Clostridiales Species Research Articles

Functional characterization of helminth-associated Clostridiales reveals covariates of Treg differentiation

BackgroundParasitic helminths influence the composition of the gut microbiome. However, the microbiomes of individuals living in helminth-endemic regions are understudied. The Orang Asli, an indigenous population in Malaysia with high burdens of the helminth Trichuris trichiura, display microbiotas enriched in Clostridiales, an order of spore-forming obligate anaerobes with immunogenic properties. We previously isolated novel Clostridiales that were enriched in these individuals and found that a subset promoted the Trichuris life cycle. In this study, we aimed to further characterize the functional properties of these bacteria.ResultsClostridiales isolates were profiled for their ability to perform 57 enzymatic reactions and produce short-chain fatty acids (SCFAs) and hydrogen sulfide, revealing that these bacteria were capable of a range of activities associated with metabolism and host response. Consistent with this finding, monocolonization of mice with individual isolates identified bacteria that were potent inducers of regulatory T-cell (Treg) differentiation in the colon. Comparisons between variables revealed by these studies identified enzymatic properties correlated with Treg induction and Trichuris egg hatching.ConclusionWe identified Clostridiales species that are sufficient to induce high levels of Tregs. We also identified a set of metabolic activities linked with Treg differentiation and Trichuris egg hatching mediated by these newly isolated bacteria. Altogether, this study provides functional insights into the microbiotas of individuals residing in a helminth-endemic region.-EyM7p7tfAvrAjtAePB85JVideo

Next-generation sequencing of the athletic gut microbiota: a systematic review.

Aim: There is growing evidence that physical activity modulates gut microbiota composition through complex interactions between diet and microbial species. On the other hand, next-generation sequencing techniques include shotgun metagenomics and 16S amplicon sequencing. These methodologies allow a comprehensive characterisation of microbial communities of athletes from different disciplines as well as non-professional players and sedentary adults exposed to training. This systematic review summarises recent applications of next-generation sequencing to characterise the athletic gut microbiome. Methods: A systematic review of microbiome research was performed to determine the association of microbiota composition profiles with sports performance. Results: Bibliographic analysis revealed the importance of a novel research trend aiming at deciphering the associations between individual microbial species and sports performance. In addition, literature review highlighted the role of butyrate-producing bacteria such as Anaerostipes hadrus, Clostridium bolteae, Faecalibacterium prausnitzii, Roseburia hominis and unidentified species belonging to Clostridiales, Lachnospiraceae and Subdoligranulum species in gut health and sports performance across several disciplines. Interestingly, metabolic activities of Prevotella copri and Veillonella atypica involved in branched amino acid and lactate metabolism may contribute to reducing muscular fatigue. Other microbial metabolic pathways of interest involved in carbohydrate metabolism showed increased proportions in athletes´ metagenomes. Conclusion: Future research will aim at developing personalised nutrition interventions to modulate key species associated with certain components of exercise.

Spiking immunotherapy with a bacterial cocktail brings T cells back to the fight

A recent study by Montalban-Arques et al.1 in Cell Host & Microbe shows that augmenting the function of the gut microbiota reduces tumor burden. Four Clostridiales species blocked tumor growth as efficient as chemotherapy or immunotherapy in colorectal cancer and melanoma.

Cultivation of Spore-Forming Gut Microbes Using a Combination of Bile Acids and Amino Acids.

Spores of certain species belonging to Firmicutes are efficiently germinated by nutrient germinators, such as amino acids, in addition to bile acid. We attempted to culture difficult-to-culture or yet-to-be cultured spore-forming intestinal bacteria, using a combination of bile acids and amino acids. The combination increased the number of colonies that formed on agar medium plated with ethanol-treated feces. The operational taxonomic units of these colonized bacteria were classified into two types. One type was colonized only by the bile acid (BA) mixture and the other type was colonized using amino acids, in addition to the BA mixture. The latter contained 13 species, in addition to 14 species of the former type, which mostly corresponds to anaerobic difficult-to-culture Clostridiales species, including several new species candidates. The use of a combination of BAs and amino acids effectively increased the culturability of spore-forming intestinal bacteria.

