A novel stepwise integrative analysis pipeline reveals distinct microbiota-host interactions and link to symptoms in irritable bowel syndrome

Although incompletely understood, microbiota-host interactions are assumed to be altered in irritable bowel syndrome (IBS). We, therefore, aimed to develop a novel analysis pipeline tailored for the integrative analysis of microbiota-host interactions and association to symptoms and prove its utility in a pilot cohort. A multilayer stepwise integrative analysis pipeline was developed to visualize complex variable associations. Application of the pipeline was demonstrated on a dataset of IBS patients and healthy controls (HC), using the R software package to analyze colonic host mRNA and mucosal microbiota (16S rRNA gene sequencing), as well as gastrointestinal (GI) and psychological symptoms. In total, 42 IBS patients (57% female, mean age 33.6 (range 18–58)) and 20 HC (60% female, mean age 26.8 (range 23–41)) were included. Only in IBS patients, mRNA expression of Toll-like receptor 4 and genes associated with barrier function (PAR2, OCLN, TJP1) intercorrelated closely, suggesting potential functional relationships. This host genes-based “permeability cluster” was associated to mucosa-adjacent Chlamydiae and Lentisphaerae, and furthermore associated to satiety as well as to anxiety, depression and fatigue. In both IBS patients and HC, chromogranins, secretogranins and TLRs clustered together. In IBS patients, this host genes-based “immune-enteroendocrine cluster” was associated to specific members of Firmicutes, and to depression and fatigue, whereas in HC no significant association to microbiota was identified. We have developed a stepwise integrative analysis pipeline that allowed identification of unique host-microbiota intercorrelation patterns and association to symptoms in IBS patients. This analysis pipeline may aid in advancing the understanding of complex variable associations in health and disease

Functional Metagenomics: A High Throughput Screening Method to Decipher Microbiota-Driven NF-κB Modulation in the Human Gut

Background/Aim: The human intestinal microbiota plays an important role in modulation of mucosal immune responses. To study interactions between intestinal epithelial cells (IECs) and commensal bacteria, a functional metagenomic approach was developed. One interest of metagenomics is to provide access to genomes of uncultured microbes. We aimed at identifying bacterial genes involved in regulation of NF-kappa B signaling in IECs. A high throughput cell-based screening assay allowing rapid detection of NF-kappa B modulation in IECs was established using the reporter-gene strategy to screen metagenomic libraries issued from the human intestinal microbiota. Methods: A plasmid containing the secreted alkaline phosphatase (SEAP) gene under the control of NF-kappa B binding elements was stably transfected in HT-29 cells. The reporter clone HT-29/kb-seap-25 was selected and characterized. Then, a first screening of a metagenomic library from Crohn's disease patients was performed to identify NF-kappa B modulating clones. Furthermore, genes potentially involved in the effect of one stimulatory metagenomic clone were determined by sequence analysis associated to mutagenesis by transposition. Results: The two proinflammatory cytokines, TNF-alpha and IL-1 beta, were able to activate the reporter system, translating the activation of the NF-kappa B signaling pathway and NF-kappa B inhibitors, BAY 11-7082, caffeic acid phenethyl ester and MG132 were efficient. A screening of 2640 metagenomic clones led to the identification of 171 modulating clones. Among them, one stimulatory metagenomic clone, 52B7, was further characterized. Sequence analysis revealed that its metagenomic DNA insert might belong to a new Bacteroides strain and we identified 2 loci encoding an ABC transport system and a putative lipoprotein potentially involved in 52B7 effect on NF-kappa B. Conclusions: We have established a robust high throughput screening assay for metagenomic libraries derived from the human intestinal microbiota to study bacteria-driven NF-kappa B regulation. This opens a strategic path toward the identification of bacterial strains and molecular patterns presenting a potential therapeutic interest

Human gut metatranscriptome changes induced by a fermented milk product are associated with improved tolerance to a flatulogenic diet

