Molecular epidemiology reveals distinct lineages and genomic profiles of avian pathogenic and commensal Escherichia coli in broiler flocks

Escherichia coli is predominantly an intestinal commensal; however, avian pathogenic E. coli (APEC) causes colibacillosis in poultry. The APEC pathotype lacks a clear genetic definition, further complicated by its opportunistic nature. To compare the genomic characteristics of avian pathogenic and commensal E. coli, isolates from diseased and healthy broiler flocks in Sweden were analysed, collected between 2022 and 2024. Clinical isolates (n=202) were collected at necropsy from 40 flocks during colibacillosis outbreaks, and non-clinical isolates (n=109) were obtained from litter using sock sampling in 60 unaffected flocks. Whole-genome sequencing was performed to determine sequence types (STs), serotypes, phylogroups, virulence-associated genes (VAGs) and to identify ColV plasmids. A five-gene APEC marker panel targeting plasmid-associated virulence genes (iutA, hlyF, iss, iroN and ompT) was used to classify isolates as APEC or non-APEC, and high-risk clones were identified according to the APECtyper scheme. Clinical isolates comprised 22 STs and 25 serotypes and were dominated (59%) by the ST23 O78:H4 clone within phylogroup C. Non-clinical isolates were more diverse (44 STs, 67 serotypes), primarily within phylogroups A (48%) and B1 (33%), with no clone predominating. Clinical isolates carried significantly more VAGs (P<0.001). Overall, 97% of clinical isolates were identified as APEC, all of which carried a ColV plasmid. Among non-clinical isolates, 28% were APEC, of which 80% were ColV-positive. However, clinical APEC isolates carried significantly more ColV-associated virulence gene clusters than non-clinical APEC isolates (P<0.001). Only 5% of non-APEC isolates were ColV-positive. High-risk clones were restricted to clinical APEC isolates (63%). These findings indicate that colibacillosis in Swedish broilers was largely driven by a dominant APEC clone during the study period, highlighting the need for coordinated surveillance and targeted control of high-risk clones. The presence of VAG reservoirs among isolates from unaffected flocks, together with the limitations of marker-based APEC typing, supports integrated frameworks combining lineage, VAG profiles and plasmid content for more reliable APEC identification and pathogenicity assessment.