Search

The human colon contains a dynamic microbial community whose composition has important implications for human health. In this work we build a process-based model of the colonic microbial ecosystem and compare with general empirical observations and the results of in-vivo experiments. Based our previous work (Kettle et al., 2015), the microbial model consists of 10 microbial functional groups, 4 substrates and 10 metabolites; to this we add the interaction with a human host to give simulations of the in-situ colonic microbial ecosystem. This model incorporates absorption of short chain fatty acids (SCFA) and water by the host through the gut wall, variations in incoming dietary substrates (in the form of “meals” whose composition varies in time), bowel movements, feedback on microbial growth from changes in pH resulting from SCFA production, and multiple compartments to represent the proximal, transverse and distal colon. We verify our model against a number of observed criteria, e.g. total SCFA concentrations, SCFA ratios, mass of bowel movements, pH and water absorption over the transit time; and then run simulations investigating the effect of colonic transit time, and the composition and amount of indigestible carbohydrate in the host diet, which we compare with in-vivo studies. Gut microbiota are highly complex and poory understood yet our work shows that it is nevertheless possible to develop predictive models of the key components of the dynamics of this ecological system. The code is available as an R package (microPopGut) to aid future research.Author SummaryKettle wrote the model code and led the writing of the manuscript. Louis and Flint both contributed to writing the manuscript and all aspects of microbiolgy. All authors contributed critically to the drafts and gave final approval for publication.

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.

Lactate accumulation in the human gut is linked to a range of deleterious health impacts. However, lactate is consumed and converted to the beneficial short-chain fatty acids butyrate and propionate by indigenous lactate-utilizing bacteria. To better understand the underlying genetic basis for lactate utilization, transcriptomic analyses were performed for two prominent lactate-utilizing species from the human gut, Anaerobutyricum soehngenii and Coprococcus catus , during growth on lactate, hexose sugar or hexose plus lactate. In A. soehngenii L2-7 six genes of the lactate utilization (lct) cluster, including NAD-independent d-lactate dehydrogenase (d-iLDH), were co-ordinately upregulated during growth on equimolar d- and l-lactate (dl-lactate). Upregulated genes included an acyl-CoA dehydrogenase related to butyryl-CoA dehydrogenase, which may play a role in transferring reducing equivalents between reduction of crotonyl-CoA and oxidation of lactate. Genes upregulated in C. catus GD/7 included a six-gene cluster (lap) encoding propionyl CoA-transferase, a putative lactoyl-CoA epimerase, lactoyl-CoA dehydratase and lactate permease, and two unlinked acyl-CoA dehydrogenase genes that are candidates for acryloyl-CoA reductase. A d-iLDH homologue in C. catus is encoded by a separate, partial lct, gene cluster, but not upregulated on lactate. While C. catus converts three mols of dl-lactate via the acrylate pathway to two mols propionate and one mol acetate, some of the acetate can be re-used with additional lactate to produce butyrate. A key regulatory difference is that while glucose partially repressed lct cluster expression in A. soehngenii , there was no repression of lactate-utilization genes by fructose in the non-glucose utilizer C. catus . This suggests that these species could occupy different ecological niches for lactate utilization in the gut, which may be important factors to consider when developing lactate-utilizing bacteria as novel candidate probiotics.

© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Lactate accumulation in the human gut is linked to a range of deleterious health impacts. However, lactate is consumed and converted to the beneficial short chain fatty acids butyrate and propionate by indigenous lactate-utilizing bacteria. To better understand the underlying genetic basis for lactate utilization, transcriptomic analysis was performed for two prominent lactate-utilizing species from the human gut, Anaerobutyricum soehngenii and Coprococcus catus, during growth on lactate, hexose sugar, or hexose plus lactate. In A. soehngenii L2-7, six genes of the lct cluster including NAD-independent D-lactate dehydrogenase (i-LDH) were co-ordinately upregulated during growth on equimolar D and L-lactate (DL-lactate). Upregulated genes included an acyl-CoA dehydrogenase related to butyryl-CoA dehydrogenase, which may play a role in transferring reducing equivalents between reduction of crotonyl-CoA and oxidation of lactate. Genes upregulated in C. catus GD/7 included a six-gene cluster (lap) encoding propionyl CoA-transferase, a putative lactoyl-CoA epimerase, lactoyl-CoA dehydratase and lactate permease, and two unlinked acyl-CoA dehydrogenase genes that are candidates for acryloyl-CoA reductase. An i-LDH homolog in C. catus is encoded by a separate, partial lct, gene cluster, but not upregulated on lactate. While C. catus converts three mols of DL-lactate via the acrylate pathway to two mols propionate and one mol acetate, some of the acetate can be re-used with additional lactate to produce butyrate. A key regulatory difference is that while glucose partially repressed lct cluster expression in A. soehngenii, there was no repression of lactate utilization genes by fructose in the non-glucose utilizer C. catus. This implies that bacteria such as C. catus might be more important in curtailing lactate accumulation in the gut.Impact statementLactate can be produced as a fermentation by-product by many gut bacteria but has the potential to perturb intestinal microbial communities by lowering luminal pH, and its accumulation has been linked to a range of deleterious health outcomes. Fortunately, in healthy individuals, lactate tends not to accumulate as it is consumed by cross-feeding lactate-utilizing bacteria, which can convert it into the beneficial short chain fatty acids butyrate and propionate. Lactate-utilizing gut bacteria are therefore promising candidates for potential development as novel probiotics. However, lactate-utilizers are taxonomically diverse, and the genes that underpin utilization of lactate by these specialized gut bacteria are not fully understood. In this study we used transcriptomics to compare gene expression profiles of Anaerobutyricum soehngenii and Coprococcus catus, two prominent lactate-utilizing species in the human gut, during growth on lactate alone, sugar alone, or sugar plus lactate. The results revealed strong upregulation of key, but distinct, gene clusters that appear to be responsible for lactate utilization by these, and other, gut bacterial species. Our results therefore increase mechanistic understanding of different lactate utilization pathways used by gut bacteria, which may help to inform selection of optimal lactate-utilizing species for development as novel therapeutics against colonic microbiota perturbations.Data summaryNovel draft genomes generated for this study have been made available from GenBank (https://www.ncbi.nlm.nih.gov/bioproject/) under BioProject number PRJNA701799. RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-10136. Further details of additional existing genomic data that were analyzed in this project are given in Table 1 and Table S2.

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., 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.

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

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.

During natural childbirth the gut of the newborn infant becomes colonized with microorganisms that come mainly from the mother, although they can also come from the environment. In non-natural (i.e. caesarean, as opposed to vaginal) births there is less opportunity for the normal, messy routes of inoculation via faecal material and body fluids and the early colonisers are more influenced by the skin surface and outside environment [1]. Which organisms become established during early life depends on the subsequent feeding regime, with breast feeding leading to different gut microbiota profiles compared with formula-based bottle feeding. In breast-fed infants, the faecal microbiota generally becomes dominated by bifidobacteria that compete most effectively for the human milk oligosaccharides (HMOs) produced in the mother’s milk [2]. In contrast, infants who are not breast-fed exhibit higher relative numbers of Firmicutes, Bacteroidetes and Proteobacteria and lower bifidobacteria [3]. The difference in gut microbiota composition between caesarian and naturally delivered babies is significant before 3 months of age, but begins to disappear thereafter [4, 5].

While all regions of our digestive tract are colonized by micro-organisms to some degree, the densities, composition and nature of the associated microbial communities change markedly as we travel down through the gut (Fig. 5.1). The mouth (oral cavity) is well colonised, particularly the surfaces of teeth and crevices between the tooth and gum. As semi-permanent features, teeth allow the development of microbial biofilms, in which different species attach to each other and to the tooth surface in a highly structured manner [1]. Microbial biofilms can develop elsewhere, for example on implanted catheters, but the rapid turnover of epithelial cells and digesta particles probably limits their formation further down the gut. Infections of teeth and gums can have a major impact on health not only through tooth decay, but also through their effects on the heart, brain and immune system. These problems are due to pathogenic oral bacteria such as Streptococcus mutans and the anaerobe Porphyromonas gingivalis, a member of the Bacteroidetes phylum. The protein-destroying enzyme gingipain, which is produced by P. gingivalis was recently proposed as a factor in the development of Alzheimer’s disease [2]. Rapid passage of gut contents after swallowing means that the healthy oesophagus shows very low numbers of micro-organisms, while the highly acidic stomach also deters most colonisers. Some specialist microorganisms do however manage to colonise the stomach wall, notably Helicobacter pylori [3].