Healthy plant-based diets rich in fermentable residues may induce gas-related symptoms, possibly mediated by the gut microbiota. We previously showed that consumption of a fermented milk product (FMP) containing Bifidobacterium animalis subsp. lactis CNCM I-2494 and lactic acid bacteria improved gastrointestinal (GI) comfort in response to a flatulogenic dietary challenge in healthy individuals. To study the effects of the FMP on gut microbiota activity from those participants, we conducted a metatranscriptomic analysis of fecal samples (n = 262), which were collected during the ingestion of a habitual diet and two series of a 3-day high-residue challenge diet, before and following 28-days of FMP consumption. Most of the FMP species were detected or found enriched upon consumption of the product. FMP mitigated the effect of a flatulogenic diet on gas-related symptoms in several ways. First, FMP consumption was associated with the depletion of gas-producing bacteria and increased hydrogen to methane conversion. It also led to the upregulation of activities such as replication and downregulation of functions related to motility and chemotaxis. Furthermore, upon FMP intake, metabolic activities such as carbohydrate metabolism, attributed to B. animalis and S. thermophilus, were enriched; these activities were coincidentally found to be negatively associated with several GI symptoms. Finally, a more connected microbial ecosystem or mutualistic relationship among microbes was found in responders to the FMP intervention. Taken together, these findings suggest that consumption of the FMP improved the tolerance of a flatulogenic diet through active interactions with the resident gut microbiota.This research was supported by a grant from Danone Nutricia Research. Danone Nutricia Research authors participated in the study design, interpretation of the data and in the writing of the report. Francisca Yáñez was supported by a fellowship from ANID, BECAS Chile, No. 72190278. Zixuan Xie received a fellowship from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Action, Innovative Training Network: FunHoMic; grant number 812969. CIBERHED is funded by the Instituto de Salud Carlos III

Identification of NF-κB Modulation Capabilities within Human Intestinal Commensal Bacteria

The intestinal microbiota plays an important role in modulation of mucosal immune responses. To seek interactions between intestinal epithelial cells (IEC) and commensal bacteria, we screened 49 commensal strains for their capacity to modulate NF-κB. We used HT-29/kb-seap-25 and Caco-2/kb-seap-7 intestinal epithelial cells and monocyte-like THP-1 blue reporter cells to measure effects of commensal bacteria on cellular expression of a reporter system for NF-κB. Bacteria conditioned media (CM) were tested alone or together with an activator of NF-κB to explore its inhibitory potentials. CM from 8 or 10 different commensal species activated NF-κB expression on HT-29 and Caco-2 cells, respectively. On THP-1, CM from all but 5 commensal strains stimulated NF-κB. Upon challenge with TNF-α or IL-1β, some CM prevented induced NF-κB activation, whereas others enhanced it. Interestingly, the enhancing effect of some CM was correlated with the presence of butyrate and propionate. Characterization of the effects of the identified bacteria and their implications in human health awaits further investigations

Individuality and temporal stability of the human gut microbiome

Introduction: The breakthrough of next generation sequencing-technologies has enabled large-scale studies of natural microbial communities and the 16S rRNA genes have been widely used as a phylogenetic marker to study community structure. However, major limitations of this approach are that neither strain-level resolution nor genomic context of microorganisms can be provided. This information, however, is crucial to answer fundamental questions about the temporal stability and distinctiveness of natural microbial communities.Material and methods: We developed a methodological framework for metagenomic single nucleotide polymorphism (SNP) variation analysis and applied it to publicly available data from 252 human fecal samples from 207 European and North American individuals. We further analyzed samples from 43 healthy subjects that were sampled at least twice over time intervals of up to one year and measured population similarities of dominant gut species.Results: We detected 10.3 million SNPs in 101 species, which nearly amounts to the number identified in more than 1,000 humans.Conclusion: The most striking result was that host-specific strains appear to be retained over long time periods. This indicates that individual-specific strains are not easily exchanged with the environment and furthermore, that an individuals appear to have a unique metagenomic genotype. This, in turn, is linked to implications for human gut physiology, such as the stability of antibiotic resistance potential

