Synergy Checkerboard Assay

Antibiotic interactions were evaluated using the checkerboard assay as previously described (Rand et al., 1993). Checkerboards were prepared by using serial dilutions of amphotericin B (0.004 to 4.0 μg/ml) and fluconazole (0.0156 to 16.0 μg/ml) in the horizontal wells and triclosan (0.25 to 16.0 μg/ml) in the vertical wells. A fungal suspension was prepared as described in broth microdilution assay (approximately 5 × 10³CFU/ml), and 100 μl was inoculated into each well of a 96-well microtiter plate. Plates were read after 48 h of incubation at 35°C, and the wells without visible signs of growth were identified by placing the plate on a mirrored surface. Each well was resuspended and agitated for 5 min, and the OD at 570 nm wavelength was determined with a spectrophotometer. The fractional inhibitory concentration index (FICI) was calculated for each drug using following formula: FICI = FIC (triclosan) + FIC (drug), where FIC equals to MIC-2 of the drug in combination divided by the MIC-2 of the drug alone. FICI ≤ 0.5 indicates synergy, FICI > 4 indicates antagonism whereas 0.5 > FICI > 4 suggests no interaction (additivity/indifference; Odds, 2003b).