Molecular and functional analysis of the novel cfr(D) linezolid resistance gene identified in Enterococcus faecium

Objectives

To characterize the novel cfr(D) gene identified in an Enterococcus faecium clinical isolate (15-307.1) collected from France.

Methods

The genome of 15-307.1 was entirely sequenced using a hybrid approach combining short-read (MiSeq, Illumina) and long-read (GridION, Oxford Nanopore Technologies) technologies in order to analyse in detail the genetic support and environment of cfr(D). Transfer of linezolid resistance from 15-307.1 to E. faecium BM4107 was attempted by filter-mating experiments. The recombinant plasmid pAT29Ωcfr(D), containing cfr(D) and its own promoter, was transferred to E. faecium HM1070, Enterococcus faecalis JH2-2 and Escherichia coli AG100A.

Results

As previously reported, 15-307.1 belonged to ST17 and was phenotypically resistant to linezolid (MIC, 16 mg/L), vancomycin and teicoplanin. A hybrid sequencing approach confirmed the presence of several resistance genes including vanA, optrA and cfr(D). Located on a 103 kb plasmid, cfr(D) encoded a 357 amino acid protein, which shared 64%, 64%, 48% and 51% amino acid identity with Cfr, Cfr(B), Cfr(C) and Cfr(E), respectively. Both optrA and cfr(D) were successfully co-transferred to E. faecium BM4107. When expressed in E. faecium HM1070 and E. faecalis JH2-2, pAT29Ωcfr(D) did not confer any resistance, whereas it was responsible for an expected PhLOPS_A resistance phenotype in E. coli AG100A. Analysis of the genetic environment of cfr(D) showed multiple IS1216 elements, putatively involved in its mobilization.

Conclusions

Cfr(D) is a novel member of the family of 23S rRNA methyltransferases. While only conferring a PhLOPS_A resistance phenotype when expressed in E. coli, enterococci could constitute an unknown reservoir of cfr(D).