Checkerboard assays

In total, 21 Col-R GNB isolates were selected for the checkerboard assays, in which effects of twofold serial dilutions of drug positioned in a 12 × 8 matrix were tested (MacNair et al., 2018; Zhang et al., 2021c). Each drug was diluted in cation-adjusted Mueller-Hinton broth (CAMHB) to a range of MIC concentrations for each experimental strain. Single bacterial colonies were cultured overnight and then diluted to the 0.5 McFarland standard in sterile saline. Next, samples were diluted in CAMHB in a 1:100 ratio. The final bacterial concentration in each well was approximately 7.5 × 10⁵ CFU/ml. The results were observed at 16–20 h at 37°C. The absorbance of the bacterial culture at 600 nm was measured with a microplate reader. All experiments were performed in triplicate with the average used for FIC calculations. The synergistic effect of NG in combination with colistin was assessed by using the fractional inhibition concentration index (FICI). The formula for calculating FICI is as follows: FICI = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone), where FICI ≤ 0.5 indicates synergism; FICI > 0.5 and ≤ 4 indicate irrelevant; and FICI > 4 was considered antagonistic (Fadwa et al., 2021).