Gorse (Ulex europeaus) wastes with 5,6-dimethyl benzimidazole supplementation can support growth of vitamin B12 producing commensal gut microbes.

Many commensal gut microbes are recognized for their potential to synthesize vitamin B12, offering a promising avenue to address deficiencies through probiotic supplementation. While bioinformatics tools aid in predicting B12 biosynthetic potential, empirical validation remains crucial to confirm production, identify cobalamin vitamers, and establish biosynthetic yields. This study investigates vitamin B12 production in three human colonic bacterial species: Anaerobutyricum hallii DSM 3353, Roseburia faecis DSM 16840, and Anaerostipes caccae DSM 14662, along with Propionibacterium freudenreichii DSM 4902 as a positive control. These strains were selected for their potential use as probiotics, based on speculated B12 production from prior bioinformatic analyses. Cultures were grown in M2GSC, chemically defined media (CDM), and Gorse extract medium (GEM). The composition of GEM was similar to CDM, except that the carbon and nitrogen sources were replaced with the protein-depleted liquid waste obtained after subjecting Gorse to a leaf protein extraction process. B12 yields were quantified using liquid chromatography with tandem mass spectrometry. The results suggested that the three butyrate-producing strains could indeed produce B12, although the yields were notably low and were detected only in the cell lysates. Furthermore, B12 production was higher in GEM compared to M2GSC medium. The positive control, P. freudenreichii DSM 4902 produced B12 at concentrations ranging from 7 ng mL-1 to 12 ng mL-1. Univariate-scaled Principal Component Analysis (PCA) of data from previous publications investigating B12 production in P. freudenreichii revealed that B12 yields diminished when the carbon source concentration was ≤30 g L-1. In conclusion, the protein-depleted wastes from the leaf protein extraction process from Gorse can be valorised as a viable substrate for culturing B12-producing colonic gut microbes. Furthermore, this is the first report attesting to the ability of A. hallii, R. faecis, and A. caccae to produce B12. However, these microbes seem unsuitable for industrial applications owing to low B12 yields.

Buckwheat (Fagopyrum esculentum) Hulls Are a Rich Source of Fermentable Dietary Fibre and Bioactive Phytochemicals.

Pseudo-cereals such as buckwheat (Fagopyrum esculentum) are valid candidates to promote diet biodiversity and nutrition security in an era of global climate change. Buckwheat hulls (BHs) are currently an unexplored source of dietary fibre and bioactive phytochemicals. This study assessed the effects of several bioprocessing treatments (using enzymes, yeast, and combinations of both) on BHs' nutrient and phytochemical content, their digestion and metabolism in vitro (using a gastrointestinal digestion model and mixed microbiota from human faeces). The metabolites were measured using targeted LC-MS/MS and GC analysis and 16S rRNA gene sequencing was used to detect the impact on microbiota composition. BHs are rich in insoluble fibre (31.09 ± 0.22% as non-starch polysaccharides), protocatechuic acid (390.71 ± 31.72 mg/kg), and syringaresinol (125.60 ± 6.76 mg/kg). The bioprocessing treatments significantly increased the extractability of gallic acid, vanillic acid, p-hydroxybenzoic acid, syringic acid, vanillin, syringaldehyde, p-coumaric acid, ferulic acid, caffeic acid, and syringaresinol in the alkaline-labile bound form, suggesting the bioaccessibility of these phytochemicals to the colon. Furthermore, one of the treatments, EC_2 treatment, increased significantly the in vitro upper gastrointestinal release of bioactive phytochemicals, especially for protocatechuic acid (p < 0.01). The BH fibre was fermentable, promoting the formation mainly of propionate and, to a lesser extent, butyrate formation. The EM_1 and EC_2 treatments effectively increased the content of insoluble fibre but had no effect on dietary fibre fermentation (p > 0.05). These findings promote the use of buckwheat hulls as a source of dietary fibre and phytochemicals to help meet dietary recommendations and needs.

Links between Diet, Intestinal Anaerobes, Microbial Metabolites and Health.

