The gut microbiome influences myriad host functions, including nutrient acquisition, immune modulation, brain development, and behavior. Although human gut microbiota are recognized to change as we age, information regarding the structure and function of the gut microbiome during childhood is limited. Using 16S rRNA gene and shotgun metagenomic sequencing, we characterized the structure, function, and variation of the healthy pediatric gut microbiome in a cohort of school-aged, pre-adolescent children (ages 7–12 years). We compared the healthy pediatric gut microbiome with that of healthy adults previously recruited from the same region (Houston, TX, USA). Although healthy children and adults harbored similar numbers of taxa and functional genes, their composition and functional potential differed significantly. Children were enriched in Bifidobacterium spp., Faecalibacterium spp., and members of the Lachnospiraceae, while adults harbored greater abundances of Bacteroides spp. From a functional perspective, significant differences were detected with respect to the relative abundances of genes involved in vitamin synthesis, amino acid degradation, oxidative phosphorylation, and triggering mucosal inflammation. Children's gut communities were enriched in functions which may support ongoing development, while adult communities were enriched in functions associated with inflammation, obesity, and increased risk of adiposity. Previous studies suggest that the human gut microbiome is relatively stable and adult-like after the first 1 to 3 years of life. Our results suggest that the healthy pediatric gut microbiome harbors compositional and functional qualities that differ from those of healthy adults and that the gut microbiome may undergo a more prolonged development than previously suspected.
Global estimations of 4 billion people living on plant-based diets signify tremendous diversity in plant consumption and their assorted miRNAs, which presents a challenging model to experimentally address how plant-based miRNAs impact the microbiome. Here we establish baseline gut microbiome composition for a mouse model deficient in the specific mammalian miR-146a shown to alter gut microbiomes. We then asses the effect on the gut microbiome when miR-146a-deficient mice are fed a transgenic plant-based diet expressing the murine-derived miR-146a. Mice deficient in miR-146a were maintained either on a baseline diet until 7 weeks of age (day 0) and then fed either vector or miR-146a-expressing plant-based diets for 21 days. The gut microbiomes of mice were examined by comparing the V4 region of 16S rRNA gene sequences of DNA isolated from fecal samples at days 0 (baseline diet) and 21 (vector or miR-146a expressing plant-based diets). Beta-diversity analysis demonstrated that the transition from baseline chow to a plant-based diet resulted in significant longitudinal shifts in microbial community structure attributable to increased fiber intake. Bipartite network analysis suggests that miR-146a-deficient mice fed a plant diet rich in miR-146a have a microbiome population modestly different than mice fed an isogenic control plant diet deficient in miR-146a. A mouse diet composed of a transgenic plant expressing a mouse miR-146a may fine tune microbial communities but does not appear to have global effects on microbiome structure and composition.
Adhesins or microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) and the ica locus help mediate S. aureus adherence to host tissue and biofilm formation and are thought to play important roles in the pathogenesis of device related infections (DRIs). We hypothesized that S. aureus isolates from pediatric DRIs differ in MSCRAMM and biofilm-associated gene profiles and display greater strain diversity compared with skin and soft-tissue infection (SSTI) isolates. Patients and isolates were identified from a prospective S. aureus surveillance study at Texas Children’s Hospital, 2008–2016. Clinical data were collected retrospectively. Age and date of infection matched SSTI control isolates were selected 4:1. Isolates were genotyped by pulsed-field gel electrophoresis. Whole genome sequencing was performed (Illumina MiSeq). Data were analyzed with CLC Genomics Workbench for the presence of MSCRAMMS (clfA, clfB, ebh, fbp, fnbpA, fnbpB, isdA, isdB, sdrC, sdrD, sdrE), biofilm-associated genes (icaA,D,B,C), accessory gene regulator group, and by multilocus-sequence typing (MLST) with eBurst analysis (www.phyloviz.net). Conditional logistic regression and Fisher’s exact were used for analysis (STATA11). Forty-five patients with 47 DRIs were identified (Table 1). Isolates from 47 DRIs and 188 SSTIs were analyzed for the presence of MSCRAMM and biofilm-associated genes. clfA, clfB, fbp, isdA, isdB, and icaA,D,B,C were present among DRIs and SSTIs more than 98% of the time. Isolates from DRIs or SSTIs did not differ significantly in carriage of MSCRAMMs or the ica locus. DRIs were MSSA (34, 72%), nonUSA300 (39, 83%), and belonged to 19 sequence types (STs). SSTIs were MSSA (79, 42%), nonUSA300 (57, 30%), and belonged to 39 STs (Table 2). Among DRI isolates, STs 5 and 8 were most common (23% each, Figure 1). SSTI isolates were predominately ST8 (68%). S. aureus isolates from DRIs were significantly more likely to be MSSA and nonUSA300 (P < 0.0001 for both) compared with SSTIs. The majority of S. aureus isolates harbored all MSCRAMM and biofilm-associated genes analyzed. Evaluating genetic polymorphisms and gene expression profiles may clarify the role of adhesion genes in the pathogenesis of DRIs vs. SSTIs. All authors: No reported disclosures.
