Genomic insights into replicons and conjugative transfer systems of blaNDM-positive plasmids in the Enterobacter cloacae complex

Abstract

The Enterobacter cloacae complex (ECC) represents a prominent opportunistic pathogen associated with multidrug-resistant nosocomial infections. The emergence of New Delhi metallo-β-lactamase (NDM)-producing ECC isolates presents a significant threat to public health, owing to the restricted available treatment regimens. Here, we identified a clinical E. hormaechei strain E20590 carrying the bla NDM-1 gene from Guangdong Province, South China, and combined it with 259 non-redundant bla NDM -harboring ECC plasmid sequences retrieved from the NCBI GenBank database (2009–2025) to form a cohort of 260 plasmids for systematic genomic characterization. Analyses of bla NDM variants, replicon types, conjugative transfer systems, clustering dendrograms and geographic distributions revealed that bla NDM was present in four ECC species, with E. hormaechei (77.31%, 201/260) as the predominant host, and bla NDM-1 (81.15%) and bla NDM-5 (14.62%) as the dominant variants. Gene-content clustering divided these plasmids into eight distinct clades, primarily differentiated by replicon types (IncX3 ~ 40%, IncF ~ 30%, IncH ~ 8%) and conjugative transfer modules. The largest clade (Clade I) comprised IncX3 single-replicon plasmids (~ 50 kb) encoding MOB P family relaxases, T4CP2 family coupling proteins and Type T T4SS, which were mainly distributed in Sichuan and Guangdong Provinces of China. The IncF-like plasmid pE20590-1 from strain E20590 belonged to Clade VIII (~ 100 kb), carrying MOB F family relaxases, T4CP2 and Type F T4SS, and was the only isolate from Guangdong in this clade with unique regional distribution characteristics. Among the analyzed GenBank-derived ECC plasmid records, China accounted for the largest proportion (49.6%), followed by Canada, Bangladesh, and Poland. Within the analyzed Chinese plasmid records, Sichuan, Guangdong, and Zhejiang contributed the largest numbers of submissions. Conjugation experiments confirmed the functional transfer of pE20590-1, and most plasmids harbored intact conjugative transfer elements, highlighting the high horizontal transmission potential of bla NDM in ECC. Our results systematically illustrate the genetic diversity, structural characteristics, and distribution patterns of bla NDM -carrying plasmids within the ECC, offering key genomic evidence to support the surveillance and control of NDM-mediated carbapenem resistance spread in clinical environments.