Co-Transfer of pK2044-Like Virulence Plasmid and Kpc Plasmid Facilitates the Evolution of Hypervirulent and Carbapenem-Resistant Klebsiella pneumoniae in the Hospitals

Background: The mechanism of the evolution of the hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is of particular interest to researchers and clinicians. Methods: We selected the hypervirulent NTUH-K2044, carbapenem-resistant JS187, and HS11286 strains to perform first-round plasmid conjugation experiments. PFGE, S1-PFGE, and draft genome sequences were performed to confirm the transconjugants. Biofilm formation, serum resistance, mucoviscosity assay, greater wax moth larvae and mice infection models, and siderophore production assays were used to determine the virulence phenotype. Antimicrobial susceptibility tests were performed to confirm the resistance phenotype. Findings: The transconjugant NTUH-K2044-pKPHS2 (CR-hvKp) could not possess both hypervirulence and carbapenem-resistance properties. Next, we performed second-round plasmid conjugation experiments, using the first-round transconjugant NTUH-K2044-pKPHS2 as a donor and carbapenem-resistant JS187 as a recipient, and we observed that pK2044 was transferred to JS187 facilitated by the conjugative KPC plasmid pKPHS2. The transconjugant JS187-pK2044 (hv-CRKP) had the dual phenotype of hypervirulence and carbapenem resistance.  Interpretation: Our study confirms an evolutionary pathway of pK2044-like virulence plasmid transfer to CRKP strains and also provides an explanation for the lower detection rate of CR-hvKp than hv-CRKP strains in clinical settings. Funding: This work was supported by National Natural Science Foundation of China under Grant 81871692 and 81572031; and Science and Technology Commission of Shanghai Municipality under Grant 19JC1413000；Shanghai Municipal Key Clinical Specialty under grant shslczdzk0330; Jiaxing Municipal Key Laboratory of Infectious Diseases and Bacterial Drug Resistance. Declaration of Interest: None to declare. Ethical Approval: Animal ethics approval was obtained from the Animal Ethics Committee of Huashan Hospital of Fudan University.