healthy donors, mainly within the Bacteroidetes phylum. These differences were highest for the B.fragilis and B.vulgatus genus-like groups. These bacteria have been implicated in IBD; antibodies against B.vulgatus were found in patients with CD.19 Moreover, B.vulgatus was found in higher abundance in feces from ileal affected CD patients, and in UC mouse models this bacterium was shown to invade host cells and trigger and immune and pro-inflammatory responses.19,35-36 Interestingly, when separating responders samples into those that will relapse or sustain remission at 1-3 years FU, these bacteria were also found at high levels in patients that relapsed. This finding
151
FMT in UC: microbiota shifts and signatures that determine success of therapy
could indicate a predisposing microbiota composition, high in groups belonging to the Bacteroidetes in UC patients at baseline for refractoriness to FMT on the long term.Similar signatures of response were observed at 12 weeks post therapy. Non-re-sponders showed significantly increased levels of Bacteroidetes but also of Proteo-bacteria as compared to healthy donors and responders, including potentially patho-genic bacteria such as Escherichia coli, found 2-fold enriched in non-responders as compared to donors or Enterococcus spp., over 7.5-fold increase in non-responders. Similarly to the B. vulgatus group, several studies involve E. coli with an abnormal immune and pro-inflammatory response in IBD.37 Furthermore, proteases secreted from these species in fecal supernatants from UC patients have been shown to induce permeability in colonic cells, suggesting this as a possible pathogenic mecha-nism in IBD.38,39
After therapy, bacteria mainly from the Clostridium cluster XIVa normalized in re-sponders’ samples (to levels similar as to those found in healthy donors) as compared to non-responders. These groups were the main drivers of difference between re-sponders and non-responders after therapy. Among these, we found species related to Anaerostipes caccae, Coprococcus eutactus or Eubacterium rectale along with other bacteria known to be involved in butyrate production. Quantification of the But-CoA gene in donors and patients before and after therapy suggested an increase of this gene after therapy in patients that responded, to levels similar to those observed for healthy donors. Furthermore, when these responders were separated into those that relapsed or sustained remission at ≥ 1 year FU, we observed that the increase was mainly in those with sustained remission, although not significantly different, possibly as a result of the large variation relative to the limited number of samples. Butyrate (and other SCFAs) is known as an energy source for colonic cells and has anti-inflammatory effects.16 It has been shown to induce an anti-inflammatory re-sponse in animal models but also in clinical trials, in concentrations similar to those found in the luminal space of the intestinal tract.17,40,41
In an in vitro dynamic gut model, colonization with microbiota of UC patients re-sulted in significantly lower levels of ButCoA and butyrate producing bacteria.42 The restoration of these bacteria as well as normal levels of ButCoA by FMT suggests a key role of butyrate in the long-term remission of UC, identifying butyrate or butyrate producing bacteria as a potential treatment strategy.Furthermore, none of the pertinent changes seen in the composition of fecal samples could be replicated in mucosal biopsies. Lack of significant differences in mucosal microbiota between responders and non-responders suggests that the efficacy of FMT was mainly driven by the luminal microbiota. Furthermore, it suggests that the cross-talk between the gut-microbiota and the mucosal immune machinery is
Chapter 7
152
mediated via signalling molecules (e.g. SCFAs), rather than a direct interaction at the mucosa adherent level.At the 12-week time point, differences between responders that relapsed and those with sustained remission were mostly seen in the Prevotella group (P.melaninogenica and P.oralis and related species) and were found significantly higher in responders with sustained remission as well as in healthy donors. These groups are of special interest as they have been characterized as bimodal distributed bacteria, and transi-tions between high and low abundance states of these groups can be associated with health implications and used for diagnostic purposes.43
In this study, a longer FU period of up to 3 years after therapy allowed us to identify potential signature microbes associated with sustained remission. In responders samples we observed that, even though considered in remission after 12 weeks, patients showing enrichment in Bacteroidetes and Proteobacteria and low levels of Clostridium cluster IV and XIVa, would relapse after time. Remarkably, relapse was also positively associated with patients that were infused with a microbiota of three donors that contained 3.8 fold higher amounts of Ruminococcus gnavus and related species, a mucolytic bacterium of the commensal microbiota. Increased levels of this bacterium have been previously reported as characteristic of the aber-rant microbiota in UC (and CD) both at the mucosal and luminal level.44–46 Excessive high levels of this group could be related to a higher mucolytic activity, resulting in tissue damage. We hypothesize that high amounts of R. gnavus and related species are predictors for unsuccessful donors.The only donor in the relapse section that was associated with a positive outcome was donor-I of patient 11. However, this patient also received a second FMT from a donor in the sustained remission quadrant, suggesting that the imprint of the donor microbiota rather originated from the latter donor. Although similarity between mi-crobial profiles of donors and non-responders was higher than between donors and responders, this comparison did not attain statistical significance (r2=0.70±0.16 and 0.75±0.09 respectively, p=0.3). A larger study may allow assessment of whether high similarity to the donor prior to treatment is a risk factor for failure of FMT, and may help in choosing an optimal donor-patient match.This study has several limitations. The associations of microbiota signature and response status are still correlative. Theoretically, it may still be possible that the microbiota changes are a consequence of attenuation of inflammation rather than cause. However, the magnitude of signature changes of sustained responders to their respective successful donors in the beneficial profile quadrant as opposed to the changes and back fall of those who relapsed, who all received feces from donors that were low in Clostridium clusters IV and XIVa, is highly suggestive of a causative mechanism. Furthermore, redundancy analysis showed that medication was one of
153
FMT in UC: microbiota shifts and signatures that determine success of therapy
the significant explanatory variables affecting the microbiota profiles of UC patients. While studies showed that mesalamine and thiopurines were shown to affect the fecal microbiota, medication use did not confound the results in the microbiota analysis of the current study.47,48 Patients were on a stable dose of medication during the first twelve weeks in the trial. Despite upscaling or ceasing of medication was allowed when the primary endpoint was attained, patients in sustained remission and patients who relapsed at 1 year follow up turned out to use the same treatment as at start of the trial except for 1 patient who started anti-TNF therapy. Moreover, medication at 12 weeks and ≥ 1 year did not differ between responders and non-responders and it was unrelated to the response success.Stability analyses revealed that FMT-D treatment had a significantly different effect from FMT-A on the microbiota of UC patients, both in responders and non-respond-ers, as these showed significantly lower similarities to their baseline composition after therapy. Moreover, responders showed a higher stability in time than non-responders, especially shortly after FMT. At the ≥1 year FU it was observed that non-responders returned to their original composition, as ≥1 year samples were more similar to baseline than after therapy. This indicated a failure of the donor microbiota to retain in the GI tract of the recipient.In conclusion, in UC patients a microbiota signature low in Clostridium clusters IV and XIVa and rich in Bacteroidetes and Proteobacteria is predictive of poor sustained response to FMT, unless they receive a successful engraftment from a Clostridium clusters IV and XIVa rich donor. This favorable signature is mainly dominated by butyr-ate producers and durable response is associated with a restoration of the signifi-cantly reduced butyrate gene counts at baseline compared with healthy donors. For donors, R. gnavus can be used as a potential biomarker species for donors that fail to induce a sustained remission. These results hold promise for novel treatment strate-gies, where individual intestinal microbiotas are analyzed and therapies are personal-ized, either by a selection of key bacteria or with an appropriate donor-patient match.
AcknowledgementsWe would like to thank all laboratory technicians who performed HITChip and Butyryl CoA experiments, the trial nurses involved in the TURN trial and all participants who either received or donated feces for FMT treatments.
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FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files
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50.
331
Bac
tero
ides
ova
tus
et r
el.
0.11
8±
0.05
00.
345
±0.
457
0.80
3±
1.35
50.
117
±0.
025
0.01
60.
194
Bac
tero
ides
spl
achn
icus
et
rel.
0.13
4±
0.03
50.
171
±0.
