Defined microbial communities and their soluble products protect mice from Clostridioides difficile infection.

Clostridioides difficile is the leading cause of antibiotic-associated infectious diarrhea. The development of C.difficile infection is tied to perturbations of the bacterial community in the gastrointestinal tract, called the gastrointestinal microbiota. Repairing the gastrointestinal microbiota by introducing lab-designed bacterial communities, or defined microbial communities, has recently shown promise as therapeutics against C.difficile infection, however, the mechanisms of action of defined microbial communities remain unclear. Using an antibiotic- C.difficile mouse model, we report the ability of an 18-member community and a refined 4-member community to protect mice from two ribotypes of C.difficile (CD027, CD078; p < 0.05). Furthermore, bacteria-free supernatant delivered orally to mice from the 4-member community proteolyzed C.difficile toxins in vitro and protected mice from C.difficile infection in vivo (p < 0.05). This study demonstrates that bacteria-free supernatant is sufficient to protect mice from C.difficile; and could be further explored as a therapeutic strategy against C.difficile infection.

The Robogut: A Bioreactor Model of the Human Colon for Evaluation of Gut Microbial Community Ecology and Function.

The human colon is inhabited by a complex community of microbes. These microbes are integral to host health and physiology. Understanding how and when the microbiome causally influences host health will require microbiome models that can be tightly controlled and manipulated. While in vivo models are unrivalled in their ability to study host-microbial interplay, in vitro models are gaining in popularity as methods to study the ecology and function of the gut microbiota, and benefit from tight controllability and reproducibility, as well as reduced ethical constraints. In this set of protocols, we describe the Robogut, a single-stage bioreactor system designed to replicate the conditions of the distal human colon, to culture whole microbial communities derived from stool and/or colonic biopsy samples, with consideration of methods to create culture medium formulations and to build, run, and sample the bioreactor apparatus. Cleaning and maintenance of the bioreactor system are also described. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth medium preparation Support Protocol 1: Preparing medium supplements Basic Protocol 2: Preparing the bioreactor vessels Support Protocol 2: Making acid and base bottles Support Protocol 3: Preparing the effluent bottles Support Protocol 4: Making acid solution Support Protocol 5: Making base solution Basic Protocol 3: Preparing inoculum and inoculating bioreactors Alternate Protocol 1: Preparing inoculum less than 0.5% (w/v) of vessel volume Alternate Protocol 2: Preparing synthetic community aliquots and inoculation via the septum Alternate Protocol 3: Preparing inoculum from a tissue sample Basic Protocol 4: Sampling the bioreactor vessel Basic Protocol 5: Harvesting bioreactor vessel contents at end of experiment Support Protocol 6: Cleaning and sterilizing sampling needles Basic Protocol 6: Cleaning the bioreactor vessel Support Protocol 7: Cleaning bioreactor support bottles.

Effects of Antibiotic Pretreatment of an Ulcerative Colitis-Derived Fecal Microbial Community on the Integration of Therapeutic Bacteria In Vitro.

Fecal microbiota transplantation (FMT) is a proposedly useful strategy for the treatment of gastrointestinal (GI) disorders through remediation of the patient gut microbiota. However, its therapeutic success has been variable, necessitating research to uncover mechanisms that improve patient response. Antibiotic pretreatment has been proposed as one method to enhance the success rate by increasing niche availability for introduced species. Several limitations hinder exploring this hypothesis in clinical studies, such as deleterious side effects and the development of antimicrobial resistance in patients. Thus, the purpose of this study was to evaluate the use of an in vitro, bioreactor-based, colonic ecosystem model as a form of preclinical testing by determining how pretreatment with the antibiotic rifaximin influenced engraftment of bacterial strains sourced from a healthy donor into an ulcerative colitis-derived defined microbial community. Distinct species integrated under the pretreated and untreated conditions, with the relative rifaximin resistance of the microbial strains being an important influencer. However, both conditions resulted in the integration of taxa from Clostridium clusters IV and XIVa, a concomitant reduction of Proteobacteria, and similar decreases in metabolites associated with poor health status. Our results agree with the findings of similar research in the clinic by others, which observed no difference in primary patient outcomes whether or not patients were given rifaximin prior to FMT. We therefore conclude that our model is useful for screening for antibiotics that could improve efficacy of FMT when used as a pretreatment.IMPORTANCE Patients with gastrointestinal disorders often exhibit derangements in their gut microbiota, which can exacerbate their symptoms. Replenishing these ecosystems with beneficial bacteria through fecal microbiota transplantation is thus a proposedly useful therapeutic; however, clinical success has varied, necessitating research into strategies to improve outcomes. Antibiotic pretreatment has been suggested as one such approach, but concerns over harmful side effects have hindered testing this hypothesis clinically. Here, we evaluate the use of bioreactors supporting defined microbial communities derived from human fecal samples as models of the colonic microbiota in determining the effectiveness of antibiotic pretreatment. We found that relative antimicrobial resistance was a key determinant of successful microbial engraftment with rifaximin (broad-spectrum antibiotic) pretreatment, despite careful timing of the application of the therapeutic agents, resulting in distinct species profiles from those of the control but with similar overall outcomes. Our model had results comparable to the clinical findings and thus can be used to screen for useful antibiotics.

Metabolomic analysis of human fecal microbiota: a comparison of feces-derived communities and defined mixed communities.

