2.2. Determination of Minimum Inhibitory Concentration (MIC) for Antibiotics

MICs of four different classes of antibiotics (polypeptide (colistin), penicillin (carbenicillin), aminoglycoside (tobramycin), and macrolide (erythromycin)) were determined using the broth microdilution method [35]. It was carried in 96-well microtiter plates (Nunc, Thermo Fisher Scientific, VIC, Australia) with an MHB overnight culture of P. aeruginosa PAO1 adjusted to OD₆₀₀ of 0.1. Two-fold serial dilutions with MHB achieved final concentrations of antibiotics (Sigma-Aldrich, Singapore): colistin (COL 27.2 to 0.2 µM), carbenicillin (CARB 1353.1 to 10.6 µM), tobramycin (TOB 13.3 to 0.1 µM), and erythromycin (ERY 1395.2 to 10.9 µM). The plate was incubated at 37 °C for 18–20 h with shaking at 180 rpm. The lowest concentration of antibiotics inhibiting growth as observed visually was recorded as the MIC for each antibiotic.