Empreinte moléculaire des communautés bactériennes et hétérogénéité intraspécifique de l'ADNr 16S: est-il raisonnable d'occulter le problème?

Abstract

Molecular fingerprinting methods are currently used to study microbial communities by culture independent approaches. They are proposed as identification tool owing to the availability of rapid automated methods. The 16S rRNA gene (16S rDNA) is an efficient marker for bacterial identification and microbial communities analysis. However, the 16S rDNA polymorphism among strains of the same species is an underestimated pitfall of the fingerprinting approaches. We studied the 16S rDNA variability among strains of three bacterial species of medical interest. Total DNA was extracted from clinical isolates of Pseudomonas aeruginosa (N=20), Clostridium difficile (N=20) and Enterobacter cloacae (N=14). The Polymerase Chain Reaction (PCR) products obtained with consensus primers flanking the 16S rDNA variable regions V3 and V6-V7-V8 were separated by Temporal Temperature Gradient gel Electrophoresis (TTGE). DNA extracted from TTGE bands were sequenced and analysed. All the isolates of P. aeruginosa and of C. difficile displayed one single TTGE band with constant migration distances suggesting that there was no 16S rDNA polymorphism among strains in these two species. Oppositely, the isolates of E. cloacae gave complex TTGE patterns formed by multiple bands with variable migration distances. These patterns corresponded to 16S rRNA genes variable in a single genome as well as among strains of the species. Intra-species and/or intragenomic variability of 16S rDNA should be taken into account for pertinent interpretation of molecular fingerprint. For this purpose, a comprehensive description of the polymorphism of this marker is necessary.