Project description:Recent advances in (meta)genomic methods have provided new opportunities to examine host-microbe-environment interactions in the human gut. While opportunities exist to extract DNA from freshly sourced colonic tissue there are potentially valuable sources of DNA from historical studies that might also be examined. We examined how four different tissue DNA extraction methods employed in past clinical trials might impact the recovery of microbial DNA from a colonic tissue sample as assessed using a custom designed phylogenetic microarray for human gut bacteria and archaebacteria. While all methods of DNA extraction produced similar phylogenetic profiles some extraction specific biases were also observed. Real time PCR analysis targeting several bacterial groups substantiated this observation. These data suggest that while the efficacy of different DNA extraction methods differs somewhat all the methods tested produce an accurate representation of microbial diversity. This suggests that DNA samples archived in biobanks should be suitable for retrospective analyses.
Project description:Recent advances in (meta)genomic methods have provided new opportunities to examine host-microbe-environment interactions in the human gut. While opportunities exist to extract DNA from freshly sourced colonic tissue there are potentially valuable sources of DNA from historical studies that might also be examined. We examined how four different tissue DNA extraction methods employed in past clinical trials might impact the recovery of microbial DNA from a colonic tissue sample as assessed using a custom designed phylogenetic microarray for human gut bacteria and archaebacteria. While all methods of DNA extraction produced similar phylogenetic profiles some extraction specific biases were also observed. Real time PCR analysis targeting several bacterial groups substantiated this observation. These data suggest that while the efficacy of different DNA extraction methods differs somewhat all the methods tested produce an accurate representation of microbial diversity. This suggests that DNA samples archived in biobanks should be suitable for retrospective analyses. Three technical replicates per sample (extraction method) were analysed
Project description:The gut microbiome is significantly altered in inflammatory bowel diseases, but the basis of these changes is not well understood. We have combined metagenomic and metatranscriptomic profiling of the gut microbiome to assess changes to both bacterial community structure and transcriptional activity in a mouse model of colitis. Gene families involved in microbial resistance to oxidative stress, including Dps/ferritin, Fe-dependent peroxidase and glutathione S-transferase, were transcriptionally up-regulated in colitis, implicating a role for increased oxygen tension in gut microbiota modulation. Transcriptional profiling of the host gut tissue and host RNA in the gut lumen revealed a marked increase in the transcription of genes with an activated macrophage and granulocyte signature, suggesting the involvement of these cell types in influencing microbial gene expression. Down-regulation of host glycosylation genes further supports a role for inflammation-driven changes to the gut niche that may impact the microbiome. We propose that members of the bacterial community react to inflammation-associated increased oxygen tension by inducing genes involved in oxidative stress resistance. Furthermore, correlated transcriptional responses between host glycosylation and bacterial glycan utilisation support a role for altered usage of host-derived carbohydrates in colitis. Complementary transcription profiling data from the mouse hosts have also been deposited at ArrayExpress under accession number E-MTAB-3590 ( http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3590/ ).
