Phenotypic and genomic analysis of antimicrobial resistant Escherichia coli isolates obtained from starter-phase broilers of commercial chicken farms in central Ethiopia

Abstract

Antimicrobials are widely used in intensive poultry production, particularly in sub-Saharan Africa, promoting the selection and spread of antimicrobial-resistant bacteria. Avian fecal Escherichia coli (AFEC) act as important reservoirs of antimicrobial resistance (AMR) genes, reflecting baseline resistance levels and facilitating dissemination of resistance and virulence determinants within poultry production systems. This study aimed to phenotypically and genotypically characterize cloacal E. coli recovered from apparently healthy broiler chickens in Ethiopia, focusing on AMR profiles and virulence-associated genes. A total of 485 cloacal swabs were collected from 52 commercial broiler farms after brooding (21 ± 1 days of age), with 9–11 swabs per farm. Antimicrobial susceptibility testing was performed on 163 E. coli isolates using the Sensititre EUVSEC3 microdilution plate against 15 antimicrobials. Whole-genome sequencing (WGS) was conducted on a subset of 20 isolates representing different resistance profiles. Overall, 256 swabs (53%) yielded E. coli , confirmed by MALDI-TOF MS. Using clinical breakpoints, resistance was most frequent to tetracycline (84%), nalidixic acid (74%), ciprofloxacin (72%), and trimethoprim (53%). No resistance was detected to ceftazidime, cefotaxime, colistin, or meropenem. Multidrug resistance (MDR) was present in (77%) of isolates, indicating a substantial resistance burden. Based on epidemiological cut-off (ECOFF) values, acquired resistance was highest for ciprofloxacin (96%), followed by tetracycline (84%), nalidixic acid (77%), and trimethoprim (54%), while resistance to aminoglycosides, azithromycin, and tigecycline remained low (<10%). WGS identified multiple AMR determinants consistent with phenotypic resistance, including frequent mutations in gyrA, parC, and parE associated with fluoroquinolone resistance. Isolates displayed considerable genetic diversity with small clusters of closely related strains. Plasmid profiling revealed recurrent IncFIB, IncFII, IncI1-I, and IncX replicon types, with most isolates carrying at least one conjugative plasmid encoding AMR genes or APEC-associated virulence determinants, particularly on IncF and IncX plasmids. Overall, Ethiopian broiler farms harbor diverse E. coli populations with high AMR prevalence and mobile genetic elements, highlighting the need for strengthened antimicrobial stewardship, improved biosecurity, enhanced diagnostics, and integrated AMU–AMR surveillance in the poultry sector.