The Microbiome and Its Therapeutic Potential in Inflammatory Bowel Diseases

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The microbiome and its therapeutic potential in inflammatory bowel diseases

Rossen, N.G.M.

Publication date 2016 Document Version Final published version

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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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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:02 Oct 2021 7 Fecal 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. Ponsioen submitted Chapter 7

Abstract

Background and aims Fecal 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.

Methods Fecal 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).

Results At 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 determined 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.

136 FMT in UC: microbiota shifts and signatures that determine success of therapy

Conclusions 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 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 response.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 difficile 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, andLactobacillus , 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

137 Chapter 7 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 complete 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 sampling Patients 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

138 FMT in UC: microbiota shifts and signatures that determine success of therapy described previously.9 Patients who achieved the primary endpoint of the trial (clinical 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 assessment 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 assessment DNA 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 concentration 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

139 Chapter 7 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 statistics Demographic 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 microbiota 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 between 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.

140 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 characteristics are shown in Table 1.

Table 1. Baseline characteristics of Donors, Non-responders and Responders. Donors* Non-responders Responders p-value n= 6 n= 22 n= 12 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 - 8 (4-11) 9 (4-11) 0.77 (range) 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).

141 Chapter 7

Shifts in microbiota composition after FMT Profiling of the intestinal microbiota at the genus-like level (130 groups) revealed significant 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

Color Key

Responders -4 -2 0 2 4 A Row Z-Score Donors Non Responders Rel SustRem Bacteroides fragilis et rel. Bacteroides intestinalis et rel. Bacteroides ovatus et rel. Bacteroides splachnicus et rel. Bacteroides stercoris et rel. Bacteroides vulgatus et rel. Prevotella ruminicola et rel. Prevotella tannerae et rel. Tannerella et rel. Sutterella wadsworthia et rel. Uncultured Selenomonadaceae Veillonella Clostridium nexile et rel. Clostridium sphenoides et rel. * Outgrouping Clostridium cluster XIVa Coprobacillus catenaformis et rel. B Responders Donors Non Responders Rel SustRem Bacteroides fragilis et rel. Bacteroides ovatus et rel. Bacteroides splachnicus et rel. Bacteroides stercoris et rel. Bacteroides vulgatus et rel. Parabacteroides distasonis et rel. Prevotella ruminicola et rel. Prevotella tannerae et rel. Tannerella et rel. Burkholderia Escherichia coli et rel. Serratia Sutterella wadsworthia et rel. Enterococcus Uncultured Selenomonadaceae Veillonella Peptostreptococcus anaerobius et rel. Peptostreptococcus micros et rel. Anaerostipes caccae et rel. * Bryantella formatexigens et rel. * Clostridium nexile et rel. * Clostridium sphenoides et rel. Coprococcus eutactus et rel. * Dorea formicigenerans et rel. Eubacterium hallii et rel. * Eubacterium rectale et rel. * Eubacterium ventriosum et rel. * Lachnospira pectinoschiza et rel. * Outgrouping clostridium cluster XIVa Ruminococcus gnavus et rel. Ruminococcus obeum et rel. * Coprobacillus catenaformis et rel.

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 remission (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 different 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.

142 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 decreased 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 FMT Similar comparisons were performed at the mucosal biopsy level at baseline and t12 weeks between responder and non-responder samples, but no significant differences between the study groups were found (FDR>0.8) (Suppl. Table 2). The microbiota composition of feces and biopsies was also compared, and differences 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 response Redundancy analysis showed an almost complete separation of patients with sustained 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-

143 Chapter 7

Sustained remission Relapse 1.0

Bacilli A p3 B Clostridium clusters IV & p8 Bacteroidetes XIVa p8 p3 p6 p10 p10 Proteobacteria p8 p8*

p3 9 . I p10 III 3 p12 3 %

p6 p6

FMT-A p12

p12 II II -1.0 FMT-D FMT-A IV t0 t0 1.0 Bacilli D t12w t12w C p1 Clostridium clusters IV & Bacteroidetes t≥1y t≥1y XIVa Donor

Dp9-I Proteobacteria p9 p1 p4

p1 9 . Dp7-I Dp7-II 3

p2 3 p7 %

p11 p9 p7 p9 FMT-D p4 p11 Dp9-II p5 p4 Dp5 p7 p2 p5** Dp11-I p11 Dp4 Dp11-II p2 Dp1 Dp2 -1.0

-1.0 1.0 -1.0 1.0 10.26% 10.26% Figure 2A. Redundancy analysis (RDA) of samples at t0, t12 weeks and t1 year of FMT-A with sustained 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. 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

144 FMT in UC: microbiota shifts and signatures that determine success of therapy

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. Associations Allistipes et rel.* Bacteroides fragilis et rel. Eubacterium siraeum et rel. Bacteroides intestinalis et rel. Bacilli Bacteroidetes Subdoligranulum variable et rel. Bacteroides stercoris et rel. Clostridium clusters IV & Proteobacteria Streptococcus intermedius et rel. Bacteroides splachnicus et rel. XIVa Aerococcus Clostridium stercorarium et rel. Eubacterium biforme et rel. Sutterella wadsworthia et rel. Veillonella Aeromonas Faecalibacterium prausnitzii et et rel. Relapse rel.* Anaerotruncus colihominis Anaerostipes caccae et rel.* Haemophilus I III Clostridium difficile et rel.* Clostridium symbiosum et rel.* Streptococcus mitis et rel. Aneurinibacillus Butyrivibrio crossotus et rel.* Uncultured Selenomonadaceae SustRem Uncultured Clostridiales I Klebisiella pneumoniae et rel. Actinomycetaceae Bulleidia moorei et rel. Donor Anaerofustis Dialister Lactobacillus plantarum et rel. Oceanospirillum Uncultured Mollicutes Bilophila et rel. Leminorella Peptostreptococcus micros et rel.