Key bacterial taxa and metabolic pathways affecting gut short-chain fatty acid profiles in early life

Infant gut microbiota development affects the host physiology throughout life, and short-chain fatty acids (SCFAs) are promising key metabolites mediating microbiota-host relationships. Here, we investigated dense longitudinally collected faecal samples from 12 subjects during the first 2 years (n = 1048) to identify early life gut SCFA patterns and their relationships with the microbiota. Our results revealed three distinct phases of progression in the SCFA profiles: early phase characterised by low acetate and high succinate, middle-phase characterised by high lactate and formate and late-phase characterised by high propionate and butyrate. Assessment of the SCFA–microbiota relationships revealed that faecal butyrate is associated with increased Clostridiales and breastfeeding cessation, and that diverse and personalised assemblage of Clostridiales species possessing the acetyl-CoA pathway play major roles in gut butyrate production. We also found an association between gut formate and some infant-type bifidobacterial species, and that human milk oligosaccharides (HMO)-derived fucose is the substrate for formate production during breastfeeding. We identified genes upregulated in fucose and fucosylated HMO utilisation in infant-type bifidobacteria. Notably, bifidobacteria showed interspecific and intraspecific variation in the gene repertoires, and cross-feeding of fucose contributed to gut formate production. This study provides an insight into early life SCFA–microbiota relationships, which is an important step for developing strategies for modulating lifelong health.

Regulatory T Cell-Derived TGF-β1 Controls Multiple Checkpoints Governing Allergy and Autoimmunity

The mechanisms by which regulatory T (Treg) cells differentially control allergic and autoimmune responses remain unclear. We show that Treg cells in food allergy (FA) had decreased expression of transforming growth factor beta 1 (TGF-β1) because of interleukin-4 (IL-4)- and signal transducer and activator of transciription-6 (STAT6)-dependent inhibition of Tgfb1 transcription. These changes were modeled by Treg cell-specific Tgfb1 monoallelic inactivation, which induced allergic dysregulation by impairing microbiota-dependent retinoic acid receptor-related orphan receptor gamma t (ROR-γt)+ Treg cell differentiation. This dysregulation was rescued by treatment with Clostridiales species, which upregulated Tgfb1 expression in Treg cells. Biallelic deficiency precipitated fatal autoimmunity with intense autoantibody production and dysregulated T follicular helper and B cell responses. These results identify a privileged role of Treg cell-derived TGF-β1 in regulating allergy and autoimmunity at distinct checkpoints in a Tgfb1 gene dose- and microbiota-dependent manner.

Donor fecal microbiota transplantation ameliorates intestinal graft-versus-host disease in allogeneic hematopoietic cell transplant recipients.

Disruption of the intestinal microbiota occurs frequently in allogeneic hematopoietic cell transplantation (allo-HCT) recipients and predisposes them to development of graft-versus-host disease (GvHD). In a prospective, single-center, single-arm study, we investigated the effect of donor fecal microbiota transplantation (FMT) on symptoms of steroid-refractory or steroid-dependent, acute or late-onset acute intestinal GvHD in 15 individuals who had undergone allo-HCT. Study participants received a fecal suspension from an unrelated healthy donor via nasoduodenal infusion. Donor FMT was well tolerated, and infection-related adverse events did not seem to be related to the FMT procedure. In 10 of 15 study participants, a complete clinical response was observed within 1 month after FMT, without additional interventions to alleviate GvHD symptoms. This response was accompanied by an increase in gut microbial α-diversity, a partial engraftment of donor bacterial species, and increased abundance of butyrate-producing bacteria, including Clostridiales and Blautia species. In 6 of the 10 responding donor FMT recipients, immunosuppressant drug therapy was successfully tapered. Durable remission of steroid-refractory or steroid-dependent GvHD after donor FMT was associated with improved survival at 24 weeks after donor FMT. This study highlights the potential of donor FMT as a treatment for steroid-refractory or steroid-dependent GvHD, but larger clinical trials are needed to confirm the safety and efficacy of this procedure.

