Acquisition of a stable and transferable plasmid coharbouring hypervirulence and MDR genes with low fitness cost: Accelerating the dissemination of ST11-KL64 CR-HvKP

Abstract

Objectives: This study aimed to delineate the ability of a plasmid, pS130–4, which harboured both hypervirulence and multidrug resistance genes, to disseminate within Klebsiella pneumoniae, as well as its potential formation mechanism. Methods: We employed whole-genome sequencing to decipher the genetic architecture of pS130–4. Its capability to conjugate and transfer was assessed through a series of experiments, including plasmid stability, competitive growth, and growth curve analysis. Its expression stability was further evaluated using drug sensitivity, larval survival, and biofilm formation tests. Results: pS130–4 contained four intact modules typical of self-transmissible plasmids. BLAST analysis revealed a sequence identity exceeding 90% with other plasmids from a variety of hosts, suggesting its broad prevalence. Our findings indicated the plasmid's formation resulted from IS26-mediated recombination, leading us to propose a model detailing the creation of this conjugative fusion plasmid housing both blaKPC-2 and hypervirulence genes. Our conjugation experiments established that pS130–4, when present in the clinical strain S130, was self-transmissible with an estimated efficiency between 10−5 and 10−4. Remarkably, pS130–4 showcased a 90% retention rate and did not impede the growth of host bacteria. Galleria mellonella larval infection assay demonstrated that S130 had pronounced toxicity when juxtaposed with high-virulence control strain NTUH-K2044 and low-toxicity control strain ATCC700603. Furthermore, pS130–4′s virulence remained intact postconjugation. Conclusion: A fusion plasmid, encompassing both hypervirulence and multidrug resistance genes, was viable within K. pneumoniae ST11-KL64 and incurred minimal fitness costs. These insights underscored the criticality of rigorous monitoring to pre-empt the escalation and distribution of this formidable super-plasmid.

Keywords

• Klebsiella pneumoniae
• Fitness cost
• Plasmid
• Transferability
• Multidrug resistance
• Hypervirulence