The outer membrane permeability was measured using a hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN, Aladdin, Shanghai, China) uptake method, as previously described (24 (link)), with some modifications. K. pneumoniae FK7921 was grown to the mid-logarithmic phase with an OD of 0.4 to 0.6 at 600 nm in LB. Equal volumes of bacterial solution were allocated to the designated tubes for the experimental groups with the desired concentrations of pentamidine monotherapy, linezolid monotherapy, or combination therapy as well as for the positive-control groups with colistin (Kangtai, Zhejiang, China), SDS (Solarbio, China), and Triton X-100 (Solarbio, China) and the negative-control group, which contained only the bacteria. The tubes were incubated at 37°C with shaking at 180 rpm for 2 h. The bacterial solutions were then pelleted and washed three times in PBS buffer (pH 7.2) with 5 mM glucose and were subsequently resuspended with 1 mL of 10 μM NPN working solution. 200 μL of the resulting cell suspension was added to each well of a black 96-well plate. After incubation at room temperature for 30 min in darkness, a multimode plate reader (VICTOR Nivo, PerkinElmer, USA) was used to measure the fluorescence at an excitation wavelength and an emission wavelength of 350 and 420 nm, respectively.
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