Early life establishment of site-specific microbial communities in the gut.
ABSTRACT: Fecal sampling is widely utilized to define small intestinal tissue-level microbial communities in healthy and diseased newborns. However, this approach may lead to inaccurate assessments of disease or therapeutics in newborns because of the assumption that the taxa in the fecal microbiota are representative of the taxa present throughout the gastrointestinal tract. To assess the stratification of microbes in the newborn gut and to evaluate the probable shortcoming of fecal sampling in place of tissue sampling, we simultaneously compared intestinal mucosa and fecal microbial communities in 15 neonates undergoing intestinal resections. We report three key results. First, when the site of fecal and mucosal samples are further apart, their microbial communities are more distinct, as indicated by low mean Sørensen similarity indices for each patient's fecal and tissue microbiota. Second, two distinct niches (intestinal mucosa and fecal microbiota) are evident by principal component analyses, demonstrating the critical role of sample source in defining microbial composition. Finally, in contrast to adult studies, intestinal bacterial diversity was higher in tissue than in fecal samples. This study represents an unprecedented map of the infant microbiota from intestinal mucosa and establishes discernable biogeography throughout the neonatal gastrointestinal tract. Our results question the reliance on fecal microbiota as a proxy for the developing intestinal microbiota. Additionally, the robust intestinal tissue-level bacterial diversity we detected at these early ages may contribute to the maturation of mucosal immunity.
Project description:The mucosal microbiota lives in close proximity with the intestinal epithelium and may interact more directly with the host immune system than the luminal/fecal bacteria. The availability of nutrients in the mucus layer of the epithelium is also very different from the gut lumen environment. Inferred metagenomic analysis for microbial function of the mucosal microbiota is possible by PICRUSt. We recently found that by using this approach, actively inflamed tissue of ulcerative colitis (UC) patients have mucosal communities enriched for genes involved in lipid and amino acid metabolism, and reduced for carbohydrate and nucleotide metabolism. Here, we find that the same bacterial taxa (e.g. Acinetobacter) and predicted microbial pathways enriched in actively inflamed colitis tissue are also enriched in the mucosa of subjects undergoing routine screening colonoscopies, when compared with paired samples of luminal/fecal bacteria. These results suggest that the mucosa of healthy individuals may be a reservoir of aerotolerant microbial communities expanded during colitis.
Project description:The gut microbiome is widely studied by fecal sampling, but the extent to which stool reflects the commensal composition at intestinal sites is poorly understood. We investigated this relationship in rhesus macaques by 16S sequencing feces and paired lumenal and mucosal samples from ten sites distal to the jejunum. Stool composition correlated highly with the colonic lumen and mucosa and moderately with the distal small intestine. The mucosal microbiota varied most based on location and was enriched in oxygen-tolerant taxa (e.g., Helicobacter and Treponema), while the lumenal microbiota showed inter-individual variation and obligate anaerobe enrichment (e.g., Firmicutes). This mucosal and lumenal community variability corresponded to functional differences, such as nutrient availability. Additionally, Helicobacter, Faecalibacterium, and Lactobacillus levels in stool were highly predictive of their abundance at most other gut sites. These results quantify the composition and biogeographic relationships between gut microbial communities in macaques and support fecal sampling for translational studies.
Project description:The gut microbiome is a community of host-associated symbiotic microbes that fulfills multiple key roles in host metabolism, immune function, and tissue development. Given the ability of the microbiome to impact host fitness, there is increasing interest in studying the microbiome of wild animals to better understand these communities in the context of host ecology and evolution. Human microbiome research protocols are well established, but wildlife microbiome research is still a developing field. Currently, there is no standardized set of best practices guiding the collection of microbiome samples from wildlife. Gut microflora are typically sampled either by fecal collection, rectal swabbing, or by destructively sampling the intestinal contents of the host animal. Studies rarely include more than one sampling technique and no comparison of these methods currently exists for a wild mammal. Although some studies have hypothesized that the fecal microbiome is a nested subset of the intestinal microbiome, this hypothesis has not been formally tested. To address these issues, we examined guano (feces) and distal intestinal mucosa from 19 species of free-ranging bats from Lamanai, Belize, using 16S rRNA amplicon sequencing to compare microbial communities across sample types. We found that the diversity and composition of intestine and guano samples differed substantially. In addition, we conclude that signatures of host evolution are retained by studying gut microbiomes based on mucosal tissue samples, but not fecal samples. Conversely, fecal samples retained more signal of host diet than intestinal samples. These results suggest that fecal and intestinal sampling methods are not interchangeable, and that these two microbiotas record different information about the host from which they are isolated.