A dense microbial community resides in the human colon, with considerable inter-individual variability in composition, although some species are relatively dominant and widespread in healthy individuals. In disease conditions, there is often a reduction in microbial diversity and perturbations in the composition of the microbiota. Dietary complex carbohydrates that reach the large intestine are important modulators of the composition of the microbiota and their primary metabolic outputs. Specialist gut bacteria may also transform plant phenolics to form a spectrum of products possessing antioxidant and anti-inflammatory activities. Consumption of diets high in animal protein and fat may lead to the formation of potentially deleterious microbial products, including nitroso compounds, hydrogen sulphide, and trimethylamine. Gut anaerobes also form a range of secondary metabolites, including polyketides that may possess antimicrobial activity and thus contribute to microbe-microbe interactions within the colon. The overall metabolic outputs of colonic microbes are derived from an intricate network of microbial metabolic pathways and interactions; however, much still needs to be learnt about the subtleties of these complex networks. In this review we consider the multi-faceted relationships between inter-individual microbiota variation, diet, and health.

Impact of changes at the Candida albicans cell surface upon immunogenicity and colonisation in the gastrointestinal tract.

The immunogenicity of Candida albicans cells is influenced by changes in the exposure of microbe-associated molecular patterns (MAMPs) on the fungal cell surface. Previously, the degree of exposure on the C. albicans cell surface of the immunoinflammatory MAMP ß-(1,3)-glucan was shown to correlate inversely with colonisation levels in the gastrointestinal (GI) tract. This is important because life-threatening systemic candidiasis in critically ill patients often arises from translocation of C. albicans strains present in the patient's GI tract. Therefore, using a murine model, we have examined the impact of gut-related factors upon ß-glucan exposure and colonisation levels in the GI tract. The degree of ß-glucan exposure was examined by imaging flow cytometry of C. albicans cells taken directly from GI compartments, and compared with colonisation levels. Fungal ß-glucan exposure was lower in the cecum than the small intestine, and fungal burdens were correspondingly higher in the cecum. This inverse correlation did not hold for the large intestine. The gut fermentation acid, lactate, triggers ß-glucan masking in vitro, leading to attenuated anti-Candida immune responses. Additional fermentation acids are present in the GI tract, including acetate, propionate, and butyrate. We show that these acids also influence ß-glucan exposure on C. albicans cells in vitro and, like lactate, they influence ß-glucan exposure via Gpr1/Gpa2-mediated signalling. Significantly, C. albicans gpr1Δ gpa2Δ cells displayed elevated ß-glucan exposure in the large intestine and a corresponding decrease in fungal burden, consistent with the idea that Gpr1/Gpa2-mediated ß-glucan masking influences colonisation of this GI compartment. Finally, extracts from the murine gut and culture supernatants from the mannan grazing gut anaerobe Bacteroides thetaiotaomicron promote ß-glucan exposure at the C. albicans cell surface. Therefore, the local microbiota influences ß-glucan exposure levels directly (via mannan grazing) and indirectly (via fermentation acids), whilst ß-glucan masking appears to promote C. albicans colonisation of the murine large intestine.

Human gut bifidobacteria inhibit the growth of the opportunistic fungal pathogen Candida albicans.

The human gut microbiota protects the host from invading pathogens and the overgrowth of indigenous opportunistic species via a process called colonization resistance. Here, we investigated the antagonistic activity of human gut bacteria towards Candida albicans, an opportunistic fungal pathogen that can cause severe infections in susceptible individuals. Coculture batch incubations of C. albicans in the presence of faecal microbiota from six healthy individuals revealed varying levels of inhibitory activity against C. albicans. 16S rRNA gene amplicon profiling of these faecal coculture bacterial communities showed that the Bifidobacteriaceae family, and Bifidobacterium adolescentis in particular, were most correlated with antagonistic activity against C. albicans. Follow-up mechanistic studies performed under anaerobic conditions confirmed that culture supernatants of Bifidobacterium species, particularly B. adolescentis, inhibited C. albicans in vitro. Fermentation acids (FA), including acetate and lactate, present in the bifidobacterial supernatants were important contributors to inhibitory activity. However, increasing the pH of both bacterial supernatants and mixtures of FA reduced their anti-Candida effects, indicating a combinatorial effect of prevailing pH and FA. This work, therefore, demonstrates potential mechanisms underpinning gut microbiome-mediated colonization resistance against C. albicans, and identifies particularly inhibitory components such as bifidobacteria and FA as targets for further study.

Pain and Opioid-Induced Gut Microbial Dysbiosis.

