Efflux pumps (EPs) are localized in the cytoplasm of bacteria and are responsible for expelling unwanted substrates like toxins and antibiotics from the cell. This ‘pumping out’ effect can be the cause of multidrug resistance (MDR) in bacterial clinical isolates from MRSA patients, tuberculosis patients, AIDS patients affected with Mycobacterium, etc. EP activity is measured using ethidium bromide (EtBr) uptake and efflux. EtBr accumulation (fluorescent increase) and expulsion (fluorescent decrease) was measured on the Rotor-Gene for 30 minutes at a constant temperature under different buffer and compound conditions. Compounds that reverse drug resistance are easily identified via real-time monitoring of efflux pump activity in living bacteria. Once suitable compounds are identified they can be combined as an adjuvant to conventional antibodies to inhibit EPs that would normally expel the antibiotic. The assay can also be used to identify overexpressed efflux activity of multidrug-resistant Gram-negative bacteria. The small volumes used consume minimal quantities of the efflux pump inhibitors under evaluation; an important advantage when screening libraries of rare or expensive compounds. References Viveiros M, et al. 2008, Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated Ethidium Bromide method. Int. J. Antimicrob. Agents Rodrigues L, et al. 2008, Thioridazine and chlorpromazine inhibition of ethidium bromide efflux in Mycobacterium avium and Mycobacterium smegmatis.J. Antimicrobial Chemotherapy Resources Download the Real-time Metabolic Analysis Application Note  (313kb)

Example data (click to enlarge):  Uptake assay: Results show accumulation of EtBr with increasing amounts of buffer reagent. Used to determine the optimal in-vitro condition for intrinsic activity. Efflux Assay: Results show efflux of EtBr with increasing amounts of inhibitory compound. Used to determine the level required to inhibit EP activity to prevent drug resistance.