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Byron P Vaughn,1 Thomas Kaiser,2,3 Christopher Staley,2,3 Matthew J Hamilton,2 Jon Reich,1 Carolyn Graiziger,1 Stephanie Singroy,2 Amanda J Kabage,1 Michael J Sadowsky,2,4,5 Alexander Khoruts1,2 1Inflammatory Bowel Program, Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota, Minneapolis, MN, USA; 2BioTechnology Institute, University of Minnesota, St Paul, MN, USA; 3Department of Surgery, University of Minnesota, Minneapolis, MN, USA; 4Department of Soil, Water, and Climate, University of Minnesota, St Paul, MN, USA; 5Department of Plant and Microbial Biology, University of Minnesota, St Paul, MN, USA Introduction: Inflammatory bowel disease (IBD) is thought to arise from an abnormal immune response to the gut microbiota. IBD is associated with altered intestinal microbial community structure and functionality, which may contribute to inflammation and complications such as colon cancer and liver disease. Primary sclerosing cholangitis (PSC) is associated with IBD and markedly increases the risk of colon cancer. We hypothesized that secondary bile acids, which are products of microbial metabolism, are increased in PSC patients.Aim: Here, we profiled the fecal bile acid composition and gut microbiota of participants with IBD and PSC, as well as healthy participants. Additionally, we tested the effects of vancomycin, a proposed treatment for PSC, on gut microbiota and fecal bile acid composition in participants with IBD and PSC.Methods: Fecal samples were collected from patients with IBD, IBD/PSC and healthy controls and fecal bile acids and DNA for microbiota analysis were extracted. Fecal bile acids were averaged over a seven-day period. For subjects with IBD/PSC, oral vancomycin 500mg twice a day was administered and fecal samples were collected for up to eleven weeks.Results: Participants with IBD and PSC had less fecal microbial diversity at baseline relative to controls. While there was some evidence of altered conversion of cholic acid to deoxycholic acid, no substantial differences were found in the fecal bile acid profiles of patients with IBD and PSC (n=7) compared to IBD alone (n=8) or healthy controls (n=8). Oral vancomycin was a potent inhibitor of secondary bile acid production in participants with IBD and PSC, particularly deoxycholic acid, although no changes in liver biochemistry patterns were noted over a two week period.Conclusion: In this pilot study, bile acid profiles were overall similar among patients with IBD and PSC, IBD alone, and healthy controls. Microbiota diversity was reduced in those with PSC and IBD compared to IBD alone or healthy controls. Keywords: vancomycin, Crohn’s disease, ulcerative colitis

Gut microbiota disruptions after allogeneic hematopoietic cell transplantation (alloHCT) are associated with increased risk of acute graft-versus-host disease (aGVHD). We designed a randomized, double-blind placebo-controlled trial to test whether healthy-donor fecal microbiota transplantation (FMT) early after alloHCT reduces the incidence of severe aGVHD. Here, we report the results from the single-arm run-in phase which identified the best of 3 stool donors for the randomized phase. The primary and key secondary endpoints were microbiota engraftment and severe aGVHD, respectively. Three cohorts of patients (20 total) received FMT, each from a different donor. FMT was safe and effective in restoring microbiota diversity and commensal species. Microbiota engraftment, determined from shotgun sequencing data, correlated with larger microbiota compositional shifts toward donor and better clinical outcomes. Donor 3 yielded a median engraftment rate of 66%, higher than donors 1 (P = 0.02) and 2 (P = 0.03) in multivariable analysis. Three patients developed severe aGVHD; all 3 had received FMT from donor 1. Donor 3 was selected as the sole donor for the randomized phase. Our findings suggest a clinically relevant donor effect and demonstrate feasibility of evidence-based donor selection. FMT is a holistic microbiota restoration approach that can be performed as a precision therapeutic. ClinicalTrials.gov identifier NCT06026371., Competing Interests: Competing interests: A.R. has received consulting fees from Seres Therapeutics (2023) related to the design of a clinical trial unrelated to the present study and using a different microbiome therapeutic. A.K. has intellectual property pertaining to extraction and cryopreservation of microbiota from stool for transplantation. The remaining authors declare no competing interests., (© 2025. The Author(s).)