Lack of efficient killing of purified dormant spores of Bacillales and Clostridiales species by glycerol monolaurate in a non-aqueous gel.

Inactivation of Bacillales and Clostridiales spores is of interest, since some cause food spoilage and human diseases. A recent publication (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of Bacillus subtilis, Bacillus cereus and Clostridioides difficile, and Bacillus anthracis spores to a lesser extent. We now show that (i) the B. subtilis spores prepared as in the prior work were impure; (ii) if spore viability was measured by diluting spores 1/10 in GMLg, serially diluting incubations 10-fold and spotting aliquots on recovery plates, there was no colony formation from the 1/10 to 1/1000 dilutions due to GMLg carryover, although thorough ethanol washes of incubated spores eliminated this problem and (iii) GMLg did not kill highly purified spores of B. subtilis, B. cereus, Bacillus megaterium and C. difficile in 3-20h in the conditions used in the recent publication. GMLg also gave no killing of crude B. subtilis spores prepared as in the recent publication in 5h but gave ~1·5 log killing at 24h. Thus, GMLg does not appear to be an effective sporicide, although the gel likely inhibits spore germination and could kill spores somewhat upon long incubations. SIGNIFICANCE AND IMPACT OF THE STUDY: Given potential deleterious effects of spores of Bacillales and Clostridiales, there is an ongoing interest in new ways of spore killing. A recent paper (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of many species. We now find that (i) the Bacillus subtilis spores prepared as in the previous report were impure and (ii) GMLg gave no killing of purified spores of Bacillales and Clostridiales species in ≤5h under the published conditions. Thus, GMLg is not an effective sporicide, though may prevent spore germination or kill germinated spores.

Physical fitness in community-dwelling older adults is linked to dietary intake, gut microbiota, and metabolomic signatures.

When humans age, changes in body composition arise along with lifestyle‐associated disorders influencing fitness and physical decline. Here we provide a comprehensive view of dietary intake, physical activity, gut microbiota (GM), and host metabolome in relation to physical fitness of 207 community‐dwelling subjects aged +65 years. Stratification on anthropometric/body composition/physical performance measurements (ABPm) variables identified two phenotypes (high/low‐fitness) clearly linked to dietary intake, physical activity, GM, and host metabolome patterns. Strikingly, despite a higher energy intake high‐fitness subjects were characterized by leaner bodies and lower fasting proinsulin‐C‐peptide/blood glucose levels in a mechanism likely driven by higher dietary fiber intake, physical activity and increased abundance of Bifidobacteriales and Clostridiales species in GM and associated metabolites (i.e., enterolactone). These factors explained 50.1% of the individual variation in physical fitness. We propose that targeting dietary strategies for modulation of GM and host metabolome interactions may allow establishing therapeutic approaches to delay and possibly revert comorbidities of aging.

P847 A pilot trial on the effect of a fibre-enriched nutritional supplement on the gut microbiota of stricturing Crohn’s disease patients

