Yanghua Xiao, Ge Zhang, Feng Nie, Keyi Li, Xingyu Tao, Tianxin Xiang, Ping Li
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.