Isolation and molecular characterization of multidrug-resistant APEC isolates from Pakistan poultry, including high-risk ST410 subclones

Colibacillosis is a systemic disease of poultry caused by avian pathogenic Escherichia coli (APEC). The prevalence rate of colibacillosis in Pakistani poultry production ranges 12.5- 89.2% with 20- 40% mortality. Many studies have reported the high prevalence of serotype O78, O1, O2, and O18 while a significant number of isolates remain non-typeable. The current study was conducted to isolate and characterize non-typeable or less commonly reported APEC isolates from Pakistani poultry. A total of ten APEC isolates were isolated from heart, liver, and lungs of morbid birds showing typical symptoms of colibacillosis. The isolates were characterized by the Congo red binding assay, phylotyping, serotyping, virulence factor profiling, and antibiotic susceptibility testing. The genomic DNA of five isolates were sequenced and characterized for virulence associated genes (VAGs), antibiotic resistance associated genes (ARAs), multi-locus sequence typing and phylogenetic analysis. The predominant virulence factors included o mpA, IbeB, mat, irp2, iucD, iroN, and iutA. All isolates were multidrug resistant with highest resistance against ampicillin/β-lactams, quinolones/fluoroquinolones, and cephems. The virulence associated genes (VAGs) of the studied APEC isolates were compared to VAGs of previously reported APEC isolates. Three isolates of high-risk ST410 representing B2/B3-H24R subclones were identified. In conclusion, these APEC isolates from diseased broiler birds exhibited high pathogenic potential and one health risks due to zoonotic MDR E. coli strains. All strains showed multidrug resistance to β-lactams, quinolones, and cephems, while pangenome clustering highlights genomic plasticity and niche specialization driving convergent pathogenicity across diverse isolates and clades in Pakistani poultry.