Background: The emergence and spread of hypervirulent Klebsiella pneumoniae (hvKp) poses a serious and growing challenge to public health worldwide. However, the knowledge regarding hvKp of animal origin remains very limited. In this study, we characterized a colistin-resistant hvKp isolate obtained from healthy chicken faeces and gained insight into the molecular basis of hypervirulence and colistin resistance.

Methods: Antimicrobial susceptibility testing was conducted by broth microdilution. The hypermucoviscous phenotype was determined by string tests. Whole-genome sequencing (WGS) was performed using a combination of Illumina NovaSeq/Oxford Nanopore PromethION platforms. Sequence alignment and complement assays were used to identify the mutations conferring colistin resistance. The virulence was investigated using both Galleria mellonella larvae and mice infection models.

Results: This strain KP20 displayed a hypermucoviscous phenotype and strong biofilm-forming capacity. WGS revealed that strain KP20 carried a pLVPK-like virulence plasmid with a novel replicon profile (IncFIB/RepB), harbouring key hypervirulence-associated genes, including iucABCD-iutA, iroBCDN, peg-344 and rmpA/rmpA2. Moreover, a plasmid harbouring the trimethoprim resistance gene dfrA50, pKP20-2, was identified as a member of the phage-like plasmids: genetic elements that function both as phages and plasmids. Sequence analysis and functional confirmation suggested that mutations in the crrB gene contributed to the colistin resistance observed in this strain. The virulence of KP20 was confirmed in the animal infection models.

Conclusions: In this study, a high-risk ST412-K57 colistin-resistant hvKp of animal origin was identified and characterized. According to the One Health concept, our findings highlight the critical need for implementing enhanced molecular surveillance of hvKp in animals.