Holtiella tumoricola gen. nov. sp. nov., isolated from a human clinical sample.

The diversity of bacteria associated with biopsy material obtained from patients with colorectal cancer was investigated using culture techniques. A novel bacterium, strain CC70AT, was isolated by diluting a sample of homogenized tissue in anaerobic medium, and then plating to yield a pure culture. Strain CC70AT was a Gram-positive, strictly anaerobic, motile, rod-shaped bacterium. Formate, but not acetate, was a fermentative end-product from growth in peptone-yeast extract and peptone-yeast-glucose broth. The G+C content of DNA from strain CC70AT was 34.9 mol%. 16S rRNA gene sequence analysis revealed that the isolate was part of the phylum Bacillota. The closest described relatives of strain CC70AT were Cellulosilyticum lentocellum (93.3 %) and Cellulosilyticum ruminicola (93.3 and 91.9% sequence similarity across 16S rRNA gene, respectively). According to the data obtained in this work, strain CC70AT represents a novel bacterium belonging to a new genus for which the name Holtiella tumoricola gen. nov., sp. nov. is proposed. The type strain for our described novel species is CC70AT (=DSM 27931T= JCM 30568T).

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.

Enterohemorrhagic Escherichia coli responds to gut microbiota metabolites by altering metabolism and activating stress responses.

Enterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC's ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC's response to metabolites from an in vitro culture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficile infection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP, spy, soxS, yhcN, and bhsA) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.

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.

Association of HLA-dependent islet autoimmunity with systemic antibody responses to intestinal commensal bacteria in children.

Microbiome sequence analyses have suggested that changes in gut bacterial composition are associated with autoimmune disease in humans and animal models. However, little is known of the mechanisms through which the gut microbiota influences autoimmune responses to distant tissues. Here, we evaluated systemic antibody responses against cultured human gut bacterial strains to determine whether observed patterns of anticommensal antibody (ACAb) responses are associated with type 1 diabetes (T1D) in two cohorts of pediatric study participants. In the first cohort, ACAb responses in sera collected from participants within 6 months of T1D diagnosis were compared with age-matched healthy controls and also with patients with recent onset Crohn's disease. ACAb responses against multiple bacterial species discriminated among these three groups. In the second cohort, we asked whether ACAb responses present before diagnosis were associated with later T1D development and with HLA genotype in participants who were discordant for subsequent progression to diabetes. Serum IgG2 antibodies against Roseburia faecis and against a bacterial consortium were associated with future T1D diagnosis in an HLA DR3/DR4 haplotype-dependent manner. These analyses reveal associations between antibody responses to intestinal microbes and HLA-DR genotype and islet autoantibody specificity and with a future diagnosis of T1D. Further, we present a platform to investigate antibacterial antibodies in biological fluids that is applicable to studies of autoimmune diseases and responses to therapeutic interventions.

Intestinal microbiota as a tetrahydrobiopterin exogenous source in hph-1 mice.

Tetrahydrobiopterin (BH4) is a cofactor of a number of regulatory enzymes. Although there are no known BH4 exogenous sources, the tissue content of this biopterin increases with age in GTP cyclohydrolase 1-deficient hyperphenylalaninemia-1 (hph-1) mice. Since certain bacteria are known to generate BH4, we hypothesize that generation of this biopterin by the intestinal microbiota contributes to its tissue increase in hph-1 adult mice. The goal of this study was to comparatively evaluate hph-1 mice and wild-type C57Bl/6 controls for the presence of intestinal BH4-producing bacteria. Newborn and adult mice fecal material was screened for 6-pyruvoyltetrahydropterin synthase (PTPS-2) an enzyme only present in BH4-generating bacteria. Adult, but not newborn, wild-type control and hph-1 mouse fecal material contained PTPS-2 mRNA indicative of the presence of BH4-generating bacteria. Utilizing chemostat-cultured human fecal bacteria, we identified the PTPS-2-producing bacteria as belonging to the Actinobacteria phylum. We further confirmed that at least two PTPS-2-producing species, Aldercreutzia equolifaciens and Microbacterium schleiferi, generate BH4 and are present in hph-1 fecal material. In conclusion, intestinal Actinobacteria generate BH4. This finding has important translational significance, since manipulation of the intestinal flora in individuals with congenital biopterin deficiency may allow for an increase in total body BH4 content.

Chemostat culture systems support diverse bacteriophage communities from human feces.

BACKGROUND: Most human microbiota studies focus on bacteria inhabiting body surfaces, but these surfaces also are home to large populations of viruses. Many are bacteriophages, and their role in driving bacterial diversity is difficult to decipher without the use of in vitro ecosystems that can reproduce human microbial communities. RESULTS: We used chemostat culture systems known to harbor diverse fecal bacteria to decipher whether these cultures also are home to phage communities. We found that there are vast viral communities inhabiting these ecosystems, with estimated concentrations similar to those found in human feces. The viral communities are composed entirely of bacteriophages and likely contain both temperate and lytic phages based on their similarities to other known phages. We examined the cultured phage communities at five separate time points over 24 days and found that they were highly individual-specific, suggesting that much of the subject-specificity found in human viromes also is captured by this culture-based system. A high proportion of the community membership is conserved over time, but the cultured communities maintain more similarity with other intra-subject cultures than they do to human feces. In four of the five subjects, estimated viral diversity between fecal and cultured communities was highly similar. CONCLUSIONS: Because the diversity of phages in these cultured fecal communities have similarities to those found in humans, we believe these communities can serve as valuable ecosystems to help uncover the role of phages in human microbial communities.

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.

Analysis of a collagen-binding trimeric autotransporter adhesin from Mannheimia haemolytica A1.

A locus that codes for a high-molecular-weight adhesin was previously isolated from Mannheimia haemolytica A1. In this study, we showed that this locus, named ahs, codes for two proteins (AhsA and AhsB) that exhibit characteristics of a trimeric autotransporter adhesin. Sequence analysis of AhsA showed the presence of 21 collagen-binding motifs in the protein. Collagen-binding assays showed that M. haemolytica A1 binds to collagen in a dose-dependent manner. This binding activity is trypsin sensitive and can be inhibited by anti-AhsA antibody. AhsB is the cognate transporter for AhsA. The C-terminal of AhsB showed highly conserved amino acids typical of trimeric autotransporters. Experimental data showed that the C-terminal 120 amino acids of AhsB could indeed form trimeric molecules. Western immunoblots showed the presence of anti-AhsA antibodies in the sera of calves that had been challenged with M. haemolytica A1, suggesting that AhsA is expressed and immunogenic in cattle.