William Tottey

Archaea and the human gut: new beginning of an old story.

Methanogenic archaea are known as human gut inhabitants since more than 30 years ago through the detection of methane in the breath and isolation of two methanogenic species belonging to the order Methanobacteriales, Methanobrevibacter smithii and Methanosphaera stadtmanae. During the last decade, diversity of archaea encountered in the human gastrointestinal tract (GIT) has been extended by sequence identification and culturing of new strains. Here we provide an updated census of the archaeal diversity associated with the human GIT and their possible role in the gut physiology and health. We particularly focus on the still poorly characterized 7th order of methanogens, the Methanomassiliicoccales, associated to aged population. While also largely distributed in non-GIT environments, our actual knowledge on this novel order of methanogens has been mainly revealed through GIT inhabitants. They enlarge the number of final electron acceptors of the gut metabolites to mono- di- and trimethylamine. Trimethylamine is exclusively a microbiota-derived product of nutrients (lecithin, choline, TMAO, L-carnitine) from normal diet, from which seems originate two diseases, trimethylaminuria (or Fish-Odor Syndrome) and cardiovascular disease through the proatherogenic property of its oxidized liver-derived form. This therefore supports interest on these methanogenic species and its use as archaebiotics, a term coined from the notion of archaea-derived probiotics.

Archaebiotics: Proposed therapeutic use of archaea to prevent trimethylaminuria and cardiovascular disease

Trimethylamine (TMA) is produced by gut bacteria from dietary ingredients. In individuals with a hereditary defect in flavin-containing monooxygenase 3, bacterial TMA production is believed to contribute to the symptoms of trimethylaminuria (TMAU; fish-odor syndrome). Intestinal microbiota TMA metabolism may also modulate atherosclerosis risk by affecting trimethylamine oxide (TMAO) production levels. We propose that reducing TMA formation in the gut by converting it to an inert molecule could be used to prevent or limit these human diseases, while avoiding the major drawbacks of other clinical interventions. Reducing TMA levels by microbiological interventions could also be helpful in some vaginoses. Particular members of a recently discovered group of methanogens, that are variably present in the human gut, are unusual in being apparently restricted to utilizing only methyl compounds including TMA as substrates. We confirmed experimentally that one of these strains tested, Methanomassiliicoccus luminyensis B10, is able to deplete TMA, by reducing it with H 2 for methanogenesis. We therefore suggest that members of this archaeal lineage could be used as treatments for metabolic disorders.

The Human Gut Chip "HuGChip'', an explorative phylogenetic microarray for determining gut microbiome diversity at family level

Evaluating the composition of the human gut microbiota greatly facilitates studies on its role in human pathophysiology, and is heavily reliant on culture-independent molecular methods. A microarray designated the Human Gut Chip (HuGChip) was developed to analyze and compare human gut microbiota samples. The PhylArray software was used to design specific and sensitive probes. The DNA chip was composed of 4,441 probes (2,442 specific and 1,919 explorative probes) targeting 66 bacterial families. A mock community composed of 16S rRNA gene sequences from intestinal species was used to define the threshold criteria to be used to analyze complex samples. This was then experimentally verified with three human faecal samples and results were compared (i) with pyrosequencing of the V4 hypervariable region of the 16S rRNA gene, (ii) metagenomic data, and (iii) qPCR analysis of three phyla. When compared at both the phylum and the family level, high Pearsons correlation coefficients were obtained between data from all methods. The HuGChip development and validation showed that it is not only able to assess the known human gut microbiota but could also detect unknown species with the explorative probes to reveal the large number of bacterial sequences not yet described in the human gut microbiota, overcoming the main inconvenience encountered when developing microarrays.

Genome Sequence of "Candidatus Methanomassiliicoccus intestinalis" Issoire-Mx1, a Third Thermoplasmatales-Related Methanogenic Archaeon from Human Feces

ABSTRACT “Candidatus Methanomassiliicoccus intestinalis” Issoire-Mx1 is a methanogenic archaeon found in the human gut and is a representative of the novel order of methanogens related to Thermoplasmatales. Its complete genome sequence is presented here.

Three-stage continuous culture system with a self-generated anaerobia to study the regionalized metabolism of the human gut microbiota

The technical and ethical difficulties in studying the gut microbiota in vivo warrant the development and improvement of in vitro systems able to simulate and control the physicochemical factors of the gut biology. Moreover, the functional regionalization of this organ implies a model simulating these differences. Here we propose an improved and alternative three-stage continuous bioreactor called 3S-ECSIM (three-stage Environmental Control System for Intestinal Microbiota) to study the human large intestine. Its main feature compared with other in vitro systems is the anaerobic atmosphere originating directly from the microbiota metabolism, leading to different gas ratios of CO2 and H2 in each compartment. Analyses of the metabolic and microbiological profiles (LC-MS and a phylogenetic microarray) show different profiles together with a maintenance of this differentiation between the three compartments, simulating respectively a proximal, a transversal and a distal colon. Moreover, the last reactor presents a high similarity with the initial fecal sample, at the microbiological diversity level. Based on our results, this in-vitro process improvement is a valuable alternative tool to dynamically study the structure and metabolism of gut microbiota, and its response to nutrients, prebiotics, probiotics, drugs or xenobiotics.

Colonic Transit Time Is a Driven Force of the Gut Microbiota Composition and Metabolism: In Vitro Evidence.

