Integrative analysis of toxin-antitoxin systems, genomics, and phenotypic traits reveal phylogroup-dependent diversity among ExPEC bloodstream isolates.

Extraintestinal pathogenic Escherichia coli (ExPEC) causing bloodstream infections encompass diverse evolutionary lineages with distinct genomic and pathogenic traits. Here, we applied an integrative strategy combining population-level analysis of type II toxin-antitoxin (TA) systems, comparative genomics, and phenotypic assays to explore ExPEC diversity. Screening of 85 ExPEC bloodstream isolates revealed phylogroup-associated TA distribution patterns, with phylogroups A and B1 exhibiting more complex and heterogeneous TA repertoires. These signatures guided the selection of two representative strains of the general TA distribution pattern, Ec182 (phylogroup A) and Ec272 (phylogroup B1), for detailed genomic and phenotypic investigation. Whole-genome sequencing demonstrated that differences in TA repertoires are embedded within broader phylogenetic and genomic contexts, including variation in virulence-associated genes and genome organization. Phenotypic analyses revealed distinct pathogenic behaviors between the two strains, mirroring their divergent genomic architectures. While direct causal links between specific TA systems and phenotypic traits were not established, the concordance between phylogroup, TA system composition, and pathogenic profiles highlights distinct adaptive strategies among ExPEC lineages. Overall, our results indicate that ExPEC bloodstream isolates comprise a spectrum of phylogroup-dependent evolutionary and pathogenic strategies. This integrative framework underscores the importance of considering evolutionary background when interpreting virulence-associated features in ExPEC.