Background

Multidrug resistance in bacteria is a major public health crisis. Gram-negative bacteria currently pose the greatest threat to public health because of the emergence and rapid spread of carbapenem resistance. Consequently, new antibacterial molecules must be identified to combat this urgent problem. The outer membrane (OM) of gram-negative bacteria is not only the target of several traditional antibiotics to exert antibacterial activities but also of novel therapeutic agents, such as antimicrobial peptides (AMPs). These molecules can disrupt the OM integrity and cause bacterial lysis through permeabilization. Thus, detecting the permeability of the outer and inner membranes is a direct and important means of assessing the efficacy of antibacterial molecules. Electron microscopy is usually applied to observe the morphological changes in OM, and the assay of minimal bactericidal concentration is used to examine bacterial viability after treatment. However, these methods are time-consuming and cannot reflect the real-time change in membrane permeability. In this protocol, we provide a convenient and fast approach for evaluating the antibacterial efficacy of agents with potential membrane-permeabilizing effect by using the fluorescent probes N-phenyl-1-naphthylamine (NPN) and propidium iodide (PI).

NPN is a hydrophobic dye that dissolves sparingly in water with very low fluorescent emission. However, the fluorescence intensity increases sharply when NPN binds with nonpolar substances. The intact OM efficiently blocks NPN out of bacteria to ensure that NPN cannot bind to the hydrophobic tail of phospholipids. By contrast, a strong fluorescence emission can be detected when an OM rupture occurs. Thus, the change in fluorescence intensity of NPN can reflect the efficacy of antibacterial molecules on increasing the permeability of OM. PI is a red-fluorescent nucleic acid stain that can bind to DNA and RNA between the bases. Binding to DNA and RNA leads to an enhancement of PI fluorescence by 20- to 30-fold compared with that in aqueous solutions. Given that PI is a membrane-impermeant stain, it can only label bacteria with a compromised inner membrane (IM). Thus, PI is used in this protocol to determine the change in IM permeability after the treatment of antibacterial molecules.

In most of the related studies, the bacterial OM/IM permeability assay is conducted in a 96-well optical-bottom black plate. Bacteria, fluorescent probe, and antibacterial molecules are mixed together and co-cultured throughout the assay, during which the fluorescence intensity is detected at each time point. To avoid the high background fluorescence emitted by the bacterial culture media, the assay buffer (5 mM HEPES, pH 7.2) is used instead. In this case, bacteria grow under an innutritious condition compared with that of the antibacterial assays such as minimum inhibitory concentration and time–kill curve assays, which may affect the physiological status of bacteria. And some antibacterial molecules may also interfere with the function and performance of the fluorescent probe. As a result, the assay may not exactly reflect the change in permeability caused by the antibacterial molecules. The current work presents an optimized procedure with respect to the previously reported protocols (Ma et al., 2016b; Yarlagadda et al., 2016; Krishnamurthy et al., 2019) by culturing bacteria and exposing them to antibacterial molecules in the same medium as those of other antibacterial assays, harvesting bacteria, and detecting the fluorescent yield in the assay buffer at each time point. This methodology can evaluate the membrane permeability more efficiently and precisely and better match the results obtained from the antibacterial assays.