4.6. Checkerboard Assay

Antibiotics from different classes whose breakpoints were non-susceptible were combined in a checkerboard style for this assay. First, each antibiotic was prepared by serially diluting in water to obtain the desired dilution folds starting from double the MIC values obtained earlier. A total of 50 µL of drug A was dispensed down each column starting from the highest concentration except for column 12. Similarly, drug B was dispensed along the rows except for row H. Then, 50 µL of each adjusted 0.5 McFarland standard was transferred into 15 mL 2× MHB and aliquoted to all the wells to obtain a final concentration of 5 × 10⁵ CFU/mL with a final volume of 150 µL per well. The last well, H12, served as the GC. The results were obtained after 24 ± 2 h of incubation at 35 ± 1 °C as described earlier. The fractional inhibitory concentration (FIC) index of the combined drugs was calculated as follows:

Synergy was defined as an FIC index value less than 0.5, while antagonism was defined for values greater than 4, and values in between were interpreted as indifferent [19,43]. The assays were duplicated, and synergy was determined when the FICI yielded values less than 0.5. When a skipped well occurred, the higher FICI was used to prevent false-positive synergy interpretations. The data were discarded if there were more than two skipped wells in a single grid or if the MIC was more than a 2-fold dilution above or below the modal MIC for that isolate, followed by a repeat of the experiment. However, the antibiotic combination for the isolate was eliminated from further investigation if the same error persisted [19].