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. Author manuscript; available in PMC: 2026 Jun 2.
Published in final edited form as: Med. 2025 Jun 2;6(9):100707. doi: 10.1016/j.medj.2025.100707

Donor Composition and Fiber Promote Strain Engraftment in a Randomized Controlled Trial of Fecal Microbiota Transplant for Ulcerative Colitis

Lasha Gogokhia 1, Nancy Tran 1, Alex Grier 2, Manabu Nagayama 2, Grace Xiang 1, Gabriela Funez-dePagnier 1, Alexa Lavergne 1, Caroline Ericsson 1, Sarah Ben Maamar 3, Mengrui Zhang 3, Robert Battat 1,4, Ellen Scherl 1, Dana J Lukin 1, Randy S Longman 1,2,*
PMCID: PMC12353604  NIHMSID: NIHMS2081305  PMID: 40460824

Summary

Background:

Fecal microbiota transplantation (FMT) is an emerging treatment for ulcerative colitis (UC), but the impact of prebiotic fiber on FMT efficacy for UC is unclear. We performed a randomized, double-blind, placebo-controlled clinical trial to examine the efficacy of FMT with and without dietary fiber supplementation in patients with UC.

Methods:

27 patients with mild to moderate UC were randomized to receive a single FMT or placebo with or without psyllium fiber supplementation for 8 weeks. The primary outcome was clinical response at week 8 and secondary outcomes included endoscopic improvement and clinical remission. Metagenomic sequencing of fecal DNA was analyzed to determine taxonomic profiles and donor strain engraftment.

Findings:

The trial was terminated early due to manufacturer discontinuation of FMT product. FMT induced clinical response, remission, and endoscopic improvement in UC patients compared to placebo (p<0.05), but fiber did not improve clinical outcomes of FMT. Recipient microbiome composition post-FMT shifted towards donor composition in responders and non-responders, but the durability of this change was stronger in responders. Clinical response and durable change in microbiome composition following FMT was donor dependent. Strain tracking analysis also demonstrated a donor dependent variability in the rate of successful engraftment and identified a consortium of engrafted bacteria associated with treatment response or fiber supplementation.

Conclusions:

Single-dose FMT demonstrated clinical efficacy for mild to moderate UC compared to placebo but revealed no benefit of fiber supplementation. These results highlight proof-of-concept that donor selection and prebiotic fiber can shape strain level engraftment. ClinicalTrials.gov, Number: NCT03998488.

Keywords: Fecal microbiota transplant, Fiber, Microbiome, Ulcerative colitis

Graphical Abstract

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eTOC blurb

The impact of dietary fiber in shaping strain engraftment and efficacy of fecal microbiota transplantation (FMT) for UC is unclear. Here, using a clinical trial of FMT with and without fiber supplementation for UC, Gogokhia et al. demonstrate that donor composition guides clinical efficacy and identify bacteria associated with treatment response and fiber supplementation.

Introduction

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon that affects millions of people worldwide1. Despite advances in immunosuppressive medications, which have significantly improved clinical outcomes by reducing inflammation and inducing remission, many individuals suffer from medically refractory disease or are unable to tolerate advanced therapies2. Long-term use of immunosuppressive medications can increase the risk for infection and malignancy, underscoring the need for safer, more effective treatment options. Achieving and maintaining remission remains a significant challenge in UC, highlighting the critical need for ongoing research to develop novel therapeutic strategies.

UC is associated with significant alterations in the gut microbiome, characterized by reduced richness and diversity compared to healthy individuals, with a notable decrease in beneficial bacteria and an increase in pathobionts3. The gut microbiome is dynamic, and microbial profiles can change rapidly with disease activity, treatment, and diet4,5. These findings highlight the potential for therapeutic interventions for UC aimed at modulating the gut microbiome, including probiotics, prebiotics, fecal microbiota transplantation, and specific dietary modifications6. However, despite these promising approaches, the role for microbial therapies for UC therapy remains elusive, necessitating further investigation to identify effective strategies for achieving sustained clinical remission and improve patient outcomes.

Emerging data from multiple randomized controlled trials (RCTs) highlight the potential therapeutic efficacy of FMT for UC7–9. Metanalysis of these RCTs reveals the overall efficacy of FMT in achieving clinical remission following short-term induction therapy with no significant signal in adverse events over placebo10. Despite the preponderance of positive data, significant heterogeneity exists in the clinical response to FMT therapy for UC, which may reflect underlying variability in product composition and preparation, dosing regimen, and microbiome-host interactions. Pilot studies suggest the higher dosing frequency improved clinical outcomes11, but the route and frequency of delivery still needs to be defined. Several studies identified the potential effect of “superdonor” material7,12. These findings highlight the potential role for donor composition and specific strains that may improve the efficacy of this therapeutic modality.

Diet is a critical regulator of the microbiome. Dietary fiber has emerged as a major regulator of both microbiome composition as well as function13. Although initial guidelines for IBD sought to reduce fiber intake14–16, studies revealing an association of UC with lower abundance of beneficial metabolites associated with fiber, including short chain fatty acids, led to a “re-thinking” of this dietary approach17,18. Experiments assessing the impact of fiber in mouse models of colitis reveal variable effects reflecting the impact of the microbiota, but only limited data exists evaluating the impact of increased fiber in UC cohort studies19–23. A recent RCT demonstrated that low fermentable fiber supplementation with FMT improved metabolic outcomes in patients with diabetes24. Moreover, combination of FMT with an anti-inflammatory diet including high fiber intake was more effective than standard medical therapy25. These findings highlight the potential impact of prebiotic manipulation of the gut microbiome in shaping microbial therapy, but the impact of prebiotic fiber in shaping strain level engraftment and clinical efficacy of FMT for UC has not been tested.

