Epidemiological characterization of chromosome-mediated colistin resistance in hypervirulent carbapenem-resistant Klebsiella pneumoniae

Hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) poses a major clinical threat. Colistin is a last-resort agent against hv-CRKP, yet resistance driven by chromosomal mutations remains poorly characterized in its prevalence and functional impact. From 2020 to 2023, 239 non-duplicate clinical hv-CRKP isolates were collected from a tertiary hospital in China. Isolates were analyzed by multilocus sequence typing, antimicrobial susceptibility testing, conventional PCR screening, and targeted sequencing of mcr, pmrAB, phoPQ, crrAB, and mgrB. RT-qPCR assessed lipid A modification gene expression. Whole-genome sequencing was performed on colistin-resistant isolates. Functional roles of novel pmrB mutations were validated using CRISPR-Cas9 genome editing. Growth kinetics, competition assays, Galleria mellonella infection models, and transcriptomic profiling were conducted to assess fitness, virulence, and gene expression changes. Most isolates (210/239, 87.87%) belonged to ST11 lineage, and no mcr genes were detected. Mutations in pmrB were highly prevalent (232/239, 97.07%), with R256G representing a lineage-associated polymorphism, while specific variants (P95L, D150H, T157P, S203P) associated with elevated colistin MICs. mgrB alterations were identified in 6.28% (15/239) of isolates and were exclusively observed in colistin-resistant strains, whereas isolates carrying crrB variants (17/239, 7.11%) remained susceptible to colistin. Mutations in the HAMP and HisKA domains of pmrB were associated with increased colistin MICs and upregulation of lipid A modification genes. Functional validation using CRISPR-Cas9 confirmed that two novel pmrB variants (D150H and S203P) conferred resistance in ST11-KL25 isolates, as reversion restored colistin susceptibility and impaired bacterial growth under colistin pressure. Transcriptomic analysis demonstrated that reversion suppressed lipid A modification pathways (arnA-D, eptA). Despite fitness advantages under colistin pressure, virulence assays in the G. mellonella infection model showed that pmrB mutants retained full pathogenic potential. Chromosomal pmrB mutations and disruptive mgrB alterations are the predominant mechanisms of colistin resistance in clinical hv-CRKP. The emergence of colistin-resistant ST11-KL64/KL25 hv-CRKP highlights the urgent need for ongoing genomic surveillance and the development of effective therapeutic strategies to mitigate their growing public health threat.