The emergence and biological characteristics of linezolid-resistant Clostridioides difficile isolates in the Asia-Pacific Region

Linezolid-resistant Clostridioides difficile, conferred by the acquisition of cfr-like genes, has been reported in Europe and America. However, the emergence of linezolid resistance in C. difficile in the Asia-Pacific region and its impacts on C. difficile pathogenicity remain unclear. In this study, 881 C. difficile isolates from the Asia-Pacific region were screened for cfr-like genes. Whole genome sequencing was performed on 16 cfr-like gene-positive isolates from four countries. Thirteen isolates possessed cfr(B), which was located within Tn6218, while three isolates possessed cfr(C), which was located within the integrative and conjugative elements (ICE) F548 and DA275. Fourteen (87.5%, 14/16) of the cfr-like gene-positive isolates were resistant to linezolid. In comparison to the two isolates susceptible to linezolid, these 14 isolates exhibited significantly higher mRNA expression levels of cfr(B) and cfr(C), along with significantly higher bacterial density at 12 h. Conversely, they demonstrated reduced abilities for sporulation and biofilm formation. After the cfr(B) gene was knocked down by the CRISPR interference, the C. difficile strain presented lower bacterial density at 12 h, higher toxin production and stronger sporulation and biofilm formation abilities. Our findings reveal the emergence of cfr-like genes C. difficile isolates in the Asia-Pacific region, highlighting that cfr-like genes not only mediate linezolid resistance but also contribute to regulating pathogenic potential. Linezolid resistance in CDI should be closely monitored in specific patients.