The effect of psyllium husk on microbiota has been studied previously, revealing its unique capacity to protect gut health through multiple mechanisms. Unlike many fermentable fibers, psyllium’s impact is not solely reliant on short-chain fatty acid (SCFA) production. It supports beneficial shifts in microbiota composition that reduce inflammation and maintain mucosal barrier integrity, particularly important in inflammatory bowel disease26,27. Psyllium has also been shown to increase bile acid excretion, which in turn activates the farnesoid X receptor (FXR), a nuclear receptor associated with anti-inflammatory responses. This FXR-mediated mechanism offers protection against colitis, providing anti-inflammatory effects even in minimal microbiota environments28. Additionally, psyllium helps counteract low-grade inflammation associated with metabolic syndrome in high-fat diets, promoting a more resilient microbiome that supports metabolic and intestinal health29. Psyllium’s distinct profile makes it a promising dietary fiber, especially for individuals sensitive to highly fermentable fibers.

Here, we designed a randomized, placebo-controlled trial of single donor FMT with or without psyllium fiber supplementation for mild to moderate ulcerative colitis (UC) to evaluate the impact of dietary fiber on clinical response and strain engraftment. Although early study termination due to manufacturer discontinuation of the product limited the study’s power to determine the impact of fiber on clinical outcomes, our study results highlight the efficacy of single dose colonoscopic FMT for the treatment of UC and provides proof-of-concept that donor composition and prebiotic fiber can shape strain-level engraftment.

Results

FMT with and without fiber induced clinical response and remission compared to placebo control

Thirty-two subjects were screened, 30 subjects were randomized and 27 subjects meeting the enrollment criteria were randomized in three groups: FMT, FMT with fiber supplementation, or placebo FMT (Fig. 1). Half of the subjects in the placebo FMT group received fiber supplementation. Baseline demographics, including age, gender, and disease activity measured by the total Mayo score were comparable across all treatment groups (Table 1). Most patients had moderate baseline disease activity with extensive/pancolitis (mean total Mayo score 7.7). There was no difference in response or remission based on baseline disease severity or endoscopic Mayo score (Fig. S1A). In addition, 18/27 (66%) were concurrently receiving advanced therapies. The placebo group had a numerically higher number of biologic-naïve participants compared with other groups as well as more patients on steroids compared with mesalamine or advanced therapies. Four patients did not undergo the week 8 flexible sigmoidoscopy due to COVID-19 restrictions (2) or withdrawal of consent to pursue alternate therapy (2) and were treated as non-responders. Due to the premature discontinuation of the FMT product by the manufacturer, the study was terminated before reaching pre-specified enrollment targets.

Figure 1: Study schematics:

Figure 1:

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Key inclusion and exclusion criteria and study workflow.

Table 1.

Baseline characteristics of study groups (also see Fig. S1)

FMT
(N=9)		 FMT+Fiber
(N=9)		 Placebo
(N=9)
Age (mean ± SD) 35 ± 15 36 ± 16 38 ± 15
Sex
Female, n (%) 3 (33%) 3 (33%) 2 (22%)
Male, n (%) 6 (67%) 6 (67%) 7 (78%)
Disease Location, n (%)
Rectosigmoiditis 2 (22%) 1 (11%) 2 (22%)
Left-Sided 1 (11%) 2 (22%) 3 (33%)
Extensive/Pancolitis 6 (67%) 6 (67%) 4 (44%)
Concomitant treatment, n (%)
Mesalamine 5 (56%) 6 (67%) 1 (11%)
Steroid 1 (11%) 3 (33%) 4 (44%)
Immunomodulator 1 (11%) 1 (11%) 0 (0%)
Biologic or Small Molecule therapy 7 (78%) 8 (89%) 3 (33%)
Biologic Naive 1 (11%) 1 (11%) 5 (56%)
Baseline Disease Activity
Mild, n (%) 1 (11%) 1 (11%) 0 (0%)
Moderate, n (%) 8 (89%) 8 (89%) 9 (100%)
Modified Mayo Score, mean (SD) 5.6 (1.4) 5.0 (0.7) 6.9 (1.2)
Partial Mayo Score, mean (SD) 5.6 (1.9) 4.2 (1.0) 6.0 (1.2)
Total Mayo Score, mean (SD) 7.4 (1.9) 6.8 (1.1) 8.9 (1.4)
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Pre-specified paired comparisons of the percentage of subjects achieving the primary endpoint of clinical response by total Mayo score revealed significant differences between FMT and placebo (56% vs. 11%, p<0.05), but not between FMT and FMT with fiber (56% vs. 22%), or FMT with fiber and placebo (22% vs. 11%) (Table 2). Three out of the 4 patients that failed to complete week 8 sigmoidoscopy were in the FMT with fiber group and were deemed non-responders for the primary endpoint analysis, further limiting the ability to determine the clinical impact of FMT with fiber (see Table S1).

Table 2. Outcome measures comparing donor fecal microbiota transplantation (FMT) with and without fiber at Week 8.

pMayo, partial Mayo score. Statistical comparisons between placebo and FMT groups were performed using chi-squared tests without Yates’ correction (also see Fig. S1)

Placebo, n (%) FMT, n (%) P value (placebo) FMT+Fiber, n (%) P value (placebo)
Primary Outcome
Clinical Response 1 (11%) 5 (56%) 0.04 2 (22%) 0.5
Secondary Outcomes
Clinical Remission 0 (0%) 3 (33%) 0.06 1 (11%) 0.3
Clinical Response, by pMayo 1 (11%) 6 (67%) 0.02 3 (33%) 0.2
Clinical Remission, by pMayo 1 (11%) 3 (33%) 0.3 2 (22%) 0.5
Endoscopic Response 0 (0%) 4 (44%) 0.02 2 (22%) 0.1
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Secondary endpoint analysis revealed higher clinical response by pMayo (p=0.02) and endoscopic response rates (p=0.02) at week 8 in the FMT group compared to the placebo group (Table 2). Analysis of clinical response determined by partial Mayo score in all patients receiving FMT (with or without fiber) revealed improvement as early as week 4 that was sustained through week 8 (Figure S1B). To evaluate the effect of psyllium on stool frequency, we compared the average stool frequency between participants who received psyllium supplementation and those who did not. The group without psyllium had a slightly higher stool frequency (2.75 vs. 2.00), but this difference was not statistically significant (p = 0.263).