Ruminococcus callidus et rel. Gemella II II Eubacterium hallii et rel.* Escherichia coli et rel. IV Peptostreptococcus anaerobius et et rel. Streptococcus bovis 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. 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 treatment, 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

145 Chapter 7

Table 2. Clinical outcome and medication use of Non-responders and Responders at 1 year Follow-up Non-responders Responders p-value n= 22 n= 12 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/ 13 (54.2) 7 (58.3) 0.81 corticosteroid rectal 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.

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 FMT Stability of the fecal microbiota was assessed by moving window correlation of samples 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

146 FMT in UC: microbiota shifts and signatures that determine success of therapy

Responders Non-Responders

FMT-A * * A B *

FMT-D C D

Figure 3. Moving window correlations indicating stability in time of patients from the FMT-A (A and B) and FMT-D (C and D) groups, separated into responders and non-responders. Y-axes indicate Pear- son’s similarity indices at the probe level of the HITChip microarray (e.g. Fig 3C: D_I-p_t0 indicates the Pearson’s similarity indices between patient samples collected at t0 and their donors at the first FMT (I). FMT, fecal microbiota transplantation; FMT-A, FMT autologous; FMT-D, FMT donor feces. * Indicates significant differences (p<0.05)

Responders Non-Responders

Similarity to t0 Similarity to t0

Figure 4. Similarity indices of samples from responders and non-responders of the FMT-D group at 12 weeks (12w) and 1 year (1y) with the baseline (t0) samples.

147 Chapter 7 FDR 0.0766 0.2436 0.2436 0.0319 0.0665 0.2436 0.0766 0.0319 0.0659 0.2775 0.0710 0.0766 0.2436 0.1133 0.0041 0.0244 0.0244 0.0003 0.0020 0.0219 0.0047 0.0005 0.0015 0.0299 0.0027 0.0041 0.0219 0.0078 p.value 1.53 2.25 1.22 3.77 2.62 4.18 1.50 1.72 2.93 1.97 2.08 1.96 1.46 0.09 Fold 4.323 0.533 0.525 3.165 2.600 2.967 0.422 0.840 5.932 2.595 0.875 0.689 1.461 0.002 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.460 0.669 2.483 3.704 1.863 0.917 3.031 7.954 2.910 1.391 1.174 2.030 0.007 Responders 10.934 7.532 0.289 0.856 0.652 1.036 0.471 0.554 1.044 2.908 0.773 0.366 0.427 1.825 0.223 Relative abundance ± SD Relative ± ± ± ± ± ± ± ± ± ± ± ± ± ± 7.155 0.204 0.548 0.658 1.416 0.446 0.611 1.761 2.719 1.477 0.668 0.599 1.387 0.081 Non-responders cluster XIVa Clostridium Ruminococcus obeum et rel. Ruminococcus Ruminococcus lactaris Ruminococcus et rel. Ruminococcus gnavus et rel. gnavus Ruminococcus Outgrouping Lachnospira pectinoschiza et rel. pectinoschiza Lachnospira Eubacterium ventriosum et rel. Eubacterium ventriosum Eubacterium hallii et rel. Dorea formicigenerans et rel. Dorea formicigenerans Coprococcus eutactus et rel. Coprococcus eutactus Clostridium symbiosum et rel. Clostridium sphenoides et rel. Clostridium nexile et rel. Clostridium nexile Anaerostipes caccae et rel. Genus-like level Genus-like anaerobius et rel. Peptostreptococcus * * * * * * * * cluster XI cluster Relative Relative abundance (± standard deviation, SD) of significant different groups (p<0.05, FDR<0.3) between responders and non-responders at t12 Table 3. Table butyrate in abundance between responders/non-responders. *Groups including known producing bacteria. differences relative shows “Fold” weeks. Phylum/Class Clostridium Clostridium XIVa

148 FMT in UC: microbiota shifts and signatures that determine success of therapy

1.60E-04

1.40E-04

1.20E-04

* p<0.05 1.00E-04

8.00E-05

ButCoA/16S rRNA 6.00E-05

4.00E-05 6.7 fold

2.00E-05

0.00E+00

t0 t12w 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.

I fecal samples II biopsy samples * * * *

t0 t12w t0 t12w

Figure 5.B. Relative abundance (%) of genus-like groups that include known butyrate producing bacteria in fecal samples of I. donors and patients at baseline (t0) and 12 weeks after treatment (t12w) and II. in biopsy samples of patients at baseline (t0) and 12 weeks after treatment (t12w).

149 Chapter 7 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 response A 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 microbiota 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 patients 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

150 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-responders showed significantly increased levels of Bacteroidetes but also of Proteo- bacteria as compared to healthy donors and responders, including potentially pathogenic 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 mechanism in IBD.38,39 After therapy, bacteria mainly from the Clostridium cluster XIVa normalized in responders’ 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 responders 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 response 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

151 Chapter 7 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 aberrant 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 microbial 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

152 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-responders, 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 butyrate producers and durable response is associated with a restoration of the significantly 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.

Acknowledgements We 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.

153 Chapter 7

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156 FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files FDR 0.151 0.151 0.333 0.333 0.331 0.331 0.175 0.151 0.151 0.194 0.175 0.151 0.151 0.194 0.331 0.355 0.006 0.007 0.041 0.039 0.036 0.035 0.009 0.006 0.005 0.016 0.011 0.004 0.005 0.016 0.035 0.012 p.value 0.044 0.022 2.187 0.327 1.278 0.022 0.008 0.041 0.054 0.002 3.573 0.012 0.024 0.025 0.005 0.023 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± SustRem Responders- 0.101 0.056 1.539 0.944 1.575 0.046 0.012 0.120 0.078 0.017 2.236 0.060 0.133 0.117 0.017 0.137 1y FU. ≥ 1.037 0.049 3.503 1.061 1.178 0.004 0.007 0.373 0.227 0.005 11.001 0.378 0.344 1.355 0.140 1.155 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.540 0.062 2.708 1.073 1.375 0.036 0.013 0.319 0.218 0.020 0.264 0.308 0.803 0.082 0.849 Responders-Rel 10.391 0.301 0.032 1.280 0.509 0.853 0.842 0.014 0.100 0.118 0.010 8.935 0.094 0.060 0.457 0.057 0.881 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Relative abundance ± SD Relative Non- responders 0.223 0.047 1.037 1.025 0.858 0.250 0.013 0.164 0.139 0.019 6.508 0.113 0.171 0.345 0.040 0.592 0.026 0.443 1.807 1.352 2.152 0.009 0.002 0.028 0.013 0.005 0.424 0.016 0.035 0.050 0.009 0.090 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Donor 0.087 0.240 2.335 1.963 2.382 0.034 0.007 0.086 0.046 0.016 0.591 0.056 0.134 0.118 0.015 0.137