060
0.30
8±
0.34
40.
133
±0.
024
0.00
50.
151
Bac
tero
ides
ste
rcor
is e
t re
l.0.
056
±0.
016
0.11
3±
0.09
40.
264
±0.
378
0.06
0±
0.01
20.
004
0.15
1
Bac
tero
ides
vul
gatu
s et
rel
.0.
591
±0.
424
6.50
8±
8.93
510
.391
±11
.001
2.23
6±
3.57
30.
011
0.17
5
Prev
otel
la ru
min
icol
a et
rel
.0.
016
±0.
005
0.01
9±
0.01
00.
020
±0.
005
0.01
7±
0.00
20.
016
0.19
4
Prev
otel
la t
anne
rae
et r
el.
0.04
6±
0.01
30.
139
±0.
118
0.21
8±
0.22
70.
078
±0.
054
0.00
50.
151
Tann
erel
la e
t re
l.0.
086
±0.
028
0.16
4±
0.10
00.
319
±0.
373
0.12
0±
0.04
10.
006
0.15
1
Firm
icut
esC
lost
ridiu
m
clus
ter
IXU
ncul
ture
d S
elen
omon
adac
eae
0.00
7±
0.00
20.
013
±0.
014
0.01
3±
0.00
70.
012
±0.
008
0.00
90.
175
Veill
onel
la0.
034
±0.
009
0.25
0±
0.84
20.
036
±0.
004
0.04
6±
0.02
20.
035
0.33
1
Clo
strid
ium
cl
uste
r X
IVa
Clo
strid
ium
nex
ile e
t re
l.2.
382
±2.
152
0.85
8±
0.85
31.
375
±1.
178
1.57
5±
1.27
80.
036
0.33
1
Clo
strid
ium
sph
enoi
des
et r
el.
1.96
3±
1.35
21.
025
±0.
509
1.07
3±
1.06
10.
944
±0.
327
0.03
90.
333
Out
grou
ping
Clo
strid
ium
cl
uste
r X
IVa
2.33
5±
1.80
71.
037
±1.
280
2.70
8±
3.50
31.
539
±2.
187
0.04
10.
333
Clo
strid
ium
cl
uste
r X
VIII
Cop
roba
cillu
s ca
tena
form
is
et r
el.
0.24
0±
0.44
30.
047
±0.
032
0.06
2±
0.04
90.
056
±0.
022
0.00
70.
151
Prot
eoba
cter
iaPr
oteo
bact
eria
Sut
tere
lla w
adsw
orth
ia e
t re
l.0.
087
±0.
026
0.22
3±
0.30
10.
540
±1.
037
0.10
1±
0.04
40.
006
0.15
1
Chapter 7
158
Kru
skal
Wal
lis -
12
wee
ks (
t12w
)R
elat
ive
abu
nd
ance
± S
tan
dar
d d
evia
tio
n
p.v
alu
eFD
RP
hylu
mP
hylu
m/C
lass
Gen
us-
like
leve
lD
on
or
No
n-
resp
on
der
sR
esp
on
der
s-R
elR
esp
on
der
s-S
ust
Rem
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
fra
gilis
et
rel.
0.13
7±
0.09
00.
810
±1.
392
0.34
7±
0.38
60.
259
±0.
158
0.01
80.
114
Bac
tero
ides
ova
tus
et r
el.
0.11
8±
0.05
00.
358
±0.
400
0.37
2±
0.42
20.
207
±0.
162
0.02
00.
119
Bac
tero
ides
spl
achn
icus
et
rel.
0.13
4±
0.03
50.
204
±0.
109
0.17
6±
0.06
70.
178
±0.
082
0.01
70.
111
Bac
tero
ides
ste
rcor
is e
t re
l.0.
056
±0.
016
0.14
8±
0.19
20.
117
±0.
062
0.07
9±
0.02
80.
004
0.05
4
Bac
tero
ides
vul
gatu
s et
rel
.0.
591
±0.
424
5.00
0±
6.13
76.
488
±5.
977
3.76
9±
4.40
00.
010
0.08
2
Para
bact
eroi
des
dist
ason
is
et r
el.
0.17
9±
0.11
30.
278
±0.
198
0.34
8±
0.22
80.
516
±0.
394
0.02
80.
137
Prev
otel
la ru
min
icol
a et
rel
.0.
016
±0.
005
0.02
3±
0.00
90.
016
±0.
004
0.01
9±
0.00
40.
005
0.05
6
Prev
otel
la t
anne
rae
et r
el.
0.04
6±
0.01
30.
138
±0.
117
0.15
6±
0.11
50.
132
±0.
108
0.00
80.
078
Tann
erel
la e
t re
l.0.
086
±0.
028
0.16
6±
0.09
70.
192
±0.
103
0.19
8±
0.13
80.
004
0.05
4
Firm
icut
esB
acill
iE
nter
ococ
cus
0.05
2±
0.01
90.
394
±1.
388
0.05
0±
0.01
00.
047
±0.
013
0.02
80.
137
Clo
strid
ium
cl
uste
r IX
Unc
ultu
red
Sel
enom
onad
acea
e0.
007
±0.
002
0.01
0±
0.00
80.
008
±0.
002
0.00
7±
0.00
40.
026
0.13
7
Veill
onel
la0.
034
±0.
009
0.28
0±
0.76
00.
040
±0.
017
0.03
7±
0.01
30.
012
0.09
8
Clo
strid
ium
cl
uste
r X
IPe
ptos
trep
toco
ccus
an
aero
bius
et
rel.
0.00
8±
0.00
30.
081
±0.
223
0.00
8±
0.00
30.
006
±0.
001
0.00
10.
030
Clo
strid
ium
cl
uste
r X
IIIPe
ptos
trep
toco
ccus
mic
ros
et r
el.
0.03
5±
0.01
20.
104
±0.
256
0.03
3±
0.00
90.
037
±0.
019
0.02
20.
124
Clo
strid
ium
cl
uste
r X
IVa
Ana
eros
tipes
cac
cae
et r
el.
1.30
4±
0.64
01.
387
±1.
825
2.37
8±
1.52
21.
682
±1.
445
0.04
00.
171
Bry
ante
lla fo
rmat
exig
ens
et
rel.
1.28
8±
0.68
40.
681
±0.
494
1.28
6±
0.72
40.
701
±0.
232
0.03
30.
152
Clo
strid
ium
nex
ile e
t re
l.2.
382
±2.
152
0.59
9±
0.42
70.
928
±0.
227
1.42
0±
0.92
00.
000
0.02
3
Clo
strid
ium
sph
enoi
des
et r
el.
1.96
3±
1.35
20.
668
±0.
366
1.92
9±
0.95
30.
854
±0.
287
0.00
10.
030
159
FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files
Kru
skal
Wal
lis -
12
wee
ks (
t12w
)R
elat
ive
abu
nd
ance
± S
tan
dar
d d
evia
tio
n
p.v
alu
eFD
RP
hylu
mP
hylu
m/C
lass
Gen
us-
like
leve
lD
on
or
No
n-
resp
on
der
sR
esp
on
der
s-R
elR
esp
on
der
s-S
ust
Rem
Cop
roco
ccus
eut
actu
s et
rel
.6.
924
±3.
565
2.71
9±
2.90
89.
324
±8.
002
6.58
5±
2.98
00.
001
0.03
0
Dor
ea fo
rmic
igen
eran
s et
rel
.3.
976
±2.
273
1.76
1±
1.04
43.
333
±0.
890
2.72
9±
0.73
40.
001
0.03
0
Eub
acte
rium
hal
lii e
t re
l.0.
968
±0.
710
0.61
1±
0.55
41.
015
±0.
489
0.81
9±
0.36
00.
015
0.10
9
Eub
acte
rium
rec
tale
et
rel.
1.39
9±
1.02
20.
579
±0.
352
2.02
1±
1.88
10.
893
±0.
648
0.04
60.
