Rapid emergence of highly variable and transferable oxazolidinone and phenicol resistance gene optrA in German Enterococcus spp. clinical isolates

Highlights

• optrA-positive and linezolid-resistant Enterococcus spp. are on the rise.

• Multiple variants of the optrA gene exist.

• optrA was detected in various plasmid backgrounds and the bacterial chromosome.

• optrA is easily transferred to other pathogenic bacteria.

ABSTRACT

The number of linezolid-resistant Enterococcus spp. isolates received by the National Reference Centre for Staphylococci and Enterococci in Germany has been increasing since 2011. Although the majority are E. faecium, clinical linezolid-resistant E. faecalis have also been isolated. With respect to the newly discovered linezolid resistance protein OptrA, the authors conducted a retrospective polymerase chain reaction screening of 698 linezolid-resistant enterococcus clinical isolates. That yielded 43 optrA-positive strains, of which a subset was analysed by whole-genome sequencing in order to infer linezolid resistance-associated mechanisms and phylogenetic relatedness, and to disclose optrA genetic environments. Multiple optrA variants were detected. The originally described variant from China (optrA_WT) was the only variant shared between the two Enterococcus spp.; however, distinct optrA_WT loci were detected for E. faecium and E. faecalis. Generally, optrA localized to a plethora of genetic backgrounds that differed even for identical optrA variants. This suggests transmission of a mobile genetic element harbouring the resistance locus. Additionally, identical optrA variants detected on presumably identical plasmids, that were present in unrelated strains, indicates dissemination of the entire optrA-containing plasmid. In accordance, in vitro conjugation experiments verified transfer of optrA plasmids between enterococci of the same and of different species. In conclusion, multiple optrA variants located on distinct plasmids and mobile genetic elements with the potential for conjugative transfer are supposedly causative for the emergence of optrA-positive enterococci. Hence, rapid dissemination of the resistance determinant under selective pressure imposed by extensive use of last-resort antibiotics in clinical settings could be expected.

Introduction

Enterococci are considered to be the second or third most common nosocomial pathogen causing life-threatening diseases amongst elderly and immunocompromised patients (https://www.ecdc.europa.eu/sites/portal/files/documents/AER_for_2015-health-care-associated-infections.pdf). The oxazolidinone linezolid represents one of the few remaining treatment options for infections caused by vancomycin-resistant enterococci (VRE) [1]. Shortly after the approval of linezolid in 2000 and following therapy, emergence of linezolid resistance has been reported for vancomycin-susceptible enterococci (VSE) and VRE [2], [3]. Nevertheless, linezolid retains its efficacy as the prevalence of linezolid-resistant enterococci (LRE) still remains at low levels worldwide [4], [5]. However, the National Reference Centre (NRC) for Staphylococci and Enterococci at the Robert Koch Institute has noted an increasing number of LRE in German hospitals in recent years [6].

Linezolid effectively inhibits bacterial protein biosynthesis [7], [8]. Resistance can either be mediated by mutations of 23S rDNA alleles or ribosomal protein genes rplC, rplD and rplV, or by acquisition of resistance determinants such as the Cfr RNA methyltransferase or the recently identified oxazolidinone and phenicol resistance protein OptrA [9], [10], [11], [12]. The latter belongs to the ATP-binding cassette-F protein subfamily and mediates resistance by executing ribosomal protection function [13]. It was first described in an Enterococcus faecalis isolate of human origin [12], but subsequently emerged in both E. faecalis and Enterococcus faecium of animals and humans alike and, moreover, was detected in individual Gram-positive bacteria such as Staphylococcus sciuri, Staphylococcus simulans and streptococci [14], [15], [16], [17], [18], [19], [20]. In a recent study, Mendes et al. observed a high frequency (53.5%) of optrA-positive enterococci, mainly amongst E. faecalis [21]. As linezolid-resistant E. faecium more often carry mutations in 23S rDNA alleles than horizontally acquired linezolid resistance determinants, species-specific resistance pathways could be assumed. It must be noted that acquisition and expression of the methyltransferase gene cfr does not necessarily result in development of linezolid resistance, nor does the sheer presence of optrA, as some isolates were tested positive for both genes but apparently lack a resistance phenotype [21], [22], [23].

A high diversity of optrA nucleotide sequences and a plethora of variable genetic environments embedding the resistance gene in either the chromosome or on diverse plasmids have been reported [16], [17], [21]. Considering the promiscuous nature of mobile genetic elements (MGEs) and conjugative plasmids, rapid dissemination of the resistance locus is highly likely. As implementation of optrA screenings of linezolid-resistant bacteria at a global level has commenced just recently, distribution of optrA may still be underestimated at the present time. In order to investigate whether optrA is already circulating in German Enterococcus spp. clinical isolates and, moreover, to assess the prevalence of the resistance locus amongst the high number of LRE received by the German NRC, the authors analysed the entire LRE strain collection retrospectively from 2007 until 2017 with respect to the presence of optrA, variants thereof, adjacent genetic loci and transferability of the resistance locus.