Genetic linkage of optrA and cfr, integrated into an unconventional circularisable structure, in Enterococcus faecium

Introduction: In enterococci nonmutational resistance to oxazolidinones can be mediated by two mostly plasmid-borne genes: cfr, encoding a ribosome-modifying enzyme and conferring resistance to linezolid but not to tedizolid; and optrA, encoding a ribosome protection mechanism and conferring resistance to both oxazolidinones. optrA was discovered in China in 2015 in enterococci of human and animal origin, where it was detected in different genetic environments. Since then optrA-positive enterococci have been described worldwide, including our report in Italy - the first outside China - in two blood isolates of Enterococcus faecium which were also positive for cfr. In that study we also noticed that, in one of those two E. faeciumisolates (strain E35048), the genetic contexts of both optrA and cfr were capable of undergoing excision to form minicircles. Now, E. faecium E35048 has been subjected to WGS and investigated for the location, characterisation, and transferability of the genetic contexts of optrA andcfr.Methods:E. faeciumE35048 underwent WGS. S1-PFGE, hybridisation assays, and BLASTN analysis were used to determine the location of optrA and cfr. Their transferability was assessed by electrotransformation and conjugation experiments. Curing assays were performed through several passages in agar without selective pressure.Results:E. faecium E35048 belonged to ST117. Both optrA and cfr probes hybridised with the chromosome and with a ~45-kb band. WGS analysis revealed that optrA and cfrwere linked, 23.1 kb apart, on a fragment of plasmid pRE25 (DNA identity, 96%), which was flanked by two extensive duplication regions (DRs) containing the erm(B) gene. Overall, the element exhibited the typical features of an unconventional circularizable structure (UCS). Neither optrA nor cfr could be transferred to enterococcal recipients by transformation or conjugation. The loss of the optrA-and cfr-carrying UCS by curing experiments was confirmed by the drop in oxazolidinone MICs and by WGS data showing only one copy of erm(B) at the empty excision site.Conclusions:The presence of the new UCS is consistent with hybridisation assays showing it both integrated into the chromosome and in circular form. The finding of a genetic linkage between optrA and cfr in Enterococcusadds to the novel finding of an UCS containing two regions - the genetic contexts of optrA and cfr- capable, in turn, of excising to form minicircles. The extensive DRs of this UCS contain erm(B), one of the most prevalent and best-conserved antibiotic resistance genes in bacteria. This, in addition to the belonging of E. faeciumE35048 to ST117, a well-known hospital-adapted clone, might favour the spread of oxazolidinone resistance in the hospital setting.