Opioid-induced dysbiosis (OID) is a specific condition describing the consequences of opioid use on the bacterial composition of the gut. Opioids have been shown to affect the epithelial barrier in the gut and modulate inflammatory pathways, possibly mediating opioid tolerance or opioid-induced hyperalgesia; in combination, these allow the invasion and proliferation of non-native bacterial colonies. There is also evidence that the gut-brain axis is linked to the emotional and cognitive aspects of the brain with intestinal function, which can be a factor that affects mental health. For example, Mycobacterium, Escherichia coli and Clostridium difficile are linked to Irritable Bowel Disease; Lactobacillaceae and Enterococcacae have associations with Parkinson's disease, and Alistipes has increased prevalence in depression. However, changes to the gut microbiome can be therapeutically influenced with treatments such as faecal microbiota transplantation, targeted antibiotic therapy and probiotics. There is also evidence of emerging therapies to combat OID. This review has collated evidence that shows that there are correlations between OID and depression, Parkinson's Disease, infection, and more. Specifically, in pain management, targeting OID deserves specific investigations.

Survival Strategies and Metabolic Interactions between Ruminococcus gauvreauii and Ruminococcoides bili, Isolated from Human Bile.

Little is known about the bacteria that reside in the human gallbladder and the mechanisms that allow them to survive within this harsh environment. Here we describe interactions between two strains from a human bile sample, one Ruminococcus gauvreauii (IPLA60001), belonging to the Lachnospiraceae family, and the other, designated as Ruminococcoides bili (IPLA60002T; DSM 110008) most closely related to Ruminococcus bromii within the family Ruminococcaceae. We provide evidence for bile salt resistance and sporulation for these new strains. Both differed markedly in their carbohydrate metabolism. The R. bili strain mainly metabolized resistant starches to form formate, lactate and acetate. R. gauvreauii mainly metabolized sugar alcohols, including inositol and also utilized formate to generate acetate employing the Wood Ljungdahl pathway. Amino acid and vitamin biosynthesis genomic profiles also differed markedly between the two isolates, likely contributing to their synergistic interactions, as revealed by transcriptomic analysis of cocultures. Transcriptome analysis also revealed that R. gauvreauii IPLA60001 is able to grow using the end-products of starch metabolism formed by the R. bili strain such as formate, and potentially other compounds (such as ethanolamine and inositol) possibly provided by the autolytic behavior of R. bili. IMPORTANCE Unique insights into metabolic interaction between two isolates; Ruminococcus gauvreauii IPLA60001 and Ruminococcoides bili IPLA60002, from the human gallbladder, are presented here. The R. bili strain metabolized resistant starches while R. gauvreauii failed to do so but grew well on sugar alcohols. Transcriptomic analysis of cocultures of these strains, provides new data on the physiology and ecology of two bacteria from human bile, with a particular focus on cross-feeding mechanisms. Both biliary strains displayed marked resistance to bile and possess many efflux transporters, potentially involved in bile export. However, they differ markedly in their amino acid catabolism and vitamin synthesis capabilities, a feature that is therefore likely to contribute to the strong synergistic interactions between these strains. This is therefore the first study that provides evidence for syntrophic metabolic cooperation between bacterial strains isolated from human bile.

Ruminococcoides bili gen. nov., sp. nov., a bile-resistant bacterium from human bile with autolytic behavior.

A strictly anaerobic, resistant starch-degrading, bile-tolerant, autolytic strain, IPLA60002T, belonging to the family Ruminococcaceae, was isolated from a human bile sample of a liver donor without hepatobiliary disease. Cells were Gram-stain-positive cocci, and 16S rRNA gene and whole genome analyses showed that Ruminococcus bromii was the phylogenetically closest related species to the novel strain IPLA60002T, though with average nucleotide identity values below 90 %. Biochemically, the new isolate has metabolic features similar to those described previously for gut R. bromii strains, including the ability to degrade a range of different starches. The new isolate, however, produces lactate and shows distinct resistance to the presence of bile salts. Additionally, the novel bile isolate displays an autolytic phenotype after growing in different media. Strain IPLA60002T is phylogenetically distinct from other species within the genus Ruminococcus. Therefore, we propose on the basis of phylogenetic, genomic and metabolic data that the novel IPLA60002T strain isolated from human bile be given the name Ruminococcoides bili gen. nov., sp. nov., within the new proposed genus Ruminococcoides and the family Ruminococcaceae. Strain IPLA60002T (=DSM 110008T=LMG 31505T) is proposed as the type strain of Ruminococcoides bili.

