Infinite m200 pro fluorescence microplate reader

1-N-phenylnaphthylamine (NPN) was also used in this assay to determine the efflux pump activity and response to treatment with PAβN. This assay is useful for the discrimination between intracellular and extracellular localization of NPN to reflect the activity of efflux pumps [51 (link)]. Previous investigators reported the use of the same method [5 (link),52 (link)], which was followed with slight modifications. A mid-logarithmic-phase bacterial suspension was adjusted to a density of 10⁹ CFU/mL (~3.0 MacFarland) in a buffer made of 50 mM K₂HPO₄ and 1 mM MgSO₄, then added into black 96-well microplates containing 10 μM NPN, along with serial dilutions of PAβN, colistin, and CCCP (both used as positive controls for outer membrane damage and efflux inhibition, respectively). The microplates were incubated at 37 °C for one hour, then properly covered to protect them from light. Efflux was initiated by adding glucose (final concentration of 1 mM), and measurements were recorded at time 0; after 10, 20, and 30 s; then every minute (from 1–5 min) using an infinite M200 PRO fluorescence microplate reader (Tecan) with excitation/emission wavelengths of 350/420 nm.

The outer membrane permeation activity of the PMPN and combinations was assessed by the 1-N-phenylnaphthylamine (NPN) assay (Sigma, United States), as described previously with slight modifications (Zhou et al., 2019 (link)). Briefly, mid-logarithmic phase bacterial cells adjusted to a density of 10⁹ CFU/ml (equivalent to 3.0 MacFarland) were added to black 96 well microplates containing 10 μM NPN and colistin, PMBN or AZT (serial dilutions), or a combination of PMBN and AZM (at FICI). The plates were incubated for 1 h at 37° C protected from light. Fluorescence intensity was measured after 1 h using infinite M200 PRO fluorescence microplate reader (Tecan, United States) at 350 nm excitation and 420 nm emission wavelengths. NPN uptake (%) was calculated for each strain as described before (MacNair et al., 2018 (link)), as follows:
NPN uptake (%) = (F_obs − F₀) / (F₁₀₀ − F₀) × 100%.
Where F_obs is the observed fluorescence at a given concentration of the drug, F₀ is the initial fluorescence of NPN with E. coli cells in the absence of any treatment, and F₁₀₀ is the fluorescence of NPN with E. coli cells upon addition of 128 μg/ml of colistin, as full NPN uptake (100%) was reported to be achieved at high concentrations of colistin (MacNair et al., 2018 (link); Zhou et al., 2019 (link)).

QR2 or NQO1 (Sigma-Aldrich) assays were conducted in 50 mm Tris, 150 mm NaCl buffer adjusted to pH 8.5 with HCl. To 100 μl reactions in a black-walled, clear bottom, 96-well plate (Thermo Fisher Scientific), QR2 or NQO1 enzyme (10 nm) was added, along with both BNAH (1-benzyl-1,4-dihydronicotinamide; Tocris Bioscience) and menadione 100 μm. The fluorescence of BNAH was monitored every 30 s by excitation with 340 nm and emission at 440 nm using a Tecan Infinite M200 PRO fluorescence microplate reader. To validate inhibitor efficacy and enzymatic activity, either S29434 (200 nm) or dicoumarol (200 μm) was added to the reaction. Each condition was done with or without enzyme, the initial linear phase of enzymatic activity was measured, and the nonenzymatic BNAH signal decay was subtracted from each relevant enzymatic reaction.