Clostridium Sutterella wadsworthia et rel. wadsworthia Sutterella Coprobacillus catenaformis Coprobacillus catenaformis et rel. Outgrouping cluster XIVa Clostridium sphenoides et rel. Clostridium nexile et rel. Clostridium nexile Veillonella Uncultured Uncultured Selenomonadaceae Tannerella et rel. Tannerella Prevotella tannerae et rel. Prevotella Prevotella ruminicola et rel. ruminicola Prevotella Bacteroides vulgatus et rel. vulgatus Bacteroides Bacteroides stercoris et rel. Bacteroides Bacteroides splachnicus et rel. splachnicus Bacteroides Bacteroides ovatus et rel. ovatus Bacteroides Bacteroides intestinalis et rel. Bacteroides Genus-like level Genus-like Bacteroides fragilis et rel. Bacteroides

Kruskal Wallis comparison of the 130 genus-like groups included in the HITChip between responders and non-responders samples at samples non-responders and responders between HITChip the in included groups genus-like 130 the of comparison Wallis Kruskal Proteobacteria Clostridium cluster XVIII Clostridium cluster XIVa Clostridium cluster IX Phylum/Class Bacteroidetes Table 1. 1. Table

Proteobacteria Firmicutes Phylum Supplementary FDR<0.33). (p<0.05; groups different significantly of Standard(± abundance deviation) relative Shown samples. donor and (t12w) weeks 12 and (t0) baseline at t or sustain remission (Responders-SustRem) are separated into those that relapse (Responders-Rel) Responders Kruskal Wallis - Baseline (t0) Wallis Kruskal Bacteroidetes

157 Chapter 7 FDR 0.030 0.023 0.152 0.098 0.030 0.124 0.171 0.054 0.137 0.137 0.078 0.056 0.137 0.082 0.054 0.111 0.119 0.114 0.001 0.000 0.033 0.012 0.001 0.022 0.040 0.004 0.028 0.026 0.008 0.005 0.028 0.010 0.004 0.017 0.020 0.018 p.value 0.287 0.920 0.232 0.013 0.001 0.019 1.445 0.138 0.013 0.004 0.108 0.004 0.394 4.400 0.028 0.082 0.162 0.158 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± SustRem Responders- 0.854 1.420 0.701 0.037 0.006 0.037 1.682 0.198 0.047 0.007 0.132 0.019 0.516 3.769 0.079 0.178 0.207 0.259 0.953 0.227 0.724 0.017 0.003 0.009 1.522 0.103 0.010 0.002 0.115 0.004 0.228 5.977 0.062 0.067 0.422 0.386 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.929 0.928 1.286 0.040 0.008 0.033 2.378 0.192 0.050 0.008 0.156 0.016 0.348 6.488 0.117 0.176 0.372 0.347 Responders-Rel 0.366 0.427 0.494 0.760 0.223 0.256 1.825 0.097 1.388 0.008 0.117 0.009 0.198 6.137 0.192 0.109 0.400 1.392 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Non- responders 0.668 0.599 0.681 0.280 0.081 0.104 1.387 0.166 0.394 0.010 0.138 0.023 0.278 5.000 0.148 0.204 0.358 0.810 Relative abundance ± Standard deviation abundance ± Standard Relative 1.352 2.152 0.684 0.009 0.003 0.012 0.640 0.028 0.019 0.002 0.013 0.005 0.113 0.424 0.016 0.035 0.050 0.090 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Donor 1.963 2.382 1.288 0.034 0.008 0.035 1.304 0.086 0.052 0.007 0.046 0.016 0.179 0.591 0.056 0.134 0.118 0.137 Clostridium sphenoides et rel. Clostridium nexile et rel. Clostridium nexile Bryantella formatexigens et Bryantella formatexigens rel. Veillonella Peptostreptococcus anaerobius et rel. micros Peptostreptococcus et rel. Anaerostipes caccae et rel. Tannerella et rel. Tannerella Enterococcus Uncultured Selenomonadaceae Prevotella tannerae et rel. Prevotella Prevotella ruminicola et rel. ruminicola Prevotella Parabacteroides distasonis Parabacteroides et rel. Bacteroides vulgatus et rel. vulgatus Bacteroides Bacteroides stercoris et rel. Bacteroides Bacteroides splachnicus et rel. splachnicus Bacteroides Genus-like level Genus-like et rel. ovatus Bacteroides Bacteroides fragilis et rel. Bacteroides

Clostridium cluster XI Clostridium cluster XIII Clostridium cluster XIVa Bacilli Clostridium cluster IX Phylum/Class Bacteroidetes Firmicutes Kruskal Wallis - 12 weeks (t12w) - 12 weeks Wallis Kruskal Phylum Bacteroidetes

