Background

Multidrug efflux pumps are widely distributed and can be found in all living species. They represent an important mechanism of antimicrobial resistance. The ability to quantify the activity of efflux pumps is necessary for understanding of their contribution to physiological processes and assessment of the validity of potential therapeutics (e.g., efflux inhibitors) (Blair and Piddock, 2016). Methods for efflux activity measurements largely rely on two different mechanisms. Some methods directly measure the substrate efflux, i.e., how much of the substrate is pumped out, and others measure substrate molecule accumulation inside the cell, the levels of which is then used to infer efflux indirectly. However, the latter is less sensitive due to variable membrane permeability that alters dye influx rates (Blair and Piddock, 2016). Accumulation of R6G in growing C. albicans cells inversely correlates with the level of the ABC transporter Candida drug resistance 1 (CDR1) mRNA expression, establishing levels of intracellular R6G accumulation can be therefore used for identification of azole-resistant strains (Maesaki et al., 1999). Historically, this was carried out by measurements of accumulated radiolabelled-substrates. More recently, fluorescence-based methods are being used. Accumulation of fluorescent dye in a single cell can also be measured by flow cytometry. The benefit of this approach lies in the ability to measure variation in efflux activity among individual cells.

The protocol of the above described method involves preloading the cell population with a fluorescent substrate prior to the efflux assay. After the loading step, substrate accumulates within the cells at maximum concentration. Cells are then washed to remove the substrate. Subsequently, glucose is supplemented to the culture as a source of energy, and the fluorescence signal of substrate is monitored. The method is suitable for use with any yeast species (Borecka-Melkusova et al., 2008).