192
Eub
acte
rium
ven
trio
sum
et
rel.
1.64
0±
1.43
10.
446
±0.
471
3.31
9±
3.77
10.
407
±0.
251
0.00
80.
078
Lach
nosp
ira p
ectin
osch
iza
et r
el.
4.31
3±
2.46
01.
416
±1.
036
5.04
6±
3.05
52.
362
±1.
100
0.00
20.
041
Out
grou
ping
Clo
strid
ium
cl
uste
r X
IVa
2.33
5±
1.80
70.
658
±0.
652
3.88
4±
4.12
81.
081
±0.
541
0.00
00.
019
Rum
inoc
occu
s gn
avus
et
rel.
1.13
3±
1.47
30.
548
±0.
856
0.93
8±
0.63
60.
400
±0.
170
0.01
40.
108
Rum
inoc
occu
s ob
eum
et
rel.
15.6
54±
6.90
67.
155
±7.
532
12.4
11±
4.60
79.
458
±3.
827
0.00
30.
049
Clo
strid
ium
cl
uste
r X
VIII
Cop
roba
cillu
s ca
tena
form
is
et r
el.
0.24
0±
0.44
30.
056
±0.
048
0.21
0±
0.32
10.
047
±0.
014
0.01
60.
111
Prot
eoba
cter
iaPr
oteo
bact
eria
Bur
khol
deria
0.00
9±
0.00
30.
013
±0.
009
0.00
9±
0.00
20.
018
±0.
010
0.03
70.
166
Esc
heric
hia
coli
et r
el.
0.09
9±
0.03
90.
205
±0.
346
0.23
7±
0.38
00.
081
±0.
020
0.04
70.
192
Ser
ratia
0.00
8±
0.00
30.
013
±0.
016
0.00
8±
0.00
20.
006
±0.
001
0.02
70.
137
Sut
tere
lla w
adsw
orth
ia e
t re
l.0.
087
±0.
026
0.26
3±
0.34
20.
148
±0.
104
0.29
1±
0.16
70.
009
0.08
2
Chapter 7
160
Su
pp
lem
enta
ry Ta
ble
2. M
ann-
Whi
tney
com
paris
on o
f the
130
gen
us-li
ke g
roup
s in
clud
ed in
the
HIT
Chi
p be
twee
n re
spon
ders
and
non
-res
pond
ers
sam
ples
at
bas
elin
e (t
0) a
nd 1
2 w
eeks
(t12
w).
Sho
wn
rela
tive
abun
danc
e (±
Sta
ndar
d de
viat
ion)
of s
elec
ted
grou
ps (p
<0.
05; F
DR
>0.
8). R
espo
nder
s ar
e se
para
ted
into
th
ose
that
rel
apse
(Res
pond
ers-
Rel
) or
sust
ain
rem
issi
on (R
espo
nder
s-S
ustR
em) a
t t≥
1y F
U.
Sho
wn
grou
ps w
ith p
<0.
05; F
DR
>0.
8.
Man
n W
hit
ney
- B
asel
ine
(t0)
Rel
ativ
e ab
un
dan
ce ±
SD
p.v
alu
eFD
RP
hylu
mP
hylu
m/C
lass
Gen
us-
like
leve
lN
on
-res
po
nd
ers
Res
po
nd
ers-
Rel
Res
po
nd
ers-
Su
stR
em
Act
inob
acte
riaA
ctin
obac
teria
Prop
ioni
bact
eriu
m0.
591
±1.
333
0.45
8±
0.45
90.
632
±0.
772
0.04
40.
813
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
inte
stin
alis
et
rel.
0.06
7±
0.13
70.
134
±0.
096
0.04
0±
0.02
80.
039
0.81
3
Firm
icut
esC
lost
ridiu
m c
lust
er X
IVa
Bry
ante
lla fo
rmat
exig
ens
et r
el.
0.61
3±
0.31
20.
890
±0.
434
0.87
0±
0.48
50.
049
0.81
3
Cop
roco
ccus
eut
actu
s et
rel
.1.
620
±1.
473
3.79
2±
2.49
12.
856
±2.
817
0.03
90.
813
Clo
strid
ium
clu
ster
XV
IIIC
lost
ridiu
m r
amos
um e
t re
l.0.
161
±0.
437
0.12
7±
0.07
10.
047
±0.
020
0.02
10.
813
Man
n W
hit
ney
- 1
2 w
eeks
(t1
2w)
Rel
ativ
e ab
un
dan
ce ±
SD
p.v
alu
eFD
RP
hylu
mP
hylu
m/C
lass
Gen
us-
like
leve
lN
on
-res
po
nd
ers
Res
po
nd
ers-
Rel
Res
po
nd
ers-
Su
stR
em
Firm
icut
esC
lost
ridiu
m c
lust
er II
IC
lost
ridiu
m s
terc
orar
ium
et
rel.
0.13
9±
0.20
90.
207
±0.
106
0.14
8±
0.05
60.
033
0.86
3
Clo
strid
ium
clu
ster
XIV
aE
ubac
teriu
m v
entr
iosu
m e
t re
l.0.
206
±0.
149
1.20
4±
0.99
80.
492
±0.
486
0.03
90.
863
Prot
eoba
cter
iaPr
oteo
bact
eria
Kle
bisi
ella
pne
umon
iae
et r
el.
0.04
8±
0.02
00.
080
±0.
027
0.06
8±
0.02
20.
023
0.86
3
Xan
thom
onad
acea
e0.
019
±0.
010
0.03
7±
0.00
70.
027
±0.
014
0.03
30.
863
161
FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files
Su
pp
lem
enta
ry A
pp
end
ix Ta
ble
3.1
. Man
n-W
hitn
ey c
ompa
rison
of f
ecal
(F) a
nd b
iops
y (B
) mic
robi
ota
from
non
-res
pond
ers
at th
e ge
nus-
like
leve
l of t
he 1
30
grou
ps in
clud
ed in
the
HIT
Chi
p. A
vera
ge r
elat
ive
abun
danc
e±S
D f
or s
igni
fican
t di
ffere
nt g
roup
s at
bas
elin
e (t
0) (
p< 0
.05
and
FDR
<0.
3).
Hig
hlig
hted
in g
rey
are
grou
ps in
hig
her
abun
danc
e in
bio
psie
s; n
on h
ighl
ight
ed g
roup
s ar
e in
hig
her
abun
danc
e in
feca
l sam
ples
. N: n
on-r
espo
nder
s; R
: res
pond
ers;
FD
R: F
alse
D
isco
very
Rat
e; S
D: s
tand
ard
devi
atio
n
Phy
lum
Phy
lum
/Cla
ssG
enu
s-lik
e g
rou
p
Rel
ativ
e ab
un
dan
ce±S
D
p.v
alu
eFD
RB
_t0_
NS
DF_
t0_N
SD
Act
inob
acte
riaA
ctin
obac
teria
Act
inom
ycet
acea
e0.
025
0.02
20.
014
0.00
50.
000
0.00
1
Act
inob
acte
riaA
ctin
obac
teria
Ato
pobi
um0.
015
0.00
50.
014
0.00
50.
013
0.03
2
Act
inob
acte
riaA
ctin
obac
teria
Cor
yneb
acte
rium
0.02
30.
009
0.01
90.
006
0.00
00.
001
Act
inob
acte
riaA
ctin
obac
teria
Mic
roco
ccac
eae
0.00
80.
004
0.00
60.
002
0.00
20.
008
Act
inob
acte
riaA
ctin
obac
teria
Prop
ioni
bact
eriu
m0.
591
1.33
30.
020
0.00
60.
000
0.00
0
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
inte
stin
alis
et
rel.
0.06
70.
137
0.04
00.
057
0.04
80.
092
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
ova
tus
et r
el.
0.57
30.
372
0.34
50.
457
0.00
80.
023
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
ple
beiu
s et
rel
.0.
338
0.28
40.
168
0.10
20.
