2.4. Antimicrobial Susceptibility Screening

The standard Kirby–Bauer agar disk diffusion method was used to screen for antimicrobial resistance following recommendations by the Clinical and Laboratory Standards Institute (2015) recommendations. A total of 12 antimicrobial discs were used based on importance in animal and human health and included: ampicillin, gentamicin, amoxicillin-clavulanic acid, streptomycin, tetracycline, tobramycin, amikacin, trimethoprim-sulphamethoxazole, nalidixic acid, meropenem, chloramphenicol, and ciprofloxacin. One confirmed E. coli isolate per animal was screened unless isolates from the same animal displayed different colony morphology on EMB (mucoid vs non mucoid) in which case both were screened. E. coli isolates were transferred to Muller Hinton broth and incubated at 37 °C to 0.5 McFarand standard turbidity. 100 µL of the broth was subsequently spread onto Muller Hinton agar 150mm plates. The twelve (12) antibiotic discs were placed using a Thermo Scientific™ Remel™ Antimicrobial Susceptibility 12-place 150 mm Disk Dispenser, incubated overnight, and zones of inhibition measured for each of the tested antibiotic. American Type Culture Collection (ATCC) E. coli 25922 was used as the quality control reference strain in all the screening tests. For each individual antibiotic tested, antimicrobial susceptibility description (“resistant”, “intermediate”, and “susceptible”) was based on the criteria outlined in the CLSI manual 2015 for Enterobacteriaceae. Isolates that were found to be resistant to any of the tested antimicrobials were further processed for determination of the commonly detected antimicrobial resistance genes/mutations, virulence genes, and also for phylogenetic grouping.