Dissemination of Linezolid Resistance Through Sex Pheromone Plasmid Transfer in Enterococcus faecalis

Abstract

Despite recent recognition of the ATP-binding cassette protein OptrA as an important mediator of linezolid resistance in Enterococcus faecalis worldwide, the mechanisms of optrA gene 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 (18/23,78%) of rep ₉-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 23 optrA-carrying isolates. Finally, functional studies of these isolates by clumping induction assay detected different degrees of clumping in 17 isolates. Our analysis suggests that optrA-mediated linezolid resistance can be widely disseminated through sex pheromone plasmid transfer.

Keywords: Enterococcus faecalis; linezolid; optrA; sex pheromone plasmid; transmission.

FIGURE 1

Determination of the location of optrA in linezolid-resistant Enterococcus faecalis isolates. (A) Representative results of S1 nuclease-pulsed-field gel electrophoresis analysis. The first lane contained XbaI-digested chromosome of Salmonella Braenderup H9812 as a DNA size marker. (B) Representative results of Southern hybridization showing the location of optrA in plasmids or chromosomes. IDs of individual isolates analyzed are indicated at the top.

FIGURE 2

Gene organization in plasmid pEF10748 from the E. faecalis clinical isolate P10748. Arrows indicate the CDSs and their transcription directions. The putative functions of CDSs are color-coded as indicated at the right.

FIGURE 3

Comparison of the sex pheromone gene cluster among known sex pheromone plasmids. Different genes are color-coded as shown in the box on the bottom. Of note, for the region shown, the gene organization in plasmid pEF10748 identified in this study is most similar to that of pKUB3006-1 reported by Kuroda et al. (2018). The bacterial origin and GenBank accession numbers of all plasmids shown are available from Supplementary Table S4.

FIGURE 4

Comparison of the genetic environment of the linezolid resistance optrA among different plasmids. Different genes are color-coded as shown in the box on the bottom. Of note, for the region shown, the gene organization in plasmid pEF10748 identified in this study is most similar to that of pXY17 reported by He et al. (2016). The bacterial origin and GenBank accession numbers of all plasmids shown are available from Supplementary Table S4.