Hfc-mediated enhancement of plasmid conjugation drives the dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae

The horizontal transfer of antimicrobial resistance (AMR) plasmids drives the global spread of multidrug-resistant Gram-negative pathogens. Among these, carbapenem-resistant Klebsiella pneumoniae (CRKP), particularly high-risk hypervirulent lineages (CR-hvKP), poses a major clinical threat due to the rapid dissemination of resistance determinants. However, the mechanisms underlying efficient plasmid transmission in these high-risk clones remain poorly understood. Here, we identify Hfc, a plasmid-encoded factor that enhances conjugative transfer in K. pneumoniae . Genomic epidemiological analyses revealed that hfc is widely distributed, predominantly associated with conjugative IncF-type plasmids, highly enriched in the ST11-KL64, the predominant epidemic CR-hvKP lineage, and has increased in prevalence alongside the recent expansion of this high-risk clone. Functional studies demonstrated that deletion of hfc markedly reduced plasmid transfer efficiency, whereas its overexpression enhanced conjugation in vitro and promoted plasmid dissemination during mouse gut colonization. Mechanistically, Hfc was found to bind the plasmid origin of transfer ( oriT ) and is associated with increased expression of a subset of T4SS-related genes, as well as enhanced pilus-like structures and elevated intracellular ROS levels. In addition, Hfc shows potential interaction with TraC, a VirB4-like ATPase involved in T4SS assembly and activity, suggesting a possible role in modulating conjugation machinery function. Notably, Hfc imposes no detectable fitness or virulence cost on the host. Together, these findings identify Hfc as a conserved enhancer of plasmid conjugation and a potential target for limiting the spread of AMR plasmids in high-risk CR-hvKP lineages.