Membrane Disruption Assay for P. aeruginosa

Disruption of the outer bacterial membrane of P. aeruginosa was measured using the fluorescent probe 1-N-phenylnaphthylamine (NPN; Merck). Late-exponential-phase cultures of P. aeruginosa were washed twice and adjusted to an OD₆₀₀ of 0.5 in 5 mM HEPES buffer. In the wells of a black microtiter plate (ThermoScientific), adjusted cultures were combined with NPN (final concentration, 10 μM) along with either no treatment (buffer only), 50 mg/L GA, 2 mg/L CST, 4 mg/L TOB, or 16 mg/L LL-37 (Merck), to a final volume of 200 μL. Agent concentrations were chosen based on EUCAST breakpoint concentrations (for CST and TOB) or previously published active concentrations (GA [18 (link)] and LL-37 [73 (link)]). Each experiment consisted of triplicate wells of each condition. Fluorescence was measured in a plate reader at excitation of 355 nm, emission at 460 nm every 30 s for 15 min. NPN uptake factor was calculated as follows: [(fluorescence of sample with NPN) − (fluorescence of sample without NPN)]/[(fluorescence of buffer with NPN) − (fluorescence of buffer without NPN)].