Project description:The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. To identify microbial genes required for establishment of human symbionts in the gut, we developed an approach (insertion-sequencing, or INSeq) based on a mutagenic transposon that allows capture of adjacent chromosomal DNA to define its genomic location. We used massively parallel sequencing to monitor the relative abundance of tens of thousands of transposon mutants of a saccharolytic human gut bacterium, Bacteroides thetaiotaomicron, as they established themselves in wild-type and immunodeficient gnotobiotic mice, in the presence or absence of other human gut commensals. In vivo selection transforms this population, revealing functions necessary for survival in the gut: we show how this selection is influenced by community composition and competition for nutrients (vitamin B12). INSeq provides a broadly applicable platform to explore microbial adaptation to the gut and other ecosystems. Keywords: Other 57 samples analyzed, 1 of these is the reference (input) sample
Project description:The gut microbiome is significantly altered in inflammatory bowel diseases, but the basis of these changes is not well understood. We have combined metagenomic and metatranscriptomic profiling of the gut microbiome to assess changes to both bacterial community structure and transcriptional activity in a mouse model of colitis. Gene families involved in microbial resistance to oxidative stress, including Dps/ferritin, Fe-dependent peroxidase and glutathione S-transferase, were transcriptionally up-regulated in colitis, implicating a role for increased oxygen tension in gut microbiota modulation. Transcriptional profiling of the host gut tissue and host RNA in the gut lumen revealed a marked increase in the transcription of genes with an activated macrophage and granulocyte signature, suggesting the involvement of these cell types in influencing microbial gene expression. Down-regulation of host glycosylation genes further supports a role for inflammation-driven changes to the gut niche that may impact the microbiome. We propose that members of the bacterial community react to inflammation-associated increased oxygen tension by inducing genes involved in oxidative stress resistance. Furthermore, correlated transcriptional responses between host glycosylation and bacterial glycan utilisation support a role for altered usage of host-derived carbohydrates in colitis. Complementary RNA-seq and DNA-seq data sets of the microbiome from this study have also been deposited at ArrayExpress under accession number E-MTAB-3562 ( http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3562/ ).
Project description:Monitoring microbial communities can aid in understanding the state of these habitats. Environmental DNA (eDNA) techniques provide efficient and comprehensive monitoring by capturing broader diversity. Besides structural profiling, eDNA methods allow the study of functional profiles, encompassing the genes within the microbial community. In this study, three methodologies were compared for functional profiling of microbial communities in estuarine and coastal sites in the Bay of Biscay. The methodologies included inference from 16S metabarcoding data using Tax4Fun, GeoChip microarrays, and shotgun metagenomics.
Project description:The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. To identify microbial genes required for establishment of human symbionts in the gut, we developed an approach (insertion-sequencing, or INSeq) based on a mutagenic transposon that allows capture of adjacent chromosomal DNA to define its genomic location. We used massively parallel sequencing to monitor the relative abundance of tens of thousands of transposon mutants of a saccharolytic human gut bacterium, Bacteroides thetaiotaomicron, as they established themselves in wild-type and immunodeficient gnotobiotic mice, in the presence or absence of other human gut commensals. In vivo selection transforms this population, revealing functions necessary for survival in the gut: we show how this selection is influenced by community composition and competition for nutrients (vitamin B12). INSeq provides a broadly applicable platform to explore microbial adaptation to the gut and other ecosystems. Keywords: Other
Project description:Background: More than 100 million Americans are living with metabolic syndrome, increasing their propensity to develop heart disease– the leading cause of death worldwide. A major contributing factor to this epidemic is caloric excess, often a result of consuming low cost, high calorie fast food. Several recent seminal studies have demonstrated the pivotal role of gut microbes contributing to cardiovascular disease in a diet-dependent manner. Given the central contributions of diet and gut microbiota to cardiometabolic disease, we hypothesized that novel microbial metabolites originating postprandially after fast food consumption may contribute to cardiometabolic disease progression. Methods: To test this hypothesis, we gave conventionally raised or antibiotic-treated mice a single oral gavage of a fast food slurry or a control rodent chow diet slurry and sacrificed the mice four hours later. Here, we coupled untargeted metabolomics in portal and peripheral blood, 16S rRNA gene sequencing, targeted liver metabolomics, and host liver RNA sequencing to identify novel fast food-derived microbial metabolites. Results: We successfully identified several metabolites that were enriched in portal blood, increased by fast food feeding, and essentially absent in antibiotic-treated mice. Strikingly, just four hours post-gavage, we found that fast food consumption resulted in rapid reorganization of the gut microbial community structure and drastically altered hepatic gene expression. Importantly, diet-driven reshaping of the microbiome and liver transcriptome was dependent on a non-antibiotic ablated gut microbial community. Conclusions: Collectively, these data suggest that single fast food meal is sufficient to reshape the gut microbial community yielding a unique signature of food-derived microbial metabolites. Future studies are warranted to determine if these metabolites are causally linked to cardiometabolic disease.