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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.

Bacterial‐derived compounds from the intestinal microbiome modulate host mucosal immunity. Identification and mechanistic studies of these compounds provide insights into host–microbial mutualism. Specific Lactobacillus reuteri strains suppress production of the proinflammatory cytokine, tumor necrosis factor (TNF), and are protective in a mouse model of colitis. Human‐derived L. reuteri strain ATCC PTA 6475 suppresses intestinal inflammation and produces 5,10‐methenyltetrahydrofolic acid polyglutamates. Insertional mutagenesis identified the bifunctional dihydrofolate synthase/folylpolyglutamate synthase type 2 (folC2) gene as essential for 5,10‐methenyltetrahydrofolic acid polyglutamate biosynthesis, as well as for suppression of TNF production by activated human monocytes, and for the anti‐inflammatory effect of L. reuteri 6475 in a trinitrobenzene sulfonic acid‐induced mouse model of acute colitis. In contrast, folC encodes the enzyme responsible for folate polyglutamylation but does not impact TNF suppression by L. reuteri. Comparative transcriptomics between wild‐type and mutant L. reuteri strains revealed additional genes involved in immunomodulation, including previously identified hdc genes involved in histidine to histamine conversion. The folC2 mutant yielded diminished hdc gene cluster expression and diminished histamine production, suggesting a link between folate and histadine/histamine metabolism. The identification of genes and gene networks regulating production of bacterial‐derived immunoregulatory molecules may lead to improved anti‐inflammatory strategies for digestive diseases.

The establishment of the infant gut microbiota is a highly dynamic process dependent on extrinsic and intrinsic factors. We characterized the faecal microbiota of 4 breastfed infants and 4 formula-fed infants at 17 consecutive time points during the first 12 weeks of life. Microbiota composition was analysed by a combination of 16S rRNA gene sequencing and quantitative PCR (qPCR). In this dataset, individuality was a major driver of microbiota composition (P = 0.002) and was more pronounced in breastfed infants. A developmental signature could be distinguished, characterized by sequential colonisation of i) intrauterine/vaginal birth associated taxa, ii) skin derived taxa and other typical early colonisers such as Streptococcus and Enterobacteriaceae, iii) domination of Bifidobacteriaceae, and iv) the appearance of adultlike taxa, particularly species associated with Blautia, Eggerthella, and the potential pathobiont Clostridium difficile. Low abundance of potential pathogens was detected by 16S profiling and confirmed by qPCR. Incidence and dominance of skin and breast milk associated microbes were increased in the gut microbiome of breastfed infants compared to formula-fed infants. The approaches in this study indicate that microbiota development of breastfed and formula-fed infants proceeds according to similar developmental stages with microbiota signatures that include stage-specific species.

The brain-gut axis allows bidirectional communication between the central nervous system (CNS) and the enteric nervous system (ENS), linking emotional and cognitive centers of the brain with peripheral intestinal functions. Recent experimental work suggests that the gut microbiota have an impact on the brain-gut axis. A group of experts convened by the International Scientific Association for Probiotics and Prebiotics (ISAPP) discussed the role of gut bacteria on brain functions and the implications for probiotic and prebiotic science. The experts reviewed and discussed current available data on the role of gut microbiota on epithelial cell function, gastrointestinal motility, visceral sensitivity, perception and behavior. Data, mostly gathered from animal studies, suggest interactions of gut microbiota not only with the enteric nervous system but also with the central nervous system via neural, neuroendocrine, neuroimmune and humoral links. Microbial colonization impacts mammalian brain development in early life and subsequent adult behavior. These findings provide novel insights for improved understanding of the potential role of gut microbial communities on psychological disorders, most particularly in the field of psychological comorbidities associated with functional bowel disorders like irritable bowel syndrome (IBS) and should present new opportunity for interventions with pro- and prebiotics.