Human Gut Faecalibacterium prausnitzii Deploys a Highly Efficient Conserved System To Cross-Feed on ß-Mannan-Derived Oligosaccharides.

ß-Mannans are hemicelluloses that are abundant in modern diets as components in seed endosperms and common additives in processed food. Currently, the collective understanding of ß-mannan saccharification in the human colon is limited to a few keystone species, which presumably liberate low-molecular-weight mannooligosaccharide fragments that become directly available to the surrounding microbial community. Here, we show that a dominant butyrate producer in the human gut, Faecalibacterium prausnitzii, is able to acquire and degrade various ß-mannooligosaccharides (ß-MOS), which are derived by the primary mannanolytic activity of neighboring gut microbiota. Detailed biochemical analyses of selected protein components from their two ß-MOS utilization loci (F. prausnitzii ß-MOS utilization loci [FpMULs]) supported a concerted model whereby the imported ß-MOS are stepwise disassembled intracellularly by highly adapted enzymes. Coculturing experiments of F. prausnitzii with the primary degraders Bacteroides ovatus and Roseburia intestinalis on polymeric ß-mannan resulted in syntrophic growth, thus confirming the high efficiency of the FpMULs' uptake system. Genomic comparison with human F. prausnitzii strains and analyses of 2,441 public human metagenomes revealed that FpMULs are highly conserved and distributed worldwide. Together, our results provide a significant advance in the knowledge of ß-mannan metabolism and the degree to which its degradation is mediated by cross-feeding interactions between prominent beneficial microbes in the human gut. IMPORTANCE Commensal butyrate-producing bacteria belonging to the Firmicutes phylum are abundant in the human gut and are crucial for maintaining health. Currently, insight is lacking into how they target otherwise indigestible dietary fibers and into the trophic interactions they establish with other glycan degraders in the competitive gut environment. By combining cultivation, genomic, and detailed biochemical analyses, this work reveals the mechanism enabling F. prausnitzii, as a model Ruminococcaceae within Firmicutes, to cross-feed and access ß-mannan-derived oligosaccharides released in the gut ecosystem by the action of primary degraders. A comprehensive survey of human gut metagenomes shows that FpMULs are ubiquitous in human populations globally, highlighting the importance of microbial metabolism of ß-mannans/ß-MOS as a common dietary component. Our findings provide a mechanistic understanding of the ß-MOS utilization capability by F. prausnitzii that may be exploited to select dietary formulations specifically boosting this beneficial symbiont, and thus butyrate production, in the gut.

Type IV pili are widespread among non-pathogenic Gram-positive gut bacteria with diverse carbohydrate utilization patterns.

Type IV pili (T4P) are bacterial surface-exposed appendages that have been extensively studied in Gram-negative pathogenic bacteria. Despite recent sequencing efforts, little is known regarding these structures in non-pathogenic anaerobic Gram-positive species, particularly commensals of the mammalian gut. Early studies revealed that T4P in two ruminal Gram-positive species are associated with growth on cellulose, suggesting possible associations of T4P with substrate utilization patterns. In the present study, genome sequences of 118 taxonomically diverse, mainly Gram-positive, bacterial strains isolated from anaerobic (gastrointestinal) environments, have been analysed. The genes likely to be associated with T4P biogenesis were analysed and grouped according to T4P genetic organization. In parallel, consortia of Carbohydrate Active enZYmes (CAZymes) were also analysed and used to predict carbohydrate utilization abilities of selected strains. The predictive power of this approach was additionally confirmed by experimental assessment of substrate-related growth patterns of selected strains. Our analysis revealed that T4P systems with diverse genetic organization are widespread among Gram-positive anaerobic non-pathogenic bacteria isolated from different environments, belonging to two phylogenetically distantly related phyla: Firmicutes and Actinobacteria.

Impact of protein on the composition and metabolism of the human gut microbiota and health.

