Evaluation of Multidrug Efflux Pump Inhibitors by a New Method Using Microfluidic Channels

Fluorescein-di-beta-D-galactopyranoside (FDG), a fluorogenic compound, is hydrolyzed by beta-galactosidase in the cytoplasm of Escherichia coli to produce a fluorescent dye, fluorescein. We found that both FDG and fluorescein were substrates of efflux pumps, and have developed a new method to evaluate efflux-inhibitory activities in E. coli using FDG and a microfluidic channel device. We used E. coli MG1655 wild-type, Delta acrB (Delta B), DtolC (Delta C) and Delta acrB Delta tolC (Delta BC) harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of Pseudomonas aeruginosa. Two inhibitors, MexB-specific pyridopyrimidine (D13-9001) and non-specific Phe-Arg-beta-naphthylamide (PAbN) were evaluated. The effects of inhibitors on pumps were observed using the microfluidic channel device under a fluorescence microscope. AcrAB-TolC and analogous pumps effectively prevented FDG influx in wild-type cells, resulting in no fluorescence. In contrast, Delta B or Delta C easily imported and hydrolyzed FDG to fluorescein, which was exported by residual pumps in Delta B. Consequently, fluorescent medium in Delta B and fluorescent cells of Delta C and Delta BC were observed in the microfluidic channels. D13-9001 substantially increased fluorescent cell number in Delta BC/pABM but not in Delta BC/pXYM. PA beta N increased medium fluorescence in all strains, especially in the pump deletion mutants, and caused fluorescein accumulation to disappear in Delta C. The checkerboard method revealed that D13-9001 acts synergistically with aztreonam, ciprofloxacin, and erythromycin only against the MexAB-OprM producer (Delta BC/pABM), and PA beta N acts synergistically, especially with erythromycin, in all strains including the pump deletion mutants. The results obtained from PA beta N were similar to the results from membrane permeabilizer, polymyxin B or polymyxin B nonapeptide by concentration. The new method clarified that D13-9001 specifically inhibited MexAB-OprM in contrast to PAbN, which appeared to be a substrate of the pumps and permeabilized the membranes in E. coli.