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

Martin P Mchugh,Joanna Walker,Katie L Hopkins,Elham Khatamzas,Geoff Toner,Benjamin J Parcell,Danièle Meunier,Noha El Sakka,Robert Weir,Kerry A Pettigrew,Neil Woodford,Kate E Templeton,Matthew T G Holden,Anne Marie Karcher,Stephen H Gillespie

doi:10.1099/mic.0.001137

Abstract

Transferable linezolid resistance due to optrA, poxtA, cfr and cfr-like genes is increasingly detected in enterococci associated with animals and humans globally. We aimed to characterize the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. Six linezolid-resistant E. faecalis isolated from urogenital samples were confirmed to carry the optrA 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 optrA gene was located on distinct plasmids in each isolate, suggesting that transfer of a single plasmid did not contribute to optrA dissemination in this collection. pTM6294-2, BX5936-1 and pWE0438-1 were similar to optrA-positive plasmids from China and Japan, while the remaining three plasmids had limited similarity to other published examples. We identified the novel Tn6993 transposon in pWE0254-1 carrying linezolid (optrA), macrolide (ermB) and spectinomycin [ANT(9)-Ia] resistance genes. OptrA amino acid sequences differed by 0–20 residues. We report multiple variants of optrA on distinct plasmids in diverse strains of E. faecalis . 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.

Highlights

Enterococcus faecalis and Enterococcus faecium are carried in the intestinal tract and are important opportunistic pathogens in humans [1]
This study found optrA present in diverse genetic lineages of E. faecalis and carried on largely unrelated plasmids in six isolates from Scotland. pTM6294-2, pBX5936-1 and pWE0438 shared homology with plasmids identified in China or Japan, highlighting the wide dispersal of optrA
We identified optrA often carried with a number of other resistance genes, including in a novel multiresistance transposon Tn6993 in pWE0254-1, and the recently described cfr(D) in pBX8117-2

Summary

Introduction

Enterococcus faecalis and Enterococcus faecium are carried in the intestinal tract and are important opportunistic pathogens in humans [1]. Among the remaining treatment options, clinical E. faecium isolates are usually resistant to amoxicillin, and resistance to vancomycin is increasingly common [2]. E. faecalis typically remains susceptible to amoxicillin and resistance to vancomycin is uncommon. Where vancomycin cannot be used, treatment options against severe enterococcal infections are largely limited to daptomycin, linezolid or combination therapy and are further complicated by issues with efficacy, susceptibility or toxicity [1]. Oxazolidinones such as linezolid block protein synthesis by binding to the 50S ribosomal subunit and inhibit formation of the initiation complex [3]. Linezolid resistance is reported in ≤1 % of bloodstream enterococcal isolates in the UK and is an important

Objectives

Methods

Results

Conclusion