The term ‘Micro-organism’ refers to any living thing that is too small to be seen with the naked eye. It covers an astonishing array of life forms that began with the earliest living occupants of our planet. Of the three recognized domains of life, two (Bacteria and Archaea) consist exclusively of micro-organisms. The third (Eukaryota) contains all of the macroscopic, multi-celled organisms that we recognize as plants and animals, but it also includes many micro-organisms. ‘Microbiome’ is a collective term for all of the micro-organisms belonging to these three domains, while the ‘Biome’ comprises all life on earth. For most of earth’s history until the evolution of multicellular Eukaryota around 600 million years ago, however, the earth’s Biome consisted only of micro-organisms (Fig. 1.1). Even now the Microbiome is estimated to represent more than half of the total living matter (biomass) on the planet [1]. Their invisibility makes it easy for us to overlook the vast impact that micro-organisms have on the sustainability of the planet and of life on earth. While this book will focus on the gut-associated Microbiome, it is important that we start with a look at the wider microbial world.

So far, we have tended to emphasise the benefits of symbiotic association between the host and its gut microbiota. On the other hand, we know only too well that micro-organisms and viruses pose a continual threat to the survival of multicellular life forms, and these micro-organisms include many that either inhabit, or pass through, our gut. A wide range of viruses, bacteria and fungi are pathogens that have the ability to infect particular host species, while many protozoal species are pathogenic or parasitic. This has imposed incredibly strong selection for systems and mechanisms that help the host animal to defend itself against attack from micro-organisms and viruses.

Nearly all animals possess a digestive tract (the exception being parasites that live within their host’s tissues or digestive tracts) and in nearly all cases this consists of an open tube with flow of contents from one end (mouth or stoma) to the other (anus). Since the animal is ingesting food from its environment, it is also ingesting all of the microbes that are associated with the food, along with those from the immediate environment. This means that, although the gut within a foetus developing in the uterus is normally sterile, the adult gut has no chance of being sterile. It will inevitably become colonised by micro-organisms that are able to take advantage of the readily available sources of energy to be found in the gut.

A significant fraction of the carbohydrates in our diet cannot be broken down by our own digestive enzymes. For simplicity, these ‘non-digestible carbohydrates’ are here referred to simply as ‘fibre’. This diet-derived fibre passes through the stomach and small intestine to reach the large intestine, where it becomes the major energy source for the resident microbiota. Most of the fibre that we eat is fermentable, meaning that it can be broken down by our gut microbiota under the anaerobic conditions that prevail in the large intestine. On the other hand, some fibre is non-fermentable and behaves simply as a ‘bulking agent’, passing right through the gut un-degraded. In addition, carbohydrates produced by our own cells can be accessed by gut micro-organisms as energy sources.

We have seen how developments in molecular biology, chemical analysis and bioinformatics provide unprecedented new tools for analysing microbial communities and their activities. We have also seen how these are revealing the impact that microbial colonisation of the gut has on many aspects of human health (Fig. 12.1) [1]. So, perhaps the most obvious question that we should be asking is—exactly what composition of the gut microbiota is most beneficial for health? This is clearly a key question but, despite a deluge of new data, it is surprisingly difficult to provide a simple, straightforward answer.

‘Molecular Biology’ has dominated the study of biology and microbiology since publication of the structure of DNA in the 1950s [1]. Right from the start, Molecular Biology, which focusses on the flow of genetic information from DNA to RNA to protein, produced staggering conceptual and technical advances. These included unravelling the genetic code by which nucleotide sequences are translated into protein sequences and the mechanisms that control when and where genes are expressed. On the technical side, the ‘recombinant DNA revolution’ was created by the ability to artificially stitch together (recombine) stretches of DNA of different origin (e.g. bacterial and mammalian) and then to propagate and manipulate them almost at will. Furthermore, in recent years the ability to sequence DNA molecules has increased in speed and decreased in cost at an astonishing pace.

In the last two chapters, we have seen that the composition of the gut microbial community responds to dietary change and that the microbiota contribute to the fermentation and transformation of dietary fibre. The gut microbiota also process other dietary components (fats and proteins) and interact with host cells, giving them the potential to influence many aspects of human nutrition and health. The possibility that our gut microbiota influence obesity and metabolic disease (diabetes) has been particularly topical in view of the increasing challenges posed by these conditions. We will start here by looking at the direct contribution of our microbiota to the recovery of energy from the diet, before moving on to consider the more complex interactions that occur with human physiology and metabolism.