The technologies of metagenomics and metabolomics are broadening our knowledge of the roles the human gut microbiota play in health and disease. For many years now, probiotics and prebiotics have been included in foods for their health benefits; however, we have only recently begun to understand their modes of action. This review highlights recent advances in deciphering the mechanisms of probiosis and prebiosis, and describes how this knowledge could be transferred to select for enhancing functional foods targeting different populations. A special focus will be given to the addition of prebiotics and probiotics in functional foods for infants and seniors.

OBJECTIVES: Exploring associations between the gut microbiota and colonic inflammation and assessing sequential changes during exclusive enteral nutrition (EEN) may offer clues into the microbial origins of Crohn’s disease (CD).\ud METHODS: Fecal samples (n=117) were collected from 23 CD and 21 healthy children. From CD children fecal samples were collected before, during EEN, and when patients returned to their habitual diets. Microbiota composition and functional capacity were characterized using sequencing of the 16S rRNA gene and shotgun metagenomics.\ud RESULTS: Microbial diversity was lower in CD than controls before EEN (P=0.006); differences were observed in 36 genera, 141 operational taxonomic units (OTUs), and 44 oligotypes. During EEN, the microbial diversity of CD children further decreased, and the community structure became even more dissimilar than that of controls. Every 10 days on EEN, 0.6 genus diversity equivalents were lost; 34 genera decreased and one increased during EEN. Fecal calprotectin correlated with 35 OTUs, 14 of which accounted for 78% of its variation. OTUs that correlated positively or negatively with calprotectin decreased during EEN. The microbiota of CD patients had a broader functional capacity than healthy controls, but diversity decreased with EEN. Genes involved in membrane transport, sulfur reduction, and nutrient biosynthesis differed between patients and controls. The abundance of genes involved in biotin (P=0.005) and thiamine biosynthesis decreased (P=0.017), whereas those involved in spermidine/putrescine biosynthesis (P=0.031), or the shikimate pathway (P=0.058), increased during EEN.\ud CONCLUSIONS: Disease improvement following treatment with EEN is associated with extensive modulation of the gut microbiome.

The vertebrate gut symbiont Lactobacillus reuteri has diversified into separate clades reflecting host origin. Strains show evidence of host adaptation, but how host–microbe coevolution influences microbial-derived effects on hosts is poorly understood. Emphasizing human-derived strains of L. reuteri, we combined comparative genomic analyses with functional assays to examine variations in host interaction among genetically distinct ecotypes. Within clade II or VI, the genomes of human-derived L. reuteri strains are highly conserved in gene content and at the nucleotide level. Nevertheless, they share only 70–90% of total gene content, indicating differences in functional capacity. Human-associated lineages are distinguished by genes related to bacteriophages, vitamin biosynthesis, antimicrobial production, and immunomodulation. Differential production of reuterin, histamine, and folate by 23 clade II and VI strains was demonstrated. These strains also differed with respect to their ability to modulate human cytokine production (tumor necrosis factor, monocyte chemoattractant protein-1, interleukin [IL]-1β, IL-5, IL-7, IL-12, and IL-13) by myeloid cells. Microarray analysis of representative clade II and clade VI strains revealed global regulation of genes within the reuterin, vitamin B12, folate, and arginine catabolism gene clusters by the AraC family transcriptional regulator, PocR. Thus, human-derived L. reuteri clade II and VI strains are genetically distinct and their differences affect their functional repertoires and probiotic features. These findings highlight the biological impact of microbe:host coevolution and illustrate the functional significance of subspecies differences in the human microbiome. Consideration of host origin and functional differences at the subspecies level may have major impacts on probiotic strain selection and considerations of microbial ecology in mammalian species.