The composition and metabolic activity of the bacteria that inhabit the large intestine can have a major impact on health. Despite considerable inter-individual variation across bacterial species, the dominant phyla are generally highly conserved. There are several exogenous and gut environmental factors that play a role in modulating the composition and activities of colonic bacteria including diet with intakes of different macronutrients, including protein, accounting for approximately 20% of the microbial variation. Certain bacterial species tend to be considered as generalists and can metabolise a broad range of substrates, including both carbohydrate- and protein-derived substrates, whilst other species are specialists with a rather limited metabolic capacity. Metabolism of peptides and amino acids by gut bacteria can result in the formation of a wide range of metabolites several of which are considered deleterious to health including nitrosamines, heterocyclic amines and hydrogen sulphide as some of these products are genotoxic and have been linked to colonic disease. Beneficial metabolites however include SCFA and certain species can use amino acids to form butyrate which is the major energy source for colonocytes. The impact on health may however depend on the source of these products. In this review, we consider the impact of diet, particularly protein diets, on modulating the composition of the gut microbiota and likely health consequences and the potential impact of climate change and food security.

Pivotal Roles for pH, Lactate, and Lactate-Utilizing Bacteria in the Stability of a Human Colonic Microbial Ecosystem.

Lactate can be produced by many gut bacteria, but in adults its accumulation in the colon is often an indicator of microbiota perturbation. Using continuous culture anaerobic fermentor systems, we found that lactate concentrations remained low in communities of human colonic bacteria maintained at pH 6.5, even when dl-lactate was infused at 10 or 20 mM. In contrast, lower pH (5.5) led to periodic lactate accumulation following lactate infusion in three fecal microbial communities examined. Lactate accumulation was concomitant with greatly reduced butyrate and propionate production and major shifts in microbiota composition, with Bacteroidetes and anaerobic Firmicutes being replaced by Actinobacteria, lactobacilli, and Proteobacteria Pure-culture experiments confirmed that Bacteroides and Firmicutes isolates were susceptible to growth inhibition by relevant concentrations of lactate and acetate, whereas the lactate-producer Bifidobacterium adolescentis was resistant. To investigate system behavior further, we used a mathematical model (microPop) based on 10 microbial functional groups. By incorporating differential growth inhibition, our model reproduced the chaotic behavior of the system, including the potential for lactate infusion both to promote and to rescue the perturbed system. The modeling revealed that system behavior is critically dependent on the proportion of the community able to convert lactate into butyrate or propionate. Communities with low numbers of lactate-utilizing bacteria are inherently less stable and more prone to lactate-induced perturbations. These findings can help us to understand the consequences of interindividual microbiota variation for dietary responses and microbiota changes associated with disease states.IMPORTANCE Lactate is formed by many species of colonic bacteria, and can accumulate to high levels in the colons of inflammatory bowel disease subjects. Conversely, in healthy colons lactate is metabolized by lactate-utilizing species to the short-chain fatty acids butyrate and propionate, which are beneficial for the host. Here, we investigated the impact of continuous lactate infusions (up to 20 mM) at two pH values (6.5 and 5.5) on human colonic microbiota responsiveness and metabolic outputs. At pH 5.5 in particular, lactate tended to accumulate in tandem with decreases in butyrate and propionate and with corresponding changes in microbial composition. Moreover, microbial communities with low numbers of lactate-utilizing bacteria were inherently less stable and therefore more prone to lactate-induced perturbations. These investigations provide clear evidence of the important role these lactate utilizers may play in health maintenance. These should therefore be considered as potential new therapeutic probiotics to combat microbiota perturbations.

Relative abundance of the Prevotella genus within the human gut microbiota of elderly volunteers determines the inter-individual responses to dietary supplementation with wheat bran arabinoxylan-oligosaccharides.