Abstract Background Diet is a potential factor that could influence the pathogenesis and activity of Inflammatory Bowel Disease(IBD). Soluble fibre is the best way to generate short-chain fatty acids such as butyrate, which has anti-inflammatory effects. Around 1/3 of Crohn’s disease (CD) patients will present with stricturing disease, most frequently in the terminal ileum. These patients often follow a very low-residue diet. CD patients present significant changes in the structure of their microbiota with a decreased prevalence of butyrate-producing bacteria such as Clostridiales species, particularly Faecalibacterium prausnitzii. Depletion of F. prausnitzii might be further enhanced in patients with a very low-residue diet. The aim of this study was to evaluate the effect of a nutritional supplement enriched with soluble fibre on the abundance of F. prausnitzii in stricturing CD. Methods We performed a single arm, pilot trial in CD patients with ileal stricturing disease who followed a very low-residue diet as assessed by a dietician. The fibre-enriched nutritional supplement (reg#26.06141/BA-72556) consisted of a 200ml vanilla-flavoured shake that included 3.4 gr of soluble fibre, omega-3 and oleic fatty acids. Patients received 2 supplements per day for 6 weeks, and were followed for 6 months. We obtained frozen faecal samples at time-points 0, 3, 6, 12 and 24 weeks. Total bacteria and F. prausnitzii counts were assessed by qPCR. Weight, BMI, CD activity index (CDAI), CRP, fatty acids, urine F2-isoprostanes and faecal calprotectin were also determined. Tolerance, palatability and acceptability of the supplement were assessed with validated questionnaires at weeks 3 and 6. Results Ten patients were included in the study with a median age of 38 [36–50] years, 60% were male, median BMI 25.2 [24.3–28.4] and 40% were current smokers. Six patients completed the 6 weeks of the supplement. Two patients did not receive the supplement because they underwent ileocecal resection, 1 patient was lost to follow-up and 1 patient dropped-out early because of poor palatability of the supplement. At baseline, all patients had F. prausnitzii levels below 109 CFU/g (median 2.02 × 106). Supplement intake did not significantly increase F. prausnitzii levels (p = 0.73) and had no effect on CDAI, CRP, urine F2-isoprostanes or faecal calprotectin. Most usual complaints associated with the supplement were abdominal bloating and flatulence. Conclusion CD patients with structuring disease and who follow a very low-residue diet have a markedly reduced abundance of F. prausnitzii. Intake of a 6.8 g/day fibre-enriched nutritional supplement was unable to significantly increase F. prausnitzii abundance in these patients. Fibre supplement was not associated with adverse events.

Genomic reconstruction of \u03c354 regulons in Clostridiales

BackgroundThe σ54 factor controls unique promoters and interacts with a specialized activator (enhancer binding proteins [EBP]) for transcription initiation. Although σ54 is present in many Clostridiales species that have great importance in human health and biotechnological applications, the cellular processes controlled by σ54 remain unknown.ResultsFor systematic analysis of the regulatory functions of σ54, we performed comparative genomic reconstruction of transcriptional regulons of σ54 in 57 species from the Clostridiales order. The EBP-binding DNA motifs and regulated genes were identified for 263 EBPs that constitute 39 distinct groups. The reconstructed σ54 regulons contain the genes involved in fermentation and amino acid catabolism. The predicted σ54 binding sites in the genomes of Clostridiales spp. were verified by in vitro binding assays. To our knowledge, this is the first report about direct regulation of the Stickland reactions and butyrate and alcohols synthesis by σ54 and the respective EBPs. Considerable variations were demonstrated in the sizes and gene contents of reconstructed σ54 regulons between different Clostridiales species. It is proposed that σ54 controls butyrate and alcohols synthesis in solvent-producing species, regulates autotrophic metabolism in acetogenic species, and affects the toxin production in pathogenic species.ConclusionsThis study reveals previously unrecognized functions of σ54 and provides novel insights into the regulation of fermentation and amino acid metabolism in Clostridiales species, which could have potential applications in guiding the treatment and efficient utilization of these species.

Analysis of RNA Methylation by Phylogenetically Diverse Cfr Radical S-Adenosylmethionine Enzymes Reveals an Iron-Binding Accessory Domain in a Clostridial Enzyme

Cfr is a radical S-adenosylmethionine (SAM) RNA methylase linked to multidrug antibiotic resistance in bacterial pathogens. It catalyzes a chemically challenging C-C bond-forming reaction to methylate C8 of A2503 (Escherichia coli numbering) of 23S rRNA during ribosome assembly. The cfr gene has been identified as a mobile genetic element in diverse bacteria and in the genome of select Bacillales and Clostridiales species. Despite the importance of Cfr, few representatives have been purified and characterized in vitro. Here we show that Cfr homologues from Bacillus amyloliquefaciens, Enterococcus faecalis, Paenibacillus lautus, and Clostridioides difficile act as C8 adenine RNA methylases in biochemical assays. C. difficile Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe2+ ion in a rubredoxin-type Cys4 motif. The C-terminal domain can be truncated with minimal impact on C. difficile Cfr activity, but the rate of turnover is decreased upon disruption of the Fe2+-binding site by Zn2+ substitution or ligand mutation. These findings indicate an important purpose for the observed C-terminal iron in the native fusion protein. Bioinformatic analysis of the C. difficile Cfr Cys-rich domain shows that it is widespread (∼1400 homologues) as a stand-alone gene in pathogenic or commensal Bacilli and Clostridia, with >10% encoded adjacent to a predicted radical SAM RNA methylase. Although the domain is not essential for in vitro C. difficile Cfr activity, the genomic co-occurrence and high abundance in the human microbiome suggest a possible functional role for a specialized rubredoxin in certain radical SAM RNA methylases that are relevant to human health.