It has become clear in recent years that the human intestinal microbiota plays an important role in maintaining health and thus is an attractive target for clinical interventions. Scientists and clinicians have become increasingly interested in assessing the ability of probiotics and prebiotics to enhance the nutritional status of malnourished children, pregnant women, the elderly, and individuals with non-communicable disease-associated malnutrition. A workshop was held by the International Scientific Association for Probiotics and Prebiotics (ISAPP), drawing on the knowledge of experts from industry, medicine, and academia, with the objective to assess the status of our understanding of the link between the microbiome and under-nutrition, specifically in relation to probiotic and prebiotic treatments for under-nourished individuals. These discussions led to four recommendations: (1) The categories of malnourished individuals need to be differentiated. To improve treatment outcomes, subjects should first be categorized based on the cause of malnutrition, additional health-concerns, differences in the gut microbiota, and sociological considerations. (2) Define a baseline >healthy> gut microbiota for each category. Altered nutrient requirement (for example, in pregnancy and old age) and individual variation may change what constitutes a healthy gut microbiota for the individual. (3) Perform studies using model systems to test the effectiveness of potential probiotics and prebiotics against these specific categories. These should illustrate how certain microbiota profiles can be altered, as members of different categories may respond differently to the same treatment. (4) Perform robust well-designed human studies with probiotics and/or prebiotics, with appropriate, defined primary outcomes and sample size. These are critical to show efficacy and understand responder and non-responder outcomes. It is hoped that these recommendations will lead to new approaches that combat malnutrition. © 2014 Landes Bioscience.

Human microbiome-derived strains of Lactobacillus reuteri potently suppress proinflammatory cytokines like human tumor necrosis factor (TNF) by converting the amino acid l -histidine to the biogenic amine histamine. Histamine suppresses mitogen-activated protein (MAP) kinase activation and cytokine production by signaling via histamine receptor type 2 (H 2 ) on myeloid cells. Investigations of the gene expression profiles of immunomodulatory L. reuteri ATCC PTA 6475 highlighted numerous genes that were highly expressed during the stationary phase of growth, when TNF suppression is most potent. One such gene was found to be a regulator of genes involved in histidine-histamine metabolism by this probiotic species. During the course of these studies, this gene was renamed the Lactobacillus reuteri -specific immunoregulatory ( rsiR ) gene. The rsiR gene is essential for human TNF suppression by L. reuteri and expression of the histidine decarboxylase ( hdc ) gene cluster on the L. reuteri chromosome. Inactivation of rsiR resulted in diminished TNF suppression in vitro and reduced anti-inflammatory effects in vivo in a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of acute colitis. A L. reuteri strain lacking an intact rsiR gene was unable to suppress colitis and resulted in greater concentrations of serum amyloid A (SAA) in the bloodstream of affected animals. The P hdcAB promoter region targeted by rsiR was defined by reporter gene experiments. These studies support the presence of a regulatory gene, rsiR , which modulates the expression of a gene cluster known to mediate immunoregulation by probiotics at the transcriptional level. These findings may point the way toward new strategies for controlling gene expression in probiotics by dietary interventions or microbiome manipulation.

Background Microbial metagenomic analyses rely on an increasing number of publicly available tools. Installation, integration, and maintenance of the tools poses significant burden on many researchers and creates a barrier to adoption of microbiome analysis, particularly in translational settings. Methods To address this need we have integrated a rich collection of microbiome analysis tools into the Genboree Microbiome Toolset and exposed them to the scientific community using the Software-as-a-Service model via the Genboree Workbench. The Genboree Microbiome Toolset provides an interactive environment for users at all bioinformatic experience levels in which to conduct microbiome analysis. The Toolset drives hypothesis generation by providing a wide range of analyses including alpha diversity and beta diversity, phylogenetic profiling, supervised machine learning, and feature selection. Results We validate the Toolset in two studies of the gut microbiota, one involving obese and lean twins, and the other involving children suffering from the irritable bowel syndrome. Conclusions By lowering the barrier to performing a comprehensive set of microbiome analyses, the Toolset empowers investigators to translate high-volume sequencing data into valuable biomedical discoveries.

