Abstract
The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP), particularly strains co-producing dual carbapenemases such as Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM), poses a significant challenge in clinical settings. This study aimed to elucidate the clinical features, molecular epidemiology, virulence phenotype, and plasmid stability of ST11-KL64-type K. pneumoniae co-producing KPC-2 and NDM-1. We retrospectively analyzed 44 non-duplicate clinical isolates of KPC-2-NDM-1-carbapenem-resistant K. pneumoniae (K2N1-CRKP) and their corresponding clinical data from patients in a Chinese teaching hospital in 2021. Comprehensive analyses included antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), whole-genome sequencing, serum killing assays, Galleria mellonella infection models, and plasmid stability experiments. All isolates were resistant to carbapenems and ceftazidime/avibactam. These strains belonged to the ST11-KL64-O1/O2v1 clone and showed high genetic relatedness (≥88% similarity) via PFGE. Among them, 79.55% (35/44) were classified as CR-hvKP, carrying the virulence genes iucABCD and rmpA/rmpA2. Genomic analysis revealed that blaKPC-2 and blaNDM-1 were located on an IncFII/IncR plasmid and an untypable plasmid, respectively, while the virulence gene cluster was identified on an IncFIB/IncHI1B virulence plasmid. The virulence phenotypes of selected strains exhibited heterogeneity. One isolate (KP1225) demonstrated high virulence in vitro and in vivo. Notably, these strains maintained their resistance plasmids without antibiotic pressure (retention rate >85% after 10 days), with no significant fitness cost observed. This study reveals a clonal dissemination of ST11-KL64-K2N1-CR-hvKP, successfully integrating carbapenem resistance and hypervirulence with high genetic stability. The potential risk of its spread warrants urgent attention and effective infection control measures. Importance: The emergence of bacterial pathogens that combine hypervirulence with advanced antimicrobial resistance poses a significant challenge for modern healthcare. This study comprehensively analyzes a concerning clinical strain: ST11-KL64 K. pneumoniae co-harboring both KPC-2 and NDM-1 carbapenemases. We demonstrate that this strain successfully integrates multiple resistance mechanisms, hypervirulence determinants, and remarkable genetic stability without observable fitness costs-a combination that significantly complicates treatment and infection control. Critically, the stable maintenance of its resistance plasmids without antibiotic selection highlights the potential for persistent colonization and transmission. These findings underscore an evolving threat in the landscape of multidrug-resistant infections and emphasize the urgent need for enhanced surveillance, robust diagnostic approaches, and novel therapeutic strategies to address such challenging pathogens.