2.3.1. Checkerboard assay

Serial dilutions of amikacin were performed in water in a 96-well plate starting with 100 μl of 64 μg/mL of amikacin in row A and diluted into 50 μl of water until row G. Only water was added to row H. In a separate plate, 200 μl of 12.4 μg/mL AgNPs was added to column 12 and then diluted in 100 μl of water from columns 11–2. Only water was added to column 1. From the AgNP plate, 50 μl was transferred and added to the amikacin dilution plate. A culture of E. coli 0494 was grown overnight as described above; the OD₆₀₀ was adjusted to 1.0, then diluted 1:500 using 2x MHB. A volume of 100 μl was added to each well bringing the final volume to 200 μl per well, the highest concentration of amikacin to 16 μg/mL, the highest concentration of AgNPs to 3.1 μg/mL, and the bacteria to a dilution of 1:1000. To test the E. coli 0346 isolate this procedure was repeated with the highest concentration of amikacin starting at 800 μg/mL, bringing the final concentration to 200 μg/mL. The plate was incubated at 37°C in the Tecan Infinite 200 Pro plate reader for 20 h. The endpoint OD₆₀₀ reading was used to determine the MIC. Experiments were performed with three independent runs. The interaction between AgNPs and amikacin was assessed by calculating the fractional inhibitory concentration index (FICI), where the FICI = FIC_AgNP + FIC_Amikacin; FIC_AgNP = MIC_AgNP combination/MIC_AgNP alone; FIC_Amikacin = MIC_Amikacin combination/MIC_Amikacin alone. The FICI values indicate synergy (FICI ≤ 0.5), partial synergy (0.5<FICI ≤ 0.75), additive (0.75<FICI ≤ 1.0), indifferent (1.0 < FICI ≤ 4.0), or antagonistic (FICI>4.0) effect.