Nicole Reichardt

Phylogenetic distribution of three pathways for propionate production within the human gut microbiota

Propionate is produced in the human large intestine by microbial fermentation and may help maintain human health. We have examined the distribution of three different pathways used by bacteria for propionate formation using genomic and metagenomic analysis of the human gut microbiota and by designing degenerate primer sets for the detection of diagnostic genes for these pathways. Degenerate primers for the acrylate pathway (detecting the lcdA gene, encoding lactoyl-CoA dehydratase) together with metagenomic mining revealed that this pathway is restricted to only a few human colonic species within the Lachnospiraceae and Negativicutes. The operation of this pathway for lactate utilisation in Coprococcus catus (Lachnospiraceae) was confirmed using stable isotope labelling. The propanediol pathway that processes deoxy sugars such as fucose and rhamnose was more abundant within the Lachnospiraceae (based on the pduP gene, which encodes propionaldehyde dehydrogenase), occurring in relatives of Ruminococcus obeum and in Roseburia inulinivorans. The dominant source of propionate from hexose sugars, however, was concluded to be the succinate pathway, as indicated by the widespread distribution of the mmdA gene that encodes methylmalonyl-CoA decarboxylase in the Bacteroidetes and in many Negativicutes. In general, the capacity to produce propionate or butyrate from hexose sugars resided in different species, although two species of Lachnospiraceae (C. catus and R. inulinivorans) are now known to be able to switch from butyrate to propionate production on different substrates. A better understanding of the microbial ecology of short-chain fatty acid formation may allow modulation of propionate formation by the human gut microbiota.

Characterization of high molecular weight coffee fractions and their fermentation by human intestinal microbiota

To investigate the structure and fermentability of high M(r) components of coffee brews by human gut bacteria Arabica coffee samples of three different degrees of roast (light, medium, and dark) were used for drip brew preparations and fractionation by ultrafiltration with different M(r) cut-offs. Total carbohydrates of the fractions ranged from 28.6 g/100 g to 56.7 g/100 g. Galactomannans and arabinogalactans were the main polysaccharides and made up between one-fourth and one-half of the respective coffee fraction. After 24 h of incubation with a human fecal suspension the polysaccharides of all fractions were extensively degraded. A decrease in the absorbance values at 405 and 280 nm, respectively, indicated that also chemically noncharacterized UV-active components such as Maillard reaction products, had been partially degraded or modified by the human gut bacteria. The remainder after 24 h of fermentation still showed antioxidant activity. Bacterial cells belonging to the Bacteroides-Prevotella group increased 2- to 40-fold during fermentation depending on the M(r) range of the fraction and the degree of roast. The production of high amounts of acetate and propionate is in accordance with a role of these bacteria in the degradation of high M(r) components from coffee.

Characterization and Fermentability of an Ethanol Soluble High Molecular Weight Coffee Fraction

Brews from differently roasted Arabica coffees were shown to contain 8-12% ethanol soluble substances with molecular masses greater than 2 kDa, possibly contributing to their dietary fiber contents. About 13% of these substances were nondigestible carbohydrates, mainly arabinogalactans. The nondigestible high molecular weight ethanol soluble fraction (HESF) of the medium roasted coffee brew was further characterized and subjected to in vitro fermentation with human fecal bacteria. In addition to carbohydrates, HESF contained proteins/peptides (approximately 20%), but the main fraction was composed of structurally unknown Maillard reaction products. From NMR spectroscopy, we conclude that intact caffeic and ferulic acid derivatives were not incorporated into the melanoidins to a significant extent. Stepwise ultrafiltration and gel filtration indicated a large variation in the molecular weights of HESF constituents. Coffee HESF was shown to be less fermentable by fecal bacteria than soluble coffee fiber isolated by the enzymatic-gravimetric methodology, and because of its lower carbohydrate content, less short-chain fatty acids were produced during the fermentation. Total cell counts, destructive chemical analysis, and NMR spectroscopy indicated that coffee carbohydrates are the preferred substrates for colonic microbiota. However, NMR spectra, absorbances at 405 nm, and nonprotein nitrogen contents showed that noncarbohydrate and nonprotein compounds were also utilized to some extent but the bacterial species involved in this degradation remain to be identified.

The Influence of Whole Grain Products and Red Meat on Intestinal Microbiota Composition in Normal Weight Adults: A Randomized Crossover Intervention Trial

Intestinal microbiota is related to obesity and serum lipid levels, both risk factors for chronic diseases constituting a challenge for public health. We investigated how a diet rich in whole grain (WG) products and red meat (RM) influences microbiota. During a 10-week crossover intervention study, 20 healthy adults consumed two isocaloric diets, one rich in WG products and one high in RM. Repeatedly data on microbiota were assessed by 16S rRNA based denaturing gradient gel electrophoresis (DGGE). A blood sample and anthropometric data were collected. Mixed models and logistic regression were used to investigate effects. Microbiota showed interindividual variability. However, dietary interventions modified microbiota appearance: 8 bands changed in at least 4 participants during the interventions. One of the bands appearing after WG and one increasing after RM remained significant in regression models and were identified as Collinsella aerofaciens and Clostridium sp. The WG intervention lowered obesity parameters, while the RM diet increased serum levels of uric acid and creatinine. The study showed that diet is a component of major relevance regarding its influence on intestinal microbiota and that WG has an important role for health. The results could guide investigations of diet and microbiota in observational prospective cohort studies. Trial registration ClinicalTrials.gov NCT01449383

