Antimicrobial assays (IC50 and MBC)

Planktonic cultures were obtained by inoculating single colonies from 18–20 h old agar cultures into 10 ml of sterile broth, followed by 20-h incubation. S. aureus and P. aeruginosa assays were performed using Mueller–Hinton broth and agar and C. albicans assays were performed using Sabaraud Dextrose broth and agar. Test organisms were isolated by centrifugation of 1 ml of the culture at 2000 × g for 3 min (MiniSpin^®; Eppendorf, Germany), and resuspending the pellet in 1 ml of fresh sterile broth, before further dilution in sterile broth, until an optical density at 600 nm (OD₆₀₀) of 0.8 was obtained. This was diluted 1:50 in sterile broth, resulting in an inoculum of ~10⁶ colony-forming units per ml (CFU/ml).

Peptide stock solutions of 800 µM were prepared by dissolving solid peptides (provided in pre-weighed aliquots by the supplier) in phosphate buffer solution (PBS) containing 5% (v/v) DMSO. Further dilutions of the stock peptides were prepared (at double the final test concentrations of 0.6–400 µM) in PBS in 5% (v/v) DMSO. All peptide stocks and dilutions were prepared in polypropylene centrifuge tubes. About 100 µl of the test inoculum was transferred into the wells of a polystyrene 96-well microtiter plate and 100 µl of each of the peptide concentrations were added into each test well, giving a final cell concentration of 5 × 10⁵ CFU/ml. Each polystyrene plate was then aerobically incubated at 37 °C for 18–20 h without shaking, before measuring OD₆₀₀ using a spectrophotometer plate reader (Nanostar; BMG Labtech, Ayelsbury). A sigmoidal function was fitted to the resulting data using the following equation:

Where x is the peptide concentration, A and B are the OD₆₀₀ of the positive and negative controls respectively, D is a scaling factor and C is the IC₅₀. The mean IC₅₀ for each peptide was calculated from the results of four independent experiments, with a single technical replicate each. Specific activity was calculated by plotting IC₅₀ (µM) multiplied by peptide length, against peptide length.

Minimum biocidal activity (MBC) for each peptide was assessed by removing 5 µl samples from all wells in the plate after overnight growth was completed (immediately after OD₆₀₀ assessment), then adding these samples to agar and incubating them at 37 °C for 18–20 h. MBC was considered to be the lowest concentration of each peptide in which no microorganism growth was observed across three technical replicates. The mean MBC was calculated using data from four independent experiments.