BACKGROUND: The human colon is colonised by a dense microbial community whose species composition and metabolism are linked to health and disease. The main energy sources for colonic bacteria are dietary polysaccharides and oligosaccharides. These play a major role in modulating gut microbial composition and metabolism, which in turn can impact on health outcomes. RESULTS: We investigated the influence of wheat bran arabinoxylan oligosaccharides (AXOS) and maltodextrin supplements in modulating the composition of the colonic microbiota and metabolites in healthy adults over the age of 60. Male and female volunteers, (n = 21, mean BMI 25.2 ± 0.7 kg/m2) participated in the double-blind, cross over supplement study. Faecal samples were collected for analysis of microbiota, short chain fatty acids levels and calprotectin. Blood samples were collected to measure glucose, cholesterol and triglycerides levels. There was no change in these markers nor in calprotectin levels in response to the supplements. Both supplements were well-tolerated by the volunteers. Microbiota analysis across the whole volunteer cohort revealed a significant increase in the proportional abundance of faecal Bifidobacterium species (P ≤ 0.01) in response to AXOS, but not maltodextrin, supplementation. There was considerable inter-individual variation in the other bacterial taxa that responded, with a clear stratification of volunteers as either Prevotella-plus (n = 8; > 0.1% proportional abundance) or Prevotella-minus (n = 13; ≤0.1% proportional abundance) subjects founded on baseline sample profiles. There was a significant increase in the proportional abundance of both faecal Bifidobacterium (P ≤ 0.01) and Prevotella species (P ≤ 0.01) in Prevotella-plus volunteers during AXOS supplementation, while Prevotella and Bacteroides relative abundances showed an inverse relationship. Proportional abundance of 26 OTUs, including bifidobacteria and Anaerostipes hadrus, differed significantly between baseline samples of Prevotella-plus compared to Prevotella-minus individuals. CONCLUSIONS: The wheat bran AXOS supplementation was bifidogenic and resulted in changes in human gut microbiota composition that depended on the initial microbiota profile, specifically the presence or absence of Prevotella spp. as a major component of the microbiota. Our data therefore suggest that initial profiling of individuals through gut microbiota analysis should be considered important when contemplating nutritional interventions that rely on prebiotics. TRIAL REGISTRATION: Clinical trial registration number: NCT02693782 . Registered 29 February 2016 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT02693782?term=NCT02693782&rank=1.

Vitamin Biosynthesis by Human Gut Butyrate-Producing Bacteria and Cross-Feeding in Synthetic Microbial Communities.

We investigated the requirement of 15 human butyrate-producing gut bacterial strains for eight B vitamins and the proteinogenic amino acids by a combination of genome sequence analysis and in vitro growth experiments. The Ruminococcaceae species Faecalibacterium prausnitzii and Subdoligranulum variabile were auxotrophic for most of the vitamins and the amino acid tryptophan. Within the Lachnospiraceae, most species were prototrophic for all amino acids and several vitamins, but biotin auxotrophy was widespread. In addition, most of the strains belonging to Eubacterium rectale and Roseburia spp., but few of the other Lachnospiraceae strains, were auxotrophic for thiamine and folate. Synthetic coculture experiments of five thiamine or folate auxotrophic strains with different prototrophic bacteria in the absence and presence of different vitamin concentrations were carried out. This demonstrated that cross-feeding between bacteria does take place and revealed differences in cross-feeding efficiency between prototrophic strains. Vitamin-independent growth stimulation in coculture compared to monococulture was also observed, in particular for F. prausnitzii A2-165, suggesting that it benefits from the provision of other growth factors from community members. The presence of multiple vitamin auxotrophies in the most abundant butyrate-producing Firmicutes species found in the healthy human colon indicates that these bacteria depend upon vitamins supplied from the diet or via cross-feeding from other members of the microbial community.IMPORTANCE Microbes in the intestinal tract have a strong influence on human health. Their fermentation of dietary nondigestible carbohydrates leads to the formation of health-promoting short-chain fatty acids, including butyrate, which is the main fuel for the colonic wall and has anticarcinogenic and anti-inflammatory properties. A good understanding of the growth requirements of butyrate-producing bacteria is important for the development of efficient strategies to promote these microbes in the gut, especially in cases where their abundance is altered. The demonstration of the inability of several dominant butyrate producers to grow in the absence of certain vitamins confirms the results of previous in silico analyses. Furthermore, establishing that strains prototrophic for thiamine or folate (butyrate producers and non-butyrate producers) were able to stimulate growth and affect the composition of auxotrophic synthetic communities suggests that the provision of prototrophic bacteria that are efficient cross feeders may stimulate butyrate-producing bacteria under certain in vivo conditions.

Analysis of 1321 Eubacterium rectale genomes from metagenomes uncovers complex phylogeographic population structure and subspecies functional adaptations.

