Genetic Characterization and Passage Instability of a Hybrid Plasmid Co-Harboring bla IMP-4 and bla NDM-1 Reveal the Contribution of Insertion Sequences During Plasmid Formation and Evolution.

Carbapenemase is the predominant enzyme in the mechanism leading to Enterobacterales resistance to carbapenems, but only a limited number of isolates harbor double classes/types of carbapenemase. Here, an IMP-4 and NDM-1 producer named Klebsiella michiganensis 7525 is reported, and the co-harboring plasmid is further characterized. K. michiganensis 7525 was positive for the bla IMP-4 and bla NDM-1 genes by the NG-Test Carba-5 method and PCR followed by sequencing, and both were located on the same plasmid (designated pKOX7525_1) according to S1-PFGE with Southern blot experiments. pKOX7525_1 was capable of transconjugation with an efficiency of 4.3 × 10 -8 in a filter mating experiment. Whole-genome sequencing and bioinformatics analysis confirmed that the plasmid was novel, clustered to the incompatibility type of IncHIB/IncFIA/IncR and presented high similarity to a bla IMP-4 -carrying IncHIB plasmid (pA) published with 79% coverage and 100% sequence identify. In contrast, a large-fragment insertion and inversion mediated by IS 26 was observed on the plasmid, which introduced a genetic hybrid zone with multiple resistance genes, including bla NDM-1 , to the plasmid. In the transconjugants, the presence of pKOX7525_1 had a negative impact on bacterial fitness. In vitro evolution experiments showed that pKOX7525_1 in the transconjugant could not be stably inherited after 10 days of passage and that bla NDM-1 could be lost during repeated laboratory passage. Our study not only reports a novel plasmid co-harboring bla IMP-4 and bla NDM-1 but also highlights the putative pathway of plasmid formation and evolution by means of genetic rearrangement through sequence insertion and homologue recombination, which may have critical value for plasmid research and increase awareness of carbapenem-resistant Enterobacteriaceae (CRE). IMPORTANCE In this study, we characterized a novel plasmid from a carbapenem-resistant K. michiganensis (CRKM) isolate, which harbors two metallo-β-lactamases (MBLs), IMP-4 and NDM-1, is capable of transconjugation and contains three replicons. Our results first expand the diversity of plasmids co-harboring carbapenemase genes in Enterobacterales , which exhibits epidemic importance in bacterial resistance. Additionally, we investigated the origin and formation of this MBL double-positive plasmid based on comparative genomics analysis, which indicated that IS 26 plays a vital role through continuous genetic rearrangements. Moreover, this plasmid is unstable in transconjugants during passage at the multidrug-resistant (MDR) region of bla NDM-1 , with fluctuating stability under varying antibiotic selection, highlighting auspicious considerations regarding recognition of the complexity and plasticity of plasmids in evolution and re-emphasizing clinical infection control inspired by CRE.