Project description:Recent research suggests that the microbial colonization of the mammalian intestine may begin before birth, but the observations are controversial due to challenges in the reliable sampling and analysis of low-abundance microbiota. We studied the perinatal microbiota of calves by sampling them immediately at birth and during the first postnatal week. The large size of the bovine newborns allows sampling directly from rectum using contamination-shielded swabs. Our 16S rDNA data, purged of potential contaminant sequences shared with negative controls, indicates the existence of a diverse low-abundance microbiota in the newborn rectal meconium and mucosa. The newborn rectal microbiota was composed of Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. The microbial profile resembled dam oral rather than fecal or vaginal vestibular microbiota, but included typical intestinal taxa. During the first postnatal day, the rectum was invaded by Escherichia/Shigella and Clostridia, and the diversity collapsed. By 7 days, diversity was again increasing. In terms of relative abundance, Proteobacteria were replaced by Firmicutes, Bacteroidetes and Actinobacteria, including Faecalibacterium, Bacteroides, Lactobacillus, Butyricicoccus and Bifidobacterium. Our observations suggest that mammals are seeded before birth with a diverse microbiota, but the microbiota changes rapidly in the early postnatal life.
Project description:Colorectal epithelial tumors occur spontaneously in dogs, and the pathogenesis seems to parallel that of humans. The development of human colorectal tumorigenesis has been linked to alterations in the composition of the intestinal microbiota. This study characterized the fecal- and mucosa-associated microbiota in dogs with colorectal epithelial tumors (n = 10). The fecal microbiota was characterized by 16S rDNA analysis and compared with that of control dogs (n = 13). We also determined the mucosa-associated microbiota composition in colonic tumor tissue (n = 8) and in adjacent non-tumor tissue (n = 5) by 16S rDNA- and rRNA profiling. The fecal microbial community structure in dogs with tumors was different from that of control samples and was distinguished by oligotypes affiliated with Enterobacteriaceae, Bacteroides, Helicobacter, Porphyromonas, Peptostreptococcus and Streptococcus, and lower abundance of Ruminococcaceae, Slackia, Clostridium XI and Faecalibacterium. The overall community structure and populations of mucosal bacteria were not different based on either the 16S rDNA or the 16S rRNA profile in tumor tissue vs. adjacent non-tumor tissue. However, the proportion of live, potentially active bacteria appeared to be higher in non-tumor tissue compared with tumor tissue and included Slackia, Roseburia, unclass. Ruminococcaeceae, unclass. Lachnospiraceae and Oscillibacter. Colorectal tumors are rarely diagnosed in dogs, but despite this limitation, we were able to show that dogs with colorectal tumors have distinct fecal microbiota profiles. These initial results support the need for future case-control studies that are adequately powered, as well as age-matched and breed-matched, in order to evaluate the influence of bacteria on colorectal cancer etiopathogenesis and to determine whether the bacteria may have potential as biomarkers in clinical settings.
