Characterization of High-Linezolid-MIC Clostridioides difficile Isolated from a Chinese Hospital: First Genomic Evidence of Cfr(B) Transmission and Tn6218 Association

Xuyan Fu,1,* Xiajing Bi,2,* Tao Lv,1 Yunbo Chen1 1State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, People’s Republic of China; 2Department of Nursing, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yunbo Chen, Email [email protected]: 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 Tn 6218, 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.Keywords: Clostridioides difficile, linezolid, cfr(B), transposon, horizontal transmission