First report of a carbapenem-resistant clinical strain co-harboring and genes in China.

Carbapenem-resistant (CRE), particularly those co-harboring multiple carbapenemase genes, pose a significant global health threat. However, the coexistence of and in has not been previously reported. This study aims to report and characterize the first carbapenem-resistant (CRSS) clinical strain MAS3954 in China co-harboring and , focusing on its genetic characteristics, plasmid stability, and transfer potential. Whole-genome analysis revealed that the and were located on two distinct plasmids. Plasmid pMAS3954-KPC (113,856 bp, IncFII/IncFIB) exhibited low similarity (<66%) to known plasmids, indicating a novel fusion event between pKPC-h2 and chromosome, while pMAS3954-NDM (55,235 bp, IncX3) was highly conserved (100% identity/coverage). Conjugation experiments showed that the was transferable, while was not. During 10 days of continuous passage, the genetic context of was gradually excised from the plasmids after the 8th day, whereas they maintained 100% retention for . MAS3954 was multidrug-resistant (MDR), including carbapenems, β-lactams, β-lactam/β-lactamase inhibitors, trimethoprim-sulfamethoxazole, tetracycline, nitrofurantoin, colistin, and fluoroquinolones, but remained susceptible to certain aminoglycosides and tigecycline. Phylogenomic analysis identified a distinct clade for MAS3954, diverging notably from other strains. Comparison of resistance genes further highlighted the unique co-harboring of and in MAS3954, absent in other strains. To our knowledge, this study represents the first characterization of clinical strain co-harboring and . The findings highlight the need for enhanced surveillance and infection control to prevent the spread of these MDR strains in healthcare settings.