(a) P. acnes (1 × 106 CFU per ml) was incubated with 0–20 µm synthetic hBD-2 peptide in 20 mm phosphate buffer, pH 6.5, containing 100 mm NaCI for 5 hours under anaerobic conditions, (b) To enable examination of the synergistic antimicrobial activity of hBD-2 and FFAs, P. acnes (1 × 106 CFU per ml) was incubated with hBD-2 (0, 2.5, 5, 10 µm) in phosphate buffer in the presence of LA, PA, or OA (25 µg ml−1) on a 96-well microplate (100 µl per well) at 37 °C for 5 hours. The control received only DMSO (0.5%(v/v)) instead of FFA. After incubation, the P. acnes suspension was diluted 1:10–1:104 with PBS, and 5 µl of the diluted solution was spotted on a Brucella broth agar plate supplemented with 5% defibrinated sheep blood and hemin and with vitamin K. After the liquid in the P. acnes suspension was absorbed into the agar, the plate was incubated under anaerobic conditions to quantify the CFUs of P. acnes. Data represent means ± SE of three individual experiments (*P<0.05, ***P<0.005 by Student’s t-test). CFU, colony-forming unit; FFA, free fatty acid; hBD-2, human β-defensin-2; LA, lauric acid; OA, oleic acid: PA, palmitic acid; PBS, phosphate-buffered saline: UD, undetectable.