The mechanisms of linezolid resistance among 13 E. faecalis and 6 E. faecium isolates,\nrecovered from six Spanish hospitals during 2017â??2018, were investigated. The presence of acquired\nlinezolid resistance genes and mutations in 23S rDNA and in genes encoding for ribosomal proteins\nwas analyzed by PCR and amplicon sequencing. Moreover, the susceptibility to 18 antimicrobial\nagents was investigated, and the respective molecular background was elucidated by PCR-amplicon\nsequencing and whole genome sequencing. The transferability of the linezolid resistance genes was\nevaluated by filter-mating experiments. The optrA gene was detected in all 13 E. faecalis isolates;\nand one optrA-positive isolate also carried the recently described cfr(D) gene. Moreover, one E. faecalis\nisolate displayed the nucleotide mutation G2576T in the 23S rDNA. This mutation was also present in\nall six E. faecium isolates. All linezolid-resistant enterococci showed a multiresistance phenotype and\nharbored several antimicrobial resistance genes, as well as many virulence determinants. The fexA\ngene was located upstream of the optrA gene in 12 of the E. faecalis isolates. Moreover, an erm(A)-like\ngene was located downstream of optrA in two isolates recovered from the same hospital. The optrA\ngene was transferable in all but one E. faecalis isolates, in all cases along with the fexA gene. The cfr(D)\ngene was not transferable. The presence of optrA and mutations in the 23S rDNA are the main\nmechanisms of linezolid resistance among E. faecalis and E. faecium, respectively. We report the first\ndescription of the cfr(D) gene in E. faecalis. The presence of the optrA and cfr(D) genes in Spanish\nhospitals is a public health concern.
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