Despite recent recognition of the ATP-binding cassette protein OptrA as an important mediator of linezolid-resistance in Enterococcus faecalis, the mechanisms of optrA acquisition and transfer remain poorly understood. In this study, we performed comprehensive molecular and phenotypic profiling of 44 optrA -carrying E. faecalis clinical isolates with linezolid-resistance. Pulse-field gel electrophoresis and DNA hybridization revealed the presence of optrA in the plasmid in 26 (59%) isolates and in the chromosome in 18 (41%) isolates. Conjugation experiments showed a successful transfer of optrA in 88.5% (23/26) of isolates carrying optrA in plasmids while no transfer occurred in any isolates carrying optrA in the chromosome (0/18). All 23 transconjugants exhibited in vitro resistance to linezolid and several other antibiotics, and were confirmed to contain optrA and other resistance genes. Plasmid typing demonstrated a predominance (18/23 or 78%) of rep9-type plasmids (pCF10 prototype) known to be the best studied sex pheromone responsive plasmids. Full plasmid genome sequencing of one isolate revealed the presence of drug resistance genes ( optrA and fexA ) and multiple sex pheromone response genes in the same plasmid, which represents the first sex pheromone responsive plasmid carrying optrA from a clinical isolate. PCR-based genotyping revealed the presence of three key sex pheromone response genes ( prgA , prgB and prgC ) in almost all 23 optrA -carrying isolates tested. Finally, functional studies of these isolates by clumping induction assay detected different degrees of clumping in most of the 23 isolates. Our analysis strongly suggests that optrA -mediated linezolid-resistance can be widely disseminated through sex pheromone plasmid transfer.

Dissemination of linezolid-resistance through sex pheromone plasmid transfer in Enterococcs faecalis