Regional Dissemination of a Carbapenemase-encoding Plasmid: Plasmidome analysis Reveals Diverse Adaptations of Carbapenemase-Producing Enterobacteriaceae

Abstract Background. The global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens human health by limiting the range of usable antibiotics even against common bacterial infections. The spread of CRE is primarily due to the transmission of carbapenemase genes located on plasmids. However, few studies have comprehensively identified regionally spreading carbapenemase-encoding plasmids because of the difficulty to determine the complete sequence of a plasmid encoding carbapenemases. In a CRE surveillance study of 1,507 patients from 43 hospitals in northern Osaka, Japan, we previously found that 12% of the patients carried CRE and 95% of CRE isolates were IMP-6 producers. This result suggested a vast horizontal spread of a clonal plasmid carrying blaIMP-6 among Enterobacteriaceae in this region. In the current study, we aimed to describe the dynamics of this regional horizontal plasmid transmission.Results. We systematically analysed the plasmids of 230 CRE isolates carrying blaIMP obtained in our previous surveillance study by using whole genome sequencing and Southern blotting. We detected a major population (187 out of 230 blaIMP-positive CRE isolates, 85.6%) that carried blaIMP-6 on the IncN plasmid pKPI-6, along with diverse minor subpopulations. Among the subpopulations, we identified a novel cluster carrying an IncF plasmid that leads to heteroresistance due to amplification of blaIMP-6, resulting in covert transmission of blaIMP-6 or occasional chromosomal integration of blaIMP-6. In addition, we detected one isolate that harboured blaIMP-1, which is identical to blaIMP-6 except for a single point mutation, on pKPI-6 and thus had acquired a broader range of antimicrobial resistance. Conclusions. Carbapenemase-encoding plasmid tracking revealed the clonal dissemination of pKPI-6 among chromosomally distinct isolates. Focusing on the mode of carbapenemase gene carriage is helpful for monitoring of horizontal spread of CRE isolates that is difficult to trace only by the comparisons of the whole genomes. A seemingly clonal horizontal dissemination of the predominant plasmid had embraced heterogenous subpopulations that contribute to diverse adaptations including covert transmission, stable chromosomal integration of blaIMP-6, or broadened antimicrobial resistance patterns, ultimately leading to treatment failure.