Characterization of a Conjugative Hybrid Plasmid Coharboring blaKPC-2 and blaIMP-4 in a Klebsiella quasipneumoniae Clinical Isolate

Abstract

Generation of hybrid MDR plasmids accelerated the evolution and transmission of resistance genes. In this study, we characterized a bla_KPC-2- and bla_IMP-4-coharboring conjugative hybrid plasmid constituted of an IncHI5 plasmid-like region, an IncFII(Yp)/IncFIA plasmid-like region, and a KPN1344 chromosome-like region from a clinical ST852-KL18 Klebsiella quasipneumoniae strain. The bla_IMP-4 gene was captured by a novel integron In1965, and the bla_KPC-2 gene was located on a new non-Tn4401 group I NTE_KPC element. Both bla_KPC-2- and bla_IMP-4-containing genetic architectures were distinguished from classical structures, highlighting the constant evolution of these genetic elements. IMPORTANCE The emergence of carbapenem-resistant Enterobacterales (CRE) that coexpress serine- and metallo-carbapenemases is a severe threat to the efficacy of ceftazidime-avibactam (CZA), which has been proven to be extremely effective against KPC-producing Enterobacterales strains. Our study described the cooccurrence of KPC-2, a serine β-lactamase, and IMP-4, a metallo-β-lactamase (MBL), on a conjugative hybrid plasmid from a clinical carbapenem-resistant K. quasipneumoniae strain, and it revealed an alternative route for IncHI5 plasmid to evolve by recombining with other plasmids to form a hybrid plasmid. Moreover, this hybrid plasmid can be transferred into other Klebsiella species and stably persist during passage. The propagation of two important carbapenemase genes with a new genetic background using well-evolved plasmids in the clinical setting promotes the emergence of superbugs that require careful monitoring.

Keywords: IncHI5 plasmid; K. quasipneumoniae; blaIMP-4; blaKPC-2; hybrid plasmid.

FIG 1

(A) PFGE of S1-digested plasmid DNA and Southern blot hybridization for bla_IMP-4 (red) and bla_KPC-2 (green). The black bands showed positive signals by Southern blot hybridization using the bla_IMP probe or bla_KPC probe. Lane M, Salmonella strain H9812 as a molecular marker. (B) Circular comparison between the hybrid plasmid pKP18-31-IMP and other reported similar plasmids or chromosomes. The plasmid pKP18-31-IMP at the outermost circle was used as the reference plasmid. Three circles with different sizes marked in blue, green, and gray dash lines represent the different genetic modules of pKP18-31-IMP. The circular map was generated using the BLAST Ring Image Generator (BRIG). (C) Linear alignment of plasmid pKP18-31-IMP with plasmid pRo24724 and plasmid pK516_KPC. The linear map was generated using EasyFig.

FIG 2

(A) Linear comparison of genetic environment of bla_IMP-4 between pKP18-31-IMP and pIMP-4-EC62. (B) Linear comparison of the genetic environment of bla_KPC-2 among pKPHS2, pKP048, pKP18-31-IMP, and pZZ40-KPC. The plasmid comparison figure was generated by EasyFig.