The safety profile in this trial of FMT with or without fiber supplementation demonstrated favorable outcomes, with no serious adverse events observed among the 27 enrolled and randomized participants across the three study groups. Although four patients did not complete the week 8 flexible sigmoidoscopy, two due to COVID-19 restrictions and two due to voluntary withdrawal to pursue alternative therapies, these discontinuations were unrelated to the tolerability of the study treatments. Throughout the study period, no additional safety concerns were identified, including in the FMT with fiber group receiving 10g of psyllium husk fiber supplementation daily.

FMT responders show durable taxonomic change in microbiome composition

Microbiome diversity correlates with disease activity, and we observed numerically reduced microbial diversity in recipients compared to donors (p=0.06, Fig. 2A). Although previous studies have highlighted the potential role for microbial diversity as a marker of success of FMT30, we observed no difference in the change in alpha diversity (measured by Shannon index) from week 0 to week 8 between study groups (Fig. 2B). In addition, there were no statistically meaningful differences in the baseline microbiome diversity (Fig. S1C) or composition (Fig. S1D) between responders and non-responders. In contrast, analysis of the change in microbiome composition using Unifrac distances to donor revealed that single dose delivery of FMT via colonoscopy with or without daily fiber supplementation led to significant shifts towards the donor microbiome compositions in comparison to those randomized to placebo (Fig. 2C). Although both responders and non-responders showed a significant shift in beta diversity toward donor at week 4, only responders sustained a robust difference up to 12 weeks (Fig. 2D). Linear discriminant analysis effect size (LEfSe) revealed the differential enrichment of taxa in both responder and non-responders at week 8 (Fig. S2A) and multivariable association modeling (using MaAsLin2) identified a distinct set of taxa durably enriched only in responders up to 12 weeks post-FMT (Fig. S2B). These taxa include Bacteroides spp., Alistipes spp, and Faecalibacterium spp consistent with responders seen in other studies30,31.

Figure 2: FMT responders show durable taxonomic change in microbiome composition.

Figure 2:

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A. Shannon diversity of donors and FMT +/− fiber recipients at weeks 0, 4, 8, and 12. B. Difference in Shannon diversity between week 8 and 0. C. Unweighted UniFrac distance to donor at week 0 and week 8 for FMT, FMT + fiber, or placebo recipients. Crossover donor assignment was used to calculate distance for placebo recipients. D. Unweighted UniFrac distance to donor for FMT +/− fiber responders and non-responders at week 0, 4, 8, and 12 (also see Fig. S3).

Donor composition drives clinical response, microbiome shift, and strain-specific transfer

Previous studies suggested the potential role for donor composition in shaping clinical response7,32. To evaluate the potential impact of donor differences, this study leveraged the use of 3 independent donors. Donor 1 induced a higher rate of clinical response, compared to donors 2 or 3 (Fig. 3A). Shannon diversity analysis revealed that donor 1 microbiome was more diverse than donors 2 and 3 (Fig. 3B). Previous reports using an enterotype classification of the gut microbiome based on the relative abundance of different microbial taxa suggested the association of a Bacteroides enterotype (called Bact2)—characterized by a high abundance of Bacteroides, reduced levels of Faecalibacterium and Akkermansia, and low microbial diversity and load—with systemic inflammation and altered gut homeostasis33. Enterotype analysis in our study showed that Donor 1 was associated with an enterotype characterized by a higher abundance of Ruminococcaceae and microbial diversity, which are linked to a more stable and anti-inflammatory gut microbiota (Fig. S3A). In contrast, Donor 2 and Donor 3 displayed features of the Bacteroides 2 (Bact2) enterotype. Composition analysis of recipients revealed that both donor 1 and 2 (but not donor 3) induced a significant reduction in Unifrac distance to donor post-FMT which was maintained until week 12; however, the magnitude and durability of the shift to donor composition (as seen in the PCoA) was strongest for donor 1 (Fig. 3C).

Figure 3: Donor composition drives clinical response and microbiome shift.

Figure 3:

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A. Clinical response at week 8 by total Mayo score stratified by donor of subjects receiving FMT alone (n=7 donor 1; n=7 donor 2, n=4 donor 3). B. Microbial diversity of donor FMT by Shannon richness. C. Change in microbiome composition measured by unweighted UniFrac distance to donor (left) and PCoA (right) for week 0, 4, 8, and 12 stratified by donor (also see Fig. S3).

Recent studies highlight the importance of strain level characteristics in determining the functional impact of the gut microbiota34. To elucidate the strain-level composition and its impact on FMT outcomes, we analyzed strain-level engraftment across various donors. This analysis leveraged the observation that microbial strains are predominantly unique to individual hosts and are infrequently shared among unrelated individuals, enabling us to determine the transfer and engraftment of the donor microbiome within the recipient35. The mutation rate (defined by the frequency of unique metagenomic single nucleotide polymorphisms (SNPs)), was used to differentiate between identical and distinct strains (Fig. S3B)36. The relative abundance of donor strain engraftment varied from 0 to 76% of detectable strains (Fig. 4A) and did not discriminate responders from non-responders. Although this finding was limited by the incomplete evaluation of three FMT + fiber patients at week 8, the total number of species-level genome bins (SGBs) engrafted in responders was numerically higher than non-responders (Fig. 4B, p=0.092). Consistent with previous reports36,37, regression analysis showed that the success of engraftment inversely correlated with the baseline diversity of recipients (Fig. S3C) and positively correlated with donor diversity (Fig. S3D).

Figure 4: Donor composition correlates with success of strain engraftment.