Project description:OBJECTIVE:Necrotizing enterocolitis (NEC) is the most common surgical emergency in preterm infants, and pathogenesis associates with changes in the fecal microbiome. As fecal samples incompletely represent microbial communities in intestinal mucosa, we sought to determine the NEC tissue-specific microbiome and assess its contribution to pathogenesis. DESIGN:We amplified and sequenced the V1-V3 hypervariable region of the bacterial 16S rRNA gene extracted from intestinal tissue and corresponding fecal samples from 12 surgical patients with NEC and 14 surgical patients without NEC. Low quality and non-bacterial sequences were removed, and taxonomic assignment was made with the Ribosomal Database Project. Operational taxonomic units were clustered at 97%. We tested for differences between NEC and non-NEC samples in microbiome alpha- and beta-diversity and differential abundance of specific taxa between NEC and non-NEC samples. Additional analyses were performed to assess the contribution of other demographic and environmental confounding factors on the infant tissue and fecal microbiome. RESULTS:The fecal and tissue microbial communities were different. NEC was associated with a distinct microbiome, which was characterized by low diversity, higher abundances of Staphylococcus and Clostridium_sensu_stricto, and lower abundances of Actinomyces and Corynebacterium. Infant age and vancomycin exposure correlated with shifts in the tissue microbiome. CONCLUSION:The observed low diversity in NEC tissues suggests that NEC is associated with a bacterial bloom and a distinct mucosal bacterial community. The exact bacterial species that constitute the bloom varied by infant and were strongly influenced by age and exposure to vancomycin.
Project description:The gut-lymph node axis is a critical player in the symbiotic relationship between gut microbiota and the host. However, little is known about the impact of diet-related bacterial shifts in the gut lumen on bacterial translocation into lymph nodes. Here, we (i) characterized changes in the viable microbiota composition along the ileal digesta-mucosa-lymph node axis and (ii) examined the effect of dietary phytase supplementation and lactic acid (LA) soaking of cereals on the bacterial taxonomy along this axis, together with their effect on the mucosal expression of innate immune and barrier function genes in pigs (n?=?8/diet). After 18?days on diets, ileal digesta, mucosa, and ileocecal lymph nodes (ICLNs) were collected for RNA isolation and 16S rRNA-based high-resolution community profiling. Bacterial communities were dominated by Lactobacillaceae and Clostridiaceae, with clearly distinguishable profiles at the three sampling sites. Specific bacterial subsampling was indicated by enrichment of the ICLNs with Lactobacillaceae, Lachnospiraceae, Veillonellaceae, and Methanobacteriaceae and less Clostridiaceae, Pasteurellaceae, Helicobacteraceae, and Enterobacteriaceae compared to that of the mucosa. LA treatment of cereals reduced proteolytic taxa in the lumen, including pathobionts like Helicobacteraceae, Campylobacteraceae, and Fusobacteriaceae When combined, phytase- and LA-treated cereals largely increased species richness, while the single treatments reduced Actinobacteria and Bacteroidetes in ICLNs and increased mucosal MUC2 expression. In contrast, phytase reduced mucosal CDH1 expression, indicating altered barrier function with potential effects on bacterial translocation. Overall, both treatments, although often differently, changed the viable microbiome along the digesta-mucosa-lymph node axis in the ileum, probably due to altered substrate availability and microbial-host interactions.IMPORTANCE A host's diet largely determines the gut microbial composition and therefore may influence bacterial translocation into ICLNs. Due to its importance for cell metabolism, the intestinal phosphorus availability, which was modified here by phytase and LA treatment of cereals, affects the intestinal microbiota. Previous studies mainly focused on bacteria in the lumen. The novelty of this work resides mainly in that we report diet-microbe effects along the digesta-mucosa-ICLN axis and linked those effects to mucosal expression of barrier function genes as crucial components for host health. Lymph nodes can serve as reservoir of pathobionts; therefore, present diet-microbiome-host interactions have implications for food safety.
Project description:Intestinal microbiota play an important role in maintaining the homeostasis of the host immune system. To analyze the alteration of the intestinal microbial community structure in Korean Crohn's disease (CD) patients, we performed a comparative metagenomic analysis between healthy people and CD patients using fecal samples and mucosal tissues of ileocecal valve.16S rRNA genes from fecal samples or mucosal tissues of 35 CD patients and 15 healthy controls (HC) were amplified using a universal primer set and sequenced with GS FLX Titanium. The microbial composition and diversity of each sample were analyzed with the mothur pipeline, and the association between microbial community and clinical characteristics of the patients were investigated.The contribution of bacterial groups to the intestinal microbial composition differed between CD and HC, especially in fecal samples. Global structure and individual bacterial abundance of intestinal microbial community were different between feces and ileocecal tissues in HC. In CD patients with active stage, relative abundances of Gammaproteobacteria and Fusobacteria were higher in both fecal and mucosal tissue samples. Moreover, the intestinal microbial community structure was altered by anti-tumor necrosis factor (anti-TNF) treatment.Our 16S rRNA sequence data demonstrate intestinal dysbiosis at the community level in Korean CD patients, which is similar to alterations of the intestinal microbial community seen in the western counterparts. Clinical disease activity and anti-TNF treatment might affect the intestinal microbial community structure in CD patients.
