Black flat bottom plates

Envelope permeability was evaluated by the Hoechst (H) 33342 dye accumulation assay described in Jimenez-Castellanos et al. [23 (link)]. Briefly, a bacterial culture was inoculated in 4 mL of LB broth and incubated in a 37 °C shaker until reaching the log phase (OD₆₀₀ 0.5–0.7). The bacterial cells were harvested by centrifugation at 6000 g for 10 min. The cell pellets were resuspended and adjusted to an OD₆₀₀ equal to 0.5 with PBS buffer. The fluorescent dye H33342 was added to the bacterial suspension at a final concentration of 2.5 μM. A 180-μL aliquot of each isolate was placed into each well of black flat-bottom plates (Costar, Corning, NY, USA). Fluorescence was measured every 5 min for 70 min by using a Varioskan Flash microplate reader (Thermo Fischer Scientific, Waltham, MA, USA) with excitation and emission filters at 355 nm and 460 nm, respectively. Two efflux pump inhibitors, phenylalanine-arginine β-naphthylamide (PAβN) and cyanide-chlorophenylhydrazone (CCCP), were separately added into the previously described bacterial suspension with the H33342 dye at final concentrations of 25 μg/mL and 25 µM, respectively. Fluorescence was measured as previously described. The relative fold changes of dye accumulation were calculated by comparing the fluorescence intensities in the presence of inhibitors to those in the absence of inhibitors for each isolate.