BACKGROUND: Eubacterium rectale is one of the most prevalent human gut bacteria, but its diversity and population genetics are not well understood because large-scale whole-genome investigations of this microbe have not been carried out. RESULTS: Here, we leverage metagenomic assembly followed by a reference-based binning strategy to screen over 6500 gut metagenomes spanning geography and lifestyle and reconstruct over 1300 E. rectale high-quality genomes from metagenomes. We extend previous results of biogeographic stratification, identifying a new subspecies predominantly found in African individuals and showing that closely related non-human primates do not harbor E. rectale. Comparison of pairwise genetic and geographic distances between subspecies suggests that isolation by distance and co-dispersal with human populations might have contributed to shaping the contemporary population structure of E. rectale. We confirm that a relatively recently diverged E. rectale subspecies specific to Europe consistently lacks motility operons and that it is immotile in vitro, probably due to ancestral genetic loss. The same subspecies exhibits expansion of its carbohydrate metabolism gene repertoire including the acquisition of a genomic island strongly enriched in glycosyltransferase genes involved in exopolysaccharide synthesis. CONCLUSIONS: Our study provides new insights into the population structure and ecology of E. rectale and shows that shotgun metagenomes can enable population genomics studies of microbiota members at a resolution and scale previously attainable only by extensive isolate sequencing.

Nondigestible Carbohydrates Affect Metabolic Health and Gut Microbiota in Overweight Adults after Weight Loss.

BACKGROUND: The composition of diets consumed following weight loss (WL) can have a significant impact on satiety and metabolic health. OBJECTIVE: This study was designed to test the effects of including a nondigestible carbohydrate to achieve weight maintenance (WM) following a period of WL. METHODS: Nineteen volunteers [11 females and 8 males, aged 20-62 y; BMI (kg/m2): 27-42] consumed a 3-d maintenance diet (15%:30%:55%), followed by a 21-d WL diet (WL; 30%:30%:40%), followed by 2 randomized 10-d WM diets (20%:30%:50% of energy from protein:fat:carbohydrate) containing either resistant starch type 3 (RS-WM; 22 or 26 g/d for females and males, respectively) or no RS (C-WM) in a within-subject crossover design without washout periods. The primary outcome, WM after WL, was analyzed by body weight. Secondary outcomes of fecal microbiota composition and microbial metabolite concentrations and gut hormones were analyzed in fecal samples and blood plasma, respectively. All outcomes were assessed at the end of each dietary period. RESULTS: Body weight was similar after the RS-WM and C-WM diets (90.7 and 90.8 kg, respectively), with no difference in subjectively rated appetite. During the WL diet period plasma ghrelin increased by 36% (P < 0.001), glucose-dependent insulinotropic polypeptide (GIP) decreased by 33% (P < 0.001), and insulin decreased by 46% (P < 0.001), but no significant differences were observed during the RS-WM and C-WM diet periods. Fasting blood glucose was lower after the RS-WM diet (5.59 ± 0.31 mmol/L) than after the C-WM diet [5.75 ± 0.49 mmol/L; P = 0.015; standard error of the difference between the means (SED): 0.09]. Dietary treatments influenced the fecal microbiota composition (R2 = 0.054, P = 0.031) but not diversity. CONCLUSIONS: The metabolic benefits, for overweight adults, from WL were maintained through a subsequent WM diet with higher total carbohydrate intake. Inclusion of resistant starch in the WM diet altered gut microbiota composition positively and resulted in lower fasting glucose compared with the control, with no apparent change in appetite. This trial was registered at clinicaltrials.gov as NCT01724411.

Evaluation of bacterial biomarkers to aid in challenging inflammatory bowel diseases diagnostics and subtype classification.