158 FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files FDR 0.082 0.137 0.192 0.049 0.111 0.166 0.108 0.019 0.041 0.078 0.192 0.109 0.030 0.030 0.009 0.027 0.047 0.003 0.016 0.037 0.014 0.000 0.002 0.008 0.046 0.015 0.001 0.001 p.value 0.167 0.001 0.020 3.827 0.014 0.010 0.170 0.541 1.100 0.251 0.648 0.360 0.734 2.980 ± ± ± ± ± ± ± ± ± ± ± ± ± ± SustRem Responders- 0.291 0.006 0.081 9.458 0.047 0.018 0.400 1.081 2.362 0.407 0.893 0.819 2.729 6.585 0.104 0.002 0.380 4.607 0.321 0.002 0.636 4.128 3.055 3.771 1.881 0.489 0.890 8.002 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.148 0.008 0.237 0.210 0.009 0.938 3.884 5.046 3.319 2.021 1.015 3.333 9.324 Responders-Rel 12.411 0.342 0.016 0.346 7.532 0.048 0.009 0.856 0.652 1.036 0.471 0.352 0.554 1.044 2.908 ± ± ± ± ± ± ± ± ± ± ± ± ± ± Non- responders 0.263 0.013 0.205 7.155 0.056 0.013 0.548 0.658 1.416 0.446 0.579 0.611 1.761 2.719 Relative abundance ± Standard deviation abundance ± Standard Relative 0.026 0.003 0.039 6.906 0.443 0.003 1.473 1.807 2.460 1.431 1.022 0.710 2.273 3.565 ± ± ± ± ± ± ± ± ± ± ± ± ± ± Donor 0.087 0.008 0.099 0.240 0.009 1.133 2.335 4.313 1.640 1.399 0.968 3.976 6.924 15.654

Clostridium Sutterella wadsworthia et rel. wadsworthia Sutterella Serratia Escherichia coli et rel. Escherichia Ruminococcus obeum et rel. Ruminococcus Coprobacillus catenaformis et rel. Burkholderia Ruminococcus gnavus et rel. gnavus Ruminococcus Outgrouping cluster XIVa Lachnospira pectinoschiza pectinoschiza Lachnospira et rel. Eubacterium ventriosum et Eubacterium ventriosum rel. Eubacterium rectale et rel. Eubacterium hallii et rel. Genus-like level Genus-like et rel. Dorea formicigenerans Coprococcus eutactus et rel. Coprococcus eutactus

Clostridium cluster XVIII Proteobacteria Phylum/Class Proteobacteria Kruskal Wallis - 12 weeks (t12w) - 12 weeks Wallis Kruskal Phylum

159 Chapter 7 FDR FDR 0.813 0.813 0.813 0.813 0.813 0.863 0.863 0.863 0.863 0.044 0.039 0.049 0.039 0.021 0.033 0.039 0.023 0.033 p.value p.value 0.772 0.028 0.485 2.817 0.020 0.056 0.486 0.022 0.014 ± ± ± ± ± ± ± ± ± SustRem SustRem Responders- Responders- 0.632 0.040 0.870 2.856 0.047 0.148 0.492 0.068 0.027 0.459 0.096 0.434 2.491 0.071 0.106 0.998 0.027 0.007 ± ± ± ± ± ± ± ± ± 0.458 0.134 0.890 3.792 0.127 0.207 1.204 0.080 0.037 Responders-Rel Responders-Rel Relative abundance ± SD Relative abundance ± SD Relative 1.333 0.137 0.312 1.473 0.437 0.209 0.149 0.020 0.010 ± ± ± ± ± ± ± ± ± 1y FU. ≥ 0.591 0.067 0.613 1.620 0.161 0.139 0.206 0.048 0.019 Non-responders Non-responders Genus-like level Genus-like Propionibacterium intestinalis et rel. Bacteroides et rel. Bryantella formatexigens et rel. Coprococcus eutactus Clostridium ramosum et rel. level Genus-like Clostridium stercorarium et rel. et rel. Eubacterium ventriosum Klebisiella pneumoniae et rel. Xanthomonadaceae cluster XIVa cluster XVIII cluster III cluster XIVa Mann-Whitney comparison of the 130 genus-like groups included in the HITChip between responders and non-responders samples non-responders and responders between HITChip the in included groups genus-like 130 the of comparison Mann-Whitney Phylum/Class Actinobacteria Bacteroidetes Clostridium Clostridium Phylum/Class Clostridium Clostridium Proteobacteria Table 2. 2. Table

Supplementary into separated are Responders FDR>0.8). (p<0.05; groups selected of Standarddeviation) (± abundance relative Shown (t12w). weeks 12 and (t0) baseline at at t or sustain remission (Responders-SustRem) those that relapse (Responders-Rel) - Baseline (t0) Whitney Mann Phylum Actinobacteria Bacteroidetes Firmicutes (t12w) - 12 weeks Whitney Mann Phylum Firmicutes Proteobacteria Shown groups with p<0.05; FDR>0.8. Shown

160 FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files FDR 0.023 0.004 0.016 0.012 0.007 0.005 0.012 0.043 0.017 0.048 0.028 0.013 0.059 0.023 0.092 0.000 0.008 0.001 0.032 0.001 0.008 0.001 0.004 0.002 0.001 0.001 0.003 0.019 0.005 0.022 0.011 0.003 0.030 0.008 0.048 0.000 0.002 0.000 0.013 0.000 p.value SD 0.007 0.002 0.002 0.003 0.100 0.118 0.010 0.261 8.935 0.410 0.094 0.060 0.102 0.457 0.057 0.006 0.002 0.006 0.005 0.005 0.020 0.007 0.006 0.007 0.164 0.139 0.019 0.292 6.508 0.284 0.113 0.171 0.168 0.345 0.040 0.020 0.006 0.019 0.014 0.014 F_t0_N SD 0.009 0.220 0.079 0.003 0.335 0.188 0.011 0.606 9.378 0.741 0.142 0.258 0.284 0.372 0.137 1.333 0.004 0.009 0.005 0.022 Relative abundance±SD Relative 0.024 0.059 0.026 0.008 0.409 0.310 0.025 0.676 0.685 0.195 0.356 0.338 0.573 0.067 0.591 0.008 0.023 0.015 0.025 12.379 B_t0_N Bacillus Aerococcus Asteroleplasma et rel. Asteroleplasma Uncultured Chroococcales Uncultured Tannerella et rel. Tannerella Prevotella tannerae et rel. Prevotella Prevotella ruminicola et rel. ruminicola Prevotella Parabacteroides distasonis et rel. Parabacteroides Bacteroides vulgatus et rel. vulgatus Bacteroides Bacteroides uniformis et rel. uniformis Bacteroides Bacteroides stercoris et rel. Bacteroides Bacteroides splachnicus et rel. splachnicus Bacteroides Bacteroides plebeius et rel. Bacteroides Bacteroides ovatus et rel. ovatus Bacteroides Bacteroides intestinalis et rel. Bacteroides Propionibacterium Micrococcaceae Corynebacterium Atopobium Actinomycetaceae Genus-like group Genus-like Mann-Whitney comparison of fecal (F) and biopsy (B) microbiota from non-responders at the genus-like level of the 130 130 the of level genus-like the at non-responders from microbiota (B) biopsy and (F) fecal of comparison Mann-Whitney Table 3.1. 3.1. Table

