Genomic characterization and global lineage–plasmid context of a blaNDM-5-harboring Escherichia coli ST3014 isolate

The global emergence and dissemination of carbapenem-resistant Enterobacterales pose a substantial public-health threat. However, Escherichia coli ( E. coli ) sequence type 3014 (ST3014), including bla NDM -positive representatives, remains poorly characterized across human, animal, and environmental interfaces. Here, we characterized the genomic features of a bla NDM-5 -harboring ST3014 isolate and its associated plasmid background to provide a lineage- and plasmid-focused framework for future surveillance. Two bla NDM -positive E. coli isolates (E44 and E56) were recovered from 60 piglet fecal samples collected from an intensive swine farm in Qionghai City, Hainan Province. Whole-genome sequencing identified E44 as a bla NDM-5 -positive ST3014 isolate and E56 as a bla NDM-1 -positive ST9547 isolate. Because bla NDM-5 differs from bla NDM-1 by amino acid substitutions associated with enhanced carbapenem hydrolytic activity and broader dissemination potential, subsequent genomic characterization focused on the bla NDM-5 -positive isolate E44. The selected isolate underwent short- and long-read sequencing, followed by hybrid assembly and functional annotation. To contextualize its genomic characteristics, a global E. coli ST3014 dataset was compiled from EnteroBase, NCBI, and published literature to assess antimicrobial resistance genes (ARGs), virulence factors (VFs), and core-genome phylogeny. Furthermore, the bla NDM-5 -carrying plasmid from E44 was queried against the PIPDB database, plasmid-level phylogenetic relatedness was inferred using Mash distances, and the bla NDM-5 genetic context of E44 was compared with those of other bla NDM-5 -carrying plasmids. The bla NDM-5 -positive ST3014 isolate E44 carried 6 ARGs and 42 VFs, with bla NDM-5 located on a 124-kb IncF multireplicon plasmid. In addition to multiple resistance determinants, this plasmid co-harbored ExPEC-associated siderophore/iron-uptake loci, including sitABCD and iucABCD - iutA . A global dataset comprising 82 ST3014 E. coli isolates demonstrated broad geographic and ecological dissemination across 23 countries, spanning human, animal, and environmental sources. A time-scaled phylogeographic reconstruction suggested that ST3014 most likely originated in Asia, with China showing the highest country-level likelihood, and indicated repeated international dissemination after 2010. Phylogenetic analysis of 153 homologous plasmids showed that related IncF plasmids are distributed across diverse Enterobacterales hosts, with E. coli as the predominant host; notably, the bla NDM-5 -harboring plasmid identified in E44 (designated E44-plasmid1) was most closely related to an E. coli -derived plasmid. Comparative analysis of bla NDM-5 genetic contexts revealed a conserved bla NDM-5 -associated core module ( ble - trpF - dsbD ) embedded in variable flanking regions, with recurrent IS26 adjacent to the upstream segment, consistent with local recombination and modular remodeling around the resistance locus. The detection of a bla NDM-5 -harboring ST3014 isolate in a breeding-farm setting, together with the broad distribution of related strains and plasmids in public datasets, suggests a potential One Health concern warranting continued surveillance.