Abstract Infants and young children receive the highest exposures to antibiotics globally. Although there is building evidence that early life exposure to antibiotics increases susceptibility to various diseases including gut disorders later in life, the lasting impact of early life antibiotics on the physiology of the gut and its enteric nervous system (ENS) remains unclear. We treated neonatal mice with the antibiotic vancomycin during their first 10 postnatal days, then examined potential lasting effects of the antibiotic treatment on their colons during young adulthood (6 weeks old). We found that neonatal vancomycin treatment disrupted the gut functions of young adult female and male mice differently. Antibiotic‐exposed females had significantly longer whole gut transit while antibiotic‐treated males had significantly lower faecal weights compared to controls. Both male and female antibiotic‐treated mice had greater percentages of faecal water content. Neonatal vancomycin treatment also had sexually dimorphic impacts on the neurochemistry and Ca 2+ activity of young adult myenteric and submucosal neurons. Myenteric neurons of male mice were more disrupted than those of females, while opposing changes in submucosal neurons were seen in each sex. Neonatal vancomycin also induced sustained changes in colonic microbiota and lasting depletion of mucosal serotonin (5‐HT) levels. Antibiotic impacts on microbiota and mucosal 5‐HT were not sex‐dependent, but we propose that the responses of the host to these changes are sex‐specific. This first demonstration of long‐term impacts of neonatal antibiotics on the ENS, gut microbiota and mucosal 5‐HT has important implications for gut function and other physiological systems of the host. image Key points Early life exposure to antibiotics can increase susceptibility to diseases including functional gastrointestinal (GI) disorders later in life. Yet, the lasting impact of this common therapy on the gut and its enteric nervous system (ENS) remains unclear. We investigated the long‐term impact of neonatal antibiotic treatment by treating mice with the antibiotic vancomycin during their neonatal period, then examining their colons during young adulthood. Adolescent female mice given neonatal vancomycin treatment had significantly longer whole gut transit times, while adolescent male and female mice treated with neonatal antibiotics had significantly wetter stools. Effects of neonatal vancomycin treatment on the neurochemistry and Ca 2+ activity of myenteric and submucosal neurons were sexually dimorphic. Neonatal vancomycin also had lasting effects on the colonic microbiome and mucosal serotonin biosynthesis that were not sex‐dependent. Different male and female responses to antibiotic‐induced disruptions of the ENS, microbiota and mucosal serotonin biosynthesis can lead to sex‐specific impacts on gut function.
Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdA and tcdB. Stool samples from hospitalized pediatric patients suspected of having C. difficile-associated disease were prospectively cultured on cycloserine-cefoxitin-fructose agar following alcohol shock. Six testing modalities were evaluated, including stool EIA, culture EIA, and real-time PCR (tcdA and tcdB) of cultured isolates and stool samples. Real-time PCR detection was performed with tcdA and tcdB gene-specific primers and hydrolysis probes using the LightCycler platforms (Roche Diagnostics, Indianapolis, IN). A total of 157 samples from 96 pediatric patients were analyzed. The sensitivities of stool real-time PCR and stool EIA were 95% and 35%, respectively, with a specificity of 100% for both methods. The lower limit of detection of the stool real-time PCR was 30 CFU/ml of stool sample per reaction for tcdA and tcdB. This study highlights the poor performance of stool toxin EIAs in pediatric settings. Direct detection of C. difficile toxin genes in stool samples by real-time PCR showed sensitivity superior to that of stool and culture EIAs and performance comparable to that of real-time PCR assay of cultured isolates. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for children that should facilitate appropriate patient management and halt the practice of serial testing by EIA.
Accumulating studies have defined a role for the intestinal microbiota in modulation of host behavior. Research using gnotobiotic mice emphasizes that early microbial colonization with a complex microbiota (conventionalization) can rescue some of the behavioral abnormalities observed in mice that grow to adulthood completely devoid of bacteria (germ-free mice). However, the human infant and adult microbiomes vary greatly, and effects of the neonatal microbiome on neurodevelopment are currently not well understood. Microbe-mediated modulation of neural circuit patterning in the brain during neurodevelopment may have significant long-term implications that we are only beginning to appreciate. Modulation of the host central nervous system by the early-life microbiota is predicted to have pervasive and lasting effects on brain function and behavior. We sought to replicate this early microbe-host interaction by colonizing gnotobiotic mice at the neonatal stage with a simplified model of the human infant gut microbiota. This model consortium consisted of four "infant-type" Bifidobacterium species known to be commensal members of the human infant microbiota present in high abundance during postnatal development. Germ-free mice and mice neonatally-colonized with a complex, conventional murine microbiota were used for comparison. Motor and non-motor behaviors of the mice were tested at 6–7 weeks of age, and colonization patterns were characterized by 16S ribosomal RNA gene sequencing. Adult germ-free mice were observed to have abnormal memory, sociability, anxiety-like behaviors, and motor performance. Conventionalization at the neonatal stage rescued these behavioral abnormalities, and mice colonized with Bifidobacterium spp. also exhibited important behavioral differences relative to the germ-free controls. The ability of Bifidobacterium spp. to improve the recognition memory of both male and female germ-free mice was a prominent finding. Together, these data demonstrate that the early-life gut microbiome, and human "infant-type" Bifidobacterium species, affect adult behavior in a strongly sex-dependent manner, and can selectively recapitulate the results observed when mice are colonized with a complex microbiota.
