Probiotics stimulate enterocyte migration and microbial diversity in the neonatal mouse intestine.

Beneficial microbes and probiotics show promise for the treatment of pediatric gastrointestinal diseases. However, basic mechanisms of probiosis are not well understood, and most investigations have been performed in germ-free or microbiome-depleted animals. We sought to functionally characterize probiotic-host interactions in the context of normal early development. Outbred CD1 neonatal mice were orally gavaged with one of two strains of human-derived Lactobacillus reuteri or an equal volume of vehicle. Transcriptome analysis was performed on enterocyte RNA isolated by laser-capture microdissection. Enterocyte migration and proliferation were assessed by labeling cells with 5-bromo-2'-deoxyuridine, and fecal microbial community composition was determined by 16S metagenomic sequencing. Probiotic ingestion altered gene expression in multiple canonical pathways involving cell motility. L. reuteri strain DSM 17938 dramatically increased enterocyte migration (3-fold), proliferation (34%), and crypt height (29%) compared to vehicle-treated mice, whereas strain ATCC PTA 6475 increased cell migration (2-fold) without affecting crypt proliferative activity. In addition, both probiotic strains increased the phylogenetic diversity and evenness between taxa of the fecal microbiome 24 h after a single probiotic gavage. These experiments identify two targets of probiosis in early development, the intestinal epithelium and the gut microbiome, and suggest novel mechanisms for probiotic strain-specific effects.

Gastrointestinal microbiome signatures of pediatric patients with irritable bowel syndrome.

BACKGROUND & AIMS: The intestinal microbiomes of healthy children and pediatric patients with irritable bowel syndrome (IBS) are not well defined. Studies in adults have indicated that the gastrointestinal microbiota could be involved in IBS. METHODS: We analyzed 71 samples from 22 children with IBS (pediatric Rome III criteria) and 22 healthy children, ages 7-12 years, by 16S ribosomal RNA gene sequencing, with an average of 54,287 reads/stool sample (average 454 read length = 503 bases). Data were analyzed using phylogenetic-based clustering (Unifrac), or an operational taxonomic unit (OTU) approach using a supervised machine learning tool (randomForest). Most samples were also hybridized to a microarray that can detect 8741 bacterial taxa (16S rRNA PhyloChip). RESULTS: Microbiomes associated with pediatric IBS were characterized by a significantly greater percentage of the class γ-proteobacteria (0.07% vs 0.89% of total bacteria, respectively; P < .05); 1 prominent component of this group was Haemophilus parainfluenzae. Differences highlighted by 454 sequencing were confirmed by high-resolution PhyloChip analysis. Using supervised learning techniques, we were able to classify different subtypes of IBS with a success rate of 98.5%, using limited sets of discriminant bacterial species. A novel Ruminococcus-like microbe was associated with IBS, indicating the potential utility of microbe discovery for gastrointestinal disorders. A greater frequency of pain correlated with an increased abundance of several bacterial taxa from the genus Alistipes. CONCLUSIONS: Using 16S metagenomics by PhyloChip DNA hybridization and deep 454 pyrosequencing, we associated specific microbiome signatures with pediatric IBS. These findings indicate the important association between gastrointestinal microbes and IBS in children; these approaches might be used in diagnosis of functional bowel disorders in pediatric patients.

Host response to probiotics determined by nutritional status of rotavirus-infected neonatal mice.

OBJECTIVES: Beneficial microbes and probiotics are promising agents for the prevention and treatment of enteric and diarrheal diseases in children; however, little is known about their in vivo mechanisms of action. We used a neonatal mouse model of rotavirus diarrhea to gain insight into how probiotics ameliorate acute gastroenteritis. METHODS: Rotavirus-infected mice were treated with 1 of 2 strains of human-derived Lactobacillus reuteri. We assessed intestinal microbiome composition with 16S metagenomic sequencing, enterocyte migration and proliferation with 5-bromo-2'-deoxyuridine, and antibody and cytokine concentrations with multiplex analyses of intestinal explant cultures. RESULTS: Probiotics reduced diarrhea duration, improved intestinal histopathology, and enhanced intestinal microbiome richness and phylogenetic diversity. The magnitude of reduction of diarrhea by probiotics was strain specific and influenced by nutritional status. L reuteri DSM 17938 reduced diarrhea duration by 0, 1, and 2 days in underweight, normal weight, and overweight pups, respectively. The magnitude of reduction of diarrhea duration correlated with increased enterocyte proliferation and migration. Strain ATCC PTA 6475 reduced diarrhea duration by 1 day in all of the mice without increasing enterocyte proliferation. Both probiotic strains decreased concentrations of proinflammatory cytokines, including macrophage inflammatory protein-1α and interleukin-1ß, in all of the animals, and increased rotavirus-specific antibodies in all but the underweight animals. Body weight also influenced the host response to rotavirus, in terms of diarrhea duration, enterocyte turnover, and antibody production. CONCLUSIONS: These data suggest that probiotic enhancement of enterocyte proliferation, villus repopulation, and virus-specific antibodies may contribute to diarrhea resolution, and that nutritional status influences the host response to both beneficial microbes and pathogens.

Functional identification in Lactobacillus reuteri of a PocR-like transcription factor regulating glycerol utilization and vitamin B12 synthesis.