Background and Aims: Microbiota transplant therapy is an emerging treatment for ulcerative colitis. One proposed mechanism for the benefit of microbiota transplant therapy is through engraftment of donor microbiota. However, the kinetics of engraftment are unknown. We identified SourceTracker as an efficient method both to determine engraftment and for the kinetic study of engrafting donor taxa to aid in determining the mechanism of how this therapy may treat ulcerative colitis., Methods: Ulcerative colitis patients were treated with either encapsulated (drug name MTP-101C) or placebo capsules daily for eight weeks followed by a four-week washout period. Amplicon sequence data from donors and patients were analyzed using the Bayesian algorithm SourceTracker., Results: Twenty-seven patients were enrolled, 14 to the placebo group and 13 to the microbiota transplant therapy group. Baseline Shannon and Chao1 indices negatively correlated with week 12 donor engraftment for patients treated with active drug capsules but not for placebo patients. SourceTracker engraftment positively correlated with the week 12 distance from donors measured using the Bray-Curtis similarity metric in treated patients but not with placebo. We identified engrafting taxa from donors in our patients as well as quantified the proportion of donor similarity or engraftment during weeks one through eight (active treatment) and week 12, four weeks after the last dose., Conclusion: SourceTracker can be used as a simple and reliable method to quantify donor microbial community engraftment and donor taxa contribution in patients with ulcerative colitis and other inflammatory conditions treated with microbiota transplant therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Acute GVHD (aGVHD) is a major complication of allogeneic hematopoietic cell transplantation (alloHCT) associated with gut microbiota disruptions. However, whether therapeutic microbiota modulation prevents aGVHD is unknown. We conducted a randomized, placebo-controlled trial of third-party fecal microbiota transplantation (FMT) administered at the peak of microbiota injury in 100 patients with acute myeloid leukemia receiving induction chemotherapy and alloHCT recipients. Despite improvements in microbiome diversity, expansion of commensals, and shrinkage of potential pathogens, aGVHD occurred more frequently after FMT than placebo. Although this unexpected finding could be explained by clinical differences between the two arms, we asked whether a microbiota explanation might be also present. To this end, we performed multi-omics analysis of preintervention and postintervention gut microbiome and serum metabolome. We found that postintervention expansion of Faecalibacterium, a commensal genus with gut-protective and anti-inflammatory properties under homeostatic conditions, predicted a higher risk for aGVHD. Faecalibacterium expansion occurred predominantly after FMT and was due to engraftment of unique donor taxa, suggesting that donor Faecalibacterium-derived antigens might have stimulated allogeneic immune cells. Faecalibacterium and ursodeoxycholic acid (an anti-inflammatory secondary bile acid) were negatively correlated, offering an alternative mechanistic explanation. In conclusion, we demonstrate context dependence of microbiota effects where a normally beneficial bacteria may become detrimental in disease. While FMT is a broad, community-level intervention, it may need precision engineering in ecologically complex settings where multiple perturbations (e.g., antibiotics, intestinal damage, alloimmunity) are concurrently in effect., Significance: Post-FMT expansion of Faecalibacterium, associated with donor microbiota engraftment, predicted a higher risk for aGVHD in alloHCT recipients. Although Faecalibacterium is a commensal genus with gut-protective and anti-inflammatory properties under homeostatic conditions, our findings suggest that it may become pathogenic in the setting of FMT after alloHCT. Our results support a future trial with precision engineering of the FMT product used as GVHD prophylaxis after alloHCT., (© 2024 The Authors; Published by the American Association for Cancer Research.)

In small series, third-party fecal microbiota transplantation (FMT) has been successful in decolonizing the gut from clinically relevant antibiotic resistance genes (ARGs). Less is known about the short- and long-term effects of FMT on larger panels of ARGs. We analyzed 226 pre- and post-treatment stool samples from a randomized placebo-controlled trial of FMT in 100 patients undergoing allogeneic hematopoietic cell transplantation or receiving anti-leukemia induction chemotherapy for 47 ARGs. These patients have heavy antibiotic exposure and a high incidence of colonization with multidrug-resistant organisms. Samples from each patient spanned a period of up to 9 months, allowing us to describe both short- and long-term effects of FMT on ARGs, while the randomized design allowed us to distinguish between spontaneous changes vs. FMT effect. We find an overall bimodal pattern. In the first phase (days to weeks after FMT), low-level transfer of ARGs largely associated with commensal healthy donor microbiota occurs. This phase is followed by long-term resistance to new ARGs as stable communities with colonization resistance are formed after FMT. The clinical implications of these findings are likely context-dependent and require further research. In the setting of cancer and intensive therapy, long-term ARG decolonization could translate into fewer downstream infections.

