Global population genomics of E. coli ST69 reveals cross-host dissemination, ecological adaptations and epidemic multidrug resistance plasmids

The extraintestinal pathogenic E. coli sequence type (ST) 69 lineage exhibits high virulence and frequent acquisition of antibiotic resistance, yet the factors underlying its global success remain incompletely understood. Here, we analyzed 160 E. coli ST69 isolates from the Czech Republic alongside 6747 publicly available ST69 genomes to investigate the genetic basis of its dissemination and transmission across diverse hosts and ecological niches. Out of 6907 isolates, 26.2% carried extended-spectrum beta-lactamases (ESBL) genes, with bla CTX-M-15 being the predominant variant. Temporal analysis revealed a marked increase in the AmpC gene bla DHA-1 and an increasing frequency of bla CTX-M-15 , whereas bla CTX-M-14 declined over time. Long-read sequencing identified conserved IncN pST1 and IncF plasmid lineages associated with the dissemination of resistance determinants. Virulence-associated plasmid profiles showed distinct source distributions, with ColV predominantly associated with poultry and pUTI89-like and senB-cjr -positive profiles with human and environmental sources. Comparative analysis indicated that ST69 carries an intermediate resistance burden while maintaining a generally high content of virulence genes among other ExPEC lineages. Network analysis revealed closely related isolates from human, animal and environmental sources across different countries, highlighting ST69 interhost transmission and One Health relevance. Genome-wide association analysis identified lineage-specific determinants linked to stress tolerance and host immunity evasion, including an esiB homolog highly conserved in ST69 and a horizontally acquired metabolism operon associated with environmental isolates. Experimental assays demonstrated that this operon enabled growth on D-glucosaminate as the sole carbon source. Together, our findings suggest that the global success of ST69 is driven by a combination of antimicrobial resistance, virulence-associated traits, ecological flexibility, and the acquisition of lineage-specific genetic determinants.