4.4. Synergy Checkerboard Assay

To assess the antimicrobial combinations, the synergy checkerboard assay was performed in 96-well microtiter plates according to a previous study with reference to the MICs of the two antimicrobial agents [59]. Briefly, bacterial cultures were suspended in TSB at a final concentration of 1 × 10⁶ CFU/mL and distributed in plates with combinations of equol and meropenem using a two-fold serial dilution method. The concentrations of equol and meropenem used were the MIC and sub-MIC concentrations (1/2, 1/4, 1/8, 1/16, and 1/32 × MIC). Plates were incubated at 37 °C for 24 h. Growth inhibition was assessed using a Multiskan GO plate reader, and the FICI for 90% inhibition was calculated as follows: FIC of equol = MIC of equol in combination/MIC of equol alone, and FIC of meropenem = MIC of meropenem in combination/MIC of meropenem alone. The FICI was defined as the FIC of equol plus the FIC of meropenem. The synergistic effect of equol in combination with meropenem against carbapenem-resistant E. coli was interpreted as follows. Synergy was indicated by a FICI of ≤0.5, no interaction was indicated by a FICI from 0.5 to 4.0, and antagonism was a FICI of ≥4.0 [60].