Most micro-organisms exist in nature as complex mixtures which are referred to as microbial communities. Members of such communities potentially interact with each other in various ways, including as competitors, predators and symbionts. Communities range from extremely dense mixtures of organisms that are held together in biofilms by binding to each other and to a surface (e.g. in dental plaque), to the highly dilute communities that are found in nutrient-poor ocean water. Finding out what organisms are present within a microbial community and how they interact with each other is one of the most intriguing challenges in biology.

In most living organisms, sugars play the central role in supplying energy for cellular activities via common pathways that start from glucose. In heterotrophs, which include all animals and many micro-organisms, sugars come from carbohydrates that are taken up from the surroundings or consumed in the diet. Autotrophic green plants, algae and cyanobacteria make their own sugars through photosynthesis by using light energy, and then use them as sources of energy to fuel cellular activities.

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 Supported by the Spanish Ministry of Education and Science, No. SAF2010-15896, No. SAF2013-43284-P and No. SAF2017-82261-P

International audience; Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

We thank the Scottish Goverment’s Rural and Environment Science and Ana-lytical Services Division (RESAS) for funding this research. Also many thanks to Rafael Munoz-Tamayo for sharing his matlab code for the rumen model

A novel lanC-like sequence was identified from the dominant human gut bacterium Blautia obeum strain A2-162. This sequence was extended to reveal a putative lantibiotic operon with biosynthetic and transport genes, two sets of regulatory genes, immunity genes, three identical copies of a nisin-like lanA gene with an unusual leader peptide, and a fourth putative lanA gene. Comparison with other nisin clusters showed that the closest relationship was to nisin U. B. obeum A2-162 demonstrated antimicrobial activity against Clostridium perfringens when grown on solid medium in the presence of trypsin. Fusions of predicted nsoA structural sequences with the nisin A leader were expressed in Lactococcus lactis containing the nisin A operon without nisA. Expression of the nisA leader sequence fused to the predicted structural nsoA1 produced a growth defect in L. lactis that was dependent upon the presence of biosynthetic genes, but failed to produce antimicrobial activity. Insertion of the nso cluster into L. lactis MG1614 gave an increased immunity to nisin A, but this was not replicated by the expression of nsoI. Nisin A induction of L. lactis containing the nso cluster and nisRK genes allowed detection of the NsoA1 pre-peptide by Western hybridization. When this heterologous producer was grown with nisin induction on solid medium, antimicrobial activity was demonstrated in the presence of trypsin against C. perfringens, Clostridium difficile and L. lactis. This research adds to evidence that lantibiotic production may be an important trait of gut bacteria and could lead to the development of novel treatments for intestinal diseases.

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.

A clone encoding carboxymethyl cellulase activity was isolated during functional screening of a human gut metagenomic library using Lactococcus lactis MG1363 as heterologous host. The insert carried a glycoside hydrolase family 9 (GH9) catalytic domain with sequence similarity to a gene from Coprococcus eutactus ART55/1. Genome surveys indicated a limited distribution of GH9 domains among dominant human colonic anaerobes. Genomes of C. eutactus-related strains harboured two GH9-encoding and four GH5-encoding genes, but the strains did not appear to degrade cellulose. Instead, they grew well on β-glucans and one of the strains also grew on galactomannan, galactan, glucomannan and starch. Coprococcus comes and Coprococcus catus strains did not harbour GH9 genes and were not able to grow on β-glucans. Gene expression and proteomic analysis of C. eutactus ART55/1 grown on cellobiose, β-glucan and lichenan revealed similar changes in expression in comparison to glucose. On β-glucan and lichenan only, one of the four GH5 genes was strongly upregulated. Growth on glucomannan led to a transcriptional response of many genes, in particular a strong upregulation of glycoside hydrolases involved in mannan degradation. Thus, β-glucans are a major growth substrate for species related to C. eutactus, with glucomannan and galactans alternative substrates for some strains.

Commensal butyrate-producing bacteria in the Firmicutes phylum are abundant in the human intestine and are important for maintaining health. However, understanding of the metabolism and host interaction of these bacteria is limited by the lack of genetic modification techniques. Here we establish a protocol enabling the transfer of autonomously-replicating shuttle vectors by conjugative plasmid transfer from an Escherichia coli donor into representatives of an important sub-group of strictly anaerobic human colonic Firmicutes. Five different plasmid shuttle vectors were tested, each carrying a different origin of replication from Gram-positive bacteria. Plasmid pMTL83151 (pCB102 replicon) were successfully transferred into two strains of Eubacterium rectale, while pMTL83151 and pMTL82151 (pBP1 replicon) were transferred into Roseburia inulinivorans A2-194. Plasmids that carried a Streptococcus bovis JB1 glycoside hydrolase family 16 β-(1,3-1,4)-glucanase gene were constructed and conjugated into Roseburia inulinivorans A2-194 and Eubacterium rectale T1-815, resulting in successful heterologous expression of this introduced enzymatic activity in these two strains of butyrate-producing Firmicutes.

