4.6. Synergy Testing via Checkerboard Assays

Synergy susceptibility testing was performed using the broth micro-diliution method as described previously [50]. Briefly, checkerboard synergy testing was performed in triplicate using 96-well microplates (Corning Corp., New York, NY, USA). Positive growth controls were performed on the same plate in wells not containing antimicrobials. Positive, negative and single treatment controls were also assayed on the same microplate. Combinations tested used 0-, 0.5-, 1-, 2-, 4-, 8-, 16-, 32-, 64-, and 128 μg/mL of all antibiotics against 0-, 0.5-, 1-, 2-, 4.1-, 8.2-, and 16.3 mg/mL of NAC_neutral. Tests were performed in triplicate for each bacterial strain tested. Microtitre plates were incubated for 24 h at 37 °C in 150 rpm shaking conditions and turbidity was assessed visually using the Tecan600 Microplate reader at OD_600nm absorbance. Interpretation of checkerboard assay follows previously described method [51]. In brief, the Mean Fractional Inhibitory Concentration (FIC) index was calculated using the concentrations in the first non-turbid (clear) well found in each set of wells with negative individual growth controls. The concentrations across replicates were then averaged [50]. The bounds of synergy have been set as follows: ≤0.5, synergy; > 0.5 ≥ 1, additive; >1–4, indifference; >4, antagonism. FICI was calculated for all treatment combinations tested.