Purpose: Gut microbiota injury in allogeneic hematopoietic cell transplantation (HCT) recipients and patients with AML has been associated with adverse clinical outcomes. Previous studies in these patients have shown improvements in various microbiome indices after fecal microbiota transplantation (FMT). However, whether microbiome improvements translate into improved clinical outcomes remains unclear. We examined this question in a randomized, double-blind, placebo-controlled phase II trial., Methods: Two independent cohorts of allogeneic HCT recipients and patients with AML receiving induction chemotherapy were randomly assigned in a 2:1 ratio to receive standardized oral encapsulated FMT versus placebo upon neutrophil recovery. After each course of antibacterial antibiotics, patients received a study treatment. Up to three treatments were administered within 3 months. The primary end point was 4-month all-cause infection rate. Patients were followed for 9 months., Results: In the HCT cohort (74 patients), 4-month infection density was 0.74 and 0.91 events per 100 patient-days in FMT and placebo arms, respectively (infection rate ratio, 0.83; 95% CI, 0.48 to 1.42; P = .49). In the AML cohort (26 patients), 4-month infection density was 0.93 in the FMT arm and 1.25 in the placebo arm, with an infection rate ratio of 0.74 (95% CI, 0.32 to 1.71; P = .48). Unique donor bacterial sequences comprised 25%-30% of the fecal microbiota after FMT. FMT improved postantibiotic recovery of microbiota diversity, restored several depleted obligate anaerobic commensals, and reduced the abundance of expanded genera Enterococcus, Streptococcus, Veillonella, and Dialister., Conclusion: In allogeneic HCT recipients and patients with AML, third-party FMT was safe and ameliorated intestinal dysbiosis, but did not decrease infections. Novel findings from this trial will inform future development of FMT trials.

Purpose: Intestinal microbiota disruptions early after allogeneic hematopoietic cell transplantation have been associated with increased risk for acute GVHD (aGVHD). In our recent randomized phase II trial of oral, encapsulated, third-party fecal microbiota transplantation (FMT) versus placebo, FMT at the time of neutrophil recovery was safe and ameliorated dysbiosis. Here, we evaluated in post hoc analysis whether donor microbiota engraftment after FMT may protect against aGVHD., Experimental Design: We analyzed pre- and post-FMT stool samples and estimated donor microbiota engraftment (a preplanned secondary endpoint) by determining the fraction of post-FMT microbiota formed by unique donor taxa (donor microbiota fraction; dMf)., Results: dMf was higher in patients who later developed grade I or no aGVHD (median 33.9%; range, 1.6%-74.3%) than those who developed grade II-IV aGVHD (median 25.3%; range, 2.2%-34.8%; P = 0.006). The cumulative incidence of grade II-IV aGVHD by day 180 was lower in the group with greater-than-median dMf than the group with less-than-median dMf [14.3% (95% confidence interval, CI, 2.1-37.5) vs. 76.9% (95% CI, 39.7-92.8), P = 0.008]. The only determinant of dMf in cross-validated least absolute shrinkage and selection operator (LASSO)-regularized regression was the patient's pre-FMT microbiota diversity (Pearson correlation coefficient -0.82, P = 1.6 × 10-9), indicating more potent microbiota modulation by FMT in patients with more severe dysbiosis. Microbiota network analysis revealed major rewiring including changes in the most central nodes, without emergence of keystone species, as a potential mechanism of FMT effect., Conclusions: FMT may have protective effects against aGVHD, especially in patients with more severe microbiota disruptions., (©2023 American Association for Cancer Research.)