030
0.05
9
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
spl
achn
icus
et
rel.
0.35
60.
258
0.17
10.
060
0.00
30.
013
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
ste
rcor
is e
t re
l.0.
195
0.14
20.
113
0.09
40.
011
0.02
8
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
uni
form
is e
t re
l.0.
685
0.74
10.
284
0.41
00.
022
0.04
8
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
vul
gatu
s et
rel
.12
.379
9.37
86.
508
8.93
50.
005
0.01
7
Bac
tero
idet
esB
acte
roid
etes
Para
bact
eroi
des
dist
ason
is e
t re
l.0.
676
0.60
60.
292
0.26
10.
019
0.04
3
Bac
tero
idet
esB
acte
roid
etes
Prev
otel
la ru
min
icol
a et
rel
.0.
025
0.01
10.
019
0.01
00.
003
0.01
2
Bac
tero
idet
esB
acte
roid
etes
Prev
otel
la t
anne
rae
et r
el.
0.31
00.
188
0.13
90.
118
0.00
10.
005
Bac
tero
idet
esB
acte
roid
etes
Tann
erel
la e
t re
l.0.
409
0.33
50.
164
0.10
00.
001
0.00
7
Cya
noba
cter
iaC
yano
bact
eria
Unc
ultu
red
Chr
ooco
ccal
es0.
008
0.00
30.
007
0.00
30.
002
0.01
2
Firm
icut
esA
ster
olep
lasm
aA
ster
olep
lasm
a et
rel
.0.
026
0.07
90.
006
0.00
20.
004
0.01
6
Firm
icut
esB
acill
iA
eroc
occu
s0.
059
0.22
00.
007
0.00
20.
001
0.00
4
Firm
icut
esB
acill
iB
acill
us0.
024
0.00
90.
020
0.00
70.
008
0.02
3
Chapter 7
162
Phy
lum
Phy
lum
/Cla
ssG
enu
s-lik
e g
rou
p
Rel
ativ
e ab
un
dan
ce±S
D
p.v
alu
eFD
RB
_t0_
NS
DF_
t0_N
SD
Firm
icut
esB
acill
iE
nter
ococ
cus
0.13
50.
278
0.17
60.
442
0.01
90.
043
Firm
icut
esB
acill
iG
ranu
licat
ella
0.03
30.
079
0.01
00.
008
0.03
70.
071
Firm
icut
esB
acill
iLa
ctob
acill
us g
asse
ri et
rel
.0.
264
0.54
60.
095
0.06
00.
019
0.04
3
Firm
icut
esB
acill
iLa
ctob
acill
us s
aliv
ariu
s et
rel
.0.
083
0.25
60.
026
0.02
60.
037
0.07
1
Firm
icut
esB
acill
iLa
ctoc
occu
s0.
044
0.09
20.
023
0.01
10.
017
0.04
3
Firm
icut
esB
acill
iS
taph
yloc
occu
s0.
035
0.05
00.
017
0.01
70.
006
0.01
9
Firm
icut
esB
acill
iW
eiss
ella
et
rel.
0.03
90.
055
0.02
40.
013
0.02
70.
057
Firm
icut
esC
lost
ridiu
m c
lust
er II
IC
lost
ridiu
m t
herm
ocel
lum
et
rel.
0.00
70.
003
0.00
60.
002
0.00
70.
022
Firm
icut
esC
lost
ridiu
m c
lust
er IX
Meg
amon
as h
yper
meg
ale
et r
el.
0.03
70.
062
0.01
90.
007
0.00
30.
012
Firm
icut
esC
lost
ridiu
m c
lust
er IX
Mits
uoke
lla m
ultia
cida
et
rel.
0.04
30.
082
0.04
40.
092
0.02
20.
048
Firm
icut
esC
lost
ridiu
m c
lust
er X
IC
lost
ridiu
m fe
lsin
eum
et
rel.
0.00
70.
003
0.00
60.
002
0.00
40.
016
Firm
icut
esC
lost
ridiu
m c
lust
er X
IIIPe
ptos
trep
toco
ccus
mic
ros
et r
el.
0.03
90.
014
0.03
40.
016
0.02
40.
051
Firm
icut
esC
lost
ridiu
m c
lust
er X
IVa
Lach
nosp
ira p
ectin
osch
iza
et r
el.
0.98
20.
650
2.44
61.
568
0.00
90.
026
Firm
icut
esC
lost
ridiu
m c
lust
er X
VE
ubac
teriu
m li
mos
um e
t re
l.0.
082
0.19
40.
030
0.03
60.
002
0.01
1
Firm
icut
esC
lost
ridiu
m c
lust
er X
VI
Bul
leid
ia m
oore
i et
rel.
0.04
20.
020
0.03
10.
014
0.01
00.
027
Firm
icut
esC
lost
ridiu
m c
lust
er X
VI
Eub
acte
rium
cyl
indr
oide
s et
rel
.0.
039
0.01
20.
033
0.01
50.
019
0.04
3
Firm
icut
esC
lost
ridiu
m c
lust
er X
VII
Cat
enib
acte
rium
mits
uoka
i et
rel.
0.04
10.
076
0.02
70.
054
0.00
10.
007
Firm
icut
esC
lost
ridiu
m c
lust
er X
VII
Lact
obac
illus
cat
enaf
orm
is e
t re
l.0.
014
0.00
50.
012
0.00
40.
030
0.05
9
Firm
icut
esC
lost
ridiu
m c
lust
er X
VIII
Clo
strid
ium
ram
osum
et
rel.
0.16
10.
437
0.03
90.
020
0.00
00.
003
Firm
icut
esC
lost
ridiu
m c
lust
er X
VIII
Cop
roba
cillu
s ca
tena
form
is e
t re
l.0.
087
0.09
20.
047
0.03
20.
003
0.01
2
Fuso
bact
eria
Fuso
bact
eria
Fuso
bact
eria
0.10
00.
150
0.05
50.
017
0.00
30.
012
Prot
eoba
cter
iaPr
oteo
bact
eria
Aer
omon
as0.
010
0.00
70.
007
0.00
20.
000
0.00
1
Prot
eoba
cter
iaPr
oteo
bact
eria
Alc
alig
enes
faec
alis
et
rel.
0.25
00.
413
0.02
60.
008
0.00
00.
000
163
FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files
Phy
lum
Phy
lum
/Cla
ssG
enu
s-lik
e g
rou
p
Rel
ativ
e ab
un
dan
ce±S
D
p.v
alu
eFD
RB
_t0_
NS
DF_
t0_N
SD
Prot
eoba
cter
iaPr
oteo
bact
eria
Ana
erob
iosp
irillu
m0.
008
0.00
40.
006
0.00
20.
000
0.00
0
Prot
eoba
cter
iaPr
oteo
bact
eria
Aqu
abac
teriu
m0.
026
0.05
20.
007
0.00
20.
000
0.00
1
Prot
eoba
cter
iaPr
oteo
bact
eria
Bilo
phila
et
rel.
0.02
40.
008
0.02
00.
006
0.01
10.
028
Prot
eoba
cter
iaPr
oteo
bact
eria
Bur
khol
deria
0.03
70.
040
0.00
90.
003
0.00
00.
000
Prot
eoba
cter
iaPr
oteo
bact
eria
Cam
pylo
bact
er0.
057
0.02
10.
048
0.01
50.
005
0.01
7
Prot
eoba
cter
iaPr
oteo
bact
eria
Des
ulfo
vibr
io e
t re
l.0.
037
0.01
30.
032
0.01
00.
011
0.02
8
Prot
eoba
cter
iaPr
oteo
bact
eria
Ent
erob
acte
r ae
roge
nes
et r
el.
0.15
60.
137
0.07
60.
030
0.00
00.
000
Prot
eoba
cter
iaPr
oteo
bact
eria
Esc
heric
hia
coli
et r
el.
0.78
71.
459
0.10
70.
068
0.00
00.