Dietary and host glycans shape the composition of the human gut microbiota with keystone carbohydrate-degrading species playing a critical role in maintaining the structure and function of gut microbial communities. Here, we focused on two major human gut symbionts, the mucin-degrader Ruminococcus gnavus ATCC 29149, and R. bromii L2-63, a keystone species for the degradation of resistant starch (RS) in human colon. Using anaerobic individual and co-cultures of R. bromii and R. gnavus grown on mucin or starch as sole carbon source, we showed that starch degradation by R. bromii supported the growth of R. gnavus whereas R. bromii did not benefit from mucin degradation by R. gnavus. Further we analyzed the growth (quantitative PCR), metabolite production (1H NMR analysis), and bacterial transcriptional response (RNA-Seq) of R. bromii cultured with RS or soluble starch (SS) in the presence or absence of R. gnavus. In co-culture fermentations on starch, 1H NMR analysis showed that R. gnavus benefits from transient glucose and malto-oligosaccharides released by R. bromii upon starch degradation, producing acetate, formate, and lactate as main fermentation end-products. Differential expression analysis (DESeq 2) on starch (SS and RS) showed that the presence of R. bromii induced changes in R. gnavus transcriptional response of genes encoding several maltose transporters and enzymes involved in its metabolism such as maltose phosphorylase, in line with the ability of R. gnavus to utilize R. bromii starch degradation products. In the RS co-culture, R. bromii showed a significant increase in the induction of tryptophan (Trp) biosynthesis genes and a decrease of vitamin B12 (VitB12)-dependent methionine biosynthesis as compared to the mono-culture, suggesting that Trp and VitB12 availability become limited in the presence of R. gnavus. Together this study showed a direct competition between R. bromii and R. gnavus on RS, suggesting that in vivo, the R. gnavus population inhabiting the mucus niche may be modulated by the supply of non-digestible carbohydrates reaching the colon such as RS.

Ruminococcus champanellensis is a keystone species in the human gut that produces an intricate cellulosome system of various architectures. A variety of cellulosomal enzymes have been identified, which exhibit a range of hydrolytic activities on lignocellulosic substrates. We describe herein a unique R. champanellensis scaffoldin, ScaK, which is expressed during growth on cellobiose and comprises a cohesin module and a family 25 glycoside hydrolase (GH25). The GH25 is non-autolytic and exhibits lysozyme-mediated lytic activity against several bacterial species. Despite the narrow acidic pH curve, the enzyme is active along a temperature range from 2 to 85°C and is stable at very high temperatures for extended incubation periods. The ScaK cohesin was shown to bind selectively to the dockerin of a monovalent scaffoldin (ScaG), thus enabling formation of a cell-free cellulosome, whereby ScaG interacts with a divalent scaffodin (ScaA) that bears the enzymes either directly or through additional monovalent scaffoldins (ScaC and ScaD). The ScaK cohesin also interacts with the dockerin of a protein comprising multiple Fn3 domains that can potentially promote adhesion to carbohydrates and the bacterial cell surface. A cell-free cellulosomal GH25 lysozyme may provide a bacterial strategy to both hydrolyze lignocellulose and repel eventual food competitors and/or cheaters.

