Global spread and evolution of KPC-2 and NDM-1-producing Gram-negative bacteria.

The co-occurrence of KPC and NDM carbapenemases in Gram-negative bacteria presents a serious and expanding global health threat. This study characterized 338 KPC-2/NDM-1 dual-positive isolates from 23 countries, including 41 clinical strains sequenced through hybrid second- and third-generation platforms from China's national surveillance network. These isolates spanned six genera, 58 species, and 138 sequence types, reflecting substantial taxonomic and geographic diversity. Molecular analysis identified IncFII(p14) plasmids as the principal vectors for cross-genus dissemination of KPC-2, while IncX3, IncN, and IncFIB(pB171)/IncFII(Yp) plasmids were dominant carriers of NDM-1 among the studied strains. Codon usage analysis indicated stronger bias in KPC-2 plasmids (effective codon number: 39.17, optimal codons: 17) compared to NDM-1 plasmids (effective codon number: 41.25, optimal codons: 12), indicating differential evolutionary pressures. Dual-positive strains exhibited significantly higher virulence scores and broader resistance profiles than reference strains (P<0.001). Notably, only 14.6% of isolates harbored Type I-E CRISPR-Cas systems, all of which encoded the anti-CRISPR protein AcrIE10. Furthermore, Type II methyltransferase numbers were significantly enriched in dual-positive strains (P<0.005), suggesting a potential role in modulating host defense evasion. We propose that in Klebsiella spp., KPC-2 plasmids are typically acquired prior to NDM-1 plasmids and can form hybrid plasmids. In non-Klebsiella genera, dual resistance is primarily driven by independent acquisition of high-risk plasmids such as IncFII(p14) and IncX3, without a fixed temporal order. These findings highlight the convergence of global plasmid-mediated resistance, host-pathogen immune interplay, and pan-resistance evolution. Targeting high-risk plasmid lineages and host defense-modulating elements may be key to forecasting resistance emergence and guiding early interventions against dual-carbapenemase-producing pathogens.