High resistance of fluoroquinolone and macrolide reported in avian pathogenic Escherichia coli isolates from the humid subtropical regions of Pakistan

•Multidrug resistance was evident in the APEC isolates, with high resistance levels to tetracycline and macrolide classes.•Around 85.7% of APEC isolates contained either blaCTX or blaTEM, indicating the high prevalence of ESBL genes.•Growth in human urine and complement resistance was evident in some APEC isolates, indicating the possibility of zoonosis.•Hybrid Escherichia coli strains were detected in APEC isolates, with APEC/EHEC (enterohemorrhagic E. coli) being the most prevalent pathotype.•ERIC-PCR fingerprinting clustered APEC isolates into 40 groups (E1–E40), confirming their diverse nature. This study aimed to assess the antimicrobial resistance profile, virulence potential, and genetic characterization of avian pathogenic Escherichia coli (APEC) that cause colibacillosis in poultry. Antibiotic susceptibility testing (AST) was measured via the Kirby-Bauer disc diffusion method against 27 commonly used antibiotics. Phylogrouping, virulence-associated gene detection, and hybrid strain detection via multiplex polymerase chain reaction (PCR) and genetic diversity were analysed via ERIC-PCR fingertyping method. AST analysis showed 100% of isolates were multidrug-resistant (MDR) and highest resistance was against penicillin, tetracycline, and macrolide classes of antibiotics. The mcr-1 gene was present in 40% of the isolates, though only 4% of isolates were showing phenotypic resistance. Despite the scarce use of fluoroquinolone, carbapenem, and cephalosporin in the poultry sector, resistance was evident because of the high prevalence of extended-spectrum β-lactamase (ESBL) (53.7%) and other β-lactamases in APEC isolates. β-lactamase genotyping of APEC isolates revealed that 85.7% of isolates contained either blaCTX or blaTEM and around 38% of isolates were complement resistant. Growth in human urine was evident in 67.3% of isolates. Phylogroup B1 (51%) was the most prevalent group followed by phylogroups A (30.6%), D (13.61%), and B2 (4.76%). The most prevalent virulence-associated genes were fimH, iss, and tatT. Results showed that 26 isolates (17.69%) can be termed hybrid strains and APEC/EHEC (enterohemorrhagic E. coli) was the most prevalent hybrid E. coli pathotype. ERIC-PCR fingerprinting genotype analysis clustered APEC isolates in 40 groups (E1-E40). This study provides insights into the antibiotic resistance and virulence profiling of the APEC isolates in Pakistan. The findings of this study provide insights into that the antibiotic resistance