Natalia Borruel

Enterotypes of the human gut microbiome

Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host–microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.

The gut microbiota in IBD

IBD—ulcerative colitis and Crohns disease—is emerging as a worldwide epidemic. An association between the increased incidence of IBD and environmental factors linked to socioeconomic development has been persistently detected in different parts of the world. The lifestyle in developed countries might impair the natural patterns of microbial colonization of the human gut. The interaction of microbes with mucosal immune compartments in the gut seems to have a major role in priming and regulating immunity. In IBD, mucosal lesions are generated by an excessive or dysregulated immune response against commensal microbes in the gut. In individuals with a genetic susceptibility to IBD, abnormal microbial colonization of the gastrointestinal tract might be the origin of such dysregulation. Developments in gene-sequencing technologies, as well as increased availability of powerful bioinformatic tools, have enabled novel insights into the microbial composition of the human gut microbiota and the effect of microbial communities on human physiology and disease. Studies that used these technologies indicate that dysbiosis (that is, abnormal microbiota composition) and decreased complexity of the gut microbial ecosystem are common features in patients with Crohns disease or ulcerative colitis. Whether such changes are a cause or a consequence of the disease remains to be elucidated.

Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes.

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

Increased mucosal tumour necrosis factor alpha production in Crohn's disease can be downregulated ex vivo by probiotic bacteria.

Background and aims: Tumour necrosis factor α (TNF-α) plays a key role in the pathogenesis of intestinal inflammation in Crohn’s disease. The effect of bacteria on TNF-α release by intestinal mucosa was investigated. Methods: Ileal specimens were obtained at surgery from 10 patients with Crohn’s disease (ileal stricture) and five disease controls undergoing right hemicolectomy (caecal cancer). Mucosal explants from each specimen were cultured for 24 hours with either non-pathogenic Escherichia coli, Lactobacillus casei DN-114001, L bulgaricus LB10, or L crispatus (each study contained blank wells with no bacteria). Tissue and bacterial viability was confirmed by lactate dehydrogenase (LDH) release and culture. Concentrations of TNF-α were measured in supernatants and the phenotype of the intestinal lymphocytes was analysed by flow cytometry. Results: Coculture of mucosa with bacteria did not modify LDH release. Release of TNF-α by inflamed Crohn’s disease mucosa was significantly reduced by coculture with L casei or L bulgaricus; changes induced by L crispatus or E coli were not significant. The effect of L casei and L bulgaricus was not prevented by protease inhibitors. Coculture with L casei and L bulgaricus reduced the number of CD4 cells as well as TNF-α expression among intraepithelial lymphocytes from Crohn’s disease mucosa. None of the bacteria induced changes in non-inflamed mucosa. Conclusions: Probiotics interact with immunocompetent cells using the mucosal interface and modulate locally the production of proinflammatory cytokines.

Unstable Composition of the Fecal Microbiota in Ulcerative Colitis During Clinical Remission

BACKGROUND AND AIM:Studies have identified abnormal characteristics of the gut microbiota in patients with active IBD, but whether the changes are causal or secondary to inflammation remains uncertain. We investigated dynamics of fecal microbiota in ulcerative colitis (UC) during remission by genomic technology.PATIENTS AND METHODS:Patients in clinical remission and on stable maintenance mesalazine therapy were recruited (N = 33). Fecal samples were collected at regular intervals over a period of 1 yr. Sixteen patients who remained in remission and eight healthy controls were included in the analysis. Variable V6 to V8 regions of the 16S rRNA gene in DNA extracts from fecal samples were amplified by polymerase chain reaction. Amplicons were separated by denaturant gradient gel electrophoresis, band profiles were compared by software, and similarity indices were calculated from densitometric curves.RESULTS:Band profiles showed unique patterns with low similarity index between individuals, suggesting host specificity in the predominant microbiota. Within the same individual, profiles were stable in controls but varied notably over time in patients. In controls, the similarity index was remarkably stable (78 ± 8% mean ± SD) over a period of 24 months. However, patients showed a steady decline in similarity index versus the initial profile, dropping down to 42 ± 24% at month 3 of follow-up and to 23 ± 19% at month 12 (P < 0.001). Biodiversity of the dominant microbiota, as estimated by number of bands, was lower in patients (17 ± 4) than controls (23 ± 4, P < 0.01).CONCLUSION:Molecular analysis of fecal bacteria in patients with inactive UC shows low biodiversity and temporal instability.

