Sonia Michail

Probiotics inhibit enteropathogenic E. coli adherence in vitro by inducing intestinal mucin gene expression

Probiotic agents, live microorganisms with beneficial effects for the host, may offer an alternative to conventional antimicrobials in the treatment and prevention of enteric infections. The probiotic agents Lactobacillus plantarum 299v and Lactobacillus rhamnosus GG quantitatively inhibited the adherence of an attaching and effacing pathogenic Escherichia coli to HT-29 intestinal epithelial cells but did not inhibit adherence to nonintestinal HEp-2 cells. HT-29 cells were grown under conditions that induced high levels of either MUC2 or MUC3 mRNA, but HEp-2 cells expressed only minimal levels of MUC2 and no MUC3 mRNA. Media enriched for MUC2 and MUC3 mucin were added exogenously to binding assays and were shown to be capable of inhibiting enteropathogen adherence to HEp-2 cells. Incubation of L. plantarum 299v with HT-29 cells increased MUC2 and MUC3 mRNA expression levels. From these in vitro studies, we propose the hypothesis that the ability of probiotic agents to inhibit adherence of attaching and effacing organisms to intestinal epithelial cells is mediated through their ability to increase expression of MUC2 and MUC3 intestinal mucins.Probiotic agents, live microorganisms with beneficial effects for the host, may offer an alternative to conventional antimicrobials in the treatment and prevention of enteric infections. The probiotic agents Lactobacillus plantarum 299v and Lactobacillus rhamnosus GG quantitatively inhibited the adherence of an attaching and effacing pathogenic Escherichia coli to HT-29 intestinal epithelial cells but did not inhibit adherence to nonintestinal HEp-2 cells. HT-29 cells were grown under conditions that induced high levels of either MUC2 or MUC3 mRNA, but HEp-2 cells expressed only minimal levels of MUC2 and no MUC3 mRNA. Media enriched for MUC2 and MUC3 mucin were added exogenously to binding assays and were shown to be capable of inhibiting enteropathogen adherence to HEp-2 cells. Incubation of L. plantarum 299v with HT-29 cells increased MUC2 and MUC3 mRNA expression levels. From these in vitro studies, we propose the hypothesis that the ability of probiotic agents to inhibit adherence of attaching and effacing organisms to intestinal epithelial cells is mediated through their ability to increase expression of MUC2 and MUC3 intestinal mucins.

Alterations in the gut microbiome of children with severe ulcerative colitis.

Background: Although the role of microbes in disease pathogenesis is well established, data describing the variability of the vast microbiome in children diagnosed with ulcerative colitis (UC) are lacking. This study characterizes the gut microbiome in hospitalized children with severe UC and determines the relationship between microbiota and response to steroid therapy. Methods: Fecal samples were collected from 26 healthy controls and 27 children hospitalized with severe UC as part of a prospective multicenter study. DNA extraction, polymerase chain reaction (PCR) amplification of bacterial 16S rRNA, and microarray hybridization were performed. Results were analyzed in GeneSpring GX 11.0 comparing healthy controls with children with UC, and steroid responsive (n = 17) with nonresponsive patients (n = 10). Results: Bacterial signal strength and distribution showed differences between UC and healthy controls (adjusted P < 0.05) for Phylum, Class, Order, Family, Genus, and Phylospecies levels with reduction in Clostridia and an increase in Gamma‐proteobacteria. The number of microbial phylospecies was reduced in UC (266 ± 69) vs. controls (758 ± 3, P < 0.001), as was the Shannon Diversity Index (6.1 ± 0.23 vs. 6.49 ± 0.04, respectively; P < 0.0001). Steroid nonresponders harbored fewer phylospecies than responders (142 ± 49 vs. 338 ± 62, P = 0.013). Conclusions: Richness, evenness, and biodiversity of the gut microbiome were remarkably reduced in children with UC compared with healthy controls. Children who did not respond to steroids harbored a microbiome that was even less rich than steroid responders. This study is the first to characterize the gut microbiome in a large cohort of pediatric patients with severe UC and describes changes in the gut microbiome as a potential prognostic feature. (Inflamm Bowel Dis 2012)

Distal gut microbiota of adolescent children is different from that of adults

Human intestinal microbiota plays a number of important roles in human health and is also implicated in several gastrointestinal disorders. Although the diversity of human gut microbiota in adults and in young children has been examined, few reports of microbiota composition are available for adolescents. In this work, we used Microbiota Array for high-throughput analysis of distal gut microbiota in adolescent children 11-18 years of age. Samples obtained from healthy adults were used for comparison. Adolescent and adult groups could be separated in the principal components analysis space based on the relative species abundance of their distal gut microbiota. All samples were dominated by class Clostridia. A core microbiome of 46 species that were detected in all examined samples was established; members of genera Ruminococcus, Faecalibacterium, and Roseburia were well represented among core species. Comparison of intestinal microbiota composition between adolescents and adults revealed a statistically significantly higher abundance of genera Bifidobacterium and Clostridium among adolescent samples. The number of detected species was similar between sample groups, indicating that it was the relative abundances of the genera and not the presence or absence of a specific genus that differentiated adolescent and adult samples. In summary, contrary to the current belief, this study suggests that the gut microbiome of adolescent children is different from that of adults.

