Molecular Characterization of Linezolid-Non-Susceptible Enterococcus faecium: Identification of optrA and vanM Co-Harboring Strain in Clinical Isolate from China

Hong Shen, Xi Chen, Jun Liu, Ming Wei, Chunxia Yang, Li Gu, Wentao Zhu,* Ran Li* Department of Infectious Diseases and Clinical Microbiology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Wentao Zhu, Department of Infectious Diseases and Clinical Microbiology, Beijing Chao-Yang Hospital, Capital Medical University, 8 Gongren Tiyuchang Nanlu, Chaoyang District, Beijing, 100020, People’s Republic of China, Email [email protected] Ran Li, Department of Infectious Diseases and Clinical Microbiology, Beijing Chao-Yang Hospital, Capital Medical University, 8 Gongren Tiyuchang Nanlu, Chaoyang District, Beijing, 100020, People’s Republic of China, Email [email protected]: This study aimed to explore the resistance mechanisms and molecular characteristics of linezolid-non-susceptible Enterococcus faecium isolates (LNSEFM) from a tertiary hospital in Beijing, China, focusing on novel findings with significant clinical and epidemiological implications.Patients and Methods: LNSEFM strains isolated from clinical specimens between January 2011 and December 2023 were collected and screened for resistance genes, including rplC, rplD, rplV, 23s rRNA, optrA, poxtA, and cfr using polymerase chain reaction (PCR) and DNA sequencing. Molecular epidemiological analysis was performed using multi-locus sequence typing (MLST). Isolates carrying optrA and those harboring poxtA were subjected to whole-genome sequencing (WGS).Results: Among 2384 clinical E. faecium isolates, 19 (0.80%) were linezolid-non-susceptible (MIC 4– 32 mg/L). Among these, two vancomycin-resistant Enterococcus (VRE) strains exhibited an intermediate susceptibility to linezolid. Two distinct optrA variants (designated as KLDK and KLDP) were detected in separate LNSEFM isolates. The KLDK-positive isolate was found to co-harbor the vanM gene cluster despite maintaining vancomycin susceptibility. Additionally, one linezolid-resistant isolate carried a G2576T mutation in the 23S rRNA gene, whereas the other harbored the poxtA gene. MLST revealed 13 sequence types (STs) among the isolates, including a novel type ST2709.Conclusion: This study identified key notable findings in LNSEFM: identification of linezolid intermediate VRE in China, clinical detection of the optrA KLDK variant in enterococci, optrA-vanM co-presence in vancomycin-susceptible E. faecium, and a novel sequence type (ST2709). These findings enrich our understanding of the molecular epidemiology of LNSEFM and provide critical insights into clinical antimicrobial management and infection control.Keywords: linezolid-non-susceptible Enterococcus, optrA, poxtA, whole genome sequencing