Antimicrobial Susceptibility Testing of Bacteria

Antimicrobial susceptibility testing (AST) was conducted by the broth microdilution method using Sensititre™ system (Thermo Fisher Scientific Inc., Waltham, USA) against a panel of 19 antibiotics on a commercially available BOPO7F Vet Antimicrobial Susceptibility Testing Plate (Table 1). In the AST procedure, 1–5 colonies of E. coli or Enterococcus were resuspended in 5 mL of demineralized water and the cell suspension adjusted to optical density of 0.5 McFarland using a nephelometer. Next, 10 μl of E. coli or Enterococcus bacterial suspension was added to 11 mL Mueller-Hinton broth (Thermo Scientific, Remel Inc., KS, USA) and mixed by repeated inversion of the tube. Fifty microliters of inoculated Mueller-Hinton broth were dispensed into each well of the 96-well BOPO7F Vet plate using Sensititre™ Autoinoculator and the plates were incubated for 18–24 h at 35°C. The minimum inhibitory concentration (MIC) was read using Sensititre™ Vizion™ Digital MIC Viewing System (Thermo Fisher Scientific Inc., Waltham, USA). The MIC values (lowest concentration of antimicrobial drug that inhibited the growth of bacteria) was interpreted following the Clinical and Laboratory Standards Institute (CLSI) guidelines [Clinical and Laboratory Standards Institute (CLSI), 2020 ; Clinical Lab Standards Institute (CLSI), 2022 ]. Quality control steps included checking for bacterial growth and colony purity by plating 1 μL of the inoculated Mueller-Hinton broth on TSA with 5% sheep blood. Contaminated or no-growth inoculated samples were not read and repeated. In addition, quality control strains (E. coli ATCC 35218 and E. coli ATCC 25922 and Enterococcus faecalis ATCC 29212) were run weekly alongside the test samples.