Quantitative Profiling of Gut Microbiota of Children with Diarrhea-Predominant Irritable Bowel Syndrome

OBJECTIVES:Human intestinal microbiota has a number of important roles in human health and is also implicated in several gastrointestinal disorders. The goal of this study was to determine the gut microbiota in two groups of pre- and adolescent children: healthy volunteers and children diagnosed with diarrhea-predominant irritable bowel syndrome (IBS-D).METHODS:Phylogenetic Microbiota Array was used to obtain quantitative measurements of bacterial presence and abundance in subjects’ fecal samples. We utilized high-throughput DNA sequencing, quantitative PCR, and fluorescent in situ hybridization to confirm microarray findings.RESULTS:Both sample groups were dominated by the phyla Firmicutes, Bacteroidetes, and Actinobacteria, which cumulatively constituted 91% of overall sample composition on average. A core microbiome shared among analyzed samples encompassed 55 bacterial phylotypes dominated by genus Ruminococcus; members of genera Clostridium, Faecalibacterium, Roseburia, Streptococcus, and Bacteroides were also present. Several genera were found to be differentially abundant in the gut of healthy and IBS groups: levels of Veillonella, Prevotella, Lactobacillus, and Parasporobacterium were increased in children diagnosed with IBS, whereas members of Bifidobacterium and Verrucomicrobium were less abundant in those individuals. By calculating a nonparametric correlation matrix among abundances of different genera in all samples, we also examined potential associations among intestinal microbes. Strong positive correlations were found between abundances of Veillonella and both Haemophilus and Streptococcus, between Anaerovorax and Verrucomicrobium, and between Tannerella and Anaerophaga.CONCLUSIONS:Although at the higher taxonomical level gut microbiota was similar between healthy and IBS-D children, specific differences in the abundances of several bacterial genera were revealed. Core microbiome in children was dominated by Clostridia. Putative relationships identified among microbial genera provide testable hypotheses of cross-species associations among members of human gut microbiota.

Clinical efficacy of probiotics: review of the evidence with focus on children.

Probiotics are marketed in several countries and widely used by pediatric health care providers. Although probiotics can be helpful for specific disorders, they have been broadly prescribed for disorders without clear evidence to support their use. Furthermore, in certain specific conditions, probiotics cause clinical deterioration. This report is a review and evaluation of the evidence or lack thereof to support a beneficial effect of probiotic agents in a variety of pediatric conditions and to review the safety and potential adverse events that may be encountered when using probiotics. It is also important to emphasize that probiotics are highly heterogeneous with differences in composition, biological activity, and dose among the different probiotic preparations.

High-Throughput Quantitative Analysis of the Human Intestinal Microbiota with a Phylogenetic Microarray

ABSTRACT Gut microbiota carry out key functions in health and participate in the pathogenesis of a growing number of diseases. The aim of this study was to develop a custom microarray that is able to identify hundreds of intestinal bacterial species. We used the Entrez nucleotide database to compile a data set of bacterial 16S rRNA gene sequences isolated from human intestinal and fecal samples. Identified sequences were clustered into separate phylospecies groups. Representative sequences from each phylospecies were used to develop a microbiota microarray based on the Affymetrix GeneChip platform. The designed microbiota array contains probes to 775 different bacterial phylospecies. In our validation experiments, the array correctly identified genomic DNA from all 15 bacterial species used. Microbiota array has a detection sensitivity of at least 1 pg of genomic DNA and can detect bacteria present at a 0.00025% level of overall sample. Using the developed microarray, fecal samples from two healthy children and two healthy adults were analyzed for bacterial presence. Between 227 and 232 species were detected in fecal samples from children, whereas 191 to 208 species were found in adult stools. The majority of identified phylospecies belonged to the classes Clostridia and Bacteroidetes. The microarray revealed putative differences between the gut microbiota of healthy children and adults: fecal samples from adults had more Clostridia and less Bacteroidetes and Proteobacteria than those from children. A number of other putative differences were found at the genus level.