Effects of Nonpathogenic Bacteria on Cytokine Secretion by Human Intestinal Mucosa

OBJECTIVE:The human intestine harbors a complex microbial ecosystem, and the mucosa is the interface between the immune system and the luminal environment. The aim of this study was to elucidate whether host–bacteria interactions influence mucosal cytokine production.METHODS:Macroscopically normal colonic specimens were obtained at surgery from eight patients with neoplasm, and inflamed ileal specimens were obtained from two patients with Crohns disease. Mucosal explants were cultured for 24 h with either nonpathogenic Escherichia coli ECOR-26, Lactobacillus casei DN-114 001, L. casei DN-114 056, L. casei ATCC-334, or Lactobacillus bulgaricus LB-10. Each study included blank wells with no bacteria. Tissue and bacteria viability were confirmed by LDH release and culture. Concentration of tumor necrosis factor (TNF)α, transforming growth factor β1, interleukin (IL)-8, and IL-10 was measured in supernatants. In parallel experiments, neutralizing anti-TNFα antibody was added to the culture.RESULTS:Co-culture of mucosa with bacteria did not modify LDH release. Co-culture with L. casei strains significantly reduced TNFα release, whereas E. coli increased it. These effects were observed both in normal and inflamed mucosa. In combination studies, L. casei DN-114 001 prevented TNFα stimulation by E. coli. L. casei DN-114 001 also reduced IL-8 release via a TNFα-independent pathway. L. casei DN-114 056 or E. coli increased IL-10 release in the presence of neutralizing anti-TNFα.CONCLUSIONS:Nonpathogenic bacteria interact with human intestinal mucosa and can induce changes in cytokine production that are strain specific.

Oral oligofructose-enriched inulin supplementation in acute ulcerative colitis is well tolerated and associated with lowered faecal calprotectin

Background Inulin and oligofructose promote selective growth of saccharolytic bacteria with low inflammatory potential.

Colonisation by Faecalibacterium prausnitzii and maintenance of clinical remission in patients with ulcerative colitis

Although incrimination of the intestinal microbiota in the pathogenesis of IBD is widely accepted, few data are available about the role of specific bacteria. Potentially, Faecalibacterium prausnitzii, bacteria with anti‐inflammatory properties, might be deficient in ulcerative colitis (UC).

Erratum: Enterotypes of the human gut microbiome (Nature (2011) 473 (174-180))

This corrects the article DOI: 10.1038/nature09944

Lactobacillus casei downregulates commensals' inflammatory signals in Crohn's disease mucosa

Background: The interaction of commensal bacteria with the intestinal immune system is an essential factor in the development of inflammatory bowel disease (IBD). The study of isolated commensal bacterias effects on the mucosal immune response might be relevant for a better understanding of pathophysiological mechanisms in IBD. Methods: We investigated the immune responses to signals from the commensal Escherichia coli ATCC 35345 and the probiotic Lactobacillus casei DN‐114 001 in Crohns disease (CD) mucosa. Ileal specimens were obtained during surgery from CD patients. Mucosal explants were incubated with L. casei or its genomic DNA; TNF‐&agr;, IFN‐&ggr;, IL‐2, IL‐6, IL‐8, and CXCL1 were measured in the supernatant. Second, tissue expression of key proinflammatory cytokines (IL‐6, TGF‐&bgr;, IL‐23p19, IL‐12p35, IL‐17F), and chemokines (IL‐8, CXCL1, CXCL2) was evaluated after incubation with L. casei or E. coli. Finally, combination experiments were carried out by incubating both strains with mucosal explants at different timepoints. Results: Live L. casei significantly decreased secretion of TNF‐&agr;, IFN‐&ggr;, IL‐2, IL‐6, IL‐8, and CXCL1 by CD mucosa, but the effect was not reproduced by L. casei DNA. Second, live L. casei downregulated expression of IL‐8, IL‐6, and CXCL1 and did not modify expression of IL‐23p19, IL‐12p35, and IL‐17F. In contrast, E. coli significantly upregulated expression of all these cytokines. Interestingly, combination experiments revealed the ability of L. casei to prevent and counteract the proinflammatory effects of E. coli. Conclusions: Live L. casei can counteract the proinflammatory effects of E. coli on CD inflamed mucosa by specific downregulation of key proinflammatory mediators.