Genomic Characterization of Linezolid-Resistant Clostridioides difficile Harboring cfr Variants

The emergence of antimicrobial resistance (AMR) in <i>Clostridium difficile</i> (<i>C. difficile</i>), particularly to last-line antibiotics such as linezolid, represents a critical challenge in clinical settings. This study investigates the genomic epidemiology of linezolid-resistant <i>C. difficile</i>, focusing on the distribution and mutational patterns of the chloramphenicol-florfenicol resistance (<i>cfr</i>) gene and its association with multidrug resistance. We analyzed 514 clinical isolates (354 from NCBI Pathogen Detection, 160 from EnteroBase), revealing distinct prevalence patterns among <i>cfr</i> subtypes: <i>cfr</i>(C) was dominant (156/354 NCBI strains; 101/160 EnteroBase strains), whereas <i>cfr</i>(B) frequently harbored missense mutations (p.R247K, p.V294I, and less commonly p.A334T). The <i>cfr</i>(E) subtype was exclusively identified in ribotype 027 (RT027) strains. Notably, <i>cfr</i>(C) exhibited a strong association with RT017, correlating with a conserved 99 bp genomic deletion. Phylogenetic analysis linked <i>cfr</i>-carriage to predominant sequence types (ST1 in NCBI strains, ST37 in EnteroBase isolates). Furthermore, the co-occurrence of <i>cfr</i> with additional AMR genes conferred resistance to macrolides (erythromycin, azithromycin) and tetracyclines, indicating a convergent evolution toward multidrug resistance. These findings underscore the interplay between <i>cfr</i> mutations, hypervirulent ribotypes, and AMR dissemination, necessitating enhanced surveillance to mitigate the spread of resistant <i>C. difficile</i> lineages.