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 (%) = (Fobs − F0) / (F100 − F0) × 100%.
Where Fobs 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)).
NPN uptake (%) = (Fobs − F0) / (F100 − F0) × 100%.
Where Fobs 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)).
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