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.

Link to publication

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

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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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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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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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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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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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Chapter 7

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Chapter 7

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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


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

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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

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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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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


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


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


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


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


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


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

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Prot

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bact

eria

Ana

erob

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m0.

007

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006

0.00

10.

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0.14

4

Prot

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cter

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oteo

bact

eria

Aqu

abac

teriu

m0.

013

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008

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006

0.06

6

Prot

eoba

cter

iaPr

oteo

bact

eria

Bur

khol

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0.02

80.

009

0.01

30.

008

0.00

20.

053

Prot

eoba

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bact

eria

Ent

erob

acte

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roge

nes

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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

The microbiome and its therapeutic potential in ... · Fecal transplantation from a healthy donor...

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