BACKGROUND: Lactobacillus reuteri harbors the genes responsible for glycerol utilization and vitamin B12 synthesis within a genetic island phylogenetically related to gamma-Proteobacteria. Within this island, resides a gene (lreu_1750) that based on its genomic context has been suggested to encode the regulatory protein PocR and presumably control the expression of the neighboring loci. However, this functional assignment is not fully supported by sequence homology, and hitherto, completely lacks experimental confirmation. RESULTS: In this contribution, we have overexpressed and inactivated the gene encoding the putative PocR in L. reuteri. The comparison of these strains provided metabolic and transcriptional evidence that this regulatory protein controls the expression of the operons encoding glycerol utilization and vitamin B12 synthesis. CONCLUSIONS: We provide clear experimental evidence for assigning Lreu_1750 as PocR in Lactobacillus reuteri. Our genome-wide transcriptional analysis further identifies the loci contained in the PocR regulon. The findings reported here could be used to improve the production-yield of vitamin B12, 1,3-propanediol and reuterin, all industrially relevant compounds.

In vitro effects of selected synbiotics on the human faecal microbiota composition.

Synbiotics are recognized means of modulating gut microbiota composition and activities. However, whether synbiotics are superior to prebiotics and probiotics alone in moderating the gut microbiota towards a purportedly healthy composition has not been determined. Eight selected synbiotics (short-chain fructooligosaccharides or fructooligosaccharides, each combined with one of four probiotics, Lactobacillus fermentum ME-3, Lactobacillus plantarum WCFS1, Lactobacillus paracasei 8700:2 or Bifidobacterium longum 46) were added to 24-h pH-controlled anaerobic faecal batch cultures. The prebiotic and probiotic components were also tested alone to determine their respective role within the synbiotic for modulation of the faecal microbiota. Effects upon major groups of the microbiota were evaluated using FISH. Rifampicin variant probiotic strains were used to assess probiotic levels. Synbiotic and prebiotics increased bifidobacteria and the Eubacterium rectale-Clostridium coccoides group. Lower levels of Escherichia coli were retrieved with these combinations after 5 and 10 h of fermentation. Probiotics alone had little effect upon the groups, however. Multivariate analysis revealed that the effect of synbiotics differed from the prebiotics as higher levels of Lactobacillus-Enterococcus were observed when the probiotic was stimulated by the prebiotic component. Here, the synbiotic approach was more effective than prebiotic or probiotic alone to modulate the gut microbiota.

Intestinal colonisation patterns in breastfed and formula-fed infants during the first 12 weeks of life reveal sequential microbiota signatures.

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.

FolC2-mediated folate metabolism contributes to suppression of inflammation by probiotic Lactobacillus reuteri.

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.

Can prebiotics and probiotics improve therapeutic outcomes for undernourished individuals?

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. This report is the result of discussion during an expert workshop titled "How do the microbiota and probiotics and/or prebiotics influence poor nutritional status?" held during the 10th Meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP) in Cork, Ireland from October 1-3, 2012. The complete list of workshop attendees is shown in Table 1.

From prediction to function using evolutionary genomics: human-specific ecotypes of Lactobacillus reuteri have diverse probiotic functions.

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]-1ß, 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.

The intestinal microbiome, probiotics and prebiotics in neurogastroenterology.

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.

Microbiome dynamics of human epidermis following skin barrier disruption.

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.

Exploring metabolic pathway reconstruction and genome-wide expression profiling in Lactobacillus reuteri to define functional probiotic features.

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.

How can probiotics and prebiotics impact mucosal immunity?

The study of probiotics and prebiotics is an expanding field of interest and scientific research that has resulted in insights related to the host immune response. Recent advances have naturally led to key questions. What are the specific probiotic components that mediate immunomodulation? Can we extrapolate the results of in vitro studies in animal and human trials? Which biomarkers and immune parameters should be measured in probiotic and prebiotic intervention studies? These questions were part of a discussion entitled "How Can Probiotics and Prebiotics Impact Mucosal Immunity" at the 2009 Annual Meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP). This review highlights recent knowledge about the modulation of mucosal immunity by probiotics and prebiotics, as well as considerations for measuring their effects on mucosal immunity. A list of biomarkers and immune parameters to be measured in human clinical trials is included.

Microbiology of the human intestinal tract and approaches for its dietary modulation.

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.

Mechanisms of probiosis and prebiosis: considerations for enhanced functional foods.

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.

Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays.

Short-chain fructooligosaccharides (scFOS) and other prebiotics are used to selectively stimulate the growth and activity of lactobacilli and bifidobacteria in the colon. However, there is little information on the mechanisms whereby prebiotics exert their specific effects upon such microorganisms. To study the genomic basis of scFOS metabolism in Lactobacillus plantarum WCFS1, two-color microarrays were used to screen for differentially expressed genes when grown on scFOS compared to glucose (control). A significant up-regulation (8- to 60-fold) was observed with a set of only five genes located in a single locus and predicted to encode a sucrose phosphoenolpyruvate transport system (PTS), a beta-fructofuranosidase, a fructokinase, an alpha-glucosidase, and a sucrose operon repressor. Several other genes were slightly overexpressed, including pyruvate dehydrogenase. For the latter, no detectable activity in L. plantarum under various growth conditions has been previously reported. A mannose-PTS likely to encode glucose uptake was 50-fold down-regulated as well as, to a lower extent, other PTSs. Chemical analysis of the different moieties of scFOS that were depleted in the growth medium revealed that the trisaccharide 1-kestose present in scFOS was preferentially utilized, in comparison with the tetrasaccharide nystose and the pentasaccharide fructofuranosylnystose. The main end products of scFOS fermentation were lactate and acetate. This is the first example in lactobacilli of the association of a sucrose PTS and a beta-fructofuranosidase that could be used for scFOS degradation.