LAUSR

Background Clostridioides difficile (C. difficile) exhibiting high linezolid minimum inhibitory concentration (>4 µg/mL) remains infrequently reported in clinical settings. Notably, the prevalence of linezolid-resistant C. difficile is exceptionally low (<3% in Chinese isolates), and the underlying genetic determinants are poorly characterized. Methods We conducted a genomic study to investigate the genetic characteristics of C. difficile with high linezolid MIC. To determine the MIC of linezolid and delineate antimicrobial resistance profiles, these isolates were systematically subjected to antimicrobial susceptibility testing. Multilocus sequence typing, antimicrobial resistance genes, and the characteristics of the cfr gene in linezolid-resistant C. difficile strains were analyzed following whole-genome sequencing. Roary was used to construct a pangenome phylogenetic tree, and a Bayesian evolutionary analysis was performed using BEAST.4. Results Among 421 screened C. difficile isolates, nine isolates (2.1%) exhibited high-linezolid MICs (≥16 μg/mL), including six ST37 (A-B+) and three ST3 strains (two A-B-). All harbored cfr(B) on Tn6218, sharing homology with E. faecium (NG_050395.1). Conclusion This study underscores the risk of cfr(B) dissemination via mobile genetic elements in clinical settings, urging surveillance of co-occurrence in Enterococcus and C. difficile to curb resistance spread.