2.5. Antibiotic susceptibility testing of isolates

Antibiotic susceptibility of the collected E. coli and enterococci isolates was determined using the disk diffusion method (Bauer et al., 1966). Briefly, isolates were grown in Mueller-Hinton broth (Hardy Diagnostics, Santa Maria, CA) until the concentration visually matched a 0.5 MacFarland standard (Hardy Diagnostics, Santa Maria, CA). Suspensions were spread onto Mueller-Hinton agar using a sterile swab, and disks containing the target antibiotics (Hardy Diagnostics, Santa Clara, CA) were placed onto each plate. Target antibiotics were chosen to include multiple antibiotic classes and mechanisms of action. E. coli isolates were tested against ampicillin (10 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), gentamycin (10 µg), tetracycline (30 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg); and enterococci isolates were tested against ampicillin (10 µg), ciprofloxacin (5 µg), erythromycin (15 µg), tetracycline (30 µg), and vancomycin (30 µg) (CLSI, 2018). Plates were inverted and incubated for 16 to 18 h at 37 °C. The zone of inhibition around the disks was measured to classify the isolates as susceptible or resistant to the tested antibiotics; isolates classified as intermediate are also reported as “resistant” in this study. Quality control checks were performed between every 25 tests with E. coli ATCC 25922 for presumptive E. coli isolates and Staphylococcus aureus ATCC 25923 for presumptive enterococci isolates (CLSI, 2018).