Use of Stable Isotopes To Measure the Metabolic Activity of the Human Intestinal Microbiota

ABSTRACT The human intestinal microbiota is a complex biological system comprising a vast repertoire of microbes with considerable metabolic activity relevant to both bacterial growth and host health. Greater strides have been made in the analysis of microbial diversity than in the measurement of functional activity, particularly in vivo. Stable isotope probing offers a new approach by coupling measurements of metabolic activity with microbial identification. Using a low-enrichment labeling strategy in vitro, this study has identified metabolically active bacterial groups via magnetic-bead capture methodology and stable isotope ratio analysis. Using five probes (EUB338, Bac303, Bif164, EREC482, and Clep866), changes in the activities of key intestinal microbial groups were successfully measured by exploiting tracers of de novo RNA synthesis. Perturbation of the nutrient source with oligofructose generated changes in the activity of bifidobacteria as expected, but also in the Bacteroides-Prevotella group, the Eubacterium rectale-Clostridium coccoides group, and the Clostridium leptum subgroup. Changes in activity were also observed in response to the medium type. This study suggests that changes in the functional activity of the gut microbiota can be assessed using tracers of de novo nucleic acid synthesis combined with measurement of low isotopic enrichment in 16S rRNA. Such tracers potentially limit substrate bias because they are universally available to bacteria. This low-enrichment labeling approach does not depend on the commercial availability of specific labeled substrates and can be easily translated to in vivo probing experiments of the functional activity of the microbiota in the human gut.

Bidirectional communication between the Aryl hydrocarbon Receptor (AhR) and the microbiome tunes host metabolism

The ligand-induced transcription factor, aryl hydrocarbon receptor (AhR) is known for its capacity to tune adaptive immunity and xenobiotic metabolism—biological properties subject to regulation by the indigenous microbiome. The objective of this study was to probe the postulated microbiome-AhR crosstalk and whether such an axis could influence metabolic homeostasis of the host. Utilising a systems-biology approach combining in-depth 1H-NMR-based metabonomics (plasma, liver and skeletal muscle) with microbiome profiling (small intestine, colon and faeces) of AhR knockout (AhR−/−) and wild-type (AhR+/+) mice, we assessed AhR function in host metabolism. Microbiome metabolites such as short-chain fatty acids were found to regulate AhR and its target genes in liver and intestine. The AhR signalling pathway, in turn, was able to influence microbiome composition in the small intestine as evident from microbiota profiling of the AhR+/+ and AhR−/− mice fed with diet enriched with a specific AhR ligand or diet depleted of any known AhR ligands. The AhR−/− mice also displayed increased levels of corticosterol and alanine in serum. In addition, activation of gluconeogenic genes in the AhR−/− mice was indicative of on-going metabolic stress. Reduced levels of ketone bodies and reduced expression of genes involved in fatty acid metabolism in the liver further underscored this observation. Interestingly, exposing AhR−/− mice to a high-fat diet showed resilience to glucose intolerance. Our data suggest the existence of a bidirectional AhR-microbiome axis, which influences host metabolic pathways.

Specific substrate-driven changes in human faecal microbiota composition contrast with functional redundancy in short-chain fatty acid production

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.

Dietary fibers inhibit obesity in mice, but host responses in the cecum and liver appear unrelated to fiber-specific changes in cecal bacterial taxonomic composition

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.

Human gut bacteria as potent class I histone deacetylase inhibitors in vitro through production of butyric acid and valeric acid

Overexpression of histone deacetylase (HDAC) isoforms has been implicated in a variety of disease pathologies, from cancer and colitis to cardiovascular disease and neurodegeneration, thus HDAC inhibitors have a long history as therapeutic targets. The gut microbiota can influence HDAC activity via microbial-derived metabolites. While HDAC inhibition (HDI) by gut commensals has long been attributed to the short chain fatty acid (SCFA) butyrate, the potent metabolic reservoir provided by the gut microbiota and its role in host physiology warrants further investigation in a variety of diseases. Cell-free supernatants (CFS) of 79 phylogenetically diverse gut commensals isolated from healthy human donors were screened for their SCFA profile and their total HDAC inhibitory properties. The three most potent HDAC inhibiting strains were further evaluated and subjected to additional analysis of specific class I and class II HDAC inhibition. All three HDAC inhibitors are butyrate producing strains, and one of these also produced substantial levels of valeric acid and hexanoic acid. Valeric acid was identified as a potential contributor to the HDAC inhibitory effects. This bacterial strain, Megasphaera massiliensis MRx0029, was added to a model microbial consortium to assess its metabolic activity in interaction with a complex community. M. massiliensis MRx0029 successfully established in the consortium and enhanced the total and specific HDAC inhibitory function by increasing the capacity of the community to produce butyrate and valeric acid. We here show that single bacterial strains from the human gut microbiota have potential as novel HDI therapeutics for disease areas involving host epigenetic aberrations.