Contemporaneous Measurement of Outer and Inner Membrane Permeability in Gram-negative Bacteria

The emergence and rapid spread of multidrug resistance in bacteria have led to the urgent need for novel antibacterial agents. Membrane permeabilization is the mechanism for many antibacterial molecules that are being developed against gram-negative bacteria. Thus, to determine the efficacy of a potential antibacterial molecule, it is important to assess the change in bacterial membrane permeability after treatment. This study describes the protocol for the assays of outer and inner membrane permeability using the fluorescent probes N-phenyl-1-naphthylamine and propidium iodide. Compared with other experiments, such as electron microscopy and the assay of minimal bactericidal concentration, this methodology provides a simpler, faster, and cost-effective way of estimating the membrane-permeabilizing effect and bactericidal efficacy of antibacterial molecules. This study presents an optimized protocol with respect to the classical protocols by incubating bacteria with antibacterial molecules in the culture condition identical to that of antibacterial assays and then detecting the signal of the fluorescent probe in the buffer without broth and antibacterial molecules. This protocol avoids the effect of nutrient deficiency on the physiological status of bacteria and the interference of antibacterial molecules towards the fluorescent probe. Thus, this method can effectively and precisely evaluate the membrane permeability and match the results obtained from other antibacterial assays, such as minimum inhibitory concentration and time-kill curve assays.