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

Abstract

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 PhLOPSA 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 PhLOPSA resistance phenotype when expressed in E. coli, enterococci could constitute an unknown reservoir of cfr(D).

<p>Molecular Investigations of Linezolid Resistance in Enterococci <em>OptrA</em> Variants from a Hospital in Shanghai</p>

“…Only one E. faecalis was detected to carry cfr(D).In this year, a study from europe characterized the novel cfr(D) gene identified in an E. faecium clinical isolate. 6 According to the results mentioned above, it might probably that the cfr(D) gene tended to transfer between two kinds of Enterococcus. Whether it played a role of conferring linezolid resistance in Enterococcus still needs to be explored.…”

“…The resistance of linezolid in enterococci can be caused by point substitutions in 23S rRNA V domain, the main reason for linezolid resistance. 4 The 23S rRNA methyltransferase coding genes cfr, 4 cfr(B), cfr(C) 5 and cfr (D) 6 and also amino acid substitutions in ribosomal proteins L3, L4, 4 L22, 7 and ABC-F family members including OptrA 8 and PoxtA. 9 How ABC-F proteins mediate antibiotic resistance has been in a long-standing dispute with both efflux and ribosome protection hypothesis competing mutually.…”

Mechanisms of Linezolid Resistance Among Enterococci of Clinical Origin in Spain—Detection of optrA- and cfr(D)-Carrying E. faecalis

“…In this study, we report, to the best of our knowledge, the first description of the cfr (D) gene in the species E. faecalis . The Cfr(D) shares 64% amino acid identity to Cfr, but there is controversy about its contribution to linezolid resistance, as it confers a PhLOPS A resistance phenotype when expressed in Escherichia coli but not in Efs or Efm [ 11 ]. The cfr (D) gene has only been detected in clinical Efm isolates from France and Australia, and, as the isolate of this study, they co-harbored the optrA gene [ 11 , 45 ].…”

“…Besides resistance towards oxazolidinones, it also confers resistance to phenicols, lincosamides, pleuromutilins, and streptogramin A antimicrobials (PhLOPS A phenotype). In addition, variants of the cfr gene [ cfr (B), cfr (D)] have been described in clinical enterococcal isolates [ 9 , 10 , 11 ]. However, the contribution of cfr -like genes to reduce the susceptibility to linezolid in enterococci is still under debate [ 11 , 12 ].…”

“…In addition, variants of the cfr gene [ cfr (B), cfr (D)] have been described in clinical enterococcal isolates [ 9 , 10 , 11 ]. However, the contribution of cfr -like genes to reduce the susceptibility to linezolid in enterococci is still under debate [ 11 , 12 ]. The optrA gene encodes an ATP-binding cassette F (ABC-F) protein [ 13 ] that confers resistance to oxazolidinones and phenicols.…”