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

Qingqing Du,1– 3,* Fen Pan,1– 3,* Fangyuan Yu,1– 3 Jie Jiang,1– 3 Chun Wang,1– 3 Huihong Qin,1– 3 Wenxin Liu,1– 3 Xiaozhou Pan,1– 3 Zhan Ma,1– 3 Wenhao Weng,1– 3 Dingding Han,1– 3 Hong Zhang1– 3 1Department of Clinical Laboratory, Shanghai Children’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China; 2Institute of Pediatric Infection, Immunity, and Critical Care Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 3College of Health Science and Technology, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Dingding Han, Email [email protected] Hong Zhang, Email [email protected]: Hypervirulent Klebsiella pneumoniae (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 Klebsiella pneumoniae strains, both of which carried the rmpA, iroB and peg344 virulence-associated genes. These strains exhibited different median death time (12 hour vs 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 rmpA gene, along with the iro operon, iutA gene and peg344 gene, was located in the integrative and conjugative element ICEKp1 in the chromosome. However, in hvKp CHK014, the rmpA gene, peg344 gene and the iro operon were located on the IncFIB/IncHI3B virulence plasmid. Furthermore, RT-qPCR results demonstrated significantly higher expression levels of the rmpA, iroB and peg344 genes in hvKp CHK014 compared to non-hvKp CHK036. Correspondingly, the capsular polysaccharide yields regulated by the rmpA gene were significantly higher in CHK014 than CHK036. Although the copy number of rmpA gene in both strains was not altered, the poly (T) track on rmpA promoter remains as P11T, which contributed to the elevated expression level in hvKp CHK014. Whereas the poly(T) track on rmpA promoter in non-hvKp CHK036 was P10T, a shorten form of poly(T). Meanwhile, the promotor of rmpA on CHK036 included 38 additional variants, compared with CHK014 and pLVPK. These findings indicated that the expression of the rmpA gene was a crucial virulence determinant, with the P11T on promoter of rmpA 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.Keywords: Klebsiella pneumoniae, hypervirulence, whole-genome sequencing, gene promotor