ST11 carbapenem-resistant Klebsiella pneumoniae integrates virulence plasmid fragments into the chromosome via insertion sequence

Virulence plasmids are key drivers of hypervirulence in Klebsiella pneumoniae. Here, we report a systematic analysis of chromosomal integration of a p17-15vir-derived fragment carrying virulence (rmpA2 and iutA-iucABCD) and resistance genes in nine bla_KPC-2-positive ST11-KL47 carbapenem-resistant K. pneumoniae (CRKP) isolates. Of these, seven exhibited a hypervirulent phenotype in a mouse infection model. Nanopore sequencing analysis revealed that these virulence-associated integration regions could be classified into three distinct groups based on their structural patterns. Notably, we investigated the mechanisms underlying the formation of the integration region and proposed an IS26-mediated model for the integration of virulence gene-carrying plasmid fragment into the chromosome. Besides, ISKpn1 was identified for the first time as a preferred insertion hotspot. Both bla_KPC-2 and rmpA2 demonstrated stable persistence in these isolates without antibiotic selection pressure, and group I integration regions displayed the capability to form circular intermediates. These findings provide critical insights into the virulence plasmid fragment integrated into their chromosomes and underscore the importance of surveillance for such hybrid threats.