Enteric and diarrheal diseases are responsible for occupying 50% of hospital beds in developing countries (1), for at least 15% of childhood deaths worldwide (2), and for predisposing children to long-term developmental deficits in immune function, fitness, and cognition (3). Present global health strategies to address enteric and diarrheal diseases include provision of potable water and sanitation, antimicrobial therapy, and enteric vaccines; however, these interventions are expensive and fraught with technical challenges. For example, in regions of the world where mortality is highest, new rotavirus vaccines are demonstrating < 50% efficacy (4–6). New low-cost solutions are needed to reduce the global burden of enteric and diarrheal diseases. Among the more promising new therapeutic strategies are probiotics, live microorganisms that confer a health benefit on the host by altering the intestinal microbial balance (7,8). Multiple randomized, placebo-controlled prospective trials with probiotics report modest benefits in patients with acute gastroenteritis (9–12); however, remarkable probiotic species and strain diversity, along with an insufficient understanding of mechanisms underlying probiosis, prevents selection of optimal therapeutic microbes for infectious diarrhea (13). Thus, it is unclear whether probiotics are ready for integration into routine clinical practice (14,15). Nonetheless, due to the low cost, ease of distribution and administration, and favorable safety profiles, probiotics-based therapies have the potential to play a critical role in comprehensive global health strategies (13). Among the most-studied probiotics for acute gastroenteritis is Lactobacillus reuteri, a commensal bacterium found in the lower gastrointestinal tracts of humans and mice (16). For children hospitalized with acute rotaviral gastroenteritis, L reuteri alone or coadministered with Lactobacillus rhamnosus reduces the duration of illness by 1 day (17,18). It is unclear how L reuteri mediates recovery from diarrhea, or whether this effect can be enhanced. Others have shown that immunomodulation could contribute to the resolution of acute rotaviral gastroenteritis by L reuteri in piglets (19-21). Furthermore, we recently found that 2 genotypically and phenotypically distinct strains of L reuteri, DSM 17938 and ATCC PTA 6475, known to be safe and effective in treating infantile colic (22,23), increase the rate of intestinal epithelial cell turnover and enhance diversity of the intestinal microbiome in uninfected mouse pups (24). Therefore, we sought to determine whether any of these mechanisms contribute to the resolution of disease by L reuteri strains 17938 and 6475 in a mouse model of acute rotaviral gastroenteritis. We also explored whether these mechanisms of probiosis are relevant to the undernourished host, which suffers a disproportional burden of global enteric and diarrheal diseases of childhood.

Contains fulltext : 108670.pdf (Publisher’s version ) (Open Access) ABSTRACT: BACKGROUND: Recent advances in sequencing technologies have enabled metagenomic analyses of many human body sites. Several studies have catalogued the composition of bacterial communities of the surface of human skin, mostly under static conditions in healthy volunteers. Skin injury will disturb the cutaneous homeostasis of the host tissue and its commensal microbiota, but the dynamics of this process have not been studied before. Here we analyzed the microbiota of the surface layer and the deeper layers of the stratum corneum of normal skin, and we investigated the dynamics of recolonization of skin microbiota following skin barrier disruption by tape stripping as a model of superficial injury. RESULTS: We observed gender differences in microbiota composition and showed that bacteria are not uniformly distributed in the stratum corneum. Phylogenetic distance analysis was employed to follow microbiota development during recolonization of injured skin. Surprisingly, the developing neo-microbiome at day 14 was more similar to that of the deeper stratum corneum layers than to the initial surface microbiome. In addition, we also observed variation in the host response towards superficial injury as assessed by the induction of antimicrobial protein expression in epidermal keratinocytes. CONCLUSIONS: We suggest that the microbiome of the deeper layers, rather than that of the superficial skin layer may be regarded as the host indigenous microbiome. Characterization of the skin microbiome under dynamic conditions, and the ensuing response of the microbial community and host tissue will shed further light on the complex interaction between resident bacteria and epidermis.

