Synergy Testing by Checkerboard Assay

The synergy of double or triple antimicrobial combinations were determined using the standard broth microdilution checkerboard assay as described previously (Berenbaum, 1978; Yoon et al., 2004). In brief, the MICs of antimicrobials were determined before the experiment. Ninety-six-well microtiter plates were arranged with increasing concentrations of one drug, ranging from 0.125 to 8 × MIC on the x-axis and increasing concentrations of the other drug ranging from 0.125 to 8 × MIC on the y-axis. When using triple antimicrobial combinations, fixed concentrations of the drugs were added into 96-well microtiter plates. The final inoculum in each well was approximately 5 × 10⁵ CFU/mL. The 96-well microtiter plates were incubated at 37°C for 24 h, and turbidity was observed by the naked eye to determine growth. The effects of the antimicrobial combinations were defined according to the fractional inhibitory concentration index (FICI). FICI = (MIC drug A/MIC drug A plus drug B) + (MIC drug B/MIC drug A plus drug B), FICI ≤ 0.5, synergism; 0.5 < FICI ≤ 4, no interaction or FICI > 4, antagonistic (Odds, 2003). With the triple antimicrobial combination, FICI < 1, synergistic, FICI = 1, additive, or FICI > 1, antagonistic (Berenbaum, 1978).