Background & Aims: Fecal microbiota transplantation (FMT) emerged as rescue treatment for multiply recurrent Clostridioides difficile infections (rCDIs) nonresponsive to standard therapy. However, estimation of FMT efficacy varies among different protocols and formulations, while placebo-controlled clinical trials have excluded most rCDI patients because of medical comorbidities. This study aimed to determine the safety and effectiveness of capsule FMT (cap-FMT) and colonoscopy FMT (colo-FMT) for rCDI using standardized products in a large, multicenter, prospective, real-world cohort., Methods: Clinical outcomes and adverse events after FMT performed for rCDI at 6 sites were captured in a prospective registry. FMT was performed using 1 of 2 standardized formulations of microbiota manufactured by the University of Minnesota Microbiota Therapeutics Program, freeze-dried/encapsulated or frozen-thawed/liquid. The FMT administration route was determined by the treating physician. The rCDI cure rate was assessed at 1 and 2 months. Safety data were collected within the first 72 hours and at 1 and 2 months. Logistic regression was used to investigate factors associated with FMT failure., Results: A total of 301 FMTs were performed in 269 unique patients. Two-thirds were cap-FMT. CDI cure rates were 86% (95% CI, 82%-90%) at 1 month and 81% (95% CI, 75%-86%) at 2 months. There was no difference in the 1-month or 2-month cure rate between cap-FMT and colo-FMT. Cap-FMT recipients were older and less likely to be immunosuppressed or have inflammatory bowel disease. Patient factors of older age and hemodialysis were associated with FMT failure by 2 months on multivariate logistic regression. In addition, post-FMT antibiotic use was associated with FMT failure at 2 months. One serious adverse event was related to colonoscopy (aspiration pneumonia), otherwise no new safety signals were identified., Conclusions: Cap-FMT using freeze-dried capsules has a similar safety and effectiveness profile compared with colo-FMT, without the procedural risks of colonoscopy. Although highly effective overall, patient selection is a key factor to optimizing FMT success., (Copyright © 2023 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Rationale: Inflammation drives pulmonary arterial hypertension (PAH). Gut dysbiosis causes immune dysregulation and systemic inflammation by altering circulating microbial metabolites; however, little is known about gut dysbiosis and microbial metabolites in PAH. Objectives: To characterize the gut microbiome and microbial metabolites in patients with PAH. Methods: We performed 16S ribosomal RNA gene and shotgun metagenomics sequencing on stool from patients with PAH, family control subjects, and healthy control subjects. We measured markers of inflammation, gut permeability, and microbial metabolites in plasma from patients with PAH, family control subjects, and healthy control subjects. Measurements and Main Results: The gut microbiome was less diverse in patients with PAH. Shannon diversity index correlated with measures of pulmonary vascular disease but not with right ventricular function. Patients with PAH had a distinct gut microbial signature at the phylogenetic level, with fewer copies of gut microbial genes that produce antiinflammatory short-chain fatty acids (SCFAs) and secondary bile acids and lower relative abundances of species encoding these genes. Consistent with the gut microbial changes, patients with PAH had relatively lower plasma concentrations of SCFAs and secondary bile acids. Patients with PAH also had enrichment of species with the microbial genes that encoded the proinflammatory microbial metabolite trimethylamine. The changes in the gut microbiome and circulating microbial metabolites between patients with PAH and family control subjects were not as substantial as the differences between patients with PAH and healthy control subjects. Conclusions: Patients with PAH have proinflammatory gut dysbiosis, in which lower circulating SCFAs and secondary bile acids may facilitate pulmonary vascular disease. These findings support investigating modulation of the gut microbiome as a potential treatment for PAH.

Immunosuppressed patients with inflammatory bowel disease (IBD) generate lower amounts of SARS-CoV-2 spike antibodies after mRNA vaccination than healthy controls. We assessed SARS-CoV-2 spike S1 receptor binding domain-specific (S1-RBD-specific) B lymphocytes to identify the underlying cellular defects. Patients with IBD produced fewer anti-S1-RBD antibody-secreting B cells than controls after the first mRNA vaccination and lower amounts of total and neutralizing antibodies after the second. S1-RBD-specific memory B cells were generated to the same degree in IBD and control groups and were numerically stable for 5 months. However, the memory B cells in patients with IBD had a lower S1-RBD-binding capacity than those in controls, which is indicative of a defect in antibody affinity maturation. Administration of a third shot to patients with IBD elevated serum antibodies and generated memory B cells with a normal antigen-binding capacity. These results show that patients with IBD have defects in the formation of antibody-secreting B cells and affinity-matured memory B cells that are corrected by a third vaccination.

