Lionel Rigottier-Gois

Reduced diversity of faecal microbiota in Crohn’s disease revealed by a metagenomic approach

Background and aim: A role for the intestinal microbial community (microbiota) in the onset and chronicity of Crohn’s disease (CD) is strongly suspected. However, investigation of such a complex ecosystem is difficult, even with culture independent molecular approaches. Methods: We used, for the first time, a comprehensive metagenomic approach to investigate the full range of intestinal microbial diversity. We used a fosmid vector to construct two libraries of genomic DNA isolated directly from faecal samples of six healthy donors and six patients with CD. Bacterial diversity was analysed by screening the two DNA libraries, each composed of 25 000 clones, for the 16S rRNA gene by DNA hybridisation. Results: Among 1190 selected clones, we identified 125 non-redundant ribotypes mainly represented by the phyla Bacteroidetes and Firmicutes. Among the Firmicutes, 43 distinct ribotypes were identified in the healthy microbiota, compared with only 13 in CD (p<0.025). Fluorescent in situ hybridisation directly targeting 16S rRNA in faecal samples analysed individually (n = 12) confirmed the significant reduction in the proportion of bacteria belonging to this phylum in CD patients (p<0.02). Conclusion: The metagenomic approach allowed us to detect a reduced complexity of the bacterial phylum Firmicutes as a signature of the faecal microbiota in patients with CD. It also indicated the presence of new bacterial species.

Alterations of the dominant faecal bacterial groups in patients with Crohn's disease of the colon

Background and aim: The colonic microflora is involved in the pathogenesis of Crohns disease (CD) but less than 30% of the microflora can be cultured. We investigated potential differences in the faecal microflora between patients with colonic CD in remission (n=9), patients with active colonic CD (n=8), and healthy volunteers (n=16) using culture independent techniques. Methods: Quantitative dot blot hybridisation with six radiolabelled 16S ribosomal ribonucleic acid (rRNA) targeting oligonucleotide probes was used to measure the proportions of rRNA corresponding to each phylogenetic group. Temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA was used to evaluate dominant species diversity. Results: Enterobacteria were significantly increased in active and quiescent CD. Probe additivity was significantly lower in patients (65 (11)% and 69 (6)% in active CD and quiescent CD) than in healthy controls (99 (7)%). TTGE profiles varied markedly between active and quiescent CD but were stable in healthy conditions. Conclusion: The biodiversity of the microflora remains high in patients with CD. Enterobacteria were observed significantly more frequently in CD than in health, and more than 30% of the dominant flora belonged to yet undefined phylogenetic groups.

Specificities of the fecal microbiota in inflammatory bowel disease.

Background: Abnormalities have been described in the fecal microbiota of patients with IBD, but it is not known whether they are specific for inflammatory bowel disease (IBD) or to some extent common to other forms of colitis. The aim of this study was to compare the bacterial composition of the dominant fecal microbiota in patients with Crohns disease (CD), ulcerative colitis (UC), infectious colitis (IC), and in healthy subjects (HS). Methods: Fluorescent in situ hybridization adapted to flow cytometry was used to analyze the bacterial composition of fecal samples from 13 patients with active CD, 13 patients with active UC, 5 patients with IC, and 13 HS. We used 6 group‐specific probes targeting 16S rRNA and spanning the main phylogenetic groups of the fecal microbiota. Results: A significantly higher proportion of the total fecal bacteria were recognized by the 6 probes in HS (86.6% ± 12.7) and in IC (84.0% ± 11.7) than in patients with IBD (70.9% ± 15 in CD and 60.1% ± 25.7 in UC). The Clostridium coccoides group was reduced in UC (20.0% ± 13.3 versus 42.0% ± 12.0 in HS; P < .001), whereas the C leptum group was reduced in CD (13.1% ± 11.9 versus 25.2% ± 14.2 in HS; P = .002). The Bacteroides group was more abundant in IC (36.4% ± 22.9) than in the other 3 groups (13.8% ± 11.8 in CD, 11.7% ± 11.7 in UC, 12.1% ± 7.0 in HS; P < .001 for all 3 comparisons). Conclusions: In IBD the dominant fecal microbiota comprises unusual bacterial species. Moreover, CD and UC fecal microbiota harbor specific discrepancies and differ from that of IC and healthy subjects.