Faecalibacterium prausnitzii depletion in intestinal diseases has been extensively reported, but little is known about intraspecies variability. This work aims to determine if subjects with gastrointestinal disease host mucosa-associated F. prausnitzii populations different from those hosted by healthy individuals. A new species-specific PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method targeting the 16S rRNA gene was developed to fingerprint F. prausnitzii populations in biopsy specimens from 31 healthy control (H) subjects and 36 Crohn's disease (CD), 23 ulcerative colitis (UC), 6 irritable bowel syndrome (IBS), and 22 colorectal cancer (CRC) patients. The richness of F. prausnitzii subtypes was lower in inflammatory bowel disease (IBD) patients than in H subjects. The most prevalent operational taxonomic units (OTUs) consisted of four phylotypes (OTUs with a 99% 16S rRNA gene sequence similarity [OTU99]), which were shared by all groups of patients. Their distribution and the presence of some disease-specific F. prausnitzii phylotypes allowed us to differentiate the populations in IBD and CRC patients from that in H subjects. At the level of a minimum similarity of 97% (OTU97), two phylogroups accounted for 98% of the sequences. Phylogroup I was found in 87% of H subjects but in under 50% of IBD patients ( P = 0.003). In contrast, phylogroup II was detected in >75% of IBD patients and in only 52% of H subjects ( P = 0.005). This study reveals that even though the main members of the F. prausnitzii population are present in both H subjects and individuals with gut diseases, richness is reduced in the latter and an altered phylotype distribution exists between diseases. This approach may serve as a basis for addressing the suitability of F. prausnitzii phylotypes to be quantified as a putative biomarker of disease and depicting the importance of the loss of these subtypes in disease pathogenesis.

Background: Faecalibacterium prausnitzii comprises 2 phylogroups, whose abundance in healthy and diseased gut and in conjunction with Escherichia coli has not yet been studied. This work aims to determine the contribution of F. prausnitzii phylogroups I and II in intestinal disease and to assess their potential diagnostic usefulness as biomarkers for gut diseases. Methods: Total F. prausnitzii, its phylogroups, and E. coli loads were determined by quantitative polymerase chain reaction targeting the 16S rRNA gene on biopsies from 31 healthy controls (H), 45 patients with Crohn's disease (CD), 25 patients with ulcerative colitis, 10 patients with irritable bowel syndrome, and 20 patients with colorectal cancer. Data were normalized to total bacterial counts and analyzed according to patients' disease location and clinical characteristics. Results: Lower levels of both total F. prausnitzii and phylogroup I were found in subjects with CD, ulcerative colitis, and colorectal cancer (P < 0.001) compared with H subjects. Phylogroup I load was a better biomarker than total F. prausnitzii to discriminate subjects with gut disorders from H. Phylogroup II depletion was observed only in patients with CD (P < 0.001) and can be potentially applied to differentiate ulcerative pancolitis from colonic CD. No statistically significant correlation between E. coli and any of the 2 F. prausnitzii phylogroups was found in any group of patients or by inflammatory bowel disease location. Phylogroup I was lower in active patients with CD, whereas those CD with intestinal resection showed a reduction in phylogroup II. Treatments with mesalazine and immunosuppressants did not result in the recovery of F. prausnitzii phylogroups abundance. Conclusions: F. prausnitzii phylogroup I was depleted in CD, ulcerative colitis, and colorectal cancer, whereas phylogroup II was specifically reduced in CD. Quantification of F. prausnitzii phylogroups and E. coli may help to identify gut disorders and to classify inflammatory bowel disease location.

Dietary fibers (DF) can prevent obesity in rodents fed a high-fat diet (HFD). Their mode of action is not fully elucidated, but the gut microbiota have been implicated. This study aimed to identify the effects of seven dietary fibers (barley beta-glucan, apple pectin, inulin, inulin acetate ester, inulin propionate ester, inulin butyrate ester or a combination of inulin propionate ester and inulin butyrate ester) effective in preventing diet-induced obesity and links to differences in cecal bacteria and host gene expression. Mice (n = 12) were fed either a low-fat diet (LFD), HFD or a HFD supplemented with the DFs, barley beta-glucan, apple pectin, inulin, inulin acetate ester, inulin propionate ester, inulin butyrate ester or a combination of inulin propionate ester and inulin butyrate ester for 8 weeks. Cecal bacteria were determined by Illumina MiSeq sequencing of 16S rRNA gene amplicons. Host responses, body composition, metabolic markers and gene transcription (cecum and liver) were assessed post intervention. HFD mice showed increased adiposity, while all of the DFs prevented weight gain. DF specific differences in cecal bacteria were observed. Results indicate that diverse DFs prevent weight gain on a HFD, despite giving rise to different cecal bacteria profiles. Conversely, common host responses to dietary fiber observed are predicted to be important in improving barrier function and genome stability in the gut, maintaining energy homeostasis and reducing HFD induced inflammatory responses in the liver.

