Studying the Human Gut Microbiota in the Trans-Omics Era - Focus on Metagenomics and Metabonomics

The human gut microbiota comprises a diverse microbial consortium closely co-evolved with the human genome and diet. The importance of the gut microbiota in regulating human health and disease has however been largely overlooked due to the inaccessibility of the intestinal habitat, the complexity of the gut microbiota itself and the fact that many of its members resist cultivation and are in fact new to science. However, with the emergence of 16S rRNA molecular tools and "post-genomics" high resolution technologies for examining microorganisms as they occur in nature without the need for prior laboratory culture, this limited view of the gut microbiota is rapidly changing. This review will discuss the application of molecular microbiological tools to study the human gut microbiota in a culture independent manner. Genomics or metagenomics approaches have a tremendous capability to generate compositional data and to measure the metabolic potential encoded by the combined genomes of the gut microbiota. Another post-genomics approach, metabonomics, has the capacity to measure the metabolic kinetic or flux of metabolites through an ecosystem at a particular point in time or over a time course. Metabonomics thus derives data on the function of the gut microbiota in situ and how it responds to different environmental stimuli e. g. substrates like prebiotics, antibiotics and other drugs and in response to disease. Recently these two culture independent, high resolution approaches have been combined into a single "transgenomic" approach which allows correlation of changes in metabolite profiles within human biofluids with microbiota compositional metagenomic data. Such approaches are providing novel insight into the composition, function and evolution of our gut microbiota.

Metabolism of Maillard reaction products by the human gut microbiota - implications for health

The human colonic microbiota imparts metabolic versatility on the colon, interacts at many levels in healthy intestinal and systemic metabolism, and plays protective roles in chronic disease and acute infection. Colonic bacterial metabolism is largely dependant on dietary residues from the upper gut. Carbohydrates, resistant to digestion, drive colonic bacterial fermentation and the resulting end products are considered beneficial. Many colonic species ferment proteins but the end products are not always beneficial and include toxic compounds, such as amines and phenols. Most components of a typical Western diet are heat processed. The Maillard reaction, involving food protein and sugar, is a complex network of reactions occurring during thermal processing. The resultant modified protein resists digestion in the small intestine but is available for colonic bacterial fermentation. Little is known about the fate of the modified protein but some Maillard reaction products (MRP) are biologically active by, e.g. altering bacterial population levels within the colon or, upon absorption, interacting with human disease mechanisms by induction of inflammatory responses. This review presents current understanding of the interactions between MRP and intestinal bacteria. Recent scientific advances offering the possibility of elucidating the consequences of microbe-MRP interactions within the gut are discussed.

Metabolism of Maillard reaction products by the human gut microbiota: implications for health

The human colonic microbiota imparts metabolic versatility on the colon, interacts at many levels in healthy intestinal and systemic metabolism, and plays protective roles in chronic disease and acute infection. Colonic bacterial metabolism is largely dependant on dietary residues from the upper gut. Carbohydrates, resistant to digestion, drive colonic bacterial fermentation and the resulting end products are considered beneficial. Many colonic species ferment proteins but the end products are not always beneficial and include toxic compounds, such as amines and phenols. Most components of a typical Western diet are heat processed. The Maillard reaction, involving food protein and sugar, is a complex network of reactions occurring during thermal processing. The resultant modified protein resists digestion in the small intestine but is available for colonic bacterial fermentation. Little is known about the fate of the modified protein but some Maillard reaction products (MRP) are biologically active by, e.g. altering bacterial population levels within the colon or, upon absorption, interacting with human disease mechanisms by induction of inflammatory responses. This review presents current understanding of the interactions between MRP and intestinal bacteria. Recent scientific advances offering the possibility of elucidating the consequences of microbe-MRP interactions within the gut are discussed.