Bacilli Bacilli Asteroleplasma Cyanobacteria Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Phylum/Class Appendix

Firmicutes Firmicutes Firmicutes Cyanobacteria Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Supplementary groups included in the relative HITChip. Average abundance±SD for significant groups different at baseline (t0) (p< 0.05 and FDR<0.3). Highlighted in grey are groups in higher abundance in biopsies; non highlighted groups are in samples. higher N: abundance non-responders; in R: fecal responders; FDR: False SD: standardDiscovery Rate; deviation Phylum

161 Chapter 7 FDR 0.000 0.001 0.012 0.012 0.003 0.059 0.007 0.043 0.027 0.011 0.026 0.051 0.016 0.048 0.012 0.022 0.057 0.019 0.043 0.071 0.043 0.071 0.043 0.000 0.000 0.003 0.003 0.000 0.030 0.001 0.019 0.010 0.002 0.009 0.024 0.004 0.022 0.003 0.007 0.027 0.006 0.017 0.037 0.019 0.037 0.019 p.value SD 0.008 0.002 0.017 0.032 0.020 0.004 0.054 0.015 0.014 0.036 1.568 0.016 0.002 0.092 0.007 0.002 0.013 0.017 0.011 0.026 0.060 0.008 0.442 0.026 0.007 0.055 0.047 0.039 0.012 0.027 0.033 0.031 0.030 2.446 0.034 0.006 0.044 0.019 0.006 0.024 0.017 0.023 0.026 0.095 0.010 0.176 F_t0_N SD 0.413 0.007 0.150 0.092 0.437 0.005 0.076 0.012 0.020 0.194 0.650 0.014 0.003 0.082 0.062 0.003 0.055 0.050 0.092 0.256 0.546 0.079 0.278 Relative abundance±SD Relative 0.250 0.010 0.100 0.087 0.161 0.014 0.041 0.039 0.042 0.082 0.982 0.039 0.007 0.043 0.037 0.007 0.039 0.035 0.044 0.083 0.264 0.033 0.135 B_t0_N Alcaligenes faecalis et rel. Alcaligenes faecalis Aeromonas Fusobacteria Coprobacillus catenaformis et rel. Coprobacillus catenaformis Clostridium ramosum et rel. Lactobacillus catenaformis et rel. Lactobacillus catenaformis Catenibacterium mitsuokai et rel. Eubacterium cylindroides et rel. Bulleidia moorei et rel. Eubacterium limosum et rel. Lachnospira pectinoschiza et rel. pectinoschiza Lachnospira Peptostreptococcus micros et rel. Peptostreptococcus Clostridium felsineum et rel. Clostridium felsineum Mitsuokella multiacida et rel. Mitsuokella Megamonas hypermegale et rel. Megamonas hypermegale Clostridium thermocellum et rel. Weissella et rel. Weissella Staphylococcus Lactococcus Lactobacillus salivarius et rel. Lactobacillus salivarius Lactobacillus gasseri et rel. Granulicatella Enterococcus Genus-like group Genus-like cluster XVIII cluster XVIII cluster XVII cluster XVII cluster XVI cluster XVI cluster XV cluster XIVa cluster XIII cluster XI cluster IX cluster IX cluster III Proteobacteria Proteobacteria Fusobacteria Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Phylum/Class Proteobacteria Proteobacteria Fusobacteria Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Phylum

162 FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files FDR 0.000 0.000 0.004 0.001 0.016 0.001 0.037 0.051 0.057 0.000 0.026 0.003 0.001 0.012 0.004 0.000 0.000 0.028 0.017 0.000 0.028 0.001 0.000 0.000 0.000 0.001 0.000 0.004 0.000 0.015 0.024 0.027 0.000 0.009 0.000 0.000 0.003 0.001 0.000 0.000 0.011 0.005 0.000 0.011 0.000 0.000 p.value SD 0.007 0.010 0.301 0.008 0.004 0.016 0.127 0.012 0.003 0.004 0.002 0.002 0.021 0.010 0.006 0.068 0.030 0.010 0.015 0.003 0.006 0.002 0.002 0.015 0.029 0.223 0.009 0.012 0.049 0.116 0.027 0.008 0.012 0.006 0.007 0.045 0.030 0.014 0.107 0.076 0.032 0.048 0.009 0.020 0.007 0.006 F_t0_N SD 0.028 0.014 0.517 0.266 0.027 0.059 0.329 0.043 0.003 0.008 0.003 0.006 0.073 0.013 0.099 1.459 0.137 0.013 0.021 0.040 0.008 0.052 0.004 Relative abundance±SD Relative 0.033 0.039 0.624 0.097 0.021 0.074 0.239 0.044 0.009 0.017 0.007 0.012 0.089 0.036 0.043 0.787 0.156 0.037 0.057 0.037 0.024 0.026 0.008 B_t0_N Xanthomonadaceae Vibrio Sutterella wadsworthia et rel. wadsworthia Sutterella Serratia Pseudomonas Proteus et rel. Proteus Oxalobacter formigenes et rel. Oxalobacter formigenes Oceanospirillum Novosphingobium Moraxellaceae Methylobacterium Leminorella Klebisiella pneumoniae et rel. Helicobacter Haemophilus Escherichia coli et rel. Escherichia Enterobacter aerogenes et rel. Desulfovibrio et rel. Desulfovibrio Campylobacter Burkholderia Bilophila et rel. Aquabacterium Anaerobiospirillum Genus-like group Genus-like Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Phylum/Class Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Phylum