Microbiota therapy acts via a regulatory T cell MyD88/RORγt pathway to suppress food allergy.

The role of dysbiosis in food allergy (FA) remains unclear. We found that dysbiotic fecal microbiota in FA infants evolved compositionally over time and failed to protect against FA in mice. Infants and mice with FA had decreased IgA and increased IgE binding to fecal bacteria, indicative of a broader breakdown of oral tolerance than hitherto appreciated. Therapy with Clostridiales species impacted by dysbiosis, either as a consortium or as monotherapy with Subdoligranulum variabile, suppressed FA in mice, as did a separate immunomodulatory Bacteroidales consortium. Bacteriotherapy induced regulatory T (Treg) cells expressing the transcription factor ROR-γt in a MyD88-dependent manner, which were deficient in FA infants and mice and ineffectively induced by their microbiota. Deletion of Myd88 or Rorc in Treg cells abrogated protection by bacteriotherapy. Thus, commensals activate a MyD88/ROR-γt pathway in nascent Treg cells to protect against FA, while dysbiosis impairs this regulatory response to promote disease.

Alterations of Gut Microbiota After Biliopancreatic Diversion with Duodenal Switch in Wistar Rats

BackgroundThe biliopancreatic diversion with duodenal switch (BPD/DS) represents the most effective surgical procedure for the treatment of severe obesity and associated type 2 diabetes. The mechanisms whereby BPD/DS exerts its positive metabolic effects have however yet to be fully delineated. The objective of this study was to distinguish the effects of the two components of BPD/DS, namely the sleeve gastrectomy (SG) and the DS derivation, on gut microbiota, and to appraise whether changes in microbial composition are linked with surgery-induced metabolic benefits.MethodsBPD/DS, DS, and SG were performed in Wistar rats fed a standard chow diet. Body weight and energy intake were measured daily during 8 weeks post-surgery, at which time glucagon-like peptide 1 (GLP-1), peptide tyrosine tyrosine (PYY), insulin, and glucose were measured. Fecal samples were collected prior to surgery and at 2 and 8 weeks post-surgery. Intraluminal contents of the alimentary, biliopancreatic, and common limbs (resulting from BPD/DS) were taken from the proximal portion of each limb. Fecal and small intestinal limb samples were analyzed by 16S ribosomal RNA gene sequencing.ResultsBPD/DS and DS led to lower digestible energy intake (P = 0.0007 and P = 0.0002, respectively), reduced weight gain (P < 0.0001) and body fat mass (P < 0.0001), improved glucose metabolism, and increased GLP-1 (P = 0.0437, SHAM versus DS) and PYY levels (P < 0.0001). These effects were associated with major alterations of both the fecal and small intestinal microbiota, as revealed by significant decrease in bacterial richness and diversity at 2 (P < 0.0001, Chao1 index; P < 0.0001, Shannon index) and 8 weeks (P = 0.0159, SHAM versus DS, Chao1 index; P = 0.0219, SHAM versus DS, P = 0.0472, SHAM versus BPD/DS, Shannon index) post-surgery in BPD/DS and DS, and increased proportions of Bifidobacteriales (a 60% increase in both groups) but reduced Clostridiales (a 50% decrease and a 90% decrease respectively), which were mostly accounted at the genus level by higher relative abundance of Bifidobacterium in both the fecal and intestinal limb samples, as well as reduced abundance of Peptostreptococcaceae and Clostridiaceae in the small intestine. Those effects were not seen in SG rats.ConclusionThe metabolic benefits following BPD/DS are seemingly due to the DS component of the surgery. Furthermore, BPD/DS causes marked alterations in fecal and small intestinal microbiota resulting in reduced bacterial diversity and richness. Our data further suggest that increased abundance of Bifidobacterium and reduced level of two Clostridiales species in the gut microbiota might contribute to the positive metabolic outcomes of BPD/DS.

