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

Abstract

Cultivation 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.