3.2. Minimum Inhibitory Concentration (MIC) Assay

The antimicrobial activity of the compounds was evaluated by MIC assay using the procedure described by Clinical and Laboratory Standards Institute (CLSI). A single colony of bacteria was cultured overnight in trypticase soy broth (TSB; Oxoid, Basingstoke, UK) at 37 °C with shaking. The resulting bacterial culture was collected by centrifugation and resuspended in TSB twice. The optical density (OD) of the resulting culture was adjusted to OD₆₆₀ = 0.1 in TSB (which is equivalent to 10⁸ colony forming unit (CFU)/mL bacteria). It was further diluted to 10⁶ CFU/mL in TSB. 100 µL of the bacterial solution was then added to wells of a 96-well plate (Costar; Sigma-Aldrich, St Louis, MO, USA) containing 100 µL serially diluted peptide mimic, with final concentration ranging from 1 to 250 µM. Wells with bacteria but no compound were used as negative control while wells with only media were set as blank. The plates were then wrapped with parafilm to prevent evaporation and incubated with shaking at 120 rpm at 37 °C for 18–24 h, and the data were recorded by measuring the OD value at 660 nm using a FLUOstar Omega (BMG Labtech, Mornington, Victoria, Australia) microplate reader. The MIC value of each compound was determined as the lowest concentration that completely inhibited the growth of bacteria. Each experiment was performed in triplicate and was repeated in three independent experiments.