Antimicrobial Efficacy Screening in Artificial Sputum

For ASM preparation, the components were dissolved into sterile water at the following concentrations: mucin (Sigma-Aldrich), 0.5% (w/v); unsheared salmon sperm DNA (Sigma-Aldrich), 0.4% (w/v); NaCl, 0.5% (w/v); KCl, 0.2% (w/v); casamino acids (Oxoid), 0.5% (w/v); egg yolk emulsion (0.15%, source of lecithin; sterile; Sigma-Aldrich). After each addition, the mixture was vigorously vortexed. The pH of the solution was adjusted at 6.8 with Tris-HCl 1 M, pH 8.5 [5 (link)]. P. aeruginosa was grown overnight in LB broth, pelleted, washed with PBS, and resuspended in ASM at a density of 10⁶ CFU/mL. A volume of 10 μL of the peptide (3.12 to 25 μg/mL) and/or 10 μL of EDTA (0.62 to 2.5 mM) were added to 80 μL of ASM containing bacteria. To check for cell viability, bacteria were suspended in ASM 80% alone. After an incubation at 37 °C with shaking for 90 min (or 24 h), the samples were diluted 10-fold in TSB and plated on TSA, to determine the number of viable cells.