First report of an Escherichia coli ST131 clinical isolate co-harboring bla KPC-2 and bla NDM-13 on an IncB/O/K/Z plasmid in China

ABSTRACT In carbapenem-resistant Enterobacteriaceae , the co-occurrence of carbapenem resistance genes poses a significant threat to global public health. This study investigated the phenotypic and genotypic characteristics of a clinical carbapenem-resistant Escherichia coli strain B5, which harbors both bla KPC-2 and bla NDM-13 . Antimicrobial susceptibility testing and plasmid conjugation assays were performed on isolate B5, using E. coli J53 (a standard recipient strain resistant to sodium azide) as the recipient, whereas passaging experiments and growth rate determination were conducted on J53 (pB5-KPC-NDM). Genetic characteristics of B5 were analyzed via whole-genome sequencing (WGS). B5 exhibits an extensive multidrug resistance phenotype, with susceptibility only to tigecycline and colistin. WGS revealed that B5 belongs to ST131, carries 11 plasmids, and co-harbors bla KPC-2 and bla NDM-13 on the IncB/O/K/Z plasmid pB5-KPC-NDM. This plasmid also exhibited considerable stability in J53 (pB5-KPC-NDM), with a retention rate of 74% (37/50) after 10 days of serial passage in antibiotic-free medium. Compared with the recipient strain J53, J53 (pB5-KPC-NDM) imposed a low fitness cost. Additionally, WGS further identified multiple additional resistance genes on pB5-KPC-NDM. Comparative analysis showed that bla KPC-2 resides within Tn 6296 derivatives and bla NDM-13 within Tn 125 derivatives on pB5-KPC-NDM, featuring both conserved and unique flanking contexts. Core structures potentially enabling horizontal transfer were identified: ∆Tn 6376-bla KPC-2 -∆IS Kpn6-korC-klcA-∆repB -∆Tn 1722 -5’ for bla KPC-2 and IS 1294 -∆IS Aba125-bla NDM-13 - ble MBL - trpF-nagA for bla NDM-13 . Notably, IS 1294 (IS 91 family), replaces IS Aba125 , is likely to mobilize bla NDM-13 . In conclusion, the pB5-KPC-NDM plasmid poses a severe threat due to its extensive resistance profile, high transferability, and low fitness cost, urging immediate intervention to prevent its dissemination. IMPORTANCE Antimicrobial resistance has become a serious global public health concern, severely limiting therapeutic options. The global proliferation of carbapenem-resistant Enterobacteriaceae , driven by plasmid-mediated horizontal gene transfer of carbapenemase-encoding elements, constitutes a critical antimicrobial resistance crisis. This study provides the first evidence of bla KPC-2 and bla NDM-13 co-occurring on an IncB/O/K/Z plasmid (pB5-KPC-NDM), as well as the first detection of these genes in a clinical Escherichia coli isolate (B5). Phenotypic and genotypic analyses demonstrate efficient horizontal transfer capacity and stability across bacterial generations of pB5-KPC-NDM, facilitating the spreading of multidrug resistance. This dual carbapenemase co-localization represents a pivotal escalation in the dissemination potential of resistance and consequently heightens the threat of its spread worldwide. These findings emphasize the critical need for enhanced genomic surveillance programs and the implementation of stringent infection control measures to mitigate the global dissemination of such multidrug-resistant plasmids carrying high-risk carbapenemase variants.