ABSTRACTThe aim of this study was to investigate the transferability of acquired linezolid resistance genes and associated mobile genetic elements in anEnterococcus faecalisisolate QZ076, cocarryingoptrA,cfr,cfr(D), andpoxtA2genes. MICs were determined by broth microdilution. Whole-genome sequencing (WGS) was performed using the Illumina and Nanopore platforms. The transfer of linezolid resistance genes was investigated by conjugation, usingE. faecalisJH2-2 and clinical methicillin-resistantStaphylococcus aureus(MRSA) 109 as recipients.E. faecalisQZ076 harbors four plasmids, designated pQZ076-1 to pQZ076-4, withoptrAlocated in the chromosomal DNA. The genecfrwas located on a novel pseudocompound transposon, designated Tn7515, integrated into the 65,961-bp pCF10-like pheromone-responsive conjugative plasmid pQZ076-1. Tn7515generated 8-bp direct target duplications (5′-GATACGTA-3′). The genescfr(D) andpoxtA2were colocated on the 16,397-bp mobilizable broad-host-range Inc18 plasmid pQZ076-4. Thecfr-carrying plasmid pQZ076-1 could transfer fromE. faecalisQZ076 toE. faecalisJH2-2, along with thecfr(D)- andpoxtA2-cocarrying plasmid pQZ076-4, conferring the corresponding resistant phenotype to the recipient. Moreover, pQZ076-4 could also transfer to MRSA 109. To the best of our knowledge, this study presented the first report of four acquired linezolid resistance genes [optrA,cfr,cfr(D), andpoxtA2] being simultaneously present in the sameE. faecalisisolate. The location of thecfrgene on a pseudocompound transposon in a pheromone-responsive conjugative plasmid will accelerate its rapid dissemination. In addition, thecfr-carrying pheromone-responsive conjugative plasmid inE. faecaliswas also able to mobilize the interspecies transfer of thecfr(D)- andpoxtA2-cocarrying plasmid between enterococci and staphylococci.IMPORTANCEIn this study, the simultaneous occurrence of four acquired oxazolidinone resistance genes [optrA,cfr,cfr(D), andpoxtA2] was identified in anE. faecalisisolate of chicken origin. The association of thecfrgene with a novel pseudocompound transposon Tn7515integrated into a pCF10-like pheromone-responsive conjugative plasmid will accelerate its dissemination. Moreover, the location of the resistance genescfr(D) andpoxtA2on a mobilizable broad-host-range Inc18 family plasmid represents the basis for their intra- and interspecies dissemination with the aid of a conjugative plasmid and further accelerates the spreading of acquired oxazolidinone resistance genes, such ascfr,cfr(D), andpoxtA2, among Gram-positive pathogens.