Background: Hydrogen sulfide (H 2 S) is a toxic end-product of microbial fermentation produced in the colon that may play a role in the pathogenesis of several diseases, including ulcerative colitis and colon cancer. However, the effect of diet interventions on intestinal burden of H 2 S gas exposure remains poorly understood., Objective: Determine the effect of short-term (1-week) plant- and animal-based eating patterns on ex vivo fecal H 2 S production in healthy human volunteers., Methods: The study design was an open-label, cross-over diet study and diets were self-administered. Each participant consumed two interventional diets: 1) an animal-based, low fiber (i.e. western) diet and 2) a plant-based, high fiber diet, separated by a two-week washout period. Participants collected full stool samples at the end of each week, which were processed within 2 h of collection to capture H 2 S production. Microfluidic qPCR (MFQPCR) was used to simultaneously quantify multiple taxonomic and functional groups involved in sulfate reduction and the fecal microbiota was characterized through high-throughput DNA sequencing., Results: Median H 2 S production was higher following the animal-based diet compared to the plant-based diet (p = 0.02; median difference 29 ppm/g, 95% CI 16-97). However, there was substantial individual variability and 2 of 11 individuals (18%) produced more H 2 S on the plant-based diet. Using the top and bottom quartiles of H 2 S percent change between animal- and plant-based diet weeks to define responders and non-responders, significant taxonomic differences were observed between the responder and non-responder cohorts., Conclusions: Here we report that substrate changes associated with a 1-week plant-based diet intervention resulted in lower ex vivo H 2 S production compared to a 1-week animal-based diet intervention in most healthy individuals. However, H 2 S responsiveness to diet was not uniform across the entire cohort, and potential H 2 S production enterotypes were characterized that may predict individualized H 2 S responsiveness to diet., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest., (Copyright © 2022 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Background: Intestinal methane (CH 4 ) gas production has been associated with a number of clinical conditions and may have important metabolic and physiological effects., Aims: In this study, taxonomic and functional gene analyses and in vitro CH 4 gas measurements were used to determine if molecular markers can potentially serve as clinical tests for colonic CH 4 production., Methods: We performed a cross-sectional study involving full stool samples collected from 33 healthy individuals. In vitro CH 4 gas measurements were obtained after 2-h incubation of stool samples and used to characterize samples as CH 4 positive (CH 4 +) and CH 4 negative (CH 4 -; n = 10 and 23, respectively). Next, we characterized the fecal microbiota through high-throughput DNA sequencing with a particular emphasis on archaeal phylum Euryarchaeota. Finally, qPCR analyses, targeting the mcrA gene, were done to determine the ability to differentiate CH 4 + versus CH 4 - samples and to delineate major methanogen species associated with CH 4 production., Results: Methanobrevibacter was found to be the most abundant methane producer and its relative abundance provides a clear distinction between CH 4 + versus CH 4 - samples. Its sequencing-based relative abundance detection threshold for CH 4 production was calculated to be 0.097%. The qPCR-based detection threshold separating CH 4 + versus CH 4 - samples, based on mcrA gene copies, was 5.2 × 10 5 copies/g., Conclusion: Given the decreased time-burden placed on patients, a qPCR-based test on a fecal sample can become a valuable tool in clinical assessment of CH 4 producing status., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Although both infections and vaccines induce memory B cell (MBC) populations that participate in secondary immune responses, the MBCs generated in each case can differ. Here, we compare SARS-CoV-2 spike receptor binding domain (S1-RBD)-specific primary MBCs that form in response to infection or a single mRNA vaccination. Both primary MBC populations have similar frequencies in the blood and respond to a second S1-RBD exposure by rapidly producing plasmablasts with an abundant immunoglobulin (Ig)A + subset and secondary MBCs that are mostly IgG + and cross-react with the B.1.351 variant. However, infection-induced primary MBCs have better antigen-binding capacity and generate more plasmablasts and secondary MBCs of the classical and atypical subsets than do vaccine-induced primary MBCs. Our results suggest that infection-induced primary MBCs have undergone more affinity maturation than vaccine-induced primary MBCs and produce more robust secondary responses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Fecal microbiota transplantation (FMT) is a highly effective treatment for recurrent Clostridioides difficile infection (rCDI). However, standardization of FMT products is essential for its broad implementation into clinical practice. We have developed an oral preparation of freeze-dried, encapsulated microbiota, which is ~ 80% clinically effective, but results in delayed engraftment of donor bacteria relative to administration via colonoscopy. Our objective was to measure the engraftment potential of freeze-dried microbiota without the complexity of variables associated with oral administration. We compared engraftment of identical preparations and doses of freeze-dried microbiota following colonoscopic (9 patients) versus oral administration (18 patients). Microbiota were characterized by sequencing of the 16S rRNA gene, and engraftment was determined using the SourceTracker algorithm. Oligotyping analysis was done to provide high-resolution patterns of microbiota engraftment. Colonoscopic FMT was associated with greater levels of donor engraftment within days following the procedure (ANOVA P = 0.035) and specific increases in the relative abundances of donor Lachnospiraceae, Bacteroidaceae, and Porphyromonadaceae (P ≤ 0.033). Lower relative abundances of Bacteroidaceae, Lachnospiraceae, and Ruminococcaceae families were associated with clinical failures. These results suggest that further optimization of oral capsule FMT may improve its engraftment efficiency and clinical efficacy.

