Broth Microdilution Assay

Minimum inhibitory concentration was determined for peptides and antibiotics in duplicate using broth microdilution assay according to CLSI guidelines (Wiegand et al., 2008). Twofold serial dilution of peptides and antibiotics were made in duplicate across the 96-well polypropylene microtiter plate using acidified water (0.01% Acetic acid and 0.1% Bovine serum albumin). This resulted in 10 μL of 10x concentration of peptides or antibiotics (gentamicin, amikacin, and vancomycin) in each well. Three to five colonies of PA strain from MHA culture plate were incubated in 5 mL MHB-II in an orbital shaker at 35°C for 16 h. The overnight cultures were sub-inoculated in fresh 5 mL MHB-II at 1 in 50 dilutions and incubated for 3–4 h in the shaking incubator at 35°C until OD₆₀₀ readings gave approximately 2 × 10⁸ CFU/mL. A suspension of 1 × 10⁶ CFU/mL in fresh MHB-II was prepared by further dilution and 90 uL of this suspension was then transferred to each well-containing 10 uL of 10x peptide or antibiotics which resulted in final inoculum of 5 × 10⁵ CFU/mL with desired final concentration of peptide (range 200 to 0.78 μg/mL) or antibiotics [gentamicin (64 to 0.5 μg/mL); amikacin (16 to 0.125 μg/mL), and vancomycin (512 to 4 μg/mL)]. Growth control (only bacteria) and sterility control (no bacteria) were also included in each plate. The plate was incubated at 37°C for 18–21 h and the MIC was determined as the lowest concentration of peptide or antibiotic that inhibited the visible growth of PA strain as observed with the unaided eye.