Francesc Casellas

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.

Diarrhoea-predominant IBS patients show mast cell activation and hyperplasia in the jejunum

Background: Increased numbers of mast cells and mast cell activation in distal gut segments are associated with symptom onset and severity in irritable bowel syndrome (IBS). Although upper gut symptoms are common, mast cells have not been thoroughly evaluated in proximal gut in IBS patients. Methods: Jejunal biopsies obtained by Watson’s capsule, aspiration of intestinal fluid and one blood sample were obtained in 20 diarrhoea-predominant patients with IBS (D-IBS) and 14 healthy volunteers (H). Psychological stress (Holmes-Rahe Scale) and depression (Beck’s Depression Inventory) were evaluated at baseline and food and respiratory allergy excluded. Biopsies were processed for H&E staining and microscopic inflammation assessed by counting intraepithelial lymphocytes. Mast cells in lamina propria were counted by immunohistochemistry with CD117 (c-kit). Tryptase concentration was measured in intestinal fluid and serum. Results: D-IBS patients showed higher psychological stress than healthy volunteers (D-IBS: 203 (SD 114) v H: 112 (SD 99); p = 0.019). Immunohistochemical staining of jejunal mucosa revealed mild increase in intraepithelial CD3+ cells in D-IBS patients (D-IBS: 15.3 (SD 5.5; 95% CI 12.7 to 17.9) v H: 10.3 (SD 3.9; 95% CI 8.0 to 12.5); p = 0.006). Moreover, D-IBS patients showed marked increase in mast cells numbers (D-IBS: 34 (SD 9.3); H: 15.3 (SD 4.4) mast cells/hpf; p<0.001) and higher tryptase concentration in jejunal fluid (D-IBS: 0.45 (SD 0.38); H: 0.09 (SD 0.10) µg/l; p = 0.005). Upper gut symptoms were not associated with gender, mast cell counts, jejunal tryptase or basal stress. Conclusion: This jejunal mucosal inflammatory profile may help identify diarrhoea-predominant IBS, a stress-related disorder.

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.

Influence of inflammatory bowel disease on different dimensions of quality of life.

Objective To establish the impairment of different dimensions of quality of life in inflammatory bowel disease (IBD). Design Prospective observational study. Participants 289 patients [160 with ulcerative colitis (UC) and 129 with Crohns disease (CD)]. Measures Health-related quality of life was assessed by means of the Inflammatory Bowel Disease Questionnaire (IBDQ) and the Psychological General Well Being Index (PGWBI). Results In active IBD, all dimensions of the quality of life scored significantly lower than in inactive IBD, indicating a poor quality of life. Social impairment was the least impaired dimension of the IBDQ in active UC and CD, compared with digestive and systemic symptoms. In inactive IBD, the systemic symptoms domain received the lowest score (P < 0.01). In a subgroup of 22 patients studied before and after remission, emotional function was the most impaired dimension after achieving remission. The Psychological General Well Being Index was significantly impaired in active UC [78.5 (range 64–89)] and CD [76.5 (range 69–97)] relative inactive IBD [104 (range 93–111)] vs 106 (95–113), respectively;P < 0.05]. Conclusions Quality of life is impaired in IBD. During relapse, clinicians should pay attention to digestive symptoms and psychological distress. In remission, they should be sensitive to systemic symptoms.

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.

Induction of nitric oxide synthase in colonic smooth muscle from patients with toxic megacolon.

BACKGROUND & AIMS Colonic inflammation may lead to motility disturbances, including severe atony. Nitric oxide is released by inflamed tissue and induces smooth muscle relaxation. The aim of this study was to analyze NO generation pathways in colonic tissue from patients who had ulcerative colitis with or without toxic megacolon and in tumor-free samples from patients with colonic neoplasm. METHODS Enzymatic activity was determined by transformation of [14C]arginine to [14C]citrulline in mucosa and muscular layer samples. Immunostaining of tissue sections with antibody against inducible NO synthase was investigated. The effects of endotoxin on NO synthase activity was tested in muscle strips from human colon. RESULTS Ca(2+)-independent NO synthase was undetectable or very low in muscularis propria from tumor and colitis controls. In contrast, specimens from patients with toxic megacolon had high activity (P < 0.05). Positive immunostaining for inducible NO synthase was found in muscular layers from patients with megacolon but not in tumor and colitis controls. Finally, endotoxin induced Ca(2+)-independent NO synthase activity in colonic muscle. CONCLUSIONS Toxic megacolon is associated with the appearance of inducible NO synthase in the colonic muscularis propria. Local generation of excessive amounts of NO may be responsible for the colonic dilatation that is the hallmark of this syndrome.