Fecal microbiota transplantation (FMT) has become a common rescue therapy for recurrent Clostridium difficile infection, and encapsulated delivery (cFMT) of healthy donor microbiota shows similar clinical efficacy as more traditional routes of administration. In this study, we characterized long-term patterns of bacterial engraftment in a cohort of 18 patients, who received capsules from one of three donors, up to 409 days post-FMT. Bacterial communities were characterized using Illumina sequencing of the V5-V6 hypervariable regions of the 16S rRNA gene, and engraftment was determined by using the Bayesian algorithm SourceTracker. All patients recovered clinically and were free of C. difficile infection following cFMT. The majority of patients (61%) showed high levels of engraftment after the first week following FMT, which were sustained throughout the year. A small subset, 22%, experienced a decline in donor engraftment after approximately 1 month, and a few patients (17%), two of whom were taking metformin, showed delayed and low levels of donor engraftment. Members of the genera Bacteroides , Parabacteroides , and Faecalibacterium were significantly and positively correlated with donor similarity (ρ = 0.237 to 0.373, P  ≤ 0.017). Furthermore, throughout the year, patient fecal communities showed significant separation based on the donor fecal microbiota that they received ( P  < 0.001). Results of this study, which characterize long-term engraftment following cFMT, suggest that numerical donor similarity is not strictly related to clinical outcome and identify a persistent donor-specific effect on patient fecal microbial communities. Furthermore, results suggest that members of the Bacteroidetes may be important targets to improve engraftment via cFMT. IMPORTANCE Recurrent Clostridium difficile infection (rCDI) is the most common cause of hospital- and community-acquired diarrheal infection associated with antibiotic use. Fecal microbiota transplantation (FMT), a treatment that involves administration of fecal bacteria from a healthy donor to a recipient patient, is a highly effective rescue therapy for rCDI that is increasingly being incorporated into standard clinical practice. Encapsulated, freeze-dried preparations of fecal microbiota, administered orally, offer the simplest and most convenient route of FMT delivery for patients (cFMT). In this study, we evaluated the extent of bacterial engraftment following cFMT and the duration of donor bacterial persistence. All patients studied recovered clinically but showed differing patterns in long-term microbial community similarity to the donor that were associated with members of the bacterial group Bacteroidetes , previously shown to be prominent contributors to rCDI resistance. Results highlight long-lasting, donor-specific effects on recipient patient microbiota and reveal potential bacterial targets to improve cFMT engraftment., (Copyright © 2019 Staley et al.)

