Divergent Virulence Traits in Klebsiella Pneumoniae Strains of Capsular Serotype K2 and ST25: Insights from Whole-Genome Sequencing

Hypervirulent <i>Klebsiella pneumoniae</i> (hvKp) poses significant challenges for clinicians, yet the virulence determinants of hvKp have not been fully ascertained. In our study, we observed a divergence in virulence between two K2-ST25 <i>Klebsiella pneumoniae</i> strains, both of which carried the <i>rmpA, iroB</i> and <i>peg344</i> virulence-associated genes. These strains exhibited different median death time (12 hour <i>vs</i> over one week in BALB/c) in the mice lethality assay. Whole genome sequencing (WGS) revealed that the virulence plasmids of hvKp CHK014 and non-hvKp CHK036, namely pCHK014 and pCHK036-1, shared 99.92% and 95.92% identity with the classic hypervirulent plasmid pLVPK, respectively. Additionally, in non-hvKp CHK036, the <i>rmpA</i> gene, along with the <i>iro</i> operon, <i>iutA</i> gene and <i>peg344</i> gene, was located in the integrative and conjugative element ICE<i>Kp1</i> in the chromosome. However, in hvKp CHK014, the <i>rmpA</i> gene, <i>peg344</i> gene and the <i>iro</i> operon were located on the IncFIB/IncHI3B virulence plasmid. Furthermore, RT-qPCR results demonstrated significantly higher expression levels of the <i>rmpA, iroB</i> and <i>peg344</i> genes in hvKp CHK014 compared to non-hvKp CHK036. Correspondingly, the capsular polysaccharide yields regulated by the <i>rmpA</i> gene were significantly higher in CHK014 than CHK036. Although the copy number of <i>rmpA</i> gene in both strains was not altered, the poly (T) track on <i>rmpA</i> promoter remains as P_11T, which contributed to the elevated expression level in hvKp CHK014. Whereas the poly(T) track on <i>rmpA</i> promoter in non-hvKp CHK036 was P_10T, a shorten form of poly(T). Meanwhile, the promotor of <i>rmpA</i> on CHK036 included 38 additional variants, compared with CHK014 and pLVPK. These findings indicated that the expression of the <i>rmpA</i> gene was a crucial virulence determinant, with the P_11T on promoter of <i>rmpA</i> gene having a higher expression potential and thus contributing to the virulence difference between hvKp CHK014 and non-hvKp CHK036 in our murine infection model.