Measuring Efflux and Permeability in Mycobacteria

Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria, active efflux systems have been widely characterized from diverse mycobacterial species in laboratory conditions, showing that they can promote resistance by extruding noxious compounds prior to their reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx, and efflux. Given the urgent need to discover and develop novel antimycobacterial compounds in order to design effective therapeutic strategies, the contributions to drug resistance made by the controlled permeabilityPermeability of the cell wall and the increased activity of effluxCell wallefflux pumps must be determined. In this chapter, we will describe a method that allows (1) the measuring of permeabilityPermeability and the quantification of general efflux activity of mycobacteria, by the study of the transportCell walltransport (influx and efflux) of fluorescent compounds, such as ethidium bromideEffluxethidium bromide (EtBr); and (2) the screening of compounds in search of agents that increase the permeabilityPermeability of the cell wall and efflux inhibitorsEffluxinhibitors that could restore the effectiveness of antimicrobials that are subject to efflux.