Su1554 – Causal Inference Reveals that Clostridiales Species are Keystone Species in Nafld Pathogenesis

Su1554 – Causal Inference Reveals that Clostridiales Species are Keystone Species in Nafld Pathogenesis

Observations on research with spores of Bacillales and Clostridiales species.

The purpose of this article is to highlight some areas of research with spores of bacteria of Firmicute species in which the methodology too commonly used is not optimal and generates misleading results. As a consequence, conclusions drawn from data obtained are often flawed or not appropriate. Topics covered in the article include the following: (i) the importance of using well-purified bacterial spores in studies on spore resistance, composition, killing, disinfection and germination; (ii) methods for obtaining good purification of spores of various species; (iii) appropriate experimental approaches to determine mechanisms of spore resistance and spore killing by a variety of agents, as well as known mechanisms of spore resistance and killing; (iv) common errors made in drawing conclusions about spore killing by various agents, including failure to neutralize chemical agents before plating for viable spore enumeration, and equating correlations between changes in spore properties accompanying spore killing with causation. It is hoped that a consideration of these topics will improve the quality of spore research going forward.

Comparative genomics identifies potential virulence factors in Clostridium tertium and C. paraputrificum

ABSTRACTSome well-known Clostridiales species such as Clostridium difficile and C. perfringens are agents of high impact diseases worldwide. Nevertheless, other foreseen Clostridiales species have recently emerged such as Clostridium tertium and C. paraputrificum. Three fecal isolates were identified as Clostridium tertium (Gcol.A2 and Gcol.A43) and C. paraputrificum (Gcol.A11) during public health screening for C. difficile infections in Colombia. C. paraputrificum genomes were highly diverse and contained large numbers of accessory genes. Genetic diversity and accessory gene percentage were lower among the C. tertium genomes than in the C. paraputrificum genomes. C. difficile tcdA and tcdB toxins encoding homologous sequences and other potential virulence factors were also identified. EndoA interferase, a toxic component of the type II toxin-antitoxin system, was found among the C. tertium genomes. toxA was the only toxin encoding gene detected in Gcol.A43, the Colombian isolate with an experimentally-determined high cytotoxic effect. Gcol.A2 and Gcol.A43 had higher sporulation efficiencies than Gcol.A11 (84.5%, 83.8% and 57.0%, respectively), as supported by the greater number of proteins associated with sporulation pathways in the C. tertium genomes compared with the C. paraputrificum genomes (33.3 and 28.4 on average, respectively). This work allowed complete genome description of two clostridiales species revealing high levels of intra-taxa diversity, accessory genomes containing virulence-factors encoding genes (especially in C. paraputrificum), with proteins involved in sporulation processes more highly represented in C. tertium. These finding suggest the need to advance in the study of those species with potential importance at public health level.

Bacterial interactions during sequential degradation of cyanobacterial necromass in a sulfidic arctic marine sediment.

SummarySeafloor microorganisms impact global carbon cycling by mineralizing vast quantities of organic matter (OM) from pelagic primary production, which is predicted to increase in the Arctic because of diminishing sea ice cover. We studied microbial interspecies‐carbon‐flow during anaerobic OM degradation in arctic marine sediment using stable isotope probing. We supplemented sediment incubations with 13C‐labeled cyanobacterial necromass (spirulina), mimicking fresh OM input, or acetate, an important OM degradation intermediate and monitored sulfate reduction rates and concentrations of volatile fatty acids (VFAs) during substrate degradation. Sequential 16S rRNA gene and transcript amplicon sequencing and fluorescence in situ hybridization combined with Raman microspectroscopy revealed that only few bacterial species were the main degraders of 13C‐spirulina necromass. Psychrilyobacter, Psychromonas, Marinifilum, Colwellia, Marinilabiaceae and Clostridiales species were likely involved in the primary hydrolysis and fermentation of spirulina. VFAs, mainly acetate, produced from spirulina degradation were mineralized by sulfate‐reducing bacteria and an Arcobacter species. Cellular activity of Desulfobacteraceae and Desulfobulbaceae species during acetoclastic sulfate reduction was largely decoupled from relative 16S rRNA gene abundance shifts. Our findings provide new insights into the identities and physiological constraints that determine the population dynamics of key microorganisms during complex OM degradation in arctic marine sediments.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd

