Genomovar-level resolution reveals rapid pathotype switching and genomovar-specific disease potential in diarrheagenic Escherichia coli populations in northern Ecuador

Diarrheagenic Escherichia coli (DEC) pathotypes are commonly defined by molecular detection of discrete virulence genes, yet how quickly these diagnostic genes emerge and move among co-circulating lineages remain unclear. Here, we classified 248 whole-genome-sequenced E. coli isolates from the EcoZUR case-control study in northern Ecuador into intra-species genomovar units using the recently described 99.5% ANI threshold. This framework exposed cryptic population structure, revealing that single sequence types, representing identical multilocus sequence types (MLST), can harbor multiple distinct genomovars. Within individual genomovars, we observed a few cases of different pathotypes among isolates showing ~99.7% ANI (and many such cases between genomovars). Coupled with synteny and phylogeny analyses that revealed pervasive incongruences between pathotype-diagnostic virulence genes and the core genome, these findings suggest recent horizontal gene transfer as the primary driver of pathotype evolution. Virulence gene profiling further revealed that accessory virulence repertoires are hierarchically structured by phylogroup across pathotypes, with genomovars assigned to phylogroups B2 and D exhibiting more conserved virulence architectures than those in phylogroup A and B1. Among DAEC isolates specifically, the B2- and D-associated genomovars showed elevated diarrhea-association rates relative to their phylogroup A counterparts. Rare virulence genes, including Type VI secretion systems, further distinguished diarrhea-associated from asymptomatic genomovars. These findings demonstrate that, although there seems to be within-lineage (phylogroup) conservation of virulence, pathotype identity is a labile state defined by horizontally acquired virulence genes at the genomovar level, and that the genomovar framework provides a biologically meaningful unit for linking intra-species diversity to pathogenic potential and outbreaks.