Checkerboard synergy testing

Synergy testing of ART and two FQs was performed in 96-well microtiter plates by checkerboard method, which was performed in triplicate with 96-well microtiter plate using an 8-by-5 well configuration.¹⁶ Positive growth controls (to assess the presence of turbidity) were performed in wells not containing drugs. In addition, negative growth controls were performed in a separate microtiter tray. Combination action of ART (64–1,024 μg/mL) with CIP or ENR was tested, respectively. Concentrations tested ranged from 0.0625 × MIC to 2 × MIC of each antibiotic. Each well was inoculated with 100 μL of a suspension of 5 × 10⁵ CFU/mL of the test strain in a final volume of 200 μL. The checkerboard plates were then incubated for 16–20 hours at 37°C. Interactions between ART and antibiotics were determined by calculating the fractional inhibitory concentration (ΣFIC) index, which is the MIC of drug in combination divided by the MIC of drug acting alone. The formula is as follows:

where MIC A + B is the MIC of ART in combination with antibiotic and MIC B + A is the MIC of antibiotic in combination with ART. Results were interpreted as follows: ΣFIC ≤0.5, synergism; 0.5 < ΣFIC <1, additive effect; 1 < ΣFIC <4, no interaction; and ΣFIC >4.0, antagonism. Data were obtained from at least two independent experiments.¹⁷