Virulence and genomic features of a bla CTX-M-3 and bla CTX-M-14 coharboring hypermucoviscous Klebsiella pneumoniae of serotype K2 and ST65

Abstract

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; ST65; blaCTX-M; comparative genome; serotype K2; virulence factor.

Figure 1

The detection of virulence potential of the KP_06. Notes: (A) Biofilm biomass expressed as crystal violet OD (OD_{570 nm}). Data are expressed as mean ± SD (error bars) of at least three independent experiments for each strain. (B) Survival of each strain was assessed by enumerating viable counts at 0, 1, 2, and 3 hours of incubation in the pooled human serum at 37°C. Data are presented as mean ± SD (n=3 for each strain). (C) The effect of 1×10⁶ CFU of each K. pneumoniae strain on the survival of G. mellonella, whereas other doses of each K. pneumoniae strain induce dose-dependent lethality are shown in Figure S2. KP_07 is a ST23:K1 strain that harbored a ~220 kbp virulence plasmid and is used as positive control. KP_36 is a bla_KPC-2-producing ST11 strain that did not harbor virulence plasmid and is used as a negative control in these studies. Abbreviations: CFU, colony-forming unit; G. mellonella, Galleria mellonella; K. pneumoniae, Klebsiella pneumoniae; ST, sequence type.

Figure 2

Genomic sequence comparative analysis of KP_06 and five HM K2-serotype K. pneumoniae strains. Notes: (A) Whole-genome sequences’ comparison of the strains. The circles from inside to outside indicate GC content of KP_06, GC skew of KP_06, and the K2-serotype K. pneumoniae strains 52.125, hvKP1, NUHL24835, RJF293, U25, and KP_06. The white and colored regions of the rings indicate absent and present, respectively. (B) Network graph shows shared orthologies and unique orthologies among the strains. Blue nodes: the name of K. pneumoniae strains; green nodes: the number of unique orthologies of the corresponding strain; the gray nodes: the number of shared orthologies among strains. Abbreviations: HM, hypermucoviscosity; K. pneumoniae, Klebsiella pneumoniae.