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

The mechanisms of linezolid resistance among 13E. faecalisand 6E. faeciumisolates, recovered from six Spanish hospitals during 2017-2018, were investigated. The presence of acquired linezolid resistance genes and mutations in 23S rDNA and in genes encoding for ribosomal proteins was analyzed by PCR and amplicon sequencing. Moreover, the susceptibility to 18 antimicrobial agents was investigated, and the respective molecular background was elucidated by PCR-amplicon sequencing and whole genome sequencing. The transferability of the linezolid resistance genes was evaluated by filter-mating experiments. TheoptrAgene was detected in all 13E. faecalisisolates; and oneoptrA-positive isolate also carried the recently describedcfr(D) gene. Moreover, oneE. faecalisisolate displayed the nucleotide mutation G2576T in the 23S rDNA. This mutation was also present in all sixE. faeciumisolates. All linezolid-resistant enterococci showed a multiresistance phenotype and harbored several antimicrobial resistance genes, as well as many virulence determinants. ThefexAgene was located upstream of theoptrAgene in 12 of theE. faecalisisolates. Moreover, anerm(A)-like gene was located downstream ofoptrAin two isolates recovered from the same hospital. TheoptrAgene was transferable in all but oneE. faecalisisolates, in all cases along with thefexAgene. Thecfr(D) gene was not transferable. The presence ofoptrAand mutations in the 23S rDNA are the main mechanisms of linezolid resistance amongE. faecalisandE. faecium, respectively. We report the first description of thecfr(D) gene inE. faecalis. The presence of theoptrAandcfr(D) genes in Spanish hospitals is a public health concern.