Elevated wzc Mutation Frequency Promotes Carbapenem Resistance Evolution in Hypervirulent Klebsiella pneumoniae ST23

AbstractThe worldwide expansion of carbapenemase-producing hypervirulent K. pneumoniae (CP-hvKP) raises serious concerns about potentially untreatable invasive infections. ST23, an archetypal hvKP clone, has been highlighted due to the rising prevalence of carbapenem resistance largely driven by horizontal plasmid transfer. However, the factors facilitating carbapenemase acquisition in this lineage remained unclear. We characterized 1159 ST23 isolates across 49 countries, including nine clinical carbapenemase-producing strains from China's national bloodstream infection surveillance network. Global phylogenetic analysis revealed two distinct sublineages, namely the globally distributed, hypervirulent ST23-I and the geographically restricted, multidrug-resistant ST23-II. Temporal analysis showed that ST23-I concurrently accumulated virulence and antimicrobial resistance traits. Among ST23-I isolates, carbapenemase plasmids exhibited notable genetic diversity and clear geographic segregation, with IncL-bla_OXA-48 dominated in Europe and IncFII-bla_KPC-2 in Asia. Conjugation assays revealed that IncFII_K34-bla_KPC-2 and IncL-bla_OXA-48 plasmids transferred more efficiently than IncX3-bla_NDM-1 and IncFII_K2-bla_NDM-1 plasmids, contributing to their high prevalence. Non-synonymous mutations in the capsular polysaccharide synthesis locus, particularly in wzc, were accumulated in ST23-KL1 CP-hvKP. Isogenic mutants carrying two identified wzc mutations (either wzc^{2042A > G} or wzc^{1738T > A}) exhibited partially reduced capsule production and enhanced conjugation efficiency of carbapenemase plasmids, confirming the functional impact of these mutations. Collectively, these findings demonstrate that an elevated wzc mutation frequency represents an adaptive evolutionary pathway that facilitates carbapenemase acquisition in hvKP ST23. The interplay between capsule-associated chromosomal mutations and plasmid-mediated horizontal gene transfer may shape the evolutionary adaptation of resistance in this clinically important pathogen.