Figure 4:

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A. Relative abundance contributed by donor (blue), recipient (pink), or new (yellow) strains post-FMT. Responder status (purple) and donor (green) is indicated. B, C. Number of engrafted species-level genome bins (SGB) in responder and nonresponders (B) or engrafted strains in recipients of specified donors (C). D. Success of strain engraftment measured as the proportion of donors strains found to be engrafted in week 8 recipients of indicated donors. E. Engraftment outcomes of week 8 recipients for strains achieving >80% overall engraftment success. Responder status and donor are indicated. F. Relative abundance of fucofuranose pathway 7312 in responders and non-responders to FMT (also see Fig. S3)

Strain level engraftment analysis showed that donors 1 and 2 had higher number of unique strains and increased frequency of engraftment compared to the donor 3 (Fig. 4C). Correspondingly, the success rate of donor 1 derived strain engraftment was higher than that of strains derived from donors 2 or 3 (Fig. 4D). To determine the identity of the bacterial species that engrafted well, we calculated the species-specific strain engraftment rates across all donors, pre-FMT, and post-FMT recipients. A strain was considered successful if its engraftment rate exceeded 80% across all opportunities (Fig. 4E). Overall, we found that the engrafted bacteria were almost evenly represented by Firmicutes and Bacteroidetes (48.1% and 51.9%, respectively) and did not include any other phyla, such as Proteobacteria. Over half of the successfully engrafted strains were associated with Donors 1 and 2 (51.8% each), with only two strains (7.4%) associated with Donor 3. Notable members of the consortia of engrafted bacteria included Faecalibacterium prausnitzii, Odoribacter splanchnicus, Ruminococcus torques, Bacteroides ovatus, and Bacteroides thetaiotaomicron. Importantly, most of these engrafted strains (81.5%) were linked to treatment response, while the remaining species had over a 65% engraftment rate in responders, indicating a significant association between successful engraftment of these bacteria and response to FMT.

Pathway analysis comparing pre- and post-FMT samples in donor 1 identified a significant increase in the D-fucofuranose biosynthesis pathway (PWY-7312), which is linked to the O-antigen component of bacterial lipopolysaccharide of gram-negative bacteria38 and known to be enriched in the cluster of bacteria with high diversity of Bacteroides. Lower diversity in this cluster is associated with higher disease prevalence39. PWY-7312 was found in all responders post-FMT (Fig. S3E) and primarily derived from Bacteroides stercoris, which remained consistently elevated in recipients of Donor 1 (Fig. 4F, S3F, G). This observation implicates Bacteroides stercoris as an important component of the donor microbiota and highlights the potential for specific donor bacteria in supporting the clinical efficacy of FMT for UC.

Fiber shapes strain level engraftment

Dietary fibers have been shown to selectively promote the growth of beneficial bacteria, leading to improved gut health outcomes and highlighting the potential for fiber supplementation to shape microbial engraftment during FMT24,40. Although our study was underpowered to determine the clinical impact of fiber due to early termination, we investigated its effect on the microbiome and strain-level engraftment in FMT recipients. Comparison of week 8 recipients receiving placebo alone or placebo + fiber revealed the ability of fiber to alter sugar biosynthesis pathways in the endogenous microbiome (Fig. S4A). Taxonomic analysis revealed the strongest increase in Phocaeicola vulgatus (previously Bacteroides vulgatus, Fig. S4B), a human gut commensal involved in degrading complex heteropolysaccharides. Strain tracking analysis revealed that fiber significantly enhanced both the absolute number of donor strains (Fig. 5A) and the overall likelihood of successful donor strain engraftment (Fig. 5B). Fiber use was associated with a distinct group of bacteria that remained persistently enriched in patients following FMT (p=0.05) (Fig. 5C). The majority of these bacteria originated from Donor 1 and Donor 2, with minimal contribution from Donor 3, including Bacteroides intestinalis, Emergencia timonensis, and Butyricimonas virosa. Remarkably, 80% of these bacterial species (12 out of 15) exhibited a 100% engraftment rate, while the remaining species had engraftment rates of 80% and 50%, respectively. Enhancement of strain engraftment was particularly pronounced in non-responders (Fig. S4C). This suggests that while fiber supplementation improves the strain-level engraftment of donor microbiota, its precise role in translating these microbiome changes into clinical benefits needs additional research.

Figure 5: Fiber shapes strain level engraftment of FMT.

Figure 5:

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A. Number of donor strains engrafted in FMT recipients with or without fiber evaluated at week 8. B. Success of strain engraftment measured as the proportion of donor strains found to be engrafted in week 8 recipients with or without fiber. P-values are indicated. C. Engraftment outcomes of week 8 recipients for strains with enhanced engraftment by fiber. Fiber status and donor are indicated (also see Fig. S4).

Discussion

Recent trials support the potential therapeutic efficacy of FMT for inducing short-term clinical remission in mild to moderate ulcerative colitis10, but heterogeneity in study outcomes have limited the overall impact of FMT therapy for UC. These initial studies highlight the potential impact of donor variability, recipient factors, dosing frequency, and route of administration6. Using a single donor approach coupled with strain-specific analysis, our results support the hypothesis that a combination of (i) the right strains in the donor material and (ii) engraftment success of those strains impact clinical outcome.

Although our study was terminated early, the clinical results support evidence for the efficacy of FMT in inducing clinical and endoscopic responses at week 8 compared to placebo. Remission rates observed here are consistent with other FMT studies requiring more intensive FMT administration8, highlighting the efficacy of single colonoscopy delivered FMT for induction therapy. The beneficial effects of FMT were apparent by week 4 post-FMT, indicating a rapid effect on clinical symptoms. While comparisons between placebo and FMT groups were performed using a chi-squared test as prespecified in our analysis plan, this approach may overestimate effect sizes in small sample sizes. Consistent with the clinical results, metagenomic analysis of the microbiome composition revealed a more robust and durable shift towards donor composition in responders compared to non-responders, which was sustained over the 12-week period, reflecting both immediate and durable impacts on disease activity.