P079 Association of gut microbiota with mucosal inflammation in ulcerative colitis

Background: Disturbance in gut microbiota (dysbiosis) is a characteristic feature of ulcerative colitis (UC). However it remains unclear whether this dysbiosis contributes to disease pathogenesis by driving immune dysregulation or is merely secondary to mucosal inflammation and/or a result of host immune response. We aimed to determine whether microbiota dysbiosis varied between areas of inflamed and non-inflamed colon in patients with UC and whether this was associated with a humoral immune response. Methods: We collected colonic biopsies from histologically confirmed areas of inflamed and non-inflamed colon from 15 patients with active left sided ulcerative colitis or proctosigmoiditis. DNA was extracted using the FASTSpin Kit and gut microbiota was characterized using 16s rRNA based analysis of the V3–V4 region (Illumina MiSeq). Quality control and operational taxonomic unit classification of sequences was executed using QIIME. As a marker of mucosal humoral immune responses, inflamed and non-inflamed colonic biopsy samples were cultured in media (RPMI + 10% FCS) for 1 to 3 days prior to measurement of immunoglobulin production (IgA, IgG and IgM) by ELISA. Results: Consistent with previous observations patients with UC demonstrated reduced bacterial diversity with an increase in Proteobacteria, Bacteroides and Clostridiales species along with a decrease in Firmicutes to Bacteroides ratio. No differences were found in the microbial diversity nor phylae and genera in mucosally adherent gut microbiota between inflamed and non-inflamed colonic segments in patients with active UC. This observation was also seen when patients were subdivided based on disease activity as defined by Mayo scoring. Total immunoglobulin production did not differ between inflamed (n=6, mean 4966 ± sd 3670 ng/ml) and non-inflamed tissue (n=11; mean 5756 ± sd 8989 ng/ml; p>0.05) suggesting that equal numbers of antibody-producing B cells are present regardless of inflammation. Conclusions: We have demonstrated that the dysbiosis observed in patients with UC is consistent and is not influenced by mucosal inflammation or disease activity. Mucosal immunoglobulin production was not upregulated at sites of inflammation possibly suggestive of a uniform humoral response across the colon; although future work may uncover differences in antibody specificity to UC-associated dysbiosis. The mechanism for the complex interplay between the host immune system and gut microbiota in contributing to mucosal inflammation remains to be understood.

A fibrolytic potential in the human ileum mucosal microbiota revealed by functional metagenomic

The digestion of dietary fibers is a major function of the human intestinal microbiota. So far this function has been attributed to the microorganisms inhabiting the colon, and many studies have focused on this distal part of the gastrointestinal tract using easily accessible fecal material. However, microbial fermentations, supported by the presence of short-chain fatty acids, are suspected to occur in the upper small intestine, particularly in the ileum. Using a fosmid library from the human ileal mucosa, we screened 20,000 clones for their activities against carboxymethylcellulose and xylans chosen as models of the major plant cell wall (PCW) polysaccharides from dietary fibres. Eleven positive clones revealed a broad range of CAZyme encoding genes from Bacteroides and Clostridiales species, as well as Polysaccharide Utilization Loci (PULs). The functional glycoside hydrolase genes were identified, and oligosaccharide break-down products examined from different polysaccharides including mixed-linkage β-glucans. CAZymes and PULs were also examined for their prevalence in human gut microbiome. Several clusters of genes of low prevalence in fecal microbiome suggested they belong to unidentified strains rather specifically established upstream the colon, in the ileum. Thus, the ileal mucosa-associated microbiota encompasses the enzymatic potential for PCW polysaccharide degradation in the small intestine.