Beyond common serotypes: Genomic characterization of diverse avian pathogenic Escherichia coli associated with broiler colibacillosis in Pakistan

Colibacillosis remains a major cause of mortality and economic loss in Pakistan’s broiler industry. Avian pathogenic Escherichia coli (APEC), genetically related to extraintestinal pathogenic E. coli (ExPEC), exhibits substantial heterogeneity in virulence and antimicrobial resistance. However, disease investigations frequently focus on a limited number of classical serotypes, potentially underestimating the contribution of diverse and non-typeable strains.

This study aimed to comprehensively characterize E. coli isolates recovered from broilers with clinical colibacillosis, irrespective of serotype, to evaluate their genomic diversity, virulence profiles, and antimicrobial resistance patterns. A total of 47 isolates were recovered from sterile organs of clinically diseased broilers. PCR-based serotyping identified only four isolates as O21, highlighting the limitation of targeted serotyping approaches. Therefore, 15 representative isolates were subjected to whole-genome sequencing for in-depth genomic analysis.

Genomic data revealed marked serotypic diversity, including O76, O11, O132, O24, O53, O71, O75, O109, and two non-typeable isolates. Multilocus sequence typing identified nine sequence types, with ST533 predominating, followed by ST457, ST117, ST9434, ST572, ST4481, ST295, ST1196, and ST2329. Phylogenetic analysis assigned isolates primarily to phylogroups A (47%), B1 (30%), and D (23%). Screening of 18 virulence-associated genes demonstrated high prevalence of ompA (98%), fimC (96%), ibeB (87%), ibeA (68%), iroN (62%), iss (62%), and iucD (53%), supporting their pathogenic potential.

All isolates exhibited phenotypic multidrug resistance determined by Kirby Bauer Disk Diffusion method, with universal resistance to erythromycin and ampicillin–sulbactam, and high resistance to tetracyclines (98%) and fluoroquinolones (71–79%). Lower resistance rates were observed for colistin (29%), ceftazidime (21%), and imipenem (5%).

These findings demonstrate that broiler colibacillosis in Pakistan is associated with genetically diverse APEC lineages beyond traditionally emphasized serotypes, accompanied by a substantial multidrug resistance burden. Comprehensive genomic surveillance that moves beyond classical serotyping is essential for accurate epidemiological understanding and development of effective control strategies.