The microbiomes of animals are complex communities that strongly affect the health of the hosts. Microbiomes on mucosal surfaces have the highest densities and most extensive biochemical exchanges with the hosts. Although antibiotics are potent tools to manage infections, they can disrupt the normal microbiota, causing numerous side effects.Taking a community ecology approach, mucosal microbiome community responses to five disruptive conditions (two broad-spectrum antibiotics, a biocide, elevated temperature, and rinsing) were analyzed. Skin of the fish Gambusia affinis was the mucosal model. Microbiome recovery was measured by culturable counts, community biochemical profiles, genetic fingerprinting, and community 16S gene sequencing (rinsing condition only).Following all disruptions, the total counts rose and then returned to the pre-treatment (PT) level. This overgrowth was confirmed via direct staining and community metabolic activity measurements. After rinsing, diversity decreased and one taxon dominated (family Aeromonadaceae) temporarily, the findings similar to numerous other studies with antibiotics. While the community did not return to the PT taxonomic composition, the biochemical profile did.This suggests that the biochemical pathways in a community are important during recovery, and a return to the original composition is not required to restore original function.
The incidence of infections due to diverse Candida species is increasing, with correspondingly different antifungal susceptibility patterns. Routine yeast identification methods cause significant delays in appropriate patient management.A DNA pyrosequencing strategy was evaluated for identification of pathogenic Candida species associated with human infections.Clinical (n = 51) and commercial (n = 9) Candida isolates were identified in a blinded, parallel study consisting of routine fungal cultures and biochemical analyses in comparison with DNA pyrosequencing.DNA pyrosequencing yielded species-level identification of all 60 Candida isolates, and sequencing interpretations agreed in all cases with results of biochemical and morphologic testing. Different Candida species were identified, such as C. albicans, C. dubliniensis, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis. Automated and manual approaches to DNA sequence interpretation, each coupled with the Identifire identification software, demonstrated 100% agreement with respect to Candida species identification. Twenty-one isolates yielded intraspecies DNA sequence differences (90%-98% nucleic acid sequence identity) by automated interpretation. Sequence differences resulted from single-nucleotide polymorphisms or single-base additions/deletions, in addition to interpretative challenges in homopolymeric tracts.DNA pyrosequencing coupled with automated DNA sequence alignment provides a practical approach for accurate and timely identification of Candida pathogens. Relatively rapid and facile genotypic studies by DNA pyrosequencing matched the effectiveness of extensive biochemical/morphologic studies for yeast identification.
Background: Little is known regarding the clinical impact of treatment and treatment duration of probiotic VSL#3 on gut and microbiome function in irritable bowel syndrome (IBS). As part of a safety trial, we assessed the effect of VSL#3 treatment duration on abdominal pain, stooling, gut permeability, microbiome composition and function. Methods: Adults with IBS were randomized into an open label trial to receive the probiotic VSL#3 for 4 or 8 weeks. Adverse events, abdominal pain, and stooling patterns were recorded daily. Gut permeability, fecal bile acid levels, and microbiome composition were profiled at baseline and after treatment. Results: Fifteen subjects completed the trial (4-week: n = 8; 8-week: n = 7). Number of pain episodes decreased in both groups (P = 0.049 and P = 0.034; 4- vs. 8-week, respectively). Probiotic organisms contained in VSL#3 were detected in feces by whole shotgun metagenomic sequencing analysis and relative abundances of Streptococcus thermophilus, Bifidobacterium animalis, Lactobacillus plantarum, and Lactobacillus casei subsp. paraccasei correlated significantly with improved abdominal pain symptoms and colonic permeability at study completion. Although abdominal pain correlated significantly with the detection of probiotic species at study completion, a composite view of gut microbiome structure showed no changes in community diversity or composition after VSL#3 treatment. Conclusions: Probiotic organisms identified in stool correlated significantly with improvement in colonic permeability and clinical symptoms, prompting future studies to investigate the mechanistic role of VSL#3 and colonic permeability in IBS pathophysiology in a larger randomized controlled trial. Clinical Trial Registration: www.clinicaltrials.gov, Identifier: NCT00971711.
