Prevalence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae under divergent evolutionary patterns

Abstract

K1/K2 hvKP strains acquire carbapenem-resistance plasmids, known as CR-hvKp, and carbapenem-resistant Klebsiella pneumoniae (CRKP) strains obtain virulence plasmids, recognized as hv-CRKP. The two different evolution patterns of hypervirulent combined carbapenem-resistant Klebsiella pneumoniae may lead to their different prevalence in hospitals. Our study aimed to investigate the prevalence of hv-CRKP and CR-hvKp strains and to analyze factors influencing their evolution and prevalence. We collected 890 K. pneumoniae genomes from GenBank and 530 clinical K. pneumoniae isolates from nine hospitals. Our study found that hv-CRKP strains were more prevalent than CR-hvKp strains and both were dominated by bla_KPC-2 gene. The bla_KPC-2-carrying plasmids could mobilize non-conjugative virulence plasmids from hvKp strains to CRKP strains. The conserved oriT of virulence plasmids and the widespread of conjugative helper plasmids were potential factors for the mobilization of non-conjugative virulence plasmids. HvKp strains with KPC plasmid could hardly simultaneously exhibit hypervirulence and carbapenem resistance as CRKP strains with virulence plasmid, and we found that rfaH mutation reduced capsular synthesis and increased carbapenem resistance of the CR-hvKp strain. In summary, this study revealed that hv-CRKP strains were more suitable for survival in hospital settings than CR-hvKp strains and the widespread conjugative KPC-producing plasmids contributed to the emergence and prevalence of hv-CRKP strains.

Keywords: Hypervirulent; carbapenem resistance; conjugation; genome evolution; oriT.

Figure 1.

The prevalence of hv-CRKP and CR-hvKp strains. Distribution of carbapenemases is depicted as below. Intensity of box shading indicates the proportion of genomes harbouring different carbapenemases (orange), as per inset legend.

Figure 2.

The mobilization of non-conjugative virulence plasmids by conjugative KPC plasmid. (A) Two rounds plasmid conjugation experiments simulating the transfer path of non-conjugative plasmid. (B) The confirmation of transconjugants by PFGE and S1-PFGE. *Virulence plasmid pK2044. ^#KPC plasmid pKPHS2. The same symbol is used to represent the parental strain and its transconjugant.

Figure 3.

The transfer potential of mobilizable virulence plasmids. (A) The mobilization of mimic virulence plasmid by conjugative KPC plasmid pKPHS2. (B) The distribution of TraM_pKPHS2-carrying plasmids in Enterobacteriaceae. (C) nic sites of pKPHS2 and pK2044. Alignment was generated with the MUSCLE sequence alignment tool.

Figure 4.

The virulence phenotypes and levels of CR-hvKp strain. (A) Mucoviscosity. (B) The relative expression of capsular synthesis genes. (C) Uronic acid. (D) Transmission electron microscopy of the capsule. (E) Biofilm formation. (F) Siderophores production. (G) Serum resistance. (H) The survival curves of infected Larvae wax. (I) The survival curves of infected mice. An unpaired two-sided Student’s t-test was performed for mucoviscosity, uronic acid, biofilm formation, siderophores production, and serum resistance. A log-rank (Mantel–Cox) test was performed for the survival curves. ***P< 0.001, **P< 0.01, *P< 0.05, ns: not significant.

Figure 5.

The influence of rfaH to carbapenem resistance and CPS production.

Figure 6.

The virulence phenotypes and levels of hv-CRKP. (A) Growth curve. (B) Siderophores production quantification. (C) Siderophores production determined by CAS agar plate. (D) Mucoviscosity. (E) Transmission electron microscopy of the capsule. (F) Uronic acid. (G) Biofilm formation. (H) Serum resistance. (I) The survival curves of infected mice. (J) The survival curves of infected larvae wax. An unpaired two-sided Student’s t-test was performed for mucoviscosity, uronic acid, biofilm formation, siderophores production, and serum resistance. A log-rank (Mantel–Cox) test was performed for the survival curves. ****P < 0.0001, ***P< 0.001, **P< 0.01, *P< 0.05, ns: not significant.