Background: Capsular serotype K2 Klebsiella pneumoniae  of sequence type (ST) 65 has been recognized as a hypervirulent clone. Simultaneous presence of different bla _CTX-M genes has never been reported in this clone. In the present study, the genetic characteristics and virulence phenotype of a CTX-M-3 and CTX-M-14 coproducing ST65 K. pneumoniae  human isolate, KP_06, that caused an intracranial infection, are evaluated.
Methods: The potential virulence of KP_06 was assayed by in vitro and in vivo methods. The molecular biology and whole-genome sequencing technology were used to analyze the genomic features associated with the virulence of this strain.
Results: The KP_06 exhibited typical features of hypervirulent K. pneumoniae  (hvKP), showing hypermucoviscosity phenotype and belonging to K2 and ST65. Apart from virulence genes linked to hvKP, including rmpA , rmpA2 , and clb  cluster and genes encoding siderophores, it was found to harbor a ~170 kb pLVPK-like virulence plasmid. In contrast to most hvKP, KP_06 was resistant to cephalosporins and the coexistence of bla _CTX-M-3 and bla _CTX-M-14 was detected. Further experiments demonstrated that this strain was classified as a nonbiofilm producer and serum sensitivity (grade 1) and killed only 30% of Galleria mellonella  inoculated with 1×106 colony-forming unit of the specimen within 48 hours, suggesting relatively low virulence. Comparative genomic analysis of KP_06 with five K2 hypermucoviscous K. pneumoniae  (HMKP) revealed seven unique orthologies with varied function in this strain. Intriguingly, the virulence genes identified in KP-06 were unexpectedly more diverse than those observed in five other K2 HMKP strains.
Conclusion: Our data support the notion that neither virulence-associated genes (clusters) nor the pLVPK-like virulence plasmid is sufficient for the hypervirulence of K. pneumoniae . Future studies aiming to explore the virulence of K. pneumoniae  should take genome-based profile together with experimental work. The detailed mechanism involving in the impaired virulence of KP_06 remains to be further explored.
Keywords: Klebsiella pneumoniae , virulence factor, serotype K2, ST65, bla _CTX-M, comparative genome