Phylogenomics, antimicrobial resistance, and mobile genetic elements in clinical Clostridioides difficile isolates from Hong Kong, China

Abstract

Whole-genome sequencing enables high-resolution characterisation of the evolutionary and accessory-genome diversity of Clostridioides difficile beyond conventional sequence typing. We analysed 118 clinical C. difficile genomes from Hong Kong using recombination-filtered phylogenomics, pangenome analysis, antimicrobial-resistance and virulence screening, mobile-element context analysis and functional annotation. The population comprised 24 sequence types and formed distinct phylogenetic clusters that were broadly consistent with multilocus sequence typing assignments. Pangenome analysis identified 9,140 gene clusters and supported an open pangenome structure, indicating ongoing gene acquisition and loss within the population. Accessory-genome divergence increased with core-genome SNP distance (Spearman's ρ = 0.437, p < 1 × 10^-5), although some closely related isolates retained substantial differences in gene content, suggesting recent accessory-genome turnover. Antimicrobial resistance determinants showed stronger associations with mobile genetic elements (MGE) than with virulence-associated loci. In particular, erm(B), AAC/APH-family genes and tet(M) were frequently detected and commonly linked to insertion-sequence- or transposon-associated contexts, whereas major toxin loci showed limited evidence of local mobility. Functional annotation revealed broadly conserved functional profiles dominated by transcription-, metabolism-, and signal transduction-related categories, alongside significant sequence type-associated variation in KEGG pathway composition (PERMANOVA R² = 0.434, p = 0.001), with ST54 forming a distinct functional cluster. Collectively, these findings demonstrate that this Hong Kong C. difficile population is structured by stable evolutionary lineages while maintaining extensive accessory-genome diversity, and highlight the prominent role of MGE in shaping antimicrobial-resistance architecture relative to the largely conserved virulence repertoire.