Variability in the clinical response to FMT therapy for UC may reflect underlying heterogeneity in product composition as well as dosing regimen. Initial studies showing the efficacy of FMT for UC revealed a clustering of responders linked to potential “superdonors”7. Subsequent larger FMT studies used pooled donors to overcome the heterogeneity of these potential donor-dependent effects9,30, but a pooled donor design is less robust for identifying composition differences impacting response6. Here, we used a single donor design distributed over the experimental groups, which enabled us to define the donor characteristics most effective in robustly and durably shifting the composition of the recipient microbiome. Consistent with pooled studies, our results similarly identify enrichment of Bacteroides spp., Faecalibacterium spp., and Odoribacter spp. in responders and E. coli in non-responders. Open label studies of pooled two-donor compositions supported the possibility that increased diversity promoted high clinical response rates12; however, recent rigorous single donor FMT trials for UC (UC-RESTORE and CRAFT-UC) failed to show improved outcomes of FMT for UC33,41. Both studies showed donor-dependent impact on clinical efficacy. The UC-RESTORE rigorously selected non-Bact2 enterotype donors and ensured high bacterial density yet still failed to demonstrate efficacy of allogeneic FMT compared to autologous FMT for UC. Like the UC-RESTORE trial, our study cohort included subjects with primarily moderate disease activity, extensive/pan-colitis, and exposure to advance therapy. The most effective donor (Donor 1) identified in our cohort was also consistent with a non-Bact2 enterotype, suggesting that additional factors may contribute to the donor effect on clinical efficacy. While our sequencing approach enabled high-resolution strain tracking, metagenomics-based methods may be limited in detecting low-abundance strains that could not be confidently assigned as donor-specific. The sequencing depth in this study (2.8 × 10⁷ reads / sample +/− standard deviation 8 × 10⁶ reads) may have also contributed to the non-detection of certain strains.

Recent studies highlight the need for strain level tracking to understand the functional impact of microbial therapies for immune mediated diseases34,36,37. Using strain tracking algorithms, we demonstrate that donor diversity increases the absolute number and success rate of strain engraftment, and this inversely correlates with recipient diversity. Our study highlights a collection of strains that are highly successful in engrafting in the recipient if they are present in the donor and these strains are enriched in responders. Proof-of-concepts studies are needed, as illustrated by the LOTUS study42, to evaluate if this consortium of bacteria is sufficient to replicate the clinical results achieved by Donor 1. Pathway analysis identified enrichment of the fucofuranose biosynthesis pathway from Bacteroides stercoris in Donor 1 and FMT responders. D-fucofuranose contributes to the biosynthesis of the O antigen component of the immunoregulatory lipopolysaccharide present in gram-negative bacteria. The presence of this pathway in communities with higher diversity of Bacteroides may act to limit disease or immune activation43,44. Additional cohorts identified strains of B. hydrogenotrophica capable of metabolizing L-arginine enriched in responders and B. fragilis and B. salyersiae capable of producing taurine and hypotaurine enriched in the lumen of non-responsive subjects post-FMT, highlighting the need for strain specific analysis to determine the mechanistic contribution in UC45.

Diet is a critical regulator of the microbiome and may play an important role in shaping the outcome of FMT. Dietary fiber impacts both microbiome composition and function13. In mouse models, highly fermentable inulin fiber promotes Bacteroides-dependent increases in primary bile acids which trigger eosinophilia and type 2 inflammation46; however, the effects of fiber in mouse models are variable, including the protective effect of low fermentable psyllium fiber in response to dextran sodium sulfate challenge27, which may reflect a unique microbe-fiber interaction. A recent RCT demonstrated that low fermentable fiber supplementation with FMT improved metabolic outcomes in patients with diabetes, indicating the potential impact of prebiotic manipulation of the gut microbiome in metabolic health24. In addition, the FMT-AID study showed that combination of FMT with an anti-inflammatory diet in an Indian cohort with high dietary fiber intake was more effective than standard medical therapy alone25. Unfortunately, due to product unavailability, early study termination prevented us from obtaining the power to assess the clinical effect of fiber in combination with FMT compared to FMT alone for the treatment of UC. Numerically, FMT with fiber supplementation resulted in lower rates of clinical response, remission and endoscopic response; however, 2/9 patients in this group were prevented from meeting the week 8 primary endpoint flexible sigmoidoscopy secondary to COVID-19 pandemic restrictions. Psyllium is known to influence bowel frequency; however, in our trial, the group without psyllium had a slightly higher stool frequency, and this difference was not statistically significant.

Despite these limitations, our study offers the first proof-of-principle that fiber can modify donor-dependent strain level engraftment and supports the rationale for future studies examining prebiotic supplementation in shaping engraftment and efficacy to optimize microbial therapy for UC. Enrichment in taxonomic pathways consistent with sugar biosynthesis in placebo recipients that received fiber compared to placebo alone highlights the functional impact psyllium supplementation on the UC microbiome. Fiber supplementation alone led to the increased abundance of Phocaeicola vulgatus, a human gut commensal involved in metabolizing complex dietary polysaccharides and producing short chain fatty acids. While it provides protection in some mouse models of colitis47,48, P. vulgatus production of dipeptidyl peptidases correlates with UC disease activity and protease activity exacerbated colitis in IL10-deficient mice49. The variable impact of P. vulgatus in pre-clinical models of IBD may reflect the impact of environmental and dietary triggers shaping its function as a commensal or opportunistic pathobiont. Fiber supplementation promoted engraftment of several distinct taxa primarily in Donor 1 and 2 recipients including Bacteroides intestinalis, Emergencia timonensis, and Butyricimonas virosa. E. timonensis is a gut commensal most closely related to Odoribacter and Butyricimonas species50, which have been shown to produce short chain fatty acids and regulate the mucosal immune response in pre-clinical models of colitis31. Emerging data suggest a potential role for xenobiotics in shaping the functional potential of specific strains51,52, but further studies are needed to define how fiber shapes their metabolic activity and the impact it may have on clinical outcomes.