High-protein diets may be linked to gut inflammation due to increased production of hydrogen sulfide (H 2 S), a potential toxin, as an end product of microbial fermentation in the colon by sulfidogenic sulfate-reducing bacteria (SRB). We hypothesized that dietary content of sulfur-containing amino acids (SAA) leads to variation in the relative abundances of intestinal SRB, which include Desulfovibrio and Bilophila taxa. To test this hypothesis we performed a pilot crossover study in four healthy volunteers, who consumed two interventional diets for 10-14 days, containing high or low SAA content. The total energy intake was similar between the two dietary extremes. Microbial communities were characterized by 16S rRNA gene amplicon and shotgun next-generation DNA sequencing. While the relative abundance of Desulfovibrio differed among participants (ANOVA P = 0.001), we could not detect a change with dietary treatments. Similarly, no differences in Bilophila abundance were observed among individuals or dietary arms. Inter-personal differences in microbial community composition and functional gene categories differed between subjects and these differences were maintained over the course of the study. These observations are consistent with re-analysis of two previously published dietary intervention studies. Finally, we found that inter-personal differences in the taxonomic composition of fecal microbiota, including the relative abundances of SRB, were maintained over time in 19 healthy individuals in our stool donor program. These results suggest that the use of dietary interventions alone may be insufficient for rapid therapeutic targeting of SRB. Nevertheless, these pilot data provide a foundation to inform future, statistically powered, studies.

Background: Dysbiosis of the human gut microbiome is defined as a maladaptive or clinically relevant deviation of the community profile from the healthy or normal state. Dysbiosis has been implicated in an extensive set of metabolic, auto-immune, and infectious diseases, and yet there is substantial inter-individual variation in microbiome composition even within body sites of healthy humans. An individual's microbiome varies over time in a high-dimensional space to form their personal microbiome cloud. This cloud may or may not be similar to that of other people, both in terms of the average microbiome profile (conformity) and the diameter of the cloud (stability). However, there is currently no robust non-parametric test that determines whether a patient's microbiome cloud is an outlier with respect to a reference group of healthy individuals with widely varying microbiome profiles., Methods: Here, we propose a test for outliers' detection in the human gut microbiome that accounts for the wide range of microbiome phenotypes observed in a typical set of healthy individuals and for intra-individual temporal variation. Our robust nonparametric outlier detection test, the CLOUD test, performs two assessments of a patient's microbiome health: conformity, the extent to which the patient's microbiome cloud is ecologically similar to a subset of healthy subjects; and stability, which compares the cloud diameter of a patient to those of healthy subjects. The CLOUD test is based on locally linear embedded ecological distances, allowing it to account for widely varying microbiome compositions among reference individuals. It also leverages temporal variability within patients and reference individuals to increase the robustness of the test., Results: We describe the CLOUD test, and we apply it to one novel and two previously published cohorts of patients receiving fecal microbiota transplantation for recurrent Clostridium difficile colitis, as well as to two known healthy cohorts, demonstrating high concordance of the CLOUD conformity and stability indices with clinical outcomes., Conclusions: Although the CLOUD test is not, on its own, a test for clinical dysbiosis, it nonetheless provides a framework for outlier testing that could be incorporated into evaluation of suspected dysbiosis, which may play a role in diagnosis and prognosis of numerous pediatric and adult diseases.

Objectives: Fecal microbiota transplantation (FMT) is increasingly being used for treatment of recurrent Clostridium difficile infection (R-CDI) that cannot be cured with antibiotics alone. In addition, FMT is being investigated for a variety of indications where restoration or restructuring of the gut microbial community is hypothesized to be beneficial. We sought to develop a stable, freeze-dried encapsulated preparation of standardized fecal microbiota that can be used for FMT with ease and convenience in clinical practice and research., Methods: We systematically developed a lyophilization protocol that preserved the viability of bacteria across the taxonomic spectrum found in fecal microbiota and yielded physicochemical properties that enabled consistent encapsulation. We also treated a cohort of R-CDI patients with a range of doses of encapsulated microbiota and analyzed the associated changes in the fecal microbiome of the recipients., Results: The optimized lyophilized preparation satisfied all our preset goals for physicochemical properties, encapsulation ease, stability at different temperatures, and microbiota viability in vitro and in vivo (germ-free mice). The capsule treatment was administered to 49 patients. Overall, 43/49 (88%) of patients achieved a clinical success, defined as no recurrence of CDI over 2 months. Analysis of the fecal microbiome demonstrated near normalization of the fecal microbial community by 1 month following FMT treatment. The simplest protocol using the lowest dose (2.1-2.5 × 10 11 bacteria in 2-3 capsules) without any colon purgative performed equally well in terms of clinical outcomes and microbiota engraftment., Conclusions: A single administration of encapsulated, freeze-dried fecal microbiota from a healthy donor was highly successful in treating antibiotic-refractory R-CDI syndrome.