| Summary: | The mechanisms of linezolid resistance among 13 <i>E. faecalis</i> and 6 <i>E. faecium</i> isolates, 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. The <i>optrA</i> gene was detected in all 13 <i>E. faecalis</i> isolates; and one <i>optrA</i>-positive isolate also carried the recently described <i>cfr</i>(D) gene. Moreover, one <i>E. faecalis</i> isolate displayed the nucleotide mutation G2576T in the 23S rDNA. This mutation was also present in all six <i>E. faecium</i> isolates. All linezolid-resistant enterococci showed a multiresistance phenotype and harbored several antimicrobial resistance genes, as well as many virulence determinants. The <i>fexA</i> gene was located upstream of the <i>optrA</i> gene in 12 of the <i>E. faecalis</i> isolates. Moreover, an <i>erm</i>(A)-like gene was located downstream of <i>optrA</i> in two isolates recovered from the same hospital. The <i>optrA</i> gene was transferable in all but one <i>E. faecalis</i> isolates, in all cases along with the <i>fexA</i> gene. The <i>cfr</i>(D) gene was not transferable. The presence of <i>optrA</i> and mutations in the 23S rDNA are the main mechanisms of linezolid resistance among <i>E. faecalis</i> and <i>E. faecium</i>, respectively. We report the first description of the <i>cfr</i>(D) gene in <i>E. faecalis</i>. The presence of the <i>optrA</i> and <i>cfr</i>(D) genes in Spanish hospitals is a public health concern.
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