BACKGROUND: The challenges for inflammatory bowel disease (IBD) diagnostics are to discriminate it from gut conditions with similar symptoms such as irritable bowel syndrome (IBS), to distinguish IBD subtypes, to predict disease progression, and to establish the risk to develop colorectal cancer (CRC). Alterations in gut microbiota have been proposed as a source of information to assist in IBD diagnostics. Faecalibacterium prausnitzii (F. prausnitzii), its phylogroups, and Escherichia coli (E. coli) have been reported as potential biomarkers, but their performance in challenging IBD diagnostic situations remains elusive. We hypothesize that bacterial biomarkers based in these species may help to discriminate these conditions of complex diagnostics. AIM: To evaluate the usefulness of indices calculated from the quantification of these species as biomarkers to aid in IBD diagnostics. METHODS: A retrospective study of 131 subjects (31 controls (H); 45 Crohn's disease (CD), 25 ulcerative colitis (UC), 10 IBS, and 20 CRC patients) was performed to assess the usefulness of bacterial biomarkers in biopsies. Further, the performance of biomarkers in faeces was studied in 29 stool samples (19 CD, 10 UC). Relative abundances of total F. prausnitzii (FP), its phylogroups (PHGI and PHGII), and E. coli (E) quantification were determined by qPCR. Loads were combined to calculate the FP-E index, the PHGI-E index and the PHGII-E index. Biomarkers accuracy to discriminate among conditions was measured by the area under the receiver operating characteristic curve (AUC). RESULTS: In biopsies, FP-E index was good for discriminating IBS from CD (AUC = 0.752) while PHGII-E index was suitable for discriminating IBS from UC (AUC = 0.632). The FP-E index would be the choice to discriminate IBD from CRC, especially from all UC subtypes (AUC ≥ 0.875), regardless of the activity status of the patient. Discrimination between UC patients that had the longest disease duration and those with CRC featured slightly lower AUC values. Concerning differentiation in IBD with shared location, PHGI-E index can establish progression from proctitis and left-sided colitis to ulcerative pancolitis (AUC ≥ 0.800). PHG I-E index analysis in tissue would be the choice to discriminate within IBD subtypes of shared location (AUC ≥ 0.712), while in non-invasive faecal samples FP or PHGI could be good indicators (AUC ≥ 0.833). CONCLUSION: F. prausnitzii phylogroups combined with E. coli offer potential to discriminate between IBD and CRC patients and can assist in IBD subtypes classification, which may help in solving IBD diagnostics challenges.

Sapogenol is a Major Microbial Metabolite in Human Plasma Associated with High Protein Soy-Based Diets: The Relevance for Functional Food Formulations.

Legumes are a source of health-promoting macro- and micronutrients, but also contain numerous phytochemicals with useful biological activities, an example of which are saponins. Epidemiological studies suggest that saponins may play a role in protection from cancer and benefit human health by lowering cholesterol. Therefore, they could represent good candidates for specialised functional foods. Following the consumption of a soya-rich high-protein weight-loss diet (SOYA HP WL), the concentrations of Soyasaponin I (SSI) and soyasapogenol B (SSB) were determined in faecal samples from human volunteers (n = 10) and found to be between 1.4 and 17.5 mg per 100 g fresh faecal sample. SSB was the major metabolite identified in volunteers' plasma (n = 10) after consumption of the soya test meal (SOYA MEAL); the postprandial (3 h after meal) plasma concentration for SSB varied between 48.5 ng/mL to 103.2 ng/mL. The metabolism of SSI by the gut microbiota (in vitro) was also confirmed. This study shows that the main systemic metabolites of soyasaponin are absorbed from the gut and that they are bioavailable in plasma predominantly as conjugates of sapogenol. The metabolism and bioavailability of biologically active molecules represent key information necessary for the efficient development of functional foods.

Comparative genetic and physiological characterisation of Pectinatus species reveals shared tolerance to beer-associated stressors but halotolerance specific to pickle-associated strains.

Obligate anaerobic bacteria from the genus Pectinatus have been known to cause beer spoilage for over 40 years. Whole genome sequencing was performed on eleven beer spoilage strains (nine Pectinatus frisingensis, one Pectinatus cerevisiiphilus and one Pectinatus haikarae isolate), as well as two pickle spoilage species (Pectinatus brassicae MB591 and Pectinatus sottacetonis MB620) and the tolerance of all species to a range of environmental conditions was tested. Exploration of metabolic pathways for carbohydrates, amino acids and vitamins showed little difference between beer spoilage- and pickle spoilage-associated strains. However, genes for certain carbohydrate- and sulphur-containing amino acid-associated enzymes were only present in the beer spoilage group and genes for specific transporters and regulatory genes were uniquely found in the pickle spoilage group. Transporters for compatible solutes, only present in pickle-associated strains, likely explain their experimentally observed higher halotolerance compared to the beer spoilers. Genes involved in biofilm formation and ATP Binding Cassette (ABC) transporters potentially capable of exporting hop-derived antimicrobial compounds were found in all strains. All species grew in the presence of alcohol up to 5% alcohol by volume (ABV) and hops extract up to 80 ppm of iso-α-acids. Therefore, the species isolated from pickle processes may pose novel hazards in brewing.