Colonic microbiota signatures across five northern European countries

ABSTRACT The composition of the colonic microbiota of 91 northern Europeans was characterized by fluorescent in situ hybridization using 18 phylogenetic probes. On average 75% of the bacteria were identified, and large interindividual variations were observed. Clostridium coccoides and Clostridium leptum were the dominant groups (28.0% and 25.2%), followed by the Bacteroides (8.5%). According to principal component analysis, no significant grouping with respect to geographic origin, age, or gender was observed.

Fusobacterium prausnitzii and related species represent a dominant group within the human fecal flora

The human gut microflora plays a key role in nutrition and health. It has been extensively studied by conventional culture techniques. However these methods are difficult, time consuming and their results not always consistent. Furthermore microscopic counts indicate that only 20 to 40% of the total flora can be cultivated. Among the predominant species of the human gut, Fusobacterium prausnitzii was reported either as one of the most frequent and numerous species or was seldom retrieved. We designed and validated a specific rRNA-targeted oligonucleotide probe, called S-*-Fprau-0645-a-A-23, to accurately detect and quantify F. prausnitzii and relatives within the human fecal microflora. The target group accounted for 5.3 +/- 3% of total bacterial 16S rRNA using dot blot hybridization (10 human fecal samples) and 16.5 +/- 7% of cells stained with Dapi using in situ hybridization (10 other human fecal samples). A specific morphology seemed to be typical and dominant: two cells forming an asymmetrical double droplet. This work showed that F. prausnitzii and phylogenetically related species represent a dominant group within the human fecal flora.

Fluorescent hybridisation combined with flow cytometry and hybridisation of total RNA to analyse the composition of microbial communities in human faeces using 16S rRNA probes

To determine the structure of human faecal microbiota, faecal samples from 23 healthy individuals were analysed with a similar set of probes targeting six phylogenetic groups using rRNA dot-blot hybridisation and whole cell fluorescent in situ hybridisation (FISH) combined with flow cytometry. When microbiota compositions derived by each method were compared, the results were not statistically different for Clostridium coccoides, Fusobacterium prausnitzii, Bifidobacterium spp. and Enterobacteria. Conversely, the proportions were significantly different for Bacteroides and Atopobium (P<0.05). The metabolic state of these bacteria within the colon could explain the discrepancy observed between the rRNA level and the actual cell proportion. However, both approaches supplied consistent and complementary information on the structure of the faecal microbiota. FISH combined with flow cytometry appears best suited to future high throughput analysis.

Intestinal Bacterial Communities That Produce Active Estrogen-Like Compounds Enterodiol and Enterolactone in Humans

ABSTRACT Lignans are dietary diphenolic compounds which require activation by intestinal bacteria to exert possible beneficial health effects. The intestinal ecosystem plays a crucial role in lignan metabolism, but the organisms involved are poorly described. To characterize the bacterial communities responsible for secoisolariciresinol (SECO) activation, i.e., the communities that produce the enterolignans enterodiol (ED) and enterolactone (EL), a study with 24 human subjects was undertaken. SECO activation was detected in all tested fecal samples. The intestinal bacteria involved in ED production were part of the dominant microbiota (6 × 108 CFU g−1), as revealed by most-probable-number enumerations. Conversely, organisms that catalyzed the formation of EL occurred at a mean concentration of approximately 3 × 105 CFU g−1. Women tended to have higher concentrations of both ED- and EL-producing organisms than men. Significantly larger amounts of EL were produced by fecal dilutions from individuals with moderate to high concentrations of EL-producing bacteria. Two organisms able to demethylate and dehydroxylate SECO were isolated from human feces. Based on 16S rRNA gene sequence analyses, they were named Peptostreptococcus productus SECO-Mt75m3 and Eggerthella lenta SECO-Mt75m2. A new 16S rRNA-targeted oligonucleotide probe specific for P. productus and related species was designed and further used in fluorescent in situ hybridization experiments, along with five additional group-specific probes. Significantly higher proportions of P. productus and related species (P = 0.012), as well as bacteria belonging to the Atopobium group (P = 0.035), were typical of individuals with moderate to high concentrations of EL-producing communities.