000
Prot
eoba
cter
iaPr
oteo
bact
eria
Hae
mop
hilu
s0.
043
0.09
90.
014
0.00
60.
001
0.00
4
Prot
eoba
cter
iaPr
oteo
bact
eria
Hel
icob
acte
r0.
036
0.01
30.
030
0.01
00.
003
0.01
2
Prot
eoba
cter
iaPr
oteo
bact
eria
Kle
bisi
ella
pne
umon
iae
et r
el.
0.08
90.
073
0.04
50.
021
0.00
00.
001
Prot
eoba
cter
iaPr
oteo
bact
eria
Lem
inor
ella
0.01
20.
006
0.00
70.
002
0.00
00.
003
Prot
eoba
cter
iaPr
oteo
bact
eria
Met
hylo
bact
eriu
m0.
007
0.00
30.
006
0.00
20.
009
0.02
6
Prot
eoba
cter
iaPr
oteo
bact
eria
Mor
axel
lace
ae0.
017
0.00
80.
012
0.00
40.
000
0.00
0
Prot
eoba
cter
iaPr
oteo
bact
eria
Nov
osph
ingo
bium
0.00
90.
003
0.00
80.
003
0.02
70.
057
Prot
eoba
cter
iaPr
oteo
bact
eria
Oce
anos
piril
lum
0.04
40.
043
0.02
70.
012
0.02
40.
051
Prot
eoba
cter
iaPr
oteo
bact
eria
Oxa
loba
cter
form
igen
es e
t re
l.0.
239
0.32
90.
116
0.12
70.
015
0.03
7
Prot
eoba
cter
iaPr
oteo
bact
eria
Prot
eus
et r
el.
0.07
40.
059
0.04
90.
016
0.00
00.
001
Prot
eoba
cter
iaPr
oteo
bact
eria
Pse
udom
onas
0.02
10.
027
0.01
20.
004
0.00
40.
016
Prot
eoba
cter
iaPr
oteo
bact
eria
Ser
ratia
0.09
70.
266
0.00
90.
008
0.00
00.
001
Prot
eoba
cter
iaPr
oteo
bact
eria
Sut
tere
lla w
adsw
orth
ia e
t re
l.0.
624
0.51
70.
223
0.30
10.
001
0.00
4
Prot
eoba
cter
iaPr
oteo
bact
eria
Vibr
io0.
039
0.01
40.
029
0.01
00.
000
0.00
0
Prot
eoba
cter
iaPr
oteo
bact
eria
Xan
thom
onad
acea
e0.
033
0.02
80.
015
0.00
70.
000
0.00
0
Chapter 7
164
Phy
lum
Phy
lum
/Cla
ssG
enu
s-lik
e g
rou
p
Rel
ativ
e ab
un
dan
ce±S
D
p.v
alu
eFD
RB
_t0_
NS
DF_
t0_N
SD
Prot
eoba
cter
iaPr
oteo
bact
eria
Yers
inia
et
rel.
0.03
40.
015
0.02
60.
009
0.00
00.
003
Spi
roch
aete
sS
piro
chae
tes
Bra
chys
pira
0.01
40.
005
0.01
20.
004
0.00
50.
017
Su
pp
lem
enta
ry A
pp
end
ix T
able
3.2
. M
ann-
Whi
tney
com
paris
on o
f fe
cal (
F) a
nd b
iops
y (B
) m
icro
biot
a fr
om r
espo
nder
s at
the
gen
us-li
ke le
vel o
f th
e 13
0 gr
oups
incl
uded
in t
he H
ITC
hip.
Ave
rage
rel
ativ
e ab
unda
nce±
SD
for
sig
nific
ant
diffe
rent
gro
ups
at b
asel
ine
(t0)
(p<
0.0
5 an
d FD
R<
0.3)
. H
ighl
ight
ed in
gre
y ar
e gr
oups
in h
ighe
r ab
unda
nce
in b
iops
ies;
non
hig
hlig
hted
gro
ups
are
in h
ighe
r ab
unda
nce
in fe
cal s
ampl
es. N
: non
-res
pond
ers;
R: r
espo
nder
s; F
DR
: Fal
se
Dis
cove
ry R
ate;
SD
: sta
ndar
d de
viat
ion
Phy
lum
Phy
lum
/Cla
ssG
enu
s-lik
e g
rou
p
Rel
ativ
e ab
un
dan
ce±S
D
B_t
0_R
SD
F_t0
_RS
Dp
.val
ue
FDR
Act
inob
acte
riaA
ctin
obac
teria
Act
inom
ycet
acea
e0.
021
0.01
90.
016
0.00
40.
019
0.03
7
Act
inob
acte
riaA
ctin
obac
teria
Ato
pobi
um0.
016
0.01
60.
015
0.00
30.
032
0.05
5
Act
inob
acte
riaA
ctin
obac
teria
Col
linse
lla0.
070
0.04
60.
307
0.31
50.
037
0.06
3
Act
inob
acte
riaA
ctin
obac
teria
Cor
yneb
acte
rium
0.02
50.
023
0.02
10.
003
0.00
10.
010
Act
inob
acte
riaA
ctin
obac
teria
Mic
roco
ccac
eae
0.00
80.
008
0.00
70.
001
0.00
40.
015
Act
inob
acte
riaA
ctin
obac
teria
Prop
ioni
bact
eriu
m0.
553
0.62
50.
022
0.00
40.
000
0.00
0
Bac
tero
idet
esB
acte
roid
etes
Alli
stip
es e
t re
l.1.
467
1.01
60.
669
0.64
00.
016
0.03
2
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
fra
gilis
et
rel.
0.86
70.
465
0.49
30.
863
0.00
60.
015
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
inte
stin
alis
et
rel.
0.08
30.
081
0.04
90.
100
0.00
60.
015
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
ova
tus
et r
el.
0.83
50.
571
0.46
00.
981
0.00
40.
015
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
ple
beiu
s et
rel
.0.
447
0.34
70.
204
0.28
20.
002
0.01
5
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
spl
achn
icus
et
rel.
0.30
80.
169
0.22
10.
250
0.00
40.
015
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
ste
rcor
is e
t re
l.0.
237
0.18
10.
162
0.27
60.
006
0.01
5
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
uni
form
is e
t re
l.0.
656
0.64
20.
241
0.25
80.
004
0.01
5
165
FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files
Phy
lum
Phy
lum
/Cla
ssG
enu
s-lik
e g
rou
p
Rel
ativ
e ab
un
dan
ce±S
D
B_t
0_R
SD
F_t0
_RS
Dp
.val
ue
FDR
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
vul
gatu
s et
rel
.16
.195
9.11
46.
313
8.88
60.
000
0.00
4
Bac
tero
idet
esB
acte
roid
etes
Para
bact
eroi
des
dist
ason
is e
t re
l.0.
935
0.55
00.
512
0.56
90.
013
0.02
8
Bac
tero
idet
esB
acte
roid
etes
Prev
otel
la m
elan
inog
enic
a et
rel
.10
.826
16.9
400.
685
1.01
30.
016
0.03
2
Bac
tero
idet
esB
acte
roid
etes
Prev
otel
la o
ralis
et
rel.
1.59
92.
407
0.13
30.
142
0.00
10.
010
Bac
tero
idet
esB
acte
roid
etes
Prev
otel
la ru
min
icol
a et
rel
.0.
025
0.02
10.
018
0.00
40.
002
0.01
5
Bac
tero
idet
esB
acte
roid
etes
Prev
otel
la t
anne
rae
et r
el.
0.39
50.
215
0.14
80.
173
0.00
10.
012
Bac
tero
idet
esB
acte
roid
etes
Tann
erel
la e
t re
l.0.
397
0.17
50.
220
0.27
40.
002
0.01
5
Bac
tero
idet
esB
acte
roid
etes
Unc
ultu
red
Bac
tero
idet
es0.
019
0.01
40.
013
0.00
60.