The extensive impact of the human gut microbiota on its human host calls for a need to understand the types of communication that occur among the bacteria and their host. A metabolomics approach can provide a snapshot of the microbe-microbe interactions occurring as well as variations in the microbes from different hosts. In this study, metabolite profiles from an anaerobic continuous stirred-tank reactors (CSTR) system supporting the growth of several consortia of bacteria representative of the human gut were established and compared. Cell-free supernatant samples were analyzed by 1D (1)H nuclear magnetic resonance (NMR) spectroscopy, producing spectra representative of the metabolic activity of a particular community at a given time. Using targeted profiling, specific metabolites were identified and quantified on the basis of NMR analyses. Metabolite profiles discriminated each bacterial community examined, demonstrating that there are significant differences in the microbiota metabolome between each cultured community. We also found unique compounds that were identifying features of individual bacterial consortia. These findings are important because they demonstrate that metabolite profiles of gut microbial ecosystems can be constructed by targeted profiling of NMR spectra. Moreover, examination of these profiles sheds light on the type of microbes present in the gut and their metabolic interactions.

Simulating distal gut mucosal and luminal communities using packed-column biofilm reactors and an in vitro chemostat model.

In vivo studies of human mucosal gut microbiota are often limited to end-point analyses and confounded by bowel cleansing procedures. Therefore, we used biofilm reactors to incorporate a simulated mucosal environment into an in vitro gut chemostat model. Communities developed were complex, reproducible, distinct, and representative of in vivo communities.

Antivirulence activity of the human gut metabolome.

The mammalian gut contains a complex assembly of commensal microbes termed microbiota. Although much has been learned about the role of these microbes in health, the mechanisms underlying these functions are ill defined. We have recently shown that the mammalian gut contains thousands of small molecules, most of which are currently unidentified. Therefore, we hypothesized that these molecules function as chemical cues used by hosts and microbes during their interactions in health and disease. Thus, a search was initiated to identify molecules produced by the microbiota that are sensed by pathogens. We found that a secreted molecule produced by clostridia acts as a strong repressor of Salmonella virulence, obliterating expression of the Salmonella pathogenicity island 1 as well as host cell invasion. It has been known for decades that the microbiota protects its hosts from invading pathogens, and these data suggest that chemical sensing may be involved in this phenomenon. Further investigations should reveal the exact biological role of this molecule as well as its therapeutic potential. Importance: Microbes can communicate through the production and sensing of small molecules. Within the complex ecosystem formed by commensal microbes living in and on the human body, it is likely that these molecular messages are used extensively during the interactions between different microbial species as well as with host cells. Deciphering such a molecular dialect will be fundamental to our understanding of host-microbe interactions in health and disease and may prove useful for the design of new therapeutic strategies that target these mechanisms of communication.

Evaluation of microbial community reproducibility, stability and composition in a human distal gut chemostat model.

In vitro gut models provide several advantages over in vivo models for the study of the human gut microbiota. However, because communities developed in these models are inevitably simplified simulations of the in vivo environment, it is necessary to broadly define the differences between in vitro consortia and the communities from which they are derived. In this study we characterized microbial community development in a twin-vessel single-stage chemostat model of the human distal gut ecosystem using both gel (Denaturing Gradient Gel Electrophoresis) and phylogenetic microarray (Human Intestinal Tract Chip) based techniques. Five different sets of twin-vessels were inoculated with feces from three different healthy adult donors and allowed to reach steady state compositions. We found that twin-vessel single-stage chemostats could develop and maintain stable, diverse, and reproducible communities that reach steady state compositions in all five runs by at most 36 days post-inoculation. As noted in other in vitro studies, steady state communities were enriched in Bacteroidetes but not Clostridium cluster XIVa, Bacilli or other Firmicutes relative to the fecal inocula. Communities developed within this model had higher within-run reproducibility than between-run repeatability when using consecutive fecal donations. Both fecal inocula and steady state chemostat communities seeded with feces from different donors had distinct compositions. We conclude that twin-vessel single-stage chemostat models represent a valid simulation of the human distal gut environment and can support complex, representative microbial communities ideal for experimental manipulation.

Stool substitute transplant therapy for the eradication of Clostridium difficile infection: 'RePOOPulating' the gut.

BACKGROUND: Fecal bacteriotherapy ('stool transplant') can be effective in treating recurrent Clostridium difficile infection, but concerns of donor infection transmission and patient acceptance limit its use. Here we describe the use of a stool substitute preparation, made from purified intestinal bacterial cultures derived from a single healthy donor, to treat recurrent C. difficile infection that had failed repeated standard antibiotics. Thirty-three isolates were recovered from a healthy donor stool sample. Two patients who had failed at least three courses of metronidazole or vancomycin underwent colonoscopy and the mixture was infused throughout the right and mid colon. Pre-treatment and post-treatment stool samples were analyzed by 16 S rRNA gene sequencing using the Ion Torrent platform. RESULTS: Both patients were infected with the hyper virulent C. difficile strain, ribotype 078. Following stool substitute treatment, each patient reverted to their normal bowel pattern within 2 to 3 days and remained symptom-free at 6 months. The analysis demonstrated that rRNA sequences found in the stool substitute were rare in the pre-treatment stool samples but constituted over 25% of the sequences up to 6 months after treatment. CONCLUSION: This proof-of-principle study demonstrates that a stool substitute mixture comprising a multi-species community of bacteria is capable of curing antibiotic-resistant C. difficile colitis. This benefit correlates with major changes in stool microbial profile and these changes reflect isolates from the synthetic mixture. CLINICAL TRIAL REGISTRATION NUMBER: CinicalTrials.gov NCT01372943.