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A direct assay of efflux byEscherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-typeE. coli . We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC4 -(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition inE. coli . We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC4 -(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of theacrB deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC4 -(3) competes with macrolides but not with Nile Red, we propose that DiBAC4 -(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux.

A Fluorescent Microplate Assay Quantifies Bacterial Efflux and Demonstrates Two Distinct Compound Binding Sites in AcrB