004
0.01
5
Cya
noba
cter
iaC
yano
bact
eria
Unc
ultu
red
Chr
ooco
ccal
es0.
008
0.00
80.
007
0.00
10.
019
0.03
7
Firm
icut
esA
ster
olep
lasm
aA
ster
olep
lasm
a et
rel
.0.
017
0.03
10.
007
0.00
10.
006
0.01
5
Firm
icut
esB
acill
iA
eroc
occu
s0.
008
0.00
80.
007
0.00
20.
006
0.01
5
Firm
icut
esB
acill
iA
neur
inib
acill
us0.
010
0.00
70.
009
0.00
20.
016
0.03
2
Firm
icut
esB
acill
iB
acill
us0.
026
0.02
50.
023
0.00
40.
009
0.02
1
Firm
icut
esB
acill
iLa
ctob
acill
us p
lant
arum
et
rel.
0.15
00.
134
0.13
30.
022
0.01
30.
028
Firm
icut
esB
acill
iLa
ctob
acill
us s
aliv
ariu
s et
rel
.0.
024
0.02
40.
022
0.00
30.
032
0.05
5
Firm
icut
esB
acill
iLa
ctoc
occu
s0.
027
0.02
40.
024
0.00
70.
003
0.01
5
Firm
icut
esB
acill
iS
taph
yloc
occu
s0.
049
0.08
80.
015
0.00
20.
003
0.01
5
Firm
icut
esB
acill
iW
eiss
ella
et
rel.
0.02
70.
023
0.02
60.
007
0.03
70.
063
Firm
icut
esC
lost
ridiu
m c
lust
er I
Clo
strid
ium
(sen
su s
tric
to)
0.30
30.
191
0.34
40.
282
0.02
70.
050
Firm
icut
esC
lost
ridiu
m c
lust
er II
IC
lost
ridiu
m t
herm
ocel
lum
et
rel.
0.00
80.
008
0.00
70.
001
0.00
70.
017
Firm
icut
esC
lost
ridiu
m c
lust
er IV
Ana
erot
runc
us c
olih
omin
is e
t re
l.0.
278
0.52
90.
145
0.09
90.
044
0.07
1
Firm
icut
esC
lost
ridiu
m c
lust
er IV
Eub
acte
rium
sira
eum
et
rel.
0.04
20.
032
0.03
90.
016
0.02
30.
043
Firm
icut
esC
lost
ridiu
m c
lust
er IV
Faec
alib
acte
rium
pra
usni
tzii
et r
el.
10.4
592.
724
8.30
25.
697
0.00
30.
015
Chapter 7
166
Phy
lum
Phy
lum
/Cla
ssG
enu
s-lik
e g
rou
p
Rel
ativ
e ab
un
dan
ce±S
D
B_t
0_R
SD
F_t0
_RS
Dp
.val
ue
FDR
Firm
icut
esC
lost
ridiu
m c
lust
er IX
Meg
amon
as h
yper
meg
ale
et r
el.
0.02
50.
024
0.02
30.
008
0.02
30.
043
Firm
icut
esC
lost
ridiu
m c
lust
er IX
Mits
uoke
lla m
ultia
cida
et
rel.
0.02
60.
025
0.02
10.
003
0.00
40.
015
Firm
icut
esC
lost
ridiu
m c
lust
er IX
Pept
ococ
cus
nige
r et
rel
.0.
038
0.03
10.
046
0.04
90.
044
0.07
1
Firm
icut
esC
lost
ridiu
m c
lust
er IX
Pha
scol
arct
obac
teriu
m fa
eciu
m e
t re
l.0.
193
0.42
50.
051
0.01
50.
011
0.02
5
Firm
icut
esC
lost
ridiu
m c
lust
er X
IC
lost
ridiu
m d
iffici
le e
t re
l.0.
859
0.57
41.
236
2.08
80.
044
0.07
1
Firm
icut
esC
lost
ridiu
m c
lust
er X
IC
lost
ridiu
m fe
lsin
eum
et
rel.
0.00
80.
008
0.00
70.
001
0.00
40.
015
Firm
icut
esC
lost
ridiu
m c
lust
er X
IVa
Clo
strid
ium
sph
enoi
des
et r
el.
1.02
90.
449
1.00
80.
752
0.03
20.
055
Firm
icut
esC
lost
ridiu
m c
lust
er X
VE
ubac
teriu
m li
mos
um e
t re
l.0.
025
0.02
40.
022
0.00
30.
003
0.01
5
Firm
icut
esC
lost
ridiu
m c
lust
er X
VI
Bul
leid
ia m
oore
i et
rel.
0.05
40.
080
0.03
70.
020
0.00
40.
015
Firm
icut
esC
lost
ridiu
m c
lust
er X
VI
Eub
acte
rium
cyl
indr
oide
s et
rel
.0.
052
0.04
40.
038
0.01
10.
016
0.03
2
Firm
icut
esC
lost
ridiu
m c
lust
er X
VII
Cat
enib
acte
rium
mits
uoka
i et
rel.
0.02
30.
019
0.01
60.
004
0.00
70.
017
Firm
icut
esC
lost
ridiu
m c
lust
er X
VII
Lact
obac
illus
cat
enaf
orm
is e
t re
l.0.
016
0.01
80.
014
0.00
20.
004
0.01
5
Firm
icut
esC
lost
ridiu
m c
lust
er X
VIII
Clo
strid
ium
ram
osum
et
rel.
0.08
30.
063
0.04
70.
020
0.00
10.
007
Firm
icut
esC
lost
ridiu
m c
lust
er X
VIII
Cop
roba
cillu
s ca
tena
form
is e
t re
l.0.
175
0.16
50.
059
0.03
60.
013
0.02
8
Firm
icut
esU
ncul
ture
d M
ollic
utes
Unc
ultu
red
Mol
licut
es0.
115
0.11
00.
107
0.04
00.
009
0.02
1
Fuso
bact
eria
Fuso
bact
eria
Fuso
bact
eria
0.06
90.
067
0.10
40.
151
0.03
20.
055
Prot
eoba
cter
iaPr
oteo
bact
eria
Aer
omon
as0.
009
0.00
80.
008
0.00
10.
006
0.01
5
Prot
eoba
cter
iaPr
oteo
bact
eria
Alc
alig
enes
faec
alis
et
rel.
0.15
00.
242
0.02
90.
005
0.00
00.
001
Prot
eoba
cter
iaPr
oteo
bact
eria
Ana
erob
iosp
irillu
m0.
008
0.00
80.
007
0.00
20.
001
0.00
7
Prot
eoba
cter
iaPr
oteo
bact
eria
Aqu
abac
teriu
m0.
0111
0.00
90.
0107
0.01
10.
003
0.01
5
Prot
eoba
cter
iaPr
oteo
bact
eria
Bur
khol
deria
0.03
10.
037
0.01
10.
004
0.00
00.
001
Prot
eoba
cter
iaPr
oteo
bact
eria
Cam
pylo
bact
er0.
067
0.06
50.
054
0.00
90.
004
0.01
5
Prot
eoba
cter
iaPr
oteo
bact
eria
Des
ulfo
vibr
io e
t re
l.0.
040
0.03
80.
037
0.00
80.
011
0.02
5
167
FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files
Phy
lum
Phy
lum
/Cla
ssG
enu
s-lik
e g
rou
p
Rel
ativ
e ab
un
dan
ce±S
D
B_t
0_R
SD
F_t0
_RS
Dp
.val
ue
FDR
Prot
eoba
cter
iaPr
oteo
bact
eria
Ent
erob
acte
r ae
roge
nes
et r
el.
0.15
20.
155
0.08
90.
018
0.00
40.
015
Prot
eoba
cter
iaPr
oteo
bact
eria
Esc
heric
hia
coli
et r
el.
0.35
10.
356
0.21
70.
375
0.00
30.
015
Prot
eoba
cter
iaPr
oteo
bact
eria
Hae
mop
hilu
s0.
053
0.08
80.
