Determination of Minimum Inhibitory Concentration (MIC) of the Peptides

For the experiment, a previously defined serial broth microdilution method was used with slight modifications.^58,59 Briefly, mid-logarithmic phase bacterial cells from the secondary culture were washed, resuspended in cation-adjusted MHB or TSB (0.25% glucose, 0.5% NaCl), and spectrophotometrically adjusted to 10⁸ CFU/mL (OD₆₀₀ of 0.5). In a polypropylene 96-well plate (Corning Incorporated, USA) containing 10 μL of serially 2-fold diluted peptides or antibiotics in 0.2% BSA (in 0.01% acetic acid), 100 μL of the bacterial suspension was added at a final density of ∼2–5 × 10⁵ CFU/mL in each well. To one column of the microtiter plate, only media were added (negative control), and another column contained bacterial cells without any treatment (growth control). The plates were incubated at 37 °C with shaking at 180 rpm for 16–18 h, and the absorbance at 600 nm was recorded using an ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA). The lowest concentration of the peptide/antibiotic completely inhibiting the bacterial growth, as observed spectrophotometrically, was taken as the MIC of that agent. The experiment was done on at least two different days in duplicate. The stability of the peptide in the presence of serum was also determined through the MIC study by diluting FBS in TSB medium to a final concentration of 25% (v/v) and then preparing the bacterial inoculum in the FBS-containing medium.