Background/Aims Human gut microbiota harbors numerous metabolic properties essential for the host’s health. Increased intestinal transit time affects a part of the population and is notably observed with human aging, which also corresponds to modifications of the gut microbiota. Thus we tested the metabolic and compositional changes of a human gut microbiota induced by an increased transit time simulated in vitro. Methods The in vitro system, Environmental Control System for Intestinal Microbiota, was used to simulate the environmental conditions of 3 different anatomical parts of the human colon in a continuous process. The retention times of the chemostat conditions were established to correspond to a typical transit time of 48 hours next increased to 96 hours. The bacterial communities, short chain fatty acids and metabolite fingerprints were determined. Results Increase of transit time resulted in a decrease of biomass and of diversity in the more distal compartments. Short chain fatty acid analyses and metabolite fingerprinting revealed increased activity corresponding to carbohydrate fermentation in the proximal compartments while protein fermentations were increased in the lower parts. Conclusions This study provides the evidence that the increase of transit time, independently of other factors, affects the composition and metabolism of the gut microbiota. The transit time is one of the factors that explain some of the modifications seen in the gut microbiota of the elderly, as well as patients with slow transit time.

Whole Rye Consumption Improves Blood and Liver n-3 Fatty Acid Profile and Gut Microbiota Composition in Rats.

Background Whole rye (WR) consumption seems to be associated with beneficial health effects. Although rye fiber and polyphenols are thought to be bioactive, the mechanisms behind the health effects of WR have yet to be fully identified. This study in rats was designed to investigate whether WR can influence the metabolism of n-3 and n-6 long-chain fatty acids (LCFA) and gut microbiota composition. Methods For 12 weeks, rats were fed a diet containing either 50% WR or 50% refined rye (RR). The WR diet provided more fiber (+21%) and polyphenols (+29%) than the RR diet. Fat intake was the same in both diets and particularly involved similar amounts of essential (18-carbon) n-3 and n-6 LCFAs. Results The WR diet significantly increased the 24-hour urinary excretion of polyphenol metabolites–including enterolactone–compared with the RR diet. The WR rats had significantly more n-3 LCFA–in particular, eicosapentanoic (EPA) and docosahexanoic (DHA) acids–in their plasma and liver. Compared with the RR diet, the WR diet brought significant changes in gut microbiota composition, with increased diversity in the feces (Shannon and Simpson indices), decreased Firmicutes/Bacteroidetes ratio and decreased proportions of uncultured Clostridiales cluster IA and Clostridium cluster IV in the feces. In contrast, no difference was found between groups with regards to cecum microbiota. The WR rats had lower concentrations of total short-chain fatty acids (SCFA) in cecum and feces (p<0.05). Finally, acetate was lower (p<0.001) in the cecum of WR rats while butyrate was lower (p<0.05) in the feces of WR rats. Interpretation This study shows for the first time that WR consumption results in major biological modifications–increased plasma and liver n-3 EPA and DHA levels and improved gut microbiota profile, notably with increased diversity–known to provide health benefits. Unexpectedly, WR decreased SCFA levels in both cecum and feces. More studies are needed to understand the interactions between whole rye (fiber and polyphenols) and gut microbiota and also the mechanisms of action responsible for stimulating n-3 fatty acid metabolism.

In-vitro model for studying methanogens in human gut microbiota.

Reported failures with gnotobiotic animal models led us to establish an in-vitro model of reciprocal conversion of methanogenic and non methanogenic microbiota from human fecal samples. Consequences on gas and microbiota compositions are reported. This should facilitate the study of the controversial role of gut methanogens in human health.

Functional amplification and preservation of human gut microbiota

ABSTRACT Background: The availability of fresh stool samples is a prerequisite in most gut microbiota functional studies. Objective: Strategies for amplification and long-term gut microbiota preservation from fecal samples would favor sample sharing, help comparisons and reproducibility over time and between laboratories, and improve the safety and ethical issues surrounding fecal microbiota transplantations. Design: Taking advantage of in vitro gut-simulating systems, we amplified the microbial repertoire of a fresh fecal sample and assessed the viability and resuscitation of microbes after preservation with some common intracellular and extracellular acting cryoprotective agents (CPAs), alone and in different combinations. Preservation efficiencies were determined after 3 and 6 months and compared with the fresh initial microbiota diversity and metabolic activity, using the chemostat-based Environmental Control System for Intestinal Microbiota (ECSIM) in vitro model of the gut environment. Microbial populations were tested for fermentation gas, short-chain fatty acids, and composition of amplified and resuscitated microbiota, encompassing methanogenic archaea. Results: Amplification of the microbial repertoire from a fresh fecal sample was achieved with high fidelity. Dimethylsulfoxide, alone or mixed with other CPAs, showed the best efficiency for functional preservation, and the duration of preservation had little effect. Conclusions: The amplification and resuscitation of fecal microbiota can be performed using specialized in vitro gut models. Correct amplification of the initial microbes should ease the sharing of clinical samples and improve the safety of fecal microbiota transplantation. Abbreviations: CDI, Clostridium difficile infection; CPA, cryoprotective agent; D, DMSO, dimethylsulfoxide; FMT, fecal microbiota transplantation; G, glycerol; IBD, inflammatory bowel disease; P, PEG-4000, polyethylene glycol 4000 g.mol−1; SCFA, short-chain fatty acid; SNR, signal-to-noise ratio