Antimicrobial Radial Diffusion Assay.

Recombined HPLC fractions were evaluated using a radial diffusion assay (RDA), as we described (6, 21). Antimicrobial activity was assessed against S. aureus (S. aureus ΔmprF). Bacteria were grown overnight in Tryptic Soy Broth (TSB) at 37°C. The overnight cultures were then diluted 1:100, grown to mid-exponential phase, and spectrophotometrically adjusted to the desired CFU concentration in TSB. Agar plates for the RDAs were prepared as previously described (24). Briefly, 0.5% tryptone medium with 1% SeaKem GTG Agarose (Cambrex Corporation) was autoclaved. The agar was cooled to 45°C and the bacteria added to an endconcentration of ~4×10⁵ CFU/ml. Immediately thereafter, 10 mL of the liquid agar with the bacteria was poured into square agar plates. After the agar solidified, the plates were refrigerated to inhibit bacterial growth. Then, 36 holes with defined size were placed into the agar plates by using sterile 1000- μL pipette tips and vacuum suction. Finally, 2 μL of the skin peptide samples were placed into the holes and the plates incubated for 24 h at 37°C. 2 μL sterile water and synthetic catestatin AMP (100 μM, GeneScript) were used as the negative and positive controls, respectively. The zone of inhibition of bacterial growth (area) was quantified using ImageJ Software (22).