Mechanistic insights into the formation and spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae via an IncHI1B/repB hybrid virulence plasmid

<b>Objectives.</b> The transfer of virulence plasmids facilitates the emergence of carbapenem-resistant hypervirulent <i>Klebsiella pneumoniae</i> (CR-hvKP), complicating infection management. This study identifies a conjugative IncHI1B/repB hybrid virulence plasmid (pHvKP51-Vir) that promotes CR-hvKP formation via recombination within a highly active mobile genetic element (MGE) region during conjugative transfer.<b>Methods.</b> The fusion of the KPC-type resistance plasmid (pKPC) with the virulence plasmid (pHvKP51-Vir) was demonstrated via conjugation experiments, leading to the formation of the hybrid plasmid pJ53-KPC_Vir. Plasmid conjugative transfer experiments, Southern blotting, <i>Galleria mellonella</i> infection assays and serum killing assays were utilized to investigate alterations in pHvKP51-Vir and its fusion with pKPC during conjugation.<b>Results.</b> The fusion plasmid pJ53-KPC_Vir co-harboured <i>blaKPC</i> and <i>rmpA2</i>, conferring both carbapenem resistance and virulence. The fusion plasmid demonstrated high transferability, facilitating the dissemination of virulence plasmids. While the fusion plasmid temporarily attenuated virulence, its dissociation restored the virulence plasmid to its original level. TnAs1 and the MGE region were critical in mediating this fusion.<b>Conclusions.</b> The TnAs1- and MGE-mediated fusion generated an unstable hybrid plasmid with high transferability. Its dissociation restored and enhanced virulence, promoting rapid dissemination of resistance and virulence, posing a substantial clinical threat.