Curated and harmonized gut microbiome 16S rRNA amplicon data from dietary fiber intervention studies in humans

Next generation amplicon sequencing has created a plethora of data from human microbiomes. The accessibility to this scientific data and its corresponding metadata is important for its reuse, to allow for new discoveries, verification of published results, and serving as path for reproducibility. Dietary fiber consumption has been associated with a variety of health benefits that are thought to be mediated by gut microbiota. To enable direct comparisons of the response of the gut microbiome to fiber, we obtained 16S rRNA sequencing data and its corresponding metadata from 11 fiber intervention studies for a total of 2,368 samples. We provide curated and pre-processed genetic data and common metadata for comparison across the different studies

Curated and Harmonized Gut Microbiome 16S rRNA Amplicon Data From Dietary Fiber Intervention Studies in Humans

Next generation amplicon sequencing has created a plethora of data from human microbiomes. The accessibility to this scientific data and its corresponding metadata is important for its reuse, to allow for new discoveries, verification of published results, and serving as path for reproducibility. Dietary fiber consumption has been associated with a variety of health benefits that are thought to be mediated by gut microbiota. To enable direct comparisons of the response of the gut microbiome to fiber, we obtained 16S rRNA sequencing data and its corresponding metadata from 11 fiber intervention studies for a total of 2,368 samples. We provide curated and pre-processed genetic data and common metadata for comparison across the different studies

Microbial Dysbiosis in Colorectal Cancer (CRC) Patients

The composition of the human intestinal microbiota is linked to health status. The aim was to analyze the microbiota of normal and colon cancer patients in order to establish cancer-related dysbiosis

Prediction of the intestinal resistome by a three-dimensional structure-based method

The intestinal microbiota is considered to be a major reservoir of antibiotic resistance determinants (ARDs) that could potentially be transferred to bacterial pathogens via mobile genetic elements. Yet, this assumption is poorly supported by empirical evidence due to the distant homologies between known ARDs (mostly from culturable bacteria) and ARDs from the intestinal microbiota. Consequently, an accurate census of intestinal ARDs (that is, the intestinal resistome) has not yet been fully determined. For this purpose, we developed and validated an annotation method (called pairwise comparative modelling) on the basis of a three-dimensional structure (homology comparative modelling), leading to the prediction of 6,095 ARDs in a catalogue of 3.9 million proteins from the human intestinal microbiota. We found that the majority of predicted ARDs (pdARDs) were distantly related to known ARDs (mean amino acid identity 29.8%) and found little evidence supporting their transfer between species. According to the composition of their resistome, we were able to cluster subjects from the MetaHIT cohort (n = 663) into six resistotypes that were connected to the previously described enterotypes. Finally, we found that the relative abundance of pdARDs was positively associated with gene richness, but not when subjects were exposed to antibiotics. Altogether, our results indicate that the majority of intestinal microbiota ARDs can be considered intrinsic to the dominant commensal microbiota and that these genes are rarely shared with bacterial pathogens

Genomic variation landscape of the human gut microbiome

While large-scale efforts have rapidly advanced the understanding and practical impact of human genomic variation, the latter is largely unexplored in the human microbiome. We therefore developed a framework for metagenomic variation analysis and applied it to 252 fecal metagenomes of 207 individuals from Europe and North America. Using 7.4 billion reads aligned to 101 reference species, we detected 10.3 million single nucleotide polymorphisms (SNPs), 107,991 short indels, and 1,051 structural variants. The average ratio of non-synonymous to synonymous polymorphism rates of 0.11 was more variable between gut microbial species than across human hosts. Subjects sampled at varying time intervals exhibited individuality and temporal stability of SNP variation patterns, despite considerable composition changes of their gut microbiota. This implies that individual-specific strains are not easily replaced and that an individual might have a unique metagenomic genotype, which may be exploitable for personalized diet or drug intake