The diet provides carbohydrates that are non-digestible in the upper gut and are major carbon and energy sources for the microbial community in the lower intestine, supporting a complex metabolic network. Fermentation produces the short-chain fatty acids (SCFAs) acetate, propionate and butyrate, which have health-promoting effects for the human host. Here we investigated microbial community changes and SCFA production during in vitro batch incubations of 15 different non-digestible carbohydrates, at two initial pH values with faecal microbiota from three different human donors. To investigate temporal stability and reproducibility, a further experiment was performed 1 year later with four of the carbohydrates. The lower pH (5.5) led to higher butyrate and the higher pH (6.5) to more propionate production. The strongest propionigenic effect was found with rhamnose, followed by galactomannans, whereas fructans and several α- and β-glucans led to higher butyrate production. 16S ribosomal RNA gene-based quantitative PCR analysis of 22 different microbial groups together with 454 sequencing revealed significant stimulation of specific bacteria in response to particular carbohydrates. Some changes were ascribed to metabolite cross-feeding, for example, utilisation by Eubacterium hallii of 1,2-propanediol produced from fermentation of rhamnose by Blautia spp. Despite marked inter-individual differences in microbiota composition, SCFA production was surprisingly reproducible for different carbohydrates, indicating a level of functional redundancy. Interestingly, butyrate formation was influenced not only by the overall % butyrate-producing bacteria in the community but also by the initial pH, consistent with a pH-dependent shift in the stoichiometry of butyrate production.

Ruminococcus champanellensis is considered a keystone species in the human gut that degrades microcrystalline cellulose efficiently and contains the genetic elements necessary for cellulosome production. The basic elements of its cellulosome architecture, mainly cohesin and dockerin modules from scaffoldins and enzyme-borne dockerins, have been characterized recently. In this study, we cloned, expressed and characterized all of the glycoside hydrolases that contain a dockerin module. Among the 25 enzymes, 10 cellulases, 4 xylanases, 3 mannanases, 2 xyloglucanases, 2 arabinofuranosidases, 2 arabinanases and one β-glucanase were assessed for their comparative enzymatic activity on their respective substrates. The dockerin specificities of the enzymes were examined by ELISA, and 80 positives out of 525 possible interactions were detected. Our analysis reveals a fine-tuned system for cohesin-dockerin specificity and the importance of diversity among the cohesin-dockerin sequences. Our results imply that cohesin-dockerin pairs are not necessarily assembled at random among the same specificity types, as generally believed for other cellulosome-producing bacteria, but reveal a more organized cellulosome architecture. Moreover, our results highlight the importance of the cellulosome paradigm for cellulose and hemicellulose degradation by R. champanellensis in the human gut.

A cellulolytic fiber-degrading bacterium, Ruminococcus champanellensis, was isolated from human faecal samples, and its genome was recently sequenced. Bioinformatic analysis of the R. champanellensis genome revealed numerous cohesin and dockerin modules, the basic elements of the cellulosome, and manual sequencing of partially sequenced genomic segments revealed two large tandem scaffoldin-coding genes that form part of a gene cluster. Representative R. champanellensis dockerins were tested against putative cohesins, and the results revealed three different cohesin-dockerin binding profiles which implied two major types of cellulosome architectures: (i) an intricate cell-bound system and (ii) a simplistic cell-free system composed of a single cohesin-containing scaffoldin. The cell-bound system can adopt various enzymatic architectures, ranging from a single enzyme to a large enzymatic complex comprising up to 11 enzymes. The variety of cellulosomal components together with adaptor proteins may infer a very tight regulation of its components. The cellulosome system of the human gut bacterium R. champanellensis closely resembles that of the bovine rumen bacterium Ruminococcus flavefaciens. The two species contain orthologous gene clusters comprising fundamental components of cellulosome architecture. Since R. champanellensis is the only human colonic bacterium known to degrade crystalline cellulose, it may thus represent a keystone species in the human gut.

Dietary plant cell wall carbohydrates are important in modulating the composition and metabolism of the complex gut microbiota, which can impact on health. Pectin is a major component of plant cell walls. Based on studies in model systems and available bacterial isolates and genomes, the capacity to utilize pectins for growth is widespread among colonic Bacteroidetes but relatively uncommon among Firmicutes. One Firmicutes species promoted by pectin is Eubacterium eligens. E. eligens DSM3376 utilizes apple pectin and encodes a broad repertoire of pectinolytic enzymes, including a highly abundant pectate lyase of around 200 kDa that is expressed constitutively. We confirmed that certain Faecalibacterium prausnitzii strains possess some ability to utilize apple pectin and report here that F. prausnitzii strains in common with E. eligens, can utilize the galacturonide oligosaccharides DP4 and DP5 derived from sugar beet pectin. F. prausnitzii strains have been shown previously to exert anti-inflammatory effects on host cells, but we show here for the first time that E. eligens strongly promotes the production of the anti-inflammatory cytokine IL-10 in in vitro cell-based assays. These findings suggest the potential to explore further the prebiotic potential of pectin and its derivatives to re-balance the microbiota towards an anti-inflammatory profile.

