Checkerboard Synergy Testing

Synergy testing of plant extracts and three antibiotics was performed in 96-well microtiter plates by following the checkerboard method (Bayer and Morrison, 1984). New Delhi metallo-β-lactamase 1 E. coli cells were inoculated in nutrient broth and incubated at 37°C for 16 h. Combinations of plant extract (10–5120 μg/ml) with colistin (0.5–8 μg/ml), meropenem (0.5–32 μg/ml), and tetracycline (1–16 μg/ml) were tested. Interactions between the antibacterial agents were determined by calculating the fractional inhibitory concentration (ΣFIC) index:

where MIC E+D is the MIC of extract in combination with antibiotic and MIC D+E is the MIC of antibiotic in combination with extract. Based on the ΣFIC, interaction between the antibiotic and the extract was inferred to have a synergistic (ΣFIC < 0.5), additive (0.5 > ΣFIC < 1), indifferent (1 > ΣFIC < 4), or antagonist (ΣFIC > 4) effects (dos Santos et al., 2015).