Molecular inventory of faecal microflora in patients with Crohn's disease

Intestinal microbial community is involved in the pathogenesis of Crohns disease, but knowledge of its potential abnormalities has been limited by the impossibility to grow many dominant intestinal bacteria. Using sequence analysis of randomly cloned bacterial 16S ribosomal DNA, the dominant faecal species from four Crohns disease patients and four controls were compared. Whereas marked inter-individual differences were observed in the faecal microflora of patients, three remained distantly related to controls on the basis of their operational taxonomic unit composition. Bacteroides vulgatus and closely related organisms represented the only molecular species shared by all patients and exhibited an unusually high rate of occurrence. Escherichia coli clones were isolated only in two patients with ileocolonic Crohns disease. Moreover, numerous clones belonged to phylogenetic groups or species that are commonly not dominant in the faecal microflora of healthy subjects: Pectinatus, Sutterella, Verrucomicrobium, Fusobacterium, Clostridium disporicum, Clostridium glycolicum, Clostridium ramosum, Clostridium innocuum and Clostridium perfringens.

Enumeration of Bacteroides Species in Human Faeces by Fluorescent in situ Hybridisation Combined with Flow Cytometry Using 16S rRNA Probes

Bacteroides is a predominant group of the faecal microbiota in healthy adults. To investigate the species composition of Bacteroides by fluorescent in situ hybridisation (FISH) combined with flow cytometry, we developed five species-specific probes targeting the 16S rRNA. Probes were designed to identify cells belonging to Bacteroides distasonis, B. fragilis, B. ovatus, B. vulgatus and B. putredinis. The species-specificity of the probes was assessed against a collection of reference strains from the Cytophaga-Flavobacterium-Bacteroides group. The results of the FISH experiments showed that the probes were specific as they only detected strains of the target species. Determining the fluorescence intensity of each probe relative to that of the EUB 338 probe (domain bacteria) further showed that each species probe easily accessed the targeted site. The probes were applied to quantify the Bacteroides species in faeces collected from 20 healthy adults. All five species were detected in the faecal samples. Cells hybridised with Bfra 998 were the most frequent as they were observed in 90% of individuals (18/20 samples, mean proportion of 3.9 +/- 2.2%). The cells hybridised with Bvulg 1017 were observed in 85% of individuals (17/20 samples) and represented with a mean proportion of 4.2 +/- 6.1%, the most abundant Bacteroides species in human faeces. Cells hybridising with probes for B. ovatus, B. distasonis and B. putredinis were less frequently detected. The large distribution of B. vulgatus and B. fragilis in human faeces is in accordance with previous reports based on culture or molecular studies. This work showed that fluorescent in situ hybridisation is a tool appropriate for a high-resolution analysis of the species composition of complex ecosystems and especially of the Bacteroides group within the faecal microbiota.

Phenotypic and Functional Similarity of Gut Intraepithelial and Systemic T Cells in a Teleost Fish

Gut-associated lymphocytes were described in fish, but their involvement in immune responses is still unknown. In rainbow trout, intraepithelial lymphocytes (IELs) are scattered between gut epithelial cells, but neither Peyer’s patches nor mesenteric lymph nodes were identified. Rainbow trout IELs contain mainly T cells, because they expressed transcripts of T cell marker homologs of CD8, CD4, CD28, CD3ε, TCRζ, TCRγ, and TCRβ and lacked IgM. However, trout IELs did not show specific homing to the gut mucosa, which in mammals defines IELs as a distinctive mucosal population. A detailed analysis of the TCRβ repertoire of rainbow trout IELs was performed in both naive and virus-infected animals. TCRβ transcripts of rainbow trout IELs were highly diverse and polyclonal in adult naive individuals, in sharp contrast with the restricted diversity of IEL oligoclonal repertoires described in birds and mammals. Significant modifications of the trout IEL TCRβ repertoire were observed after a systemic infection with a fish rhabdovirus and were especially marked for Vβ4-bearing receptors as previously reported for spleen cells. Thus, we could not find any specific properties of the trout IEL TCRβ repertoire compared with the spleen and pronephros TCRβ repertoire, which questions the reality of a distinct IEL compartment in teleosts. Our findings suggest that a highly diversified αβ ΤCR repertoire is maintained in fish IELs in the absence of Peyer’s patches and mesenteric lymph nodes, whereas the restricted diversity of mouse αβ IELs is attributed to multiple cycles of activation and recirculation, allowing a progressive narrowing of the repertoire.