Carbapenem-resistant Citrobacter freundii harboring bla(KPC−2) and bla(NDM−1): a study on their transferability and potential dissemination via generating a transferrable hybrid plasmid mediated by IS6100

INTRODUCTION: The increase in clinical Enterobacteriaceae with dual carbapenemase has become a serious healthcare concern. It is essential to characterize the transferability and potential dissemination of bla(KPC−2)- and bla(NDM−1)-coharboring carbapenem-resistant Citrobacter freundii (CRCF). METHODS: Four bla(KPC−2)- and bla(NDM−1)-coharboring CRCF strains were collected from our surveillance of the prevalence of carbapenem-resistant Enterobacteriaceae. The isolates were assessed using species identification, antimicrobial susceptibility testing, conjugation assays, whole-genome sequencing, plasmid stability, and fitness costs. Clonality, genome, plasmidome, and phylogeny were analyzed to reveal potential dissemination. RESULTS: Three ST523 bla(KPC−2)- and bla(NDM−1)-coharboring CRCF strains, collected from the same hospital within 1 month, exhibited high homology (both identity and coverage >99%), implying clonal dissemination and a small-scale outbreak. Moreover, the bla(KPC−2) and bla(NDM−1) genes were coharbored on an IncR plasmid, probably generated by a bla(KPC−2)-harboring plasmid acquiring bla(NDM−1), in these three strains. Importantly, the IncR plasmid may form a transferable hybrid plasmid, mediated by IS6100 via transposition, with another IncFII plasmid included in the same C. freundii strain. Furthermore, the bla(KPC−2) and bla(NDM−1) of the fourth CRCF strain are located on two different non-transferable plasmids lacking complete transfer elements. Additionally, throughout the course of the 10-day continuous passage, the genetic surroundings of bla(NDM−1) in four CRCF strains were gradually excised from their plasmids after the 8th day, whereas they maintained 100% retention for bla(KPC−2). Genome and plasmidome analyses revealed that bla(KPC−2)- or bla(NDM−1)-harboring C. freundii were divergent, and these plasmids have high homology to plasmids of other Enterobacteriaceae. CONCLUSION: Clonal dissemination of ST523 bla(KPC−2)- and bla(NDM−1)-coharboring CRCF strains was detected, and we first reported bla(KPC−2) and bla(NDM−1) concomitantly located on one plasmid, which could be transferred with mediation by IS6100 via transposition. Continued surveillance should urgently be implemented.