163 Chapter 7 FDR FDR 0.015 0.015 0.015 0.015 0.015 0.015 0.015 0.032 0.000 0.015 0.010 0.063 0.055 0.017 0.037 0.003 0.004 0.006 0.004 0.002 0.004 0.006 0.006 0.016 0.000 0.004 0.001 0.037 0.032 0.005 0.019 0.000 p.value p.value SD SD 0.258 0.276 0.250 0.282 0.981 0.100 0.863 0.640 0.004 0.001 0.003 0.315 0.003 0.004 0.004 0.009 0.241 0.162 0.221 0.204 0.460 0.049 0.493 0.669 0.022 0.007 0.021 0.307 0.015 0.012 0.016 0.026 F_t0_R F_t0_N SD SD 0.642 0.181 0.169 0.347 0.571 0.081 0.465 1.016 0.625 0.008 0.023 0.046 0.016 0.005 0.019 0.015 Relative abundance±SD Relative Relative abundance±SD Relative 0.656 0.237 0.308 0.447 0.835 0.083 0.867 1.467 0.553 0.008 0.025 0.070 0.016 0.014 0.021 0.034 B_t0_R B_t0_N Bacteroides uniformis et rel. uniformis Bacteroides Bacteroides stercoris et rel. Bacteroides Bacteroides splachnicus et rel. splachnicus Bacteroides Bacteroides plebeius et rel. Bacteroides Bacteroides ovatus et rel. ovatus Bacteroides Bacteroides intestinalis et rel. Bacteroides Bacteroides fragilis et rel. Bacteroides Allistipes et rel. Propionibacterium Micrococcaceae Corynebacterium Collinsella Atopobium Brachyspira Actinomycetaceae Yersinia et rel. Yersinia Genus-like group Genus-like Genus-like group Genus-like Mann-Whitney Mann-Whitney comparison of fecal (F) and biopsy (B) microbiota from responders at the genus-like level of the 130 Table 3.2. Table

Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Spirochaetes groups included in the relative HITChip. Average abundance±SD for significant groups different at baseline (t0) (p< 0.05 and FDR<0.3). Highlighted in grey are groups in higher abundance in biopsies; non highlighted groups are in samples. higher N: abundance non-responders; in R: fecal responders; FDR: False SD: standardDiscovery Rate; deviation Actinobacteria Proteobacteria Supplementary Phylum Phylum

164 FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files FDR 0.015 0.043 0.071 0.017 0.050 0.063 0.015 0.015 0.055 0.028 0.021 0.032 0.015 0.015 0.037 0.015 0.015 0.012 0.015 0.010 0.032 0.028 0.004 0.003 0.023 0.044 0.007 0.027 0.037 0.003 0.003 0.032 0.013 0.009 0.016 0.006 0.006 0.019 0.004 0.002 0.001 0.002 0.001 0.016 0.013 0.000 p.value SD 5.697 0.016 0.099 0.001 0.282 0.007 0.002 0.007 0.003 0.022 0.004 0.002 0.002 0.001 0.001 0.006 0.274 0.173 0.004 0.142 1.013 0.569 8.886 8.302 0.039 0.145 0.007 0.344 0.026 0.015 0.024 0.022 0.133 0.023 0.009 0.007 0.007 0.007 0.013 0.220 0.148 0.018 0.133 0.685 0.512 6.313 F_t0_R SD 2.724 0.032 0.529 0.008 0.191 0.023 0.088 0.024 0.024 0.134 0.025 0.007 0.008 0.031 0.008 0.014 0.175 0.215 0.021 2.407 0.550 9.114 16.940 Relative abundance±SD Relative 0.042 0.278 0.008 0.303 0.027 0.049 0.027 0.024 0.150 0.026 0.010 0.008 0.017 0.008 0.019 0.397 0.395 0.025 1.599 0.935 10.459 10.826 16.195 B_t0_R Faecalibacterium prausnitzii et rel. Faecalibacterium Eubacterium siraeum et rel. Anaerotruncus colihominis et rel. Anaerotruncus Clostridium thermocellum et rel. Clostridium (sensu stricto) Weissella et rel. Weissella Staphylococcus Lactococcus Lactobacillus salivarius et rel. Lactobacillus salivarius Lactobacillus plantarum et rel. Lactobacillus plantarum Bacillus Aneurinibacillus Aerococcus Asteroleplasma et rel. Asteroleplasma Uncultured Chroococcales Uncultured Uncultured Bacteroidetes Uncultured Tannerella et rel. Tannerella Prevotella tannerae et rel. Prevotella Prevotella ruminicola et rel. ruminicola Prevotella Prevotella oralis et rel. Prevotella Prevotella melaninogenica et rel. Prevotella Parabacteroides distasonis et rel. Parabacteroides Bacteroides vulgatus et rel. vulgatus Bacteroides Genus-like group Genus-like cluster IV cluster IV cluster IV cluster III cluster I Clostridium Clostridium Clostridium Clostridium Clostridium Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Asteroleplasma Cyanobacteria Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Phylum/Class Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Cyanobacteria Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Phylum

