Stocks of antibiotics to test were prepared by dissolving them according to CLSI M100 Performance Standards for Antimicrobial Susceptibility Testing41 in the indicated solvent and diluent to a final concentration of 5.12 mg/ml. Salts were corrected for their mass. Used antibiotics: Ciprofloxacin hydrochloride monohydrate (Sigma-Aldrich; European Pharmacopoeia Reference Standard), piperacillin sodium (Sigma-Aldrich, analytical standard), imipenem (Sigma-Aldrich; European Pharmacopoeia Reference Standard), ceftazidime pentahydrate (Sigma-Aldrich; European Pharmacopoeia Reference Standard) and aztreonam (United States Pharmacopeia Reference Standard).
Working stocks were then prepared by serial dilution in MHB II medium. Plates for checkerboards were prepared by adding 25 µl of each antibiotic at 4x the final concentration to be tested in the respective well in a flat bottom, transparent 96 well plate (Greiner). In one column a growth control was prepared by adding 50 µl of MHB II medium. A sterility control was prepared in a second column by adding 100 µl of MHB II medium. Inocula of the strain to be tested were prepared by inoculating physiological NaCl solution to a McFarland standard of 0.5 from overnight cultures. In total, 125 µl of this solution were then added to 15 mL of MHB II medium and 50 µl of this inoculum added to the wells containing antibiotics as well as to the growth control wells. Plates were incubated for 20 h at 37 °C. After incubation the OD600 values were determined using a Tecan Infinite® 200 PRO. Each assay was prepared in duplicate. For each replicate, the ratio of signal for each well and the mean of the sterility control was calculated. The mean value of both replicates was calculated. If the value was smaller than 1.5, this concentration was considered to be inhibitive. From these values, the MIC and FIC-I for the tested antibiotics were calculated as follows:
Interpretation of FIC-I: ≤ 0.5: Synergism; >0.5 to 1: additive effect; >1 to 4: indifferent effect; >4: Antagonism
Working stocks were then prepared by serial dilution in MHB II medium. Plates for checkerboards were prepared by adding 25 µl of each antibiotic at 4x the final concentration to be tested in the respective well in a flat bottom, transparent 96 well plate (Greiner). In one column a growth control was prepared by adding 50 µl of MHB II medium. A sterility control was prepared in a second column by adding 100 µl of MHB II medium. Inocula of the strain to be tested were prepared by inoculating physiological NaCl solution to a McFarland standard of 0.5 from overnight cultures. In total, 125 µl of this solution were then added to 15 mL of MHB II medium and 50 µl of this inoculum added to the wells containing antibiotics as well as to the growth control wells. Plates were incubated for 20 h at 37 °C. After incubation the OD600 values were determined using a Tecan Infinite® 200 PRO. Each assay was prepared in duplicate. For each replicate, the ratio of signal for each well and the mean of the sterility control was calculated. The mean value of both replicates was calculated. If the value was smaller than 1.5, this concentration was considered to be inhibitive. From these values, the MIC and FIC-I for the tested antibiotics were calculated as follows:
Interpretation of FIC-I: ≤ 0.5: Synergism; >0.5 to 1: additive effect; >1 to 4: indifferent effect; >4: Antagonism
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