Genomic Characterization of Linezolid-Resistant Clostridioides difficile Harboring cfr Variants

Abstract

The emergence of antimicrobial resistance (AMR) in Clostridium difficile (C. difficile), particularly to last-line antibiotics such as linezolid, represents a critical challenge in clinical settings. This study investigates the genomic epidemiology of linezolid-resistant C. difficile, focusing on the distribution and mutational patterns of the chloramphenicol-florfenicol resistance (cfr) 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 cfr subtypes: cfr(C) was dominant (156/354 NCBI strains; 101/160 EnteroBase strains), whereas cfr(B) frequently harbored missense mutations (p.R247K, p.V294I, and less commonly p.A334T). The cfr(E) subtype was exclusively identified in ribotype 027 (RT027) strains. Notably, cfr(C) exhibited a strong association with RT017, correlating with a conserved 99 bp genomic deletion. Phylogenetic analysis linked cfr-carriage to predominant sequence types (ST1 in NCBI strains, ST37 in EnteroBase isolates). Furthermore, the co-occurrence of cfr 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 cfr mutations, hypervirulent ribotypes, and AMR dissemination, necessitating enhanced surveillance to mitigate the spread of resistant C. difficile lineages.

Keywords: C. difficile; Cfr subtypes; ST; antimicrobial resistance genes; linezolid.

Figure 1

Linezolid-resistant C. difficile strains: (a) country of isolation for linezolid-resistant strains of NCBI Pathogen Detection; (b) distribution of ST types and cfr gene in linezolid-resistant strains of NCBI Pathogen Detection; (c) distribution of STs and cfr gene in linezolid-resistant strains of EnteroBase; (d) distribution of PCR ribotypes and cfr gene in linezolid-resistant strains of EnteroBase.

Figure 2

(a) Most frequent STs in susceptible C. difficile strains. (b) Distribution of different STs in strains with cfr gene (x axis) and in strains lacking AMR genes (y axis).

Figure 3

Polymorphic loci and conservation level of cfr(B) gene. From left to right, mutant amino acids p.R247K, p.V294I, and p.A334T are shown, and the corresponding nucleotide substitutions.

Figure 4

(a) Phylogenetic tree of 44 cfr(B) gene: red: gene subtypes of strains with RT27, RT78, RT126; black and blue: gene subtypes of strains with no hypervirulent STs. Green clade includes strains with p.R247K and p.V294I mutations, pink clade includes strains with p.V294I mutation and yellow clade includes strains with p.A334T mutation. Blue clade includes the rest of the strains. (b) Phylogenetic tree of 99 cfr(C) gene. The gene subtypes with RT17 are colored pink, the gene subtypes with the p.C111Y mutation are purple, the gene subtypes with the p.I134N and p.C142Y mutations are grey, and the gene subtypes with no important difference in alignment are colored yellow.