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

Abstract

24Despite recent recognition of the ATP-binding cassette protein OptrA as an important mediator of 25 linezolid-resistance in Enterococcus faecalis worldwide, the mechanisms of optrA gene 26 acquisition and transfer remain poorly understood. In this study, we performed comprehensive 27 molecular and phenotypic profiling of 44 optrA-carrying E. faecalis clinical isolates with 28 linezolid-resistance. Pulse-field gel electrophoresis and DNA hybridization revealed the presence 29 of optrA in the plasmid in 26 (59%) isolates and in the chromosome in 18 (41%) isolates. 30Conjugation experiments showed a successful transfer of optrA in 88.5% (23/26) of isolates 31 carrying optrA in plasmids while no transfer occurred in any isolates carrying optrA in the 32 chromosome (0/18). All 23 transconjugants exhibited in vitro resistance to linezolid and several 33 other antibiotics, and were confirmed to contain optrA and other resistance genes. Plasmid typing 34 demonstrated a predominance (18/23 or 78%) of rep 9 -type plasmids (pCF10 prototype) known to 35 be the best studied sex pheromone responsive plasmids. Full plasmid genome sequencing of one 36 isolate revealed the presence of drug resistance genes (optrA and fexA) and multiple sex 37 pheromone response genes in the same plasmid, which represents the first sex pheromone 38 responsive plasmid carrying optrA from a clinical isolate. PCR-based genotyping revealed the 39 presence of three key sex pheromone response genes (prgA, prgB and prgC) in almost all 23 40 optrA-carrying isolates tested. Finally, functional studies of these isolates by clumping induction 41 assay detected different degrees of clumping in most of the 23 isolates. Our analysis strongly 42 suggests that optrA-mediated linezolid-resistance can be widely disseminated through sex 43 pheromone plasmid transfer. 44 45