Limitations of the study

There are several limitations of this study. Due to manufacturer discontinuation of FMT production, we were unable to complete target enrollment. While the results demonstrate clinical efficacy of FMT, we were underpowered to detect clinical differences between FMT alone compared to FMT with fiber. Three out of the 4 patients that failed to complete week 8 sigmoidoscopy were in the FMT with fiber group and were deemed non-responders for the primary endpoint analysis, further limiting the ability to determine the clinical impact of FMT with fiber. The absence of sex- and gender-based analyses, as well as the lack of socioeconomic data, is also noted as a limitation, as these gaps may impact the study’s ability to fully generalize findings across diverse populations. While this study was not specifically designed to assess differences in stool frequency, the potential impact of psyllium and other dietary fibers on bowel habits is an important consideration for future investigations in IBD. Finally, functional analysis of individual strains is needed to define a mechanistic contribution to their therapeutic utility as live biotherapeutics for the treatment of UC.

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Randy Longman (ral2006@med.cornell.edu)

Materials availability

This study did not generate unique reagents.

Data and Code availability

All sequencing data are deposited in the NCBI BioProject repository under accession number PRJNA1169096. Clinical metadata are provided in supplemental table 1. Demographic data is reported in aggregate in Table 1. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Star Methods

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Study Population

This phase 2 trial was approved by the Weill Cornell Institutional Review Board (#1904020045) and registered with ClinicalTrials.gov (NCT03998488). Patients ≥ 18 years old with a history of ulcerative colitis were eligible for screening, which comprised an interview, physical examination, and screening colonoscopy (Fig. S1). Criteria for inclusion were defined by a total Mayo score of 4–10 with an endoscopic sub-score ≥ 1, a stool frequency or rectal bleeding sub-score of ≥ 1, and confirmed, active ulcerative colitis ≥ 15 centimeters at the screening colonoscopy. Patients were also required to have an intact colon, and patients taking steroid or biologic therapy must have been on a stable dose for 4 weeks prior to screening and agree to maintain such dosing throughout the trial. Additionally, patients were required to undergo serological and fecal screening testing prior to the screening colonoscopy to rule out active infections, pregnancy, as well as discontinue antibiotics 48 hours prior to the screening colonoscopy. Patients were excluded if they met any of the following criteria: biopsy proven Crohn’s disease, severe ulcerative colitis defined by a total mayo score > 10, clinical complications requiring emergent management (e.g. stricture, bowel obstruction, perforation and/or abscess), concurrent C. difficile or other infections, primary sclerosing cholangitis, prior history of FMT, treatment for malignancy within the past 5 years excluding non-melanoma skin cancers, active or latent tuberculosis, clinically meaningful laboratory abnormalities (Hb: < 8 g/dL, ALT: greater than 3x the upper limit of normal), history of anaphylactic reactions to food allergens or allergy to psyllium husk, pregnancy or lactation, probiotic use in the 4 weeks prior to screening and for the duration of the trial, or any other condition that, in the opinion of the investigator, would jeopardize the safety or rights of the subject participating in the study, would make it unlikely for the subject to complete the study. To ensure inclusivity and provide context for study generalizability, participant demographics (e.g., age and sex) are summarized in Table 1. Among the participants, 88.9% identified as White, while 3.7% each identified as Black or African American, Middle Eastern or North African, and Other. Furthermore, 96.3% identified as not Hispanic or Latino. Information about socioeconomic factors was not collected, which is recognized as a limitation. Ethical compliance included obtaining written informed consent from all participants prior to study enrollment, and the absence of sex- and gender-based analyses is acknowledged as a limitation in the discussion of the study’s generalizability.

Study Design

This is a double-blind, randomized, placebo-controlled trial investigating fecal microbiota transplantation (FMT) with or without fiber supplementation. Eligible patients were randomized at the screening visit into one of three groups (FMT with Fiber, FMT without Fiber, and Placebo with or without Fiber) using a 1:1:1 allocation ratio. A system of randomized blocks with fixed, blinded block sizes ensured equal distribution among the groups without unblinding before reaching the primary endpoint (Fig. S1). Only the statistician and study coordinator had access to the randomization list, while the research coordinator assigned coded identifiers to each patient. At Week 0, each participant received their initially assigned treatment (FMT or placebo) in a blinded manner. Blinding was maintained for both participants and investigators at Weeks 0 and 8. All participants initially randomized to the placebo arm received FMT at week 8; however, their data were excluded from the primary endpoint analysis and were only incorporated into the microbiome analysis. Clinicians were unblinded only after all evaluations were completed to reduce bias. Fiber supplementation was not blinded; patients randomized to the fiber groups received two bottles of 10g psyllium husk powder (NOW Foods) and began supplementation three days before their screening colonoscopy (Week 0). Before starting fiber, participants met with a registered dietitian to review fiber dosing (10g per day) and completed a diet recall to document typical dietary intake. The trial aimed to enroll 135 subjects (45 per group) to achieve sufficient power, based on an expected 55% clinical response rate in the FMT + Fiber group compared to a 27% response rate in the FMT alone group, assuming 80% power and a two-sided significance level of 0.05. However, the study was terminated early due to product unavailability, which prevented full enrollment.

Fecal Microbiota Preparation

All investigational fecal microbiota preparation (FMP250) were biologically active fecal material from rigorously screened, healthy human donors. All units were pre-screened, tested, quarantined, stored, packaged, and labeled by a universal stool bank (OpenBiome). Placebo FMP250 were used as control units and made of glycerol, saline, and food dye that was stored, packaged, and labeled identically to the investigational FMP250 to ensure blinding during delivery. Approximate FMP250 density was 1011 CFU/mL. Product from 3 distinct donors was allocated evenly over the 3 groups.