Studying the Human Gut Microbiota in the Trans-Omics Era - Focus on Metagenomics and Metabonomics

The human gut microbiota comprises a diverse microbial consortium closely co-evolved with the human genome and diet. The importance of the gut microbiota in regulating human health and disease has however been largely overlooked due to the inaccessibility of the intestinal habitat, the complexity of the gut microbiota itself and the fact that many of its members resist cultivation and are in fact new to science. However, with the emergence of 16S rRNA molecular tools and "post-genomics" high resolution technologies for examining microorganisms as they occur in nature without the need for prior laboratory culture, this limited view of the gut microbiota is rapidly changing. This review will discuss the application of molecular microbiological tools to study the human gut microbiota in a culture independent manner. Genomics or metagenomics approaches have a tremendous capability to generate compositional data and to measure the metabolic potential encoded by the combined genomes of the gut microbiota. Another post-genomics approach, metabonomics, has the capacity to measure the metabolic kinetic or flux of metabolites through an ecosystem at a particular point in time or over a time course. Metabonomics thus derives data on the function of the gut microbiota in situ and how it responds to different environmental stimuli e.g. substrates like prebiotics, antibiotics and other drugs and in response to disease. Recently these two culture independent, high resolution approaches have been combined into a single "transgenomic" approach which allows correlation of changes in metabolite profiles within human biofluids with microbiota compositional metagenomic data. Such approaches are providing novel insight into the composition, function and evolution of our gut microbiota.

In vitro studies on colonization resistance of the human gut microbiota to Candida albicans and the effects of tetracycline and Lactobacillus plantarum LPK

An anaerobic three-vessel continuous-flow culture system, which models the three major anatomical regions of the human colon, was used to study the persistence of Candida albicans in the presence of a faecal microbiota. During steady state conditions, overgrowth of C. albicans was prevented by commensal bacteria indigenous to the system. However antibiotics, such as tetracycline have the ability to disrupt the bacterial populations within the gut. Thus, colonization resistance can be compromised and overgrowth of undesirable microorganisms like C. albicans can then occur. In this study, growth of C. albicans was not observed in the presence of an established faecal microbiota. However, following the addition of tetracycline to the growth medium, significant growth of C. albicans occurred. A probiotic Lactobacillus plantarum LPK culture was added to the system to investigate whether this organism had any effects upon the Candida populations. Although C. albicans was not completely eradicated in the presence of this bacterium, cell counts were markedly reduced, indicating a compromised physiological function. This study shows that the normal gut flora can exert 'natural' resistance to C. albicans, however this may be diminished during antibiotic intake. The use of probiotics can help fortify natural resistance.

Whole-grain wheat breakfast cereal has a prebiotic effect on the human gut microbiota: a double-blind, placebo-controlled, crossover study

Epidemiological studies have shown an inverse association between dietary intake of whole grains and the risk of chronic disease. This may be related to the ability to mediate a prebiotic modulation of gut microbiota. However, no studies have been conducted on the microbiota modulatory capability of whole-grain (WG) cereals. In the present study, the impact of WG wheat on the human intestinal microbiota compared to wheat bran (WB) was determined. A double-blind, randomised, crossover study was carried out in thirty-one volunteers who were randomised into two groups and consumed daily 48g breakfast cereals, either WG or WB, in two 3-week study periods, separated by a 2-week washout period. Numbers of faecal bifidobacteria and lactobacilli (the target genera for prebiotic intake), were significantly higher upon WG ingestion compared with WB. Ingestion of both breakfast cereals resulted in a significant increase in ferulic acid concentrations in blood but no discernible difference in faeces or urine. No significant differences in faecal SCFA, fasting blood glucose, insulin, total cholesterol (TC), TAG or HDL-cholesterol were observed upon ingestion of WG compared with WB. However, a significant reduction in TC was observed in volunteers in the top quartile of TC concentrations upon ingestion of either cereal. No adverse intestinal symptoms were reported and WB ingestion increased stool frequency. Daily consumption of WG wheat exerted a pronounced prebiotic effect on the human gut microbiota composition. This prebiotic activity may contribute towards the beneficial physiological effects of WG wheat.

Use of a continuous culture fermentation system to investigate the effect of GanedenBC30 (Bacillus coagulans GBI-30, 6086) supplementation on pathogen survival in the human gut microbiota

Single-stage continuous fermentation systems were employed to examine the effects of GanedenBC30 supplementation on the human gastrointestinal microbiota in relation to pathogen challenge in vitro. Denaturing gradient gel electrophoresis analysis demonstrated that GanedenBC30 supplementation modified the microbial profiles in the fermentation systems compared with controls, with profiles clustering according to treatment. Overall, GanedenBC30 supplementation did not elicit major changes in bacterial population counts in vitro, although notably higher Bcoa191 counts were seen following probiotic supplementation (compared to the controls). Pathogen challenge did not elicit significant modification of the microbial counts in vitro, although notably higher Clit135 counts were seen in the control system post-Clostridium difficile challenge than in the corresponding GanedenBC30-supplemented systems. Sporulation appears to be associated with the anti-microbial activity of GanedenBC30, suggesting that a bi-modal lifecycle of GanedenBC30 in vivo may lead to anti-microbial activity in distal regions of the gastrointestinal tract.

