Genomic and phenotypic characterization of a carbapenem-resistant hypervirulent Klebsiella pneumoniae strain with chromosomal wcaj inactivation and reduced virulence

The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) represents an important clinical concern. However, the presence of canonical hypervirulence-associated determinants does not always correspond to the expected virulence phenotype. In this study, we combined multiregional molecular epidemiology, comparative genomics, and phenotypic assays to characterize the clinical isolate D209. Among 424 isolates carrying canonical hypervirulence-associated markers, ST11 and K64 were most frequently detected in bronchoalveolar lavage fluid, blood, and urine. In contrast, ST23 and K1 predominated in pus, whereas sputum isolates showed more heterogeneous sequence-type and K-type distributions. D209, an ST23-K1 isolate, carried a bla KPC-2 -bearing resistance plasmid and a virulence plasmid containing rmpA / rmpA2 , iuc , and iro , thereby fulfilling commonly used marker-based genotypic criteria for CR-hvKP. Nevertheless, D209 exhibited markedly reduced capsular viscosity and lower virulence than NTUH-K2044. D209 showed no net proliferation in normal human serum and substantially lower lethality than NTUH-K2044 in the Galleria mellonella and murine infection models, while retaining measurable lethality in the larval model. D209 also exhibited higher adhesion to and invasion of Caco-2 and HepG2 cells than the two reference strains. Comparative genomic analysis identified a 1,056-bp IS903B -like element belonging to the IS5 family inserted between nucleotides 460 and 461 of the chromosomal capsule biosynthesis gene wcaJ , without deletion of any wcaJ sequence. This chromosomal alteration was associated with markedly reduced capsular viscosity in D209. The virulence plasmid additionally carried the copper- and silver-resistance operons pco and sil and the plasmid-borne toxin-antitoxin system higAB . These findings indicate that canonical plasmid-borne hypervirulence markers alone may not fully predict the expressed virulence phenotype and support consideration of chromosomal capsule-associated loci and experimentally measured phenotypes when evaluating CR-hvKP isolates.