165 Chapter 7 FDR 0.025 0.015 0.001 0.015 0.007 0.001 0.015 0.055 0.021 0.028 0.007 0.015 0.017 0.032 0.015 0.015 0.055 0.015 0.071 0.025 0.071 0.015 0.043 0.011 0.004 0.000 0.003 0.001 0.000 0.006 0.032 0.009 0.013 0.001 0.004 0.007 0.016 0.004 0.003 0.032 0.004 0.044 0.011 0.044 0.004 0.023 p.value SD 0.008 0.009 0.004 0.011 0.002 0.005 0.001 0.151 0.040 0.036 0.020 0.002 0.004 0.011 0.020 0.003 0.752 0.001 2.088 0.015 0.049 0.003 0.008 0.037 0.054 0.011 0.007 0.029 0.008 0.104 0.107 0.059 0.047 0.014 0.016 0.038 0.037 0.022 1.008 0.007 1.236 0.051 0.046 0.021 0.023 0.0107 F_t0_R SD 0.038 0.065 0.037 0.009 0.008 0.242 0.008 0.067 0.110 0.165 0.063 0.018 0.019 0.044 0.080 0.024 0.449 0.008 0.574 0.425 0.031 0.025 0.024 Relative abundance±SD Relative 0.040 0.067 0.031 0.008 0.150 0.009 0.069 0.115 0.175 0.083 0.016 0.023 0.052 0.054 0.025 1.029 0.008 0.859 0.193 0.038 0.026 0.025 0.0111 B_t0_R Desulfovibrio et rel. Desulfovibrio Campylobacter Burkholderia Aquabacterium Anaerobiospirillum Alcaligenes faecalis et rel. Alcaligenes faecalis Aeromonas Fusobacteria Uncultured Mollicutes Uncultured Coprobacillus catenaformis et rel. Coprobacillus catenaformis Clostridium ramosum et rel. Lactobacillus catenaformis et rel. Lactobacillus catenaformis Catenibacterium mitsuokai et rel. Eubacterium cylindroides et rel. Bulleidia moorei et rel. Eubacterium limosum et rel. Clostridium sphenoides et rel. Clostridium felsineum et rel. Clostridium felsineum Clostridium difficile et rel. Phascolarctobacterium faecium et rel. Phascolarctobacterium faecium Peptococcus niger et rel. Peptococcus Mitsuokella multiacida et rel. Mitsuokella Megamonas hypermegale et rel. Megamonas hypermegale Genus-like group Genus-like cluster XVIII cluster XVIII cluster XVII cluster XVII cluster XVI cluster XVI cluster XV cluster XIVa cluster XI cluster XI cluster IX cluster IX cluster IX cluster IX Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Fusobacteria Uncultured Mollicutes Uncultured Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Phylum/Class Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Fusobacteria Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Phylum

166 FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files FDR 0.015 0.015 0.015 0.000 0.009 0.017 0.000 0.015 0.007 0.050 0.028 0.055 0.001 0.015 0.015 0.015 0.015 0.015 0.015 0.015 0.004 0.004 0.004 0.000 0.001 0.007 0.000 0.004 0.001 0.027 0.013 0.032 0.000 0.006 0.006 0.006 0.004 0.004 0.003 0.004 p.value SD 0.023 0.002 0.005 0.002 0.010 0.736 0.002 0.003 0.009 0.081 0.022 0.001 0.002 0.001 0.002 0.009 0.006 0.002 0.375 0.018 0.022 0.013 0.030 0.016 0.034 0.320 0.008 0.014 0.055 0.109 0.034 0.009 0.014 0.007 0.008 0.049 0.034 0.014 0.217 0.089 F_t0_R SD 0.037 0.014 0.031 0.042 0.034 0.678 0.086 0.016 0.064 0.179 0.114 0.010 0.016 0.008 0.007 0.075 0.040 0.088 0.356 0.155 Relative abundance±SD Relative 0.030 0.015 0.034 0.034 0.040 0.439 0.060 0.018 0.068 0.200 0.073 0.010 0.019 0.008 0.011 0.078 0.039 0.053 0.351 0.152 B_t0_R Akkermansia Brachyspira Yersinia et rel. Yersinia Xanthomonadaceae Vibrio Sutterella wadsworthia et rel. wadsworthia Sutterella Serratia Pseudomonas Proteus et rel. Proteus Oxalobacter formigenes et rel. Oxalobacter formigenes Oceanospirillum Novosphingobium Moraxellaceae Methylobacterium Leminorella Klebisiella pneumoniae et rel. Helicobacter Haemophilus Escherichia coli et rel. Escherichia Enterobacter aerogenes et rel. Genus-like group Genus-like Verrucomicrobia Spirochaetes Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Phylum/Class Verrucomicrobia Spirochaetes Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Phylum

167 Chapter 7 FDR 0.246 0.231 0.021 0.231 0.040 0.113 0.017 0.073 0.006 0.101 0.000 0.311 0.246 0.254 0.101 0.161 0.231 0.127 0.000 0.143 0.034 0.028 0.001 0.028 0.002 0.009 0.001 0.004 0.000 0.007 0.000 0.048 0.034 0.037 0.007 0.016 0.028 0.011 0.000 0.013 p.value SD 0.010 0.014 0.016 0.100 0.006 0.003 0.346 0.044 0.009 0.003 0.015 0.016 0.097 0.117 2.724 6.137 0.051 0.010 0.009 18.996 0.020 0.034 0.013 0.096 0.014 0.007 0.205 0.094 0.013 0.008 0.031 0.019 0.166 0.138 2.048 5.000 0.033 0.023 0.021 13.449 F_t12_N SD 0.010 0.014 0.116 0.165 0.013 0.005 0.393 0.055 0.017 0.015 0.224 0.037 0.213 0.114 2.841 5.595 0.038 1.701 0.009 20.722 Relative abundance±SD Relative 0.019 0.031 0.071 0.200 0.017 0.008 0.315 0.096 0.022 0.013 0.173 0.028 0.259 0.192 3.828 7.301 0.037 1.020 0.019 26.502 B_t12_N Xanthomonadaceae Vibrio Serratia Oxalobacter formigenes et rel. Oxalobacter formigenes Moraxellaceae Leminorella Escherichia coli et rel. Escherichia Enterobacter aerogenes et rel. Burkholderia Aquabacterium Alcaligenes faecalis et rel. Alcaligenes faecalis Staphylococcus Tannerella et rel. Tannerella Prevotella tannerae et rel. Prevotella Prevotella oralis et rel. Prevotella Prevotella melaninogenica et rel. Prevotella Bacteroides vulgatus et rel. vulgatus Bacteroides Bacteroides intestinalis et rel. Bacteroides Propionibacterium Corynebacterium Genus-like group Genus-like Mann-Whitney comparison of fecal (F) and biopsy (B) microbiota from non-responders at the genus-like level of the 130 130 the of level genus-like the at non-responders from microbiota (B) biopsy and (F) fecal of comparison Mann-Whitney Table 3.3. 3.3. Table

Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Firmicutes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Actinobacteria groups responders; included R: in non-responders; the N: HITChip. relative Average samples. abundance±SD for fecal significantin groups different abundance at12higher weeks in after FMTare (t12) (p

168 FMT in UC: microbiota shifts and signatures that determine success of therapy – supplementary files FDR 0.105 0.020 0.087 0.156 0.062 0.144 0.076 0.156 0.144 0.142 0.067 0.062 0.092 0.075 0.156 0.092 0.062 0.002 0.144 0.031 0.023 0.000 0.015 0.049 0.004 0.041 0.012 0.049 0.041 0.034 0.007 0.004 0.018 0.009 0.049 0.018 0.004 0.000 0.041 0.001 p.value SD 0.002 0.003 0.011 0.001 0.116 0.107 0.004 0.320 5.201 0.269 0.050 0.071 0.236 0.317 0.018 0.285 0.552 0.004 0.001 0.277 0.007 0.013 0.048 0.006 0.195 0.144 0.018 0.432 5.129 0.192 0.098 0.177 0.222 0.290 0.027 0.303 0.731 0.020 0.006 0.324 F_t12_R SD 0.008 0.028 0.041 0.004 0.169 0.185 0.008 1.070 9.055 0.448 0.121 0.234 0.516 0.492 0.035 0.419 1.078 0.629 0.004 0.031 Relative abundance±SD Relative 0.010 0.031 0.065 0.008 0.396 0.299 0.024 1.328 0.506 0.194 0.399 0.520 0.741 0.055 0.675 2.292 0.372 0.007 0.071 11.678 B_t12_R Peptostreptococcus anaerobius et rel. Peptostreptococcus Staphylococcus Enterococcus Aerococcus Tannerella et rel. Tannerella Prevotella tannerae et rel. Prevotella Prevotella ruminicola et rel. ruminicola Prevotella Parabacteroides distasonis et rel. Parabacteroides Bacteroides vulgatus et rel. vulgatus Bacteroides Bacteroides uniformis et rel. uniformis Bacteroides Bacteroides stercoris et rel. Bacteroides Bacteroides splachnicus et rel. splachnicus Bacteroides Bacteroides plebeius et rel. Bacteroides Bacteroides ovatus et rel. ovatus Bacteroides Bacteroides intestinalis et rel. Bacteroides Bacteroides fragilis et rel. Bacteroides Allistipes et rel. Propionibacterium Micrococcaceae Collinsella Genus-like group Genus-like Mann-Whitney Mann-Whitney comparison of fecal and biopsy microbiota from responders at the genus-like level of the 130 groups cluster XI Table 3.4. Table

Clostridium Bacilli Bacilli Bacilli Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Actinobacteria Actinobacteria Actinobacteria Phylum/Class Appendix

Firmicutes Firmicutes Firmicutes Firmicutes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Actinobacteria Actinobacteria included in the relative abundance±SD Average HITChip. for groupssignificant12 at different weeks FMT after (p< (t12) 0.05 and FDR<0.3). Highlighted in grey are groups in higher abundance in biopsies; non highlighted groups are in higher abundance in samples. fecal N: non-responders; R: responders; FDR: SD: standard Discovery Rate; deviation False Actinobacteria Supplementary Phylum

169 Chapter 7 FDR 0.144 0.087 0.067 0.066 0.075 0.092 0.076 0.047 0.144 0.067 0.092 0.053 0.066 0.144 0.020 0.121 0.156 0.105 0.121 0.076 0.076 0.041 0.015 0.007 0.006 0.009 0.018 0.012 0.002 0.041 0.007 0.018 0.002 0.006 0.041 0.000 0.028 0.049 0.023 0.028 0.012 0.012 p.value SD 0.037 0.006 0.009 0.008 0.002 0.010 0.002 0.012 0.003 0.269 0.033 0.008 0.003 0.001 0.007 0.009 4.323 3.165 2.600 0.840 0.689 0.023 0.026 0.019 0.030 0.007 0.049 0.012 0.048 0.013 0.159 0.094 0.013 0.008 0.006 0.028 0.016 2.483 3.704 3.031 1.174 10.934 F_t12_R SD 0.028 0.016 0.012 0.018 0.054 0.042 0.008 0.024 0.032 1.260 0.049 0.009 0.008 0.004 0.084 0.018 3.372 2.291 1.189 1.036 0.312 Relative abundance±SD Relative 0.024 0.031 0.032 0.037 0.037 0.064 0.015 0.073 0.031 0.625 0.142 0.028 0.013 0.007 0.094 0.026 5.819 1.287 1.677 1.619 0.470 B_t12_R Akkermansia Yersinia et rel. Yersinia Xanthomonadaceae Vibrio Serratia Proteus et rel. Proteus Moraxellaceae Klebisiella pneumoniae et rel. Haemophilus Escherichia coli et rel. Escherichia Enterobacter aerogenes et rel. Burkholderia Aquabacterium Anaerobiospirillum Alcaligenes faecalis et rel. Alcaligenes faecalis Catenibacterium mitsuokai et rel. Ruminococcus obeum et rel. Ruminococcus Outgrouping clostridium cluster XIVa Lachnospira pectinoschiza et rel. pectinoschiza Lachnospira Dorea formicigenerans et rel. Dorea formicigenerans Clostridium nexile et rel. Clostridium nexile Genus-like group Genus-like cluster XVII cluster XIVa cluster XIVa cluster XIVa cluster XIVa cluster XIVa Verrucomicrobia Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Phylum/Class Verrucomicrobia Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Phylum

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