014
0.00
20.
004
0.01
5
Prot
eoba
cter
iaPr
oteo
bact
eria
Hel
icob
acte
r0.
039
0.04
00.
034
0.00
60.
004
0.01
5
Prot
eoba
cter
iaPr
oteo
bact
eria
Kle
bisi
ella
pne
umon
iae
et r
el.
0.07
80.
075
0.04
90.
009
0.00
60.
015
Prot
eoba
cter
iaPr
oteo
bact
eria
Lem
inor
ella
0.01
10.
007
0.00
80.
002
0.00
60.
015
Prot
eoba
cter
iaPr
oteo
bact
eria
Met
hylo
bact
eriu
m0.
008
0.00
80.
007
0.00
10.
006
0.01
5
Prot
eoba
cter
iaPr
oteo
bact
eria
Mor
axel
lace
ae0.
019
0.01
60.
014
0.00
20.
000
0.00
1
Prot
eoba
cter
iaPr
oteo
bact
eria
Nov
osph
ingo
bium
0.01
00.
010
0.00
90.
001
0.03
20.
055
Prot
eoba
cter
iaPr
oteo
bact
eria
Oce
anos
piril
lum
0.07
30.
114
0.03
40.
022
0.01
30.
028
Prot
eoba
cter
iaPr
oteo
bact
eria
Oxa
loba
cter
form
igen
es e
t re
l.0.
200
0.17
90.
109
0.08
10.
027
0.05
0
Prot
eoba
cter
iaPr
oteo
bact
eria
Prot
eus
et r
el.
0.06
80.
064
0.05
50.
009
0.00
10.
007
Prot
eoba
cter
iaPr
oteo
bact
eria
Pse
udom
onas
0.01
80.
016
0.01
40.
003
0.00
40.
015
Prot
eoba
cter
iaPr
oteo
bact
eria
Ser
ratia
0.06
00.
086
0.00
80.
002
0.00
00.
000
Prot
eoba
cter
iaPr
oteo
bact
eria
Sut
tere
lla w
adsw
orth
ia e
t re
l.0.
439
0.67
80.
320
0.73
60.
007
0.01
7
Prot
eoba
cter
iaPr
oteo
bact
eria
Vibr
io0.
040
0.03
40.
034
0.01
00.
001
0.00
9
Prot
eoba
cter
iaPr
oteo
bact
eria
Xan
thom
onad
acea
e0.
034
0.04
20.
016
0.00
20.
000
0.00
0
Prot
eoba
cter
iaPr
oteo
bact
eria
Yers
inia
et
rel.
0.03
40.
031
0.03
00.
005
0.00
40.
015
Spi
roch
aete
sS
piro
chae
tes
Bra
chys
pira
0.01
50.
014
0.01
30.
002
0.00
40.
015
Verr
ucom
icro
bia
Verr
ucom
icro
bia
Akk
erm
ansi
a0.
030
0.03
70.
022
0.02
30.
004
0.01
5
Chapter 7
168
Su
pp
lem
enta
ry A
pp
end
ix Ta
ble
3.3
. Man
n-W
hitn
ey c
ompa
rison
of f
ecal
(F) a
nd b
iops
y (B
) mic
robi
ota
from
non
-res
pond
ers
at th
e ge
nus-
like
leve
l of t
he 1
30
grou
ps in
clud
ed in
the
HIT
Chi
p. A
vera
ge r
elat
ive
abun
danc
e±S
D f
or s
igni
fican
t di
ffere
nt g
roup
s at
12
wee
ks a
fter
FM
T (t
12)
(p<
0.0
5 an
d FD
R<
0.3)
. H
igh-
light
ed in
gre
y ar
e gr
oups
in h
ighe
r abu
ndan
ce in
bio
psie
s; n
on h
ighl
ight
ed g
roup
s ar
e in
hig
her a
bund
ance
in fe
cal s
ampl
es. N
: non
-res
pond
ers;
R: r
espo
nder
s;
FDR
: Fal
se D
isco
very
Rat
e; S
D: s
tand
ard
devi
atio
n
Rel
ativ
e ab
un
dan
ce±S
D
Phy
lum
Phy
lum
/Cla
ssG
enu
s-lik
e g
rou
pB
_t12
_NS
DF_
t12_
NS
Dp
.val
ue
FDR
Act
inob
acte
riaA
ctin
obac
teria
Cor
yneb
acte
rium
0.01
90.
009
0.02
10.
009
0.01
30.
143
Act
inob
acte
riaA
ctin
obac
teria
Prop
ioni
bact
eriu
m1.
020
1.70
10.
023
0.01
00.
000
0.00
0
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
inte
stin
alis
et
rel.
0.03
70.
038
0.03
30.
051
0.01
10.
127
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
vul
gatu
s et
rel
.7.
301
5.59
55.
000
6.13
70.
028
0.23
1
Bac
tero
idet
esB
acte
roid
etes
Prev
otel
la m
elan
inog
enic
a et
rel
.26
.502
20.7
2213
.449
18.9
960.
016
0.16
1
Bac
tero
idet
esB
acte
roid
etes
Prev
otel
la o
ralis
et
rel.
3.82
82.
841
2.04
82.
724
0.00
70.
101
Bac
tero
idet
esB
acte
roid
etes
Prev
otel
la t
anne
rae
et r
el.
0.19
20.
114
0.13
80.
117
0.03
70.
254
Bac
tero
idet
esB
acte
roid
etes
Tann
erel
la e
t re
l.0.
259
0.21
30.
166
0.09
70.
034
0.24
6
Firm
icut
esB
acill
iS
taph
yloc
occu
s0.
028
0.03
70.
019
0.01
60.
048
0.31
1
Prot
eoba
cter
iaPr
oteo
bact
eria
Alc
alig
enes
faec
alis
et
rel.
0.17
30.
224
0.03
10.
015
0.00
00.
000
Prot
eoba
cter
iaPr
oteo
bact
eria
Aqu
abac
teriu
m0.
013
0.01
50.
008
0.00
30.
007
0.10
1
Prot
eoba
cter
iaPr
oteo
bact
eria
Bur
khol
deria
0.02
20.
017
0.01
30.
009
0.00
00.
006
Prot
eoba
cter
iaPr
oteo
bact
eria
Ent
erob
acte
r ae
roge
nes
et r
el.
0.09
60.
055
0.09
40.
044
0.00
40.
073
Prot
eoba
cter
iaPr
oteo
bact
eria
Esc
heric
hia
coli
et r
el.
0.31
50.
393
0.20
50.
346
0.00
10.
017
Prot
eoba
cter
iaPr
oteo
bact
eria
Lem
inor
ella
0.00
80.
005
0.00
70.
003
0.00
90.
113
Prot
eoba
cter
iaPr
oteo
bact
eria
Mor
axel
lace
ae0.
017
0.01
30.
014
0.00
60.
002
0.04
0
Prot
eoba
cter
iaPr
oteo
bact
eria
Oxa
loba
cter
form
igen
es e
t re
l.0.
200
0.16
50.
096
0.10
00.
028
0.23
1
Prot
eoba
cter
iaPr
oteo
bact
eria
Ser
ratia
0.07
10.
116
0.01
30.
016
0.00
10.
021
Prot
eoba
cter
iaPr
oteo
bact
eria
Vibr
io0.
031
0.01
40.
034
0.01
40.
028
0.23
1
Prot
eoba
cter
iaPr
oteo
bact
eria
Xan
thom
onad
acea
e0.
019
0.01
00.
020
0.01
00.
034
0.24
6
169
FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files
Su
pp
lem
enta
ry A
pp
end
ix T
able
3.4
. Man
n-W
hitn
ey c
ompa
rison
of
feca
l and
bio
psy
mic
robi
ota
from
res
pond
ers
at t
he g
enus
-like
leve
l of
the
130
grou
ps
incl
uded
in t
he H
ITC
hip.
