Checkerboard Assay

The checkerboard broth microdilution assay was performed in 96-well microtiter plates using 2-fold dilutions of two antibiotics diluted in cation-adjusted Mueller–Hinton broth (CAMHB). AZM ranging from 1/64 × MIC to 2× MIC was dispensed in every row. TGC ranging between 1/64× MIC and 2× MIC was added in each column. An equal volume of standardized bacterial suspension of 1 × 10⁶ CFU/mL was added and then all plates were incubated at 37°C in an aerobic atmosphere for 24 h. Fractional inhibitory concentration index (FICI) values were calculated as FICI = FIC A (MIC antibiotic A in combination)/(MIC antibiotic A) + FIC B (MIC antibiotic B in combination)/(MIC antibiotic B). To categorize the drug combination that consistently generated the lowest FICI after repeating the experiment in duplicate on two further occasions, the results were grouped as follows: FICIs of ≤0.5 were interpreted as synergistic; FICIs of >0.5 but ≤1 were considered additive; FICIs of >1 but ≤4 were considered indifferent; and FICIs >4 were interpreted as antagonistic.¹⁶ The measurements were performed on three different samples of each.