The genomes of four Lactobacillus reuteri strains isolated from human breast milk and the gastrointestinal tract have been recently sequenced as part of the Human Microbiome Project. Preliminary genome comparisons suggested that these strains belong to two different clades, previously shown to differ with respect to antimicrobial production, biofilm formation, and immunomodulation. To explain possible mechanisms of survival in the host and probiosis, we completed a detailed genomic comparison of two breast milk-derived isolates representative of each group: an established probiotic strain (L. reuteri ATCC 55730) and a strain with promising probiotic features (L. reuteri ATCC PTA 6475). Transcriptomes of L. reuteri strains in different growth phases were monitored using strain-specific microarrays, and compared using a pan-metabolic model representing all known metabolic reactions present in these strains. Both strains contained candidate genes involved in the survival and persistence in the gut such as mucus-binding proteins and enzymes scavenging reactive oxygen species. A large operon predicted to encode the synthesis of an exopolysaccharide was identified in strain 55730. Both strains were predicted to produce health-promoting factors, including antimicrobial agents and vitamins (folate, vitamin B(12)). Additionally, a complete pathway for thiamine biosynthesis was predicted in strain 55730 for the first time in this species. Candidate genes responsible for immunomodulatory properties of each strain were identified by transcriptomic comparisons. The production of bioactive metabolites by human-derived probiotics may be predicted using metabolic modeling and transcriptomics. Such strategies may facilitate selection and optimization of probiotics for health promotion, disease prevention and amelioration.

Although commensal microbes have been shown to modulate host immune responses, many of the bacterial factors that mediate immune regulation remain unidentified. Select strains of human-derived Lactobacillus reuteri synthesize immunomodulins that potently inhibit production of the inflammatory cytokine TNF. In this study, genetic and genomic approaches were used to identify and investigate L. reuteri genes required for human TNF immunomodulatory activity. Analysis of membrane fatty acids from multiple L. reuteri strains cultured in MRS medium showed that only TNF inhibitory strains produced the cyclopropane fatty acid (CFA) lactobacillic acid. The enzyme cyclopropane fatty acid synthase is required for synthesis of CFAs such as lactobacillic acid, therefore the cfa gene was inactivated and supernatants from the cfa mutant strain were assayed for TNF inhibitory activity. We found that supernatants from the wild-type strain, but not the cfa mutant, suppressed TNF production by activated THP-1 human monocytoid cells. Although this suggested a direct role for lactobacillic acid in immunomodulation, purified lactobacillic acid did not suppress TNF at physiologically relevant concentrations. We further analyzed TNF inhibitory and TNF non-inhibitory strains under different growth conditions and found that lactobacillic acid production did not correlate with TNF inhibition. These results indicate that cfa indirectly contributed to L. reuteri immunomodulatory activity and suggest that other mechanisms, such as decreased membrane fluidity or altered expression of immunomodulins, result in the loss of TNF inhibitory activity. By increasing our understanding of immunomodulation by probiotic species, beneficial microbes can be rationally selected to alleviate intestinal inflammation.

Gut bacteria can be categorised as being either beneficial or potentially pathogenic due to their metabolic activities and fermentation end-products. Health-promoting effects of the microflora may include immunostimulation, improved digestion and absorption, vitamin synthesis, inhibition of the growth of potential pathogens and lowering of gas distension. Detrimental effects are carcinogen production, intestinal putrefaction, toxin production, diarrhoea/constipation and intestinal infections. Certain indigenous bacteria such as bifidobacteria and lactobacilli are considered to be examples of health-promoting constituents of the microflora. They may aid digestion of lactose in lactose-intolerant individuals, reduce diarrhoea, help resist infections and assist in inflammatory conditions. Probiotics, prebiotics and synbiotics are functional foods that fortify the lactate producing microflora of the human or animal gut.