Fecal Microbiota Transplantation

Subjects underwent a bowel preparation (polyethylene glycol 3350 split prep) on the day prior to the Week 0 colonoscopy. The morning of the colonoscopy, subjects were administered 4 mg of loperamide to assist with FMT retention. If subjects met eligibility criteria of active ulcerative colitis ≥ 15 centimeters, mayo endoscopic sub-score ≥ 1, and total mayo score 4–10, then the blinded FMT was delivered. Prior to administration of FMT into the terminal ileum, cecum and ascending colon, mucosal biopsies were obtained from the rectum and sigmoid for analysis. At Week 8, subjects were instructed to undergo a clear liquid diet on the day prior to the Week 8 flexible sigmoidoscopy and use two water enemas. The morning of the flexible sigmoidoscopy, subjects were administered 4 mg of loperamide to assist with FMT retention. Coded FMT and placebo material was delivered by the investigator in a blinded fashion. Prior to administration, mucosal biopsies were obtained from the rectum and sigmoid for analysis.

METHOD DETAILS

Shotgun Metagenomics Sequencing

DNA Extraction

Stool samples collected from subjects and donors were stored in 2 mL cryovials and frozen at −80°C. A single fecal pellet is deposited into a Qiagen PowerBead glass 0.1 mm tube (13118–50). Using a Promega Maxwell RSC PureFood GMO and Authentication Kit (AS1600), 1mL of CTAB buffer & 20μl of RNAse A Solution is added to the PowerBead tube containing the sample. The sample/buffer is mixed for 10 seconds on a Vortex Genie2 and then incubated at 95°C for 5 minutes on an Eppendorf ThermoMixer F2.0, shaking at 1500 rpm. The tube is removed and clipped to a horizontal microtube attachment on a Vortex Genie2 (SI-H524) and vortexed at high-speed for 20 minutes. The sample is removed from the Vortex and centrifuged on an Eppendorf Centrifuge 5430R at 40°C, 12700 rpm for 10 minutes. Upon completion, the sample is centrifuged again for an ADDITIONAL 10 minutes to eliminate foam. The sample is checked for foam and particulates (they will clog the robotic instrument tips). If foam or particulates are found in the sample, they are carefully removed using P1000 pipette. DNA extraction is then processed via the Promega MaxPrep Liquid Handler and Promega Maxwell RSC 48 instruments. DNA is quantified using Quant-iT dsDNA High Sensitivity Assay Kit using Promega GloMax plate reader on a microplate (655087).

Metagenomic Shotgun Sequencing Library Generation

Library generation follows the Illumina Nextera XT DNA Library Prep Kit Reference Guide: https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera-xt/nextera-xt-library-prep-reference-guide-15031942-05.pdf

Library Verification, Quality Check, Pooling

DNA sequencing libraries are washed using Beckman Coulter AMPure XP magnetic beads. Library quality & size verification is performed using PerkinElmer LabChip GXII instrument with DNA 1K Reagent Kit (CLS760673). Library concentrations are quantified using Quant-iT dsDNA High Sensitivity Assay Kit using Promega GloMax plate reader on a microplate (655087). Library molarity is calculated based on library peak size & concentration. Libraries are normalized to 2nM using the PerkinElmer Zephyr G3 NGS Workstation (133750) and pooled together using the same volume across all normalized libraries into a 1.5ml Eppendorf DNA tube (022431021).

Sequencing

Pooled libraries are sequenced on the Illumina HiSeqX instrument at loading concentration of 10pM, paired-end 150bp. High-throughput sequencing was performed on all stool samples to ensure adequate coverage for strain tracking analyses. The sequencing generated an average of 2.8 × 107 reads per sample, with a standard deviation of 8 × 106 reads. This robust coverage meets current standards for high-resolution strain tracking, ensuring reliable data quality.

Data Processing

Quality-based filtering and removal of host sequences and adapter and quality trimming was performed using kneadData (https://huttenhower.sph.harvard.edu/kneaddata/) version 0.10.0, using the hg37 human reference genome. Taxonomic composition was calculated using MetaPhlAn453 with reference database version mpa_vOct22_CHOCOPhlAnSGB_202212. Abundance of gene families and metabolic pathways were quantified using HUMAnN3 (version 3.6)54. Gene families and pathways were renormalized to relative abundances after removing unmapped/ungrouped counts.

Strain Level Analysis

Strain level profiling was performed using StrainPhlAn55. Marker genes present in fewer than 50% of samples and samples with fewer than 50% of marker genes for a given species were filtered out. Post-FMT engraftment events were assessed using all available post-FMT samples. A donor strain was classified as engrafted if it was detected in any post-FMT sample and had not been observed in the recipient prior to FMT. This approach allowed for a comprehensive assessment of donor strain acquisition over time55. To establish the cutoff for same strain tracking, we analyzed mutation rates in two distinct groups: (1) mutation rates observed within the same species from longitudinal samples collected from the same subjects, representing cases where the same strain was likely retained over time, and (2) mutation rates observed within a species between subjects who did not share a donor and were not part of donor-recipient pairs, representing cases where the same strain was unlikely to be present. The resulting histogram displayed a clear bimodal distribution, aligning with our expectation of ‘likely same strain’ and ‘unlikely same strain’ categories (Fig. S3B). The mutation rate cutoff was selected based on this distribution, ensuring clear separation between the two modes. To assess strain transfer, we then generated an additional histogram incorporating a third group: mutation rates observed within a species between donor-recipient pairs, with recipient samples obtained post-FMT. As anticipated, this group exhibited a distribution spanning both sides of the cutoff, reflecting a mixture of strains that were either below the cutoff (same strain) or above the cutoff (unlikely same strain). Strains with mutation rates below the cutoff were classified as engraftments, indicating successful strain transmission from donor to recipient. To assess variables that may influence the success rate of engraftment, a binomial family generalized mixed effects linear regression model was fit using the variable(s) of interest as fixed effects and subject and species as random effects.

