Screening for efflux pump systems of bacteria by the new acridine orange agar method.

AIM: Development of a non-toxic, fluorescent-based, agar system for the screening of overexpressed bacterial efflux pump systems with common, inexpensive UV accessories.
MATERIALS AND METHODS: Wild type Gram-negative and positive bacteria expressing intrinsic efflux pumps and their progeny that overexpress a specific efflux pump were selected for evaluation of efflux pump activity in a Mueller-Hinton agar, containing increasing concentrations of the non-toxic fluorescent chromophore acridine orange (AO). The method is based on the same principle as the first-generation ethidium bromide method, according to which the concentration of the fluorescent dye that first produces fluorescence of the overlying bacterial colony represents the maximum concentration of the dye that the bacterium can extrude. The higher the concentration needed to produce fluorescence, the greater the ability of the bacterial efflux pump to extrude the dye.
RESULTS: Progeny of Escherichia coli, Salmonella enterica serovar Enteritidis and Staphylococcus aureus that over-expressed a given efflux pump fluoresced (i.e. accumulated AO) at concentrations of AO that were much greater than the ones required for the emission of fluorescence by their corresponding wild-type counterpart which expressed an intrinsic efflux pump.
CONCLUSION: The AO agar method readily identifies strains of Gram-negative and Gram-positive bacteria that overexpress efflux pump systems compared to their wild-type progeny.