Efficacy of probiotics in the treatment of pediatric atopic dermatitis: a meta-analysis of randomized controlled trials

BACKGROUND Several articles describing the efficacy of probiotics in atopic dermatitis (AD) have been published. However, not all studies support a similar outcome. OBJECTIVE To determine whether probiotics are efficacious in treating AD and to explore whether type of probiotic used, duration of therapy, patient age, severity of disease, and IgE sensitization are factors in determining efficacy. METHODS For this meta-analysis of randomized controlled trials describing the efficacy of probiotics in AD, a comprehensive search was performed of databases through January 2008. Three reviewers independently evaluated the studies for methodological qualities. All the data were analyzed, and forest plots were evaluated for the overall efficacy of probiotics in AD and the therapeutic benefit to subgroups of selected patient populations. RESULTS Eleven studies were identified, and data from 10 studies (n = 678) were available to analyze. There was an overall statistically significant difference favoring probiotics compared with placebo in reducing the Scoring of Atopic Dermatitis Severity Index score (mean change from baseline, -3.01; 95% confidence interval, -5.36 to -0.66; P = .01). Children with moderately severe disease were more likely to benefit. Duration of probiotic administration, age, and type of probiotic used did not affect outcome. CONCLUSION Data from this meta-analysis suggest a modestrole for probiotics in pediatric AD. The effect is seen in moderately severe rather than mild disease.

Lactobacillus plantarum reduces the in vitro secretory response of intestinal epithelial cells to Enteropathogenic Escherichia coli infection

Objective Enteropathogenic Escherichia coli (EPEC) is a Gram-negative bacillus that causes diarrhea. Secretory responses of intestinal epithelial cells can be seen after EPEC infection. Probiotics, which are live bacteria that have proven benefit to the host, play a role in the treatment and prevention of the different enteric pathogens. The goals of the study were to determine whether the probiotic agent Lactobacillus plantarum (LBP) strain 299v alters the secretory changes seen in EPEC infection and, if so, what underlying mechanism is possible. Methods Caco-2 cell monolayers were rapidly infected with EPEC strain E2348/69 and immediately mounted in Ussing chambers. The monolayers were exposed to LBP before, after, and simultaneously with EPEC infection. Short circuit current (Isc) was measured in the Ussing chamber. Results EPEC infection caused an increase in short circuit current that was reduced by preincubation with LBP (P < 0.01). No direct bactericidal effect was observed, but LBP reduced the attachment of EPEC to Caco-2 cells. Conclusion LBP can play an important role in reducing the secretory change in response to EPEC infection, possibly through inhibition of its binding. However, the presence of the probiotic agent before the infection is necessary. In this setting, its role is more preventive rather than therapeutic.

Altered gut microbial energy and metabolism in children with non-alcoholic fatty liver disease.

Obesity is becoming the new pediatric epidemic. Non-alcoholic fatty liver disease (NAFLD) is frequently associated with obesity and has become the most common cause of pediatric liver disease. The gut microbiome is the major metabolic organ and determines how calories are processed, serving as a caloric gate and contributing towards the pathogenesis of NAFLD. The goal of this study is to examine gut microbial profiles in children with NAFLD using phylogenetic, metabolomic, metagenomic and proteomic approaches. Fecal samples were obtained from obese children with or without NAFLD and healthy lean children. Stool specimens were subjected to 16S rRNA gene microarray, shotgun sequencing, mass spectroscopy for proteomics and NMR spectroscopy for metabolite analysis. Children with NAFLD had more abundant Gammaproteobacteria and Prevotella and significantly higher levels of ethanol, with differential effects on short chain fatty acids. This group also had increased genomic and protein abundance for energy production with a reduction in carbohydrate and amino acid metabolism and urea cycle and urea transport systems. The metaproteome and metagenome showed similar findings. The gut microbiome in pediatric NAFLD is distinct from lean healthy children with more alcohol production and pathways allocated to energy metabolism over carbohydrate and amino acid metabolism, which would contribute to development of disease.

Polyethylene glycol for constipation in children younger than eighteen months old.

Background: Polyethylene glycol (PEG) is a safe and effective treatment for constipation in children older than 18 months. Data on its safety and efficacy in infants are lacking. The goal of this study was to determine safety, efficacy, and optimal dose of polyethylene glycol powder for treatment of constipation in patients younger than 18 months. Methods: The authors reviewed the charts of patients younger than 18 months treated with PEG 3350 for constipation. The initial dose, effective maintenance dose, response to therapy, duration of therapy, and side effects were recorded. Results: Twenty-eight patients younger than 18 months of age treated with PEG were identified (3, age 0–5 months; 9, age 6–11 months; 16, age 12–17 months). Mean duration of therapy was 6.2 ± 5 months (range, 3 weeks–21 months). Mean initial dose was 0.88 g/kg/day (range, 0.26–2.14 g/kg/day). Mean effective maintenance dose was 0.78 g/kg/day (range, 0.26–1.26 g/kg/day). PEG relieved constipation in 97.6% of patients. One infant experienced increased gas per rectum and four others experienced transient diarrhea that resolved after adjusting the dose. Conclusion: Oral powdered polyethylene glycol at a maintenance dose of 0.78 g/kg/day is safe and effective for patients younger than 18 months. Dose and safety profiles are similar for those reported in older children.