QUANTIFICATION AND STATISTICAL ANALYSIS

Outcome Measures

The primary endpoint was clinical response at Week 8 assessed by flexible sigmoidoscopy, physical examination, and medical interview to evaluate mucosal appearance and assess for UC activity. Clinical response was defined by a reduction in the total Mayo score from baseline by ≥ 3 points (≥ 30% reduction), with a decrease in the rectal bleeding sub-score by ≥ 1 point. The secondary endpoint was clinical remission, endoscopic response, and endoscopic remission at Week 8. Clinical remission was defined by a total Mayo score ≤ 2, with no individual score ≥ 1. Endoscopic response was defined by a reduction in the endoscopic sub-score by ≥ 1, and endoscopic remission was defined by an endoscopic sub-score ≤ 1. The participants with a baseline endoscopic Mayo score of 1 were required to drop at least one point (achieving a score of 0) in order to be classified as in endoscopic remission. The secondary endpoint also included safety of FMT and was evaluated by adverse events assessment during in-person visits at Week 4, Week 8, and Week 12. Safety-phone calls were conducted at Day 1 post-FMT, Week 2, 6, and 10 to assess for additional adverse events. Subjects were also instructed to notify the treating physician at any time post-FMT if they were to develop any symptoms or new medical conditions. Adverse event severity was graded using the NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. Additionally, secondary outcomes of clinical response and remission at Week 8 were evaluated using the partial Mayo score. Response was defined by a reduction in the partial Mayo score by ≥ 2 from baseline and remission was defined as a partial Mayo score ≤ 2, with no subscore ≥ 1. With subjects receiving the crossover treatment at Week 8, exploratory analysis between Week 8 and Week 12 was similarly conducted using the partial Mayo score.

Statistical Analysis

Primary endpoint analysis was performed based on intent to treat. Therefore, any subject with missing data at Week 8 was labeled as a non-responder (no change in disease activity from baseline). For the primary endpoint, pairwise comparisons between the three groups (FMT, FMT with fiber supplementation, and placebo) were conducted using chi-square tests. The same analysis was applied for categorical variables of the secondary and exploratory endpoints. All p-value statistical significances were evaluated at the 0.05 alpha level.

Microbiome Analysis

Alpha diversity (Shannon Index) was calculated for all samples using the unfiltered abundance matrix, while beta diversity (Bray-Curtis) was calculated using the unfiltered relative abundance matrix. Both alpha and beta diversity were calculated at the species level using the ‘vegan’A package in R. Statistical comparisons of alpha diversity were done using the Wilcoxon rank sum test, while comparisons of beta diversity were done using the permutational multivariate analysis of variance (PERMANOVA). Unweighted UniFrac distances were calculated for each subject’s samples to their respective donor’s sample using the ‘phyloseq’ package. Paired analysis for unweighted UniFrac distances were done using the Wilcoxon signed rank test. Additionally, the Dirichlet Multinomial Mixture method was used to enterotype the three donors at the genus level. This was done with the “DirichletMultinomial” package. Differential abundance of taxa and pathways was assessed using MaAsLin256 and LEfSe57. For longitudinal comparisons, a random effect for subject was included. LEfSe was only used for cross-sectional comparisons.

Supplementary Material

1

Key resources table

REAGENT or RESOURCE SOURCE IDENTIFIER
Deposited data
BioProject repository NCBI PRJNA1169096
Biological samples
Human fecal samples This study N/A
Commercial assays for DNA library prep
Qiagen PowerBead Qiagen Cat# 13118–50
Promega Maxwell RSC PureFood GMO Promega AS1600
Nextera XT DNA Library Prep Kit Illumina 15032355
Quant-iT dsDNA High Sensitivity Assay Kit Fisher 655087
Software and algorithms
kneadData https://huttenhower.sph.harvard.edu/kneaddata/ Version 0.10.0
MetaPhlAn4 mpa_vOct22_CHOCOPhlAnSGB_202212 Ref. 53
HUMAnN3 version 3.6 Ref. 54
StrainPhlAn N/A Ref. 55
MaAsLin2 N/A Ref. 56
LEfSe N/A Ref. 57
R R Core Team N/A
GraphPad Prism 10 GraphPad Software N/A
Open in a new tab

Context and Significance.

Fecal microbiota transplantation (FMT) is under investigation as a therapeutic strategy for ulcerative colitis (UC), though the roles of specific microbial taxa and dietary factors in mediating treatment efficacy remain unclear. In this randomized controlled trial, the authors evaluated the efficacy of FMT with and without dietary fiber supplementation in patients with UC. The study demonstrated that FMT significantly improved clinical outcomes compared to placebo. However, fiber supplementation did not enhance the therapeutic benefit of FMT. Treatment response was influenced by the composition of the donor microbiota. Longitudinal metagenomic sequencing enabled strain-level tracking, revealing specific bacterial strains associated with clinical response or fiber exposure. These findings may inform the development of future precision microbiome-based therapies for UC.

Highlights.

  • FMT induced clinical improvement in UC, but fiber did not enhance the clinical benefit.

  • Clinical response and strain engraftment was donor dependent.

  • Strain tracking identified engrafted bacteria in clinical response or fiber exposure.

Acknowledgements

This study was funded by the NIH/NIDDK (R01 DK128257 to RSL). Funding was also provided by CURE for IBD and the Mishaan family.

Declaration of Interests:

R.L: Consultant for Pfizer and Sanofi. Scientific Advisory Board of CJ Biosciences and Ancilia Biosciences. Grant recipient from Boehringer Ingelheim.

D.L: Consultant for Abbvie, Boehringer Ingelheim, Bristol Meyers Squibb, Eli Lilly, Fresenius Kabi, Janssen, Palatin Technologies, Pfizer, Prometheus Laboratories. Grants: Abbvie, Janssen, Takeda.

R.B: Speaking/Consulting/Advertising Boards: Prometheus Laboratories, Bristol Myers Squibb, Janssen, Abbvie, Takeda, Pfizer, Eli Lilly.

All other authors have no conflicts to disclose.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The authors are dedicated to sharing anonymized data from the clinical trials while ensuring the privacy of trial participants is protected. ClinicalTrials.gov, Number: NCT03998488.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2081305-supplement-1.pdf (1.4MB, pdf)

Data Availability Statement

All sequencing data are deposited in the NCBI BioProject repository under accession number PRJNA1169096. Clinical metadata are provided in supplemental table 1. Demographic data is reported in aggregate in Table 1. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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