Molecular characterization and resistance mechanisms of linezolid-resistant enterococci clinical isolates and identification of a clinical Enterococcus faecium strain co-harboring optrA, poxtA, and cfr(D) in a tertiary hospital in China

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• The emergence of linezolid-resistant enterococci has become a significant global health concern. • It is important to understand the epidemiology and mechanisms of linezolid-resistant enterococci . • The mechanisms of linezolid resistance in enterococci exhibit diversity in our unit, including acquisition of resistance genes ( optrA, poxtA , and cfr (D)), point mutations in domain V of the 23S rRNA gene, and co-occurrence of multiple mechanisms. • Our findings highlight the need for enhanced surveillance of linezolid resistance in our clinical setting. The emergence of linezolid-resistant enterococci has become a significant global health concern. This study aimed to investigate the molecular characterization and resistance mechanisms in clinical isolates in a tertiary hospital in China. The VITEK®2 Compact system, VITEK® MS, and whole-genome sequencing (WGS) were used to identify 59 linezolid-resistant enterococci isolates. Antimicrobial susceptibilities were assessed by broth microdilution. WGS and bioinformatics tools analyzed resistance mechanisms and molecular characteristics. Plasmid-borne linezolid resistance gene transfer was assessed via conjugation and transformation assays. We collected 59 linezolid-resistant enterococci isolates, including 54 Enterococcus faecalis and 5 Enterococcus faecium strains. One strain carried the poxtA gene, one isolate carried the G2576T mutation in the 23S rRNA gene, and 56 isolates harbored the optrA gene. Notably, one isolate co-harbored the optrA, poxtA , and cfr (D) genes. Twenty-three sequence types (STs) were identified. ST16 (48.1%, 26/54) predominated in E. faecalis . Multilocus sequence typing (MLST) and evolutionary analysis indicated that E. faecalis strains of the same ST were genetically related. Among the seven strains (two E. faecium and five E. faecalis strains) selected for further investigation, IS 1216E was detected on plasmids carrying linezolid resistance genes in all five strains. Additionally, transposon Tn 554 was detected on the chromosome of an optrA -carrying strain. This study describes the molecular characteristics and resistance mechanisms of linezolid-resistant enterococci in our hospital, including a novel E. faecium isolate with optrA, poxtA, and cfr (D), highlighting the need for enhanced surveillance to control the dissemination of resistant strains.

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