In vitro fermentation of a red wine extract by human gut microbiota: changes in microbial groups and formation of phenolic metabolites

An in vitro batch culture fermentation experiment was conducted with fecal inocula from three healthy volunteers in the presence and absence of a red wine extract. Changes in main bacterial groups were determined by FISH during a 48 h fermentation period. The catabolism of main flavonoids (i.e., flavan-3-ols and anthocyanins) and the formation of a wide a range of phenolic microbial metabolites were determined by a targeted UPLC-PAD-ESI-TQ MS method. Statistical analysis revealed that catechol/pyrocatechol, as well as 4-hydroxy-5-(phenyl)-valeric, 3- and 4-hydroxyphenylacetic, phenylacetic, phenylpropionic, and benzoic acids, showed the greatest increases in concentration during fermentation, whereas 5-(3′-hydroxyphenyl)-γ-valerolactone, its open form 4-hydroxy-5-(3′-hydroxyphenyl)-valeric acid, and 3,4-dihydroxyphenylacetic acid represented the largest interindividual variations in the catabolism of red wine polyphenols. Despite these changes, microbial catabolism did not produce significant changes in the main bacterial groups detected, although a slight inhibition of the Clostridium histolyticum group was observed.

Effect of prebiotics on the human gut microbiota of elderly persons

The colonic microbiota undergoes certain age related changes that may affect health. For example, above the age of 55–65 y, populations of bifidobacteria are known to decrease markedly. Bifidobacteria are known inhibitors of pathogenic microbes and a decrease in their activities may increase susceptibility to infections. There is therefore interest in trying to reverse their decline in aged persons. As the gut microbiota responds to dietary intervention, both probiotics and prebiotics have been tested in this regard. Probiotics are live microbes in the diet, whereas prebiotics are fermentable ingredients that specifically target components of the indigenous microbiota seen to be beneficial. We have published a recent paper demonstrating that prebiotic galactooligosaccharides can exert power effects upon bifidobacteria in the gut flora of elderly persons (both in vivo and in vitro). This addendum summarizes research that led up to this study and discusses the possible impact of prebiotics in impacting upon the gut health of aged persons.

Human gut microbiota and its relationship to health and disease

Probiotics are live microorganisms that confer a health benefit on the host when administered in appropriate amounts. Over 700 randomized, controlled, human studies have been conducted with probiotics thus far, with the results providing strong support for the use of probiotics in the clinical prevention or treatment of gastrointestinal tract disorders and metabolic syndrome. The present review is based on webinar presentations that were developed by the American Gastroenterological Association (AGA) in partnership with the International Scientific Association for Probiotics and Prebiotics (ISAPP) and the North American branch of the International Life Sciences Institute (ILSI North America). The presentations provided gastroenterologists and researchers with fundamental and current scientific information on the influence of gut microbiota on human health and disease, as well as clinical intervention strategies and practical guidelines for the use of probiotics and prebiotics.

Prebiotic potential of a maize-based soluble fibre and impact of dose on the human gut microbiota

