Bacterial viability assay

Cell viability of biofilms was assessed using an adapted method of the ATP assay for biofilms⁶². Briefly, P. aeruginosa isolates were grown to stationary phase at 37 °C with shaking at 200 RPM in cation-adjusted Muller-Hinton broth (CAMHB-Sigma-Aldrich.). This culture was then diluted to an OD₆₀₀ of 0.05 and grown to an OD of 0.1 (early exponential phase) prior to plating. 100 µL of this culture were added to a white Grenier LUMITRAC medium binding plate (Sigma-Aldrich, city, country) in triplicate and incubated for 24 hours at 37 °C without shaking. After 24 hours, media were removed, and cells were gently washed 2X with fresh media. 100 µL of fresh CAMHB was added back to the wells along with 100 µL of antibiotic, cationic peptide 6K-F17, or antibiotic plus peptide, for a final volume of 200 µL and mixed gently for 5 minutes on an orbital shaker. The plate was then incubated for 16 hours at 37 °C. Following these procedures, 100 µL from each well was removed and placed into an empty well of a 96-well plate to monitor ATP in the planktonic or detached fraction of the wells. 100 µL of the Bac-titer glo ATP cell viability solution (Promega, Madison, WI) was added to each well. Plates were gently mixed on an orbital shaker for 10 minutes prior to luminescence reading as per manufacturer’s suggested protocol.