Ave
rage
rel
ativ
e ab
unda
nce±
SD
for
sig
nific
ant
diffe
rent
gro
ups
at 1
2 w
eeks
aft
er F
MT
(t12
) (p<
0.0
5 an
d FD
R<
0.3)
. Hig
hlig
hted
in
grey
are
gro
ups
in h
ighe
r ab
unda
nce
in b
iops
ies;
non
hig
hlig
hted
gro
ups
are
in h
ighe
r ab
unda
nce
in fe
cal s
ampl
es. N
: non
-res
pond
ers;
R: r
espo
nder
s; F
DR
: Fa
lse
Dis
cove
ry R
ate;
SD
: sta
ndar
d de
viat
ion
Phy
lum
Phy
lum
/Cla
ssG
enu
s-lik
e g
rou
p
Rel
ativ
e ab
un
dan
ce±S
D
p.v
alu
eFD
RB
_t12
_RS
DF_
t12_
RS
D
Act
inob
acte
riaA
ctin
obac
teria
Col
linse
lla0.
071
0.03
10.
324
0.27
70.
001
0.03
1
Act
inob
acte
riaA
ctin
obac
teria
Mic
roco
ccac
eae
0.00
70.
004
0.00
60.
001
0.04
10.
144
Act
inob
acte
riaA
ctin
obac
teria
Prop
ioni
bact
eriu
m0.
372
0.62
90.
020
0.00
40.
000
0.00
2
Bac
tero
idet
esB
acte
roid
etes
Alli
stip
es e
t re
l.2.
292
1.07
80.
731
0.55
20.
004
0.06
2
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
fra
gilis
et
rel.
0.67
50.
419
0.30
30.
285
0.01
80.
092
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
inte
stin
alis
et
rel.
0.05
50.
035
0.02
70.
018
0.04
90.
156
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
ova
tus
et r
el.
0.74
10.
492
0.29
00.
317
0.00
90.
075
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
ple
beiu
s et
rel
.0.
520
0.51
60.
222
0.23
60.
018
0.09
2
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
spl
achn
icus
et
rel.
0.39
90.
234
0.17
70.
071
0.00
40.
062
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
ste
rcor
is e
t re
l.0.
194
0.12
10.
098
0.05
00.
007
0.06
7
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
uni
form
is e
t re
l.0.
506
0.44
80.
192
0.26
90.
034
0.14
2
Bac
tero
idet
esB
acte
roid
etes
Bac
tero
ides
vul
gatu
s et
rel
.11
.678
9.05
55.
129
5.20
10.
041
0.14
4
Bac
tero
idet
esB
acte
roid
etes
Para
bact
eroi
des
dist
ason
is e
t re
l.1.
328
1.07
00.
432
0.32
00.
049
0.15
6
Bac
tero
idet
esB
acte
roid
etes
Prev
otel
la ru
min
icol
a et
rel
.0.
024
0.00
80.
018
0.00
40.
012
0.07
6
Bac
tero
idet
esB
acte
roid
etes
Prev
otel
la t
anne
rae
et r
el.
0.29
90.
185
0.14
40.
107
0.04
10.
144
Bac
tero
idet
esB
acte
roid
etes
Tann
erel
la e
t re
l.0.
396
0.16
90.
195
0.11
60.
004
0.06
2
Firm
icut
esB
acill
iA
eroc
occu
s0.
008
0.00
40.
006
0.00
10.
049
0.15
6
Firm
icut
esB
acill
iE
nter
ococ
cus
0.06
50.
041
0.04
80.
011
0.01
50.
087
Firm
icut
esB
acill
iS
taph
yloc
occu
s0.
031
0.02
80.
013
0.00
30.
000
0.02
0
Firm
icut
esC
lost
ridiu
m c
lust
er X
IPe
ptos
trep
toco
ccus
ana
erob
ius
et r
el.
0.01
00.
008
0.00
70.
002
0.02
30.
105
Chapter 7
170
Phy
lum
Phy
lum
/Cla
ssG
enu
s-lik
e g
rou
p
Rel
ativ
e ab
un
dan
ce±S
D
p.v
alu
eFD
RB
_t12
_RS
DF_
t12_
RS
D
Firm
icut
esC
lost
ridiu
m c
lust
er X
IVa
Clo
strid
ium
nex
ile e
t re
l.0.
470
0.31
21.
174
0.68
90.
012
0.07
6
Firm
icut
esC
lost
ridiu
m c
lust
er X
IVa
Dor
ea fo
rmic
igen
eran
s et
rel
.1.
619
1.03
63.
031
0.84
00.
012
0.07
6
Firm
icut
esC
lost
ridiu
m c
lust
er X
IVa
Lach
nosp
ira p
ectin
osch
iza
et r
el.
1.67
71.
189
3.70
42.
600
0.02
80.
121
Firm
icut
esC
lost
ridiu
m c
lust
er X
IVa
Out
grou
ping
clo
strid
ium
clu
ster
XIV
a1.
287
2.29
12.
483
3.16
50.
023
0.10
5
Firm
icut
esC
lost
ridiu
m c
lust
er X
IVa
Rum
inoc
occu
s ob
eum
et
rel.
5.81
93.
372
10.9
344.
323
0.04
90.
156
Firm
icut
esC
lost
ridiu
m c
lust
er X
VII
Cat
enib
acte
rium
mits
uoka
i et
rel.
0.02
60.
018
0.01
60.
009
0.02
80.
121
Prot
eoba
cter
iaPr
oteo
bact
eria
Alc
alig
enes
faec
alis
et
rel.
0.09
40.
084
0.02
80.
007
0.00
00.
020
Prot
eoba
cter
iaPr
oteo
bact
eria
Ana
erob
iosp
irillu
m0.
007
0.00
40.
006
0.00
10.
041
0.14
4
Prot
eoba
cter
iaPr
oteo
bact
eria
Aqu
abac
teriu
m0.
013
0.00
80.
008
0.00
30.
006
0.06
6
Prot
eoba
cter
iaPr
oteo
bact
eria
Bur
khol
deria
0.02
80.
009
0.01
30.
008
0.00
20.
053
Prot
eoba
cter
iaPr
oteo
bact
eria
Ent
erob
acte
r ae
roge
nes
et r
el.
0.14
20.
049
0.09
40.
033
0.01
80.
092
Prot
eoba
cter
iaPr
oteo
bact
eria
Esc
heric
hia
coli
et r
el.
0.62
51.
260
0.15
90.
269
0.00
70.
067
Prot
eoba
cter
iaPr
oteo
bact
eria
Hae
mop
hilu
s0.
031
0.03
20.
013
0.00
30.
041
0.14
4
Prot
eoba
cter
iaPr
oteo
bact
eria
Kle
bisi
ella
pne
umon
iae
et r
el.
0.07
30.
024
0.04
80.
012
0.00
20.
047
Prot
eoba
cter
iaPr
oteo
bact
eria
Mor
axel
lace
ae0.
015
0.00
80.
012
0.00
20.
012
0.07
6
Prot
eoba
cter
iaPr
oteo
bact
eria
Prot
eus
et r
el.
0.06
40.
042
0.04
90.
010
0.01
80.
092
Prot
eoba
cter
iaPr
oteo
bact
eria
Ser
ratia
0.03
70.
054
0.00
70.
002
0.00
90.
075
Prot
eoba
cter
iaPr
oteo
bact
eria
Vibr
io0.
037
0.01
80.
030
0.00
80.
006
0.06
6
Prot
eoba
cter
iaPr
oteo
bact
eria
Xan
thom
onad
acea
e0.
032
0.01
20.
019
0.00
90.
007
0.06
7
Prot
eoba
cter
iaPr
oteo
bact
eria
Yers
inia
et
rel.
0.03
10.
016
0.02
60.
006
0.01
50.
087
Verr
ucom
icro
bia
Verr
ucom
icro
bia
Akk
erm
ansi
a0.
024
0.02
80.
023
0.03
70.
041
0.14
4