Protein-protein interactions play a vital role in cellular processes as exemplified by assembly of the intricate multi-enzyme cellulosome complex. Cellulosomes are assembled by selective high-affinity binding of enzyme-borne dockerin modules to repeated cohesin modules of structural proteins termed scaffoldins. Recent sequencing of the fiber-degrading Ruminococcus flavefaciens FD-1 genome revealed a particularly elaborate cellulosome system. In total, 223 dockerin-bearing ORFs potentially involved in cellulosome assembly and a variety of multi-modular scaffoldins were identified, and the dockerins were classified into six major groups. Here, extensive screening employing three complementary medium- to high-throughput platforms was used to characterize the different cohesin-dockerin specificities. The platforms included (i) cellulose-coated microarray assay, (ii) enzyme-linked immunosorbent assay (ELISA) and (iii) in-vivo co-expression and screening in Escherichia coli. The data revealed a collection of unique cohesin-dockerin interactions and support the functional relevance of dockerin classification into groups. In contrast to observations reported previously, a dual-binding mode is involved in cellulosome cell-surface attachment, whereas single-binding interactions operate for cellulosome integration of enzymes. This sui generis cellulosome model enhances our understanding of the mechanisms governing the remarkable ability of R. flavefaciens to degrade carbohydrates in the bovine rumen and provides a basis for constructing efficient nano-machines applied to biological processes.

What we eat influences the species composition of our gut microbiota. This is not only because diet composition determines the supply of substrates for microbial growth (in the form of dietary residue, mainly fibre, that reaches the large intestine) but also because of impacts on gut transit and the gut environment. In turn the metabolic activities of the gut microbiota, which have important health consequences, are influenced by diet and diet-driven changes in microbiota composition. Better understanding of the metabolic capabilities and host-interactions of dominant members of the gut microbiota will aid our ability to improve human health through diet.

We present here a first attempt at modelling microbial dynamics in the human colon incorporating both uncertainty and adaptation. This is based on the development of a Monod-equation based, differential equation model, which produces computer simulations of the population dynamics and major metabolites of microbial communities from the human colon. To reduce the complexity of the system, we divide the bacterial community into 10 bacterial functional groups (BFGs) each distinguished by its substrate preferences, metabolic pathways and its preferred pH range. The model simulates the growth of a large number of bacterial strains and incorporates variation in microbiota composition between people, while also allowing succession and enabling adaptation to environmental changes. The model is shown to reproduce many of the observed changes in major phylogenetic groups and key metabolites such as butyrate, acetate and propionate in response to a one unit pH shift in experimental continuous flow fermentors inoculated with human faecal microbiota. Nevertheless, it should be regarded as a learning tool to be updated as our knowledge of bacterial groups and their interactions expands. Given the difficulty of accessing the colon, modelling can play an extremely important role in interpreting experimental data and predicting the consequences of dietary modulation.

The gut microbiota and its metabolic products interact with the host in many different ways, influencing gut homoeostasis and health outcomes. The species composition of the gut microbiota has been shown to respond to dietary change, determined by competition for substrates and by tolerance of gut conditions. Meanwhile, the metabolic outputs of the microbiota, such as SCFA, are influenced both by the supply of dietary components and via diet-mediated changes in microbiota composition. There has been significant progress in identifying the phylogenetic distribution of pathways responsible for formation of particular metabolites among human colonic bacteria, based on combining cultural microbiology and sequence-based approaches. Formation of butyrate and propionate from hexose sugars, for example, can be ascribed to different bacterial groups, although propionate can be formed via alternative pathways from deoxy-sugars and from lactate by a few species. Lactate, which is produced by many gut bacteria in pure culture, can also be utilised by certain Firmicutes to form butyrate, and its consumption may be important for maintaining a stable community. Predicting the impact of diet upon such a complex and interactive system as the human gut microbiota not only requires more information on the component groups involved but, increasingly, the integration of such information through modelling approaches.