Evolution and transmission potential of iuc3-positive virulence plasmids in hypervirulent Klebsiella pneumoniae

The global emergence of hypervirulent Klebsiella pneumoniae (hvKP) poses significant threats to public health. The iucABCD (iuc) operon, a plasmid-borne genetic determinant encoding the siderophore aerobactin, serves as a reliable biomarker for hvKP identification. This study aimed to characterize the iuc3 (type 3 iuc)-positive K.pneumoniae isolates from diseased cows as hvKP strains and to elucidate the contribution of iuc3-positive plasmids to the hypervirulence phenotype, the evolutionary pathways and the spreading risk of the iuc3-positive plasmids. Eight of 108 K. pneumoniae isolates from 763 cow milk samples contained the iuc3 operon, and all iuc3 operons were located on T4SS-bearing plasmids. Notably, these isolates showed hypervirulent phenotype compared to the iuc3 knockout and plasmid-cured strains, including severe tissue damage, high bacterial loads in typical organs and high mortality in animal models. Analysis of NCBI database showed iuc3-positive K. pneumoniae strains have been widely distributed across animal, food, and humans, and exhibit an independent evolutionary pathways compared to the human-associated iuc1-type hvKP. The IS elements likely drive the acquisition of various resistance genes and the emergence of iuc3-positive resistance plasmids. The iuc3-bearing plasmids in this study demonstrated highly conjugative ability, low fitness costs, and the ability to replace the iuc1-bearing classical virulence plasmid in human hvKP strain. Besides, they could completely fuse with broad-host-range resistance plasmid (IncX3-bla_NDM-5), becoming carbapenem-resistant hvKP strains. The iuc3- and bla_NDM-5- fusion plasmids showed strong in vivo retention and dissemination capabilities in mouse models. Our findings emphasize the necessity for long-term monitoring of iuc3-positive plasmids in K. pneumoniae isolates from both animals and humans.