Project description:An imbalance in the bacterial species resulting in the loss of intestinal homeostasis has been described in inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS). In this prospective study, we investigated whether IBD and IBS patients exhibit specific changes in richness and distribution of fecal and mucosal-associated microbiota. Additionally, we assessed potential 16S rRNA gene amplicons biomarkers for IBD, IBS, and controls (CTRLs) by comparison of taxonomic composition. The relative abundance of bacteria, at phylum and genus/species levels, and the bacterial diversity were determined through 16S rRNA sequence-based fecal and mucosal microbiota analysis. Linear discriminant analysis effect size (LEfSe) was used for biomarker discovery associated to IBD and IBS as compared to CTRLs. In fecal and mucosal samples, the microbiota richness was characterized by a microbial diversity reduction, going from CTRLs to IBS to IBD. ?-diversity analysis showed a clear separation between IBD and CTRLs and between IBD and IBS with no significant separation between IBS and CTRLs. ?-diversity showed a clear separation between mucosa and stool samples in all the groups. In IBD, there was no difference between inflamed and not inflamed mucosa. Based upon the LEfSe data, the Anaerostipes and Ruminococcaceae were identified as the most differentially abundant bacterial taxa in CTRLs. Erysipelotrichi was identified as potential biomarker for IBS, while Gammaproteobacteria, Enterococcus, and Enterococcaceae for IBD. This study provides an overview of the alterations of microbiota and may aid in identifying potential 16S rRNA gene amplicons mucosal biomarkers for IBD and IBS.
Project description:The mammalian intestinal tract is heavily colonized with a dense, complex, and diversified microbial populations. In healthy individuals, an array of epithelial antimicrobial agents is secreted in the gut to aid intestinal homeostasis. Enterochromaffin cells (EC) in the intestinal epithelium are a major source of chromogranin A (CgA), which is a pro-hormone and can be cleaved into many bioactive peptides that include catestatin (CST). This study was carried out to evaluate the possible impact of CST on gut microbiota in vivo using a mouse model. The CST (Human CgA352-372) or normal saline was intrarectally administered in C57BL/6 male mice for 6 days and then sacrificed. Feces and colonic mucosa tissue samples were collected, DNA was extracted, the V4 region of bacterial 16S rRNA gene was amplified and subjected to MiSeq Illumina sequencing. The α-diversity was calculated using Chao 1 and β-diversity was determined using QIIME. Differences at the genus level were determined using partial least square discriminant analysis (PLS-DA). Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was used to predict functional capacity of bacterial community. CST treatment did not modify bacterial richness in fecal and colonic mucosa-associated microbiota; however, treatment significantly modified bacterial community composition between the groups. Also, CST-treated mice had a significantly lower relative abundance of Firmicutes and higher abundance of Bacteroidetes, observed only in fecal samples. However, at lower phylogenetic levels, PLS-DA analysis revealed that some bacterial taxa were significantly associated with the CST-treated mice in both fecal and colonic mucosa samples. In addition, differences in predicted microbial functional pathways in both fecal and colonic mucosa samples were detected. The results support the hypothesis that CST treatment modulates gut microbiota composition under non-pathophysiological conditions, however, the result of this study needs to be further validated in a larger experiment. The data may open new avenues for the development of a potential new line of antimicrobial peptides and their use as therapeutic agents to treat several inflammatory conditions of the gastrointestinal tract, such as inflammatory bowel disease (IBD), inflammatory bowel syndrome (IBS), or other health conditions.