Comparative Genomic Analysis of Clostridioides difficile Strains in Mexico: Insights into Virulence and Resistance

Clostridioides difficile infection (CDI) remains a major global health threat due to the emergence of hypervirulent, multidrug-resistant lineages. However, the evolutionary dynamics and resistance associated genomic profiles of strains circulating in underrepresented regions, such as Mexico, remain poorly characterized. Here, we present a comprehensive genomic and phylogenetic analysis of 77 Mexican C. difficile strains compared with 74 strains from other parts of the world. Using whole-genome sequencing (WGS) and core-genome multilocus sequence typing (cgMLST), we identified 19 sequence types (STs) grouped across three clades, with hypervirulent ST01 dominating clade 2. Virulome analysis showed conserved toxin gene profiles (tcdA, tcdB, cdtAB) across strains, while clade-specific differences were observed in adhesion and survival genes. These variations, particularly pronounced in clade 2 strains from both global and Mexican collections, may contribute to enhanced persistence and transmissibility.  Pangenome analysis of 151 genomes highlighted distinct genomic architectures. Clade 2, enriched in ST01 epidemic lineages, contained 5,480 genes (58% core, 42% accessory), showing a compact structure consistent with recent clonal expansion. In contrast, Clade 1 displayed the highest diversity, with 8,584 genes (30% core, 70% accessory), indicative of an open and dynamic pangenome, while Clade 4 showed a smaller, more conserved profile (4,784 genes, 63% core). These findings underscore the contrasting evolutionary strategies among clades. Notably, Mexican ST01 strains exhibited a distinct resistome, including the high prevalence of the vanG operon and the VanR T115A substitution (94% vs. 23% globally), as well as near complete prevalence of the PnimBG mutation associated with reduced metronidazole susceptibility. This pattern may reflect local selective pressures associated with antimicrobial exposure. Phenotypic susceptibility testing of newly sequenced isolates showed that most ST01 strains remained susceptible to metronidazole and vancomycin despite carrying resistance-associated determinants. Our findings highlight the urgent need to recognize hypervirulent and resistant C. difficile lineages arising outside traditional surveillance regions. These Mexican strains not only reflect regional antibiotic usage patterns but also represent a potential reservoir of globally significant resistance traits. This work underscores the importance of integrating genomic surveillance across all continents to refine treatment protocols, prevent outbreaks, and contain the spread of resistant CDI.