High transconjugation efficiency of fusion plasmid pNDM_KPC in carbapenem-resistant <i>Citrobacter freundii</i> and its formation driven by IS <i>26</i> -mediated integration

Carbapenem-resistant <i>Enterobacteriales</i> (CRE) are increasingly recognized as reemerging nosocomial pathogens due to the dissemination of dual carbapenemase production. This study investigates the coexistence of <i>bla</i>_KPC-2 and <i>bla</i>_NDM-1 in CRE isolates, harbored on plasmids either separately and integratively, and examines the underlying mechanisms of plasmid formation and resistance dissemination. Two <i>Citrobacter freundii</i> isolates co-producing <i>Klebsiella pneumoniae</i> carbapenemase (KPC) and New Delhi metallo-beta-lactamase (NDM) carbapenemases were analyzed. S1-PFGE, Southern blot, and mating conjugation experiments were conducted to determine gene location, transferability, and transconjugation efficiency. Whole-genome sequencing (WGS) was used to elucidate plasmid evolution mechanisms. Specific PCR followed by sequencing was performed to identify hotspot integration sites. Experiments of checkerboard microdilution and time-kill were completed to test the treatment option based on aztreonam. Three plasmids were identified in <i>C. freundii,</i> pNDM, pKPC, and a fusion plasmid (pNDM_KPC) co-harboring <i>bla</i>_KPC-2 and <i>bla</i>_NDM-1. All plasmids exhibited transconjugation capability, with significant differences in transfer efficiency (<i>P</i> < 0.05). pKPC demonstrated the highest efficiency (ranging from 10⁻¹ to 10⁻²), followed by pNDM_KPC (10⁻⁴ to 10⁻⁶) and then pNDM (around 10⁻⁸). IS<i>26</i>, located downstream of the <i>intI</i> site, was identified as a critical hotspot mediating the disintegration and integration of pNDM_KPC. Synergy effect and bactericidal activity were observed on aztreonam plus ceftazidime/avibactam or meropenem/vaborbactam or imipenem/relebactam. The presence of CRE isolates harboring fusion plasmid pNDM_KPC would pose a significant threat in nosocomial infections and should arise wide attention. IS<i>26</i> downstream of IntI would be the hotspot driving the integration state of pNDM_KPC and contributing to complex resistance mechanisms.IMPORTANCEThe global rise of carbapenem-resistant <i>Enterobacterales</i> (CRE) poses a critical threat to public health. Our study identifies <i>Citrobacter freundii</i> strains co-harboring three distinct carbapenemase-encoding plasmids: pNDM, pKPC, and a novel fusion plasmid (pNDM_KPC). We reveal significant differences in conjugation efficiency among these plasmids, with the fusion plasmid demonstrating a moderate yet concerning ability to spread dual carbapenemase genes. This finding highlights the fusion plasmid as a potential vector for the co-dissemination of <i>bla</i>_NDM-1 and <i>bla</i>_KPC-2, emphasizing the need for enhanced surveillance. Furthermore, we identify the IS<i>26</i> element downstream of the <i>intI</i> site as a potential recombination hotspot driving plasmid fusion. These insights deepen our understanding of carbapenemase gene transmission and call for global attention to the fusion plasmid-mediated resistance spread.