Caractérisation de la microflore digestive de l'homme par des approches moléculaires (application dans le cadre d'études nutritionnelles évaluant l'impact de souches probiotiques)

La microflore du tractus digestif de l'homme est très dense et diversifiée. Elle exerce d'importantes fonctions physiologiques qui contribuent au maintien d'un bon état de santé. L'identification et la quantification des groupes bactériens qui la composent sont indispensables à la compréhension de leurs rôles. Les méthodes d'étude de la microflore digestive classiquement utilisées impliquaient la mise en culture d'échantillons fécaux et ont montré que, seule, une faible fraction de la biomasse présente est cultivée. Dans ce contexte, nous avons développé et validé l'hybridation in situ fluorescente combinée à une détection par cytométrie de flux, méthode indépendante de la culture. Nous avons également défini des sondes d'hybridation ciblant des groupes phylogénétiques dominants jusqu'alors non reconnus. Nous avons appliqué cette méthode à la description de la microflore digestive de l'homme et dans le cadre d'une étude nutritionnelle impliquant l'ingestion d'une souche probiotique.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Long-term storage of human fecal samples for in situ hybridization analysis of gut microbiota composition

International audienc

Analyse de la microflore intestinale humaine par hybridation in situ par des sondes fluorescentes ciblant l'ARNr 16S

National audienc

Analysis of human fecal microflora by in situ hybridisation and image analysis

National audienc

Analysis of human fecal microflora by in situ hybridisation and image analysis

International audienc

Composition of human intestinal flora analysed by fluorescent in situ hybridisation using group-specific 16S rRNA-targeted oligonucleotide probes

(Meeting on Current Knowledge and Management of Genetic Resources, TOULOUSE, OCT 09-11, 2000)International audienceCultivation methods classically used to describe the faecal flora composition are often too selective for certain groups of bacteria. This study was conducted to determine the human faecal flora composition by fluorescent in situ hybridisation, a direct and culture-independent method. Four group-specific probes and a domain probe Eub 338 targeting the 16S rRNA were used to analyse fixed faecal bacterial suspensions from nine healthy adult volunteers. Epifluorescence microscopy and image analysis were performed to evaluate the relative proportion of cells from each group. After optimisation of hybridisation conditions, the reproducibility of the protocol was evaluated to validate the FISH procedure. The domain probe Eub 338 labelled an average of 80 +/- 11% total faecal bacteria. The panel of four probes revealed more than 75% of the flora. The Clostridium coccoides-Eubacterium rectale group was the most represented, accounting for 36 +/- 7% of the bacteria. The three other probes used, Bacto 1080, Bif 164 and Fprau 645 labelled 17 +/- 5%, 15 +/- 9% and 23 +/- 5% of the total flora, respectively. The two dominant groups belonging to Clostridium and relatives constituted nearly 60% of the total flora. FISH coupled with image analysis is a direct and powerful molecular tool to assess the composition of the human faecal flora

Gnotobiotic rats harboring human intestinal microbiota as a model for studying cholesterol-to-coprostanol conversion

The efficiency of microbial reduction of cholesterol to coprostanol in human gut is highly variable among population and mechanisms remain unexplored. In the present study, we investigated whether microbial communities and their cholesterol metabolism characteristics can be transferred to germ-free rats. Two groups of six, initially germ-free rats were associated with two different human microbiota, exhibiting high and low cholesterol-reducing activities. Four months after inoculation, enumeration of coprostanoligenic bacteria, fecal coprostanol levels and composition of the fecal microbial communities were studied in gnotobiotic rats and compared with those of the human donors. Combination of culture (most probable number enumeration of active bacteria) and biochemical approaches (extraction followed by gas chromatography of sterols) showed that gnotobiotic rats harbored a coprostanoligenic bacterial population level and exhibited coprostanoligenic activities similar to those of the corresponding human donor. On the other hand, molecular approaches (whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes, and temporal temperature gradient gel electrophoresis of bacterial 16S rRNA gene amplicons) demonstrated that gnotobiotic rats reproduced a stable microbial community, close to the human donor microbiota at the group or genus levels but different at the dominant species level. These results suggest that the gnotobiotic rat model can be used to explore the still unknown human intestinal microbiota involved in luminal cholesterol metabolism, including regulation of expression of its activity and impact on health

Effet de la prise d'un lait fermenté contenant Lactobacillus casei DN-114 001 sur la microflore fécale de volontaires sains

National audienc