Preparation of artificial sputum medium (ASMDM)

ASMDM was prepared in 30 mL aliquots as described by Fung et al. [14]. Porcine stomach mucin (Sigma-Aldrich, St. Louis, MO), DNA from salmon testes (Sigma-Aldrich, St. Louis, MO), potassium chloride (Fisher Scientific, Hanover Park, IL), sodium chloride (Fisher Scientific, Hanover Park, IL), diethylene triamine pentaacetic acid (Fluka Analytical, St. Louis, MO), casamino acids (Acros, Hanover Park, IL), and bovine serum albumin (Sigma-Aldrich, St. Louis, MO) were dissolved in 25 mL sterile dH₂O (final concentrations: 10 mg/mL, 1.4 mg/mL, 2.2 mg/mL, 5 mg/mL, 5.9 mg/mL, 5 mg/mL, and 10 mg/mL, respectively). Egg yolk emulsion (0.15 mL) (Becton Dickinson) and the antibiotics ampicillin (1 ug/mL; (Fisher Scientific, Hanover Park, IL) and penicillin (1 ug/mL; Sigma-Aldrich, St. Louis, MO) were added to revent contamination. Tetracycline was omitted because B. cenocepacia is sensitive to this antibiotic [18]. The suspension was stirred for 5 min at room temperature to dissolve the DNA and mucin. The pH was adjusted to 6.5, and sterile dH₂O was added to 30 mL ASMDM was stored at 4 °C, but warmed to 37 °C before addition to cells.