Presence of <i>optrA-</i>mediated linezolid resistance in multiple lineages and plasmids of <i>Enterococcus faecalis</i> revealed by long read sequencing

Transferable linezolid resistance due to <i>optrA</i>, <i>poxtA</i>, <i>cfr</i> and <i>cfr</i>-like genes is increasingly detected in enterococci associated with animals and humans globally. We aimed to characterize the genetic environment of <i>optrA</i> in linezolid-resistant <i>Enterococcus faecalis</i> isolates from Scotland. Six linezolid-resistant <i>E. faecalis</i> isolated from urogenital samples were confirmed to carry the <i>optrA</i> gene by PCR. Short read (Illumina) sequencing showed the isolates were genetically distinct (>13900 core SNPs) and belonged to different MLST sequence types. Plasmid contents were examined using hybrid assembly of short and long read (Oxford Nanopore MinION) sequencing technologies. The <i>optrA</i> gene was located on distinct plasmids in each isolate, suggesting that transfer of a single plasmid did not contribute to <i>optrA</i> dissemination in this collection. pTM6294-2, BX5936-1 and pWE0438-1 were similar to <i>optrA</i>-positive plasmids from China and Japan, while the remaining three plasmids had limited similarity to other published examples. We identified the novel Tn<i>6993</i> transposon in pWE0254-1 carrying linezolid (<i>optrA</i>), macrolide (<i>ermB</i>) and spectinomycin [ANT(9)-Ia] resistance genes. OptrA amino acid sequences differed by 0-20 residues. We report multiple variants of <i>optrA</i> on distinct plasmids in diverse strains of <i>E. faecalis</i>. It is important to identify the selection pressures driving the emergence and maintenance of resistance against linezolid to retain the clinical utility of this antibiotic.