Global emergence of Carbapenem-resistant Hypervirulent Klebsiella pneumoniae driven by an IncFII_K34 KPC-2 plasmid

Background: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) has been increasingly reported worldwide, posing a severe challenge to public health; however, the mechanisms driving its emergence and global dissemination remain unclear. Methods: We analysed CR-hvKp strains derived from canonical hvKp backgrounds, and acquired a carbapenemase-encoding gene. These strains were identified from 485 CRKp isolates in the CRACKLE-2 China cohort, 259 CRKp isolates from a multi-centre study, and 67,631 K. pneumoniae genomes available in GenBank. Clinical isolates harbouring the IncFII_K34 KPC-2 plasmid were selected for genome sequencing, RNA-Seq, conjugation assays, in vivo, ex vivo, and in vitro phenotypic characterisation. Findings: Analysis of clinical CR-hvKp isolates and the 414 genomes from 24 countries available in GenBank identified an IncFII_K34 KPC-2 plasmid as the prevalent KPC plasmid (detected in 25%, 45/178 of KPC-producing CR-hvKp). Compared with the epidemic IncFII_K2 KPC-2 plasmid, the IncFII_K34 KPC-2 plasmid exhibited a 100- to 1000-fold increase in conjugation frequency (10⁻⁴–10⁻⁵ vs. 10⁻⁷) and an in vitro growth advantage under meropenem challenge–likely due to the overexpression of conjugation-related genes and an increased bla_KPC copy number and expression. CR-hvKp isolates and hvKp transconjugants carrying this plasmid often exhibited reduced mucoviscosity, while retaining hypervirulence in both murine models and human neutrophil assays. Interpretation: The IncFII_K34 plasmid may be a key factor driving the global dissemination of CR-hvKp, underscoring the urgent need for enhanced molecular surveillance of this emerging pathogen. Funding: National Natural Science Foundation of China and National Institutes of Health.