Dietary management of the human gut microbiota towards a more beneficial composition is one approach that may improve host health. To date, a large number of human intervention studies have demonstrated that dietary consumption of certain food products can result in significant changes in the composition of the gut microbiota i.e. the prebiotic concept. Thus the prebiotic effect is now established as a dietary approach to increase beneficial gut bacteria and it has been associated with modulation of health biomarkers and modulation of the immune system. Promitor™ Soluble Corn Fibre (SCF) is a well-known maize-derived source of dietary fibre with potential selective fermentation properties. Our aim was to determine the optimum prebiotic dose of tolerance, desired changes to microbiota and fermentation of SCF in healthy adult subjects. A double-blind, randomised, parallel study was completed where volunteers (n = 8/treatment group) consumed 8, 14 or 21 g from SCF (6, 12 and 18 g/fibre delivered respectively) over 14-d. Over the range of doses studied, SCF was well tolerated Numbers of bifidobacteria were significantly higher for the 6 g/fibre/day compared to 12g and 18g/fibre delivered/day (mean 9.25 and 9.73 Log10 cells/g fresh faeces in the pre-treatment and treatment periods respectively). Such a numerical change of 0.5 Log10 bifidobacteria/g fresh faeces is consistent with those changes observed for inulin-type fructans, which are recognised prebiotics. A possible prebiotic effect of SCF was therefore demonstrated by its stimulation of bifidobacteria numbers in the overall gut microbiota during a short-term intervention.

Impact of increasing fruit and vegetables and flavonoid intake on the human gut microbiota

Epidemiological studies have shown protective effects of fruits and vegetables (F&V) in lowering the risk of developing cardiovascular diseases (CVD) and cancers. Plant-derived dietary fibre (non-digestible polysaccharides) and/or flavonoids may mediate the observed protective effects particularly through their interaction with the gut microbiota. The aim of this study was to assess the impact of fruit and vegetable (F&V) intake on gut microbiota, with an emphasis on the role of flavonoids, and further to explore relationships between microbiota and factors associated with CVD risk. In the study, a parallel design with 3 study groups, participants in the two intervention groups representing high-flavonoid (HF) and low flavonoid (LF) intakes were asked to increase their daily F&V intake by 2, 4 and 6 portions for a duration of 6 weeks each, while a third (control) group continued with their habitual diet. Faecal samples were collected at baseline and after each dose from 122 subjects. Faecal bacteria enumeration was performed by fluorescence in situ hybridisation (FISH). Correlations of dietary components, flavonoid intake and markers of CVD with bacterial numbers were also performed. A significant dose X treatment interaction was only found for Clostidium leptum-Ruminococcus bromii/flavefaciens with a significant increase after intake of 6 additional portions in the LF group. Correlation analysis of the data from all 122 subjects independent from dietary intervention indicated an inhibitory role of F&V intake, flavonoid content and sugars against the growth of potentially pathogenic clostridia. Additionally, we observed associations between certain bacterial populations and CVD risk factors including plasma TNF-α, plasma lipids and BMI/waist circumference.

Molecular characterization of the human gut microbiota: the effect of aging

Age-related physiological changes in the gastrointestinal tract, as well as modification in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbiota. The study presented here is focused on the application and comparison of two different microarray approaches for the characterization of the human gut microbiota, the HITChip and the HTF-Microb.Array, with particular attention to the effects of the aging process on the composition of this ecosystem. By using the Human Intestinal Tract Chip (HITChip), recently developed at the Wageningen University, The Netherland, we explored the age-related changes of gut microbiota during the whole adult lifespan, from young adults, through elderly to centenarians. We observed that the microbial composition and diversity of the gut ecosystem of young adults and seventy-years old people is highly similar but differs significantly from that of the centenarians. After 100 years of symbiotic association with the human host, the microbiota is characterized by a rearrangement in the Firmicutes population and an enrichment of facultative anaerobes. The presence of such a compromised microbiota in the centenarians is associated with an increased inflammation status, also known as inflamm-aging, as determined by a range of peripheral blood inflammatory markers. In parallel, we overtook the development of our own phylogenetic microarray with a lower number of targets, aiming the description of the human gut microbiota structure at high taxonomic level. The resulting chip was called High Taxonomic level Fingerprinting Microbiota Array (HTF-Microb.Array), and was based on the Ligase Detection Reaction (LDR) technology, which allowed us to develop a fast and sensitive tool for the fingerprint of the human gut microbiota in terms of presence/absence of the principal groups. The validation on artificial DNA mixes, as well as the pilot study involving eight healthy young adults, demonstrated that the HTF-Microb.Array can be used to successfully characterize the human gut microbiota, allowing us to obtain results which are in approximate accordance with the most recent characterizations. Conversely, the evaluation of the relative abundance of the target groups on the bases of the relative fluorescence intensity probes response still has some hindrances, as demonstrated by comparing the HTF.Microb.Array and HITChip high taxonomic level fingerprints of the same centenarians.