Distinct and Shared Impacts of Virulence Plasmids on the Phenotype and Transcriptome in Convergent Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae

The global rise of convergent carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) represents a major clinical challenge, yet the role of virulence plasmids (pVirs) in shaping bacterial physiology and pathogenicity remains incompletely understood. Using a CRISPR-Cas9-based curing system, we precisely eliminated pVirs from two clinical CR-hvKp strains with distinct genetic backgrounds, ST23-KL1 (blaNDM-1) and ST11-KL64 (blaKPC-2). Loss of pVir conferred fitness advantages in vitro, reduced capsule production and hypermucoviscosity, and promoted biofilm formation, while markedly attenuating virulence in murine sepsis models. Despite this reduction, the pVir-cured ST23-KL1 strain retained higher virulence than the pVir-cured ST11-KL64 strain, underscoring the contribution of chromosomal background to pathogenic potential. Transcriptomic profiling revealed both shared and strain-specific transcriptional responses to pVir deletion, with broader perturbations observed in the ST11-KL64 strain. pVir removal had limited effects on antibiotic MICs. Complementation experiments further demonstrated differential regulatory roles of the rmpADC and rmpA2D2 operons in capsule expression and hypermucoviscosity across the two strains. Together, these findings establish pVirs as central determinants of CR-hvKp virulence and highlight complex host-plasmid interactions that influence bacterial adaptation and pathogenicity. IMPORTANCEThe emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) poses a critical threat to global health, yet the contribution of virulence plasmids (pVirs) to bacterial fitness and pathogenicity remains poorly defined. By employing a CRISPR-Cas9-based curing strategy, we dissected the role of pVirs in two genetically distinct CR-hvKp strains and uncovered their multifaceted impact on capsule production, hypermucoviscosity, biofilm formation, and virulence. Our findings reveal that pVir loss confers fitness advantages in vitro while attenuating virulence in vivo, with strain-specific transcriptional responses and differential regulation by rmp operons. These results underscore the complex interplay between plasmid-encoded and chromosomal determinants in shaping CR-hvKp pathogenicity and adaptation, offering mechanistic insights that may inform future therapeutic strategies targeting plasmid-mediated virulence.