title: Evaluation of multidrug efflux pump inhibitors by a new method using microfluidic channels.
abstract: Fluorescein-di-β-D-galactopyranoside (FDG), a fluorogenic compound, is hydrolyzed by β-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, ΔacrB (ΔB), ΔtolC (ΔC) and ΔacrBΔtolC (ΔBC) 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-β-naphthylamide (PAβN) 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, ΔB or ΔC easily imported and hydrolyzed FDG to fluorescein, which was exported by residual pumps in ΔB. Consequently, fluorescent medium in ΔB and fluorescent cells of ΔC and ΔBC were observed in the microfluidic channels. D13-9001 substantially increased fluorescent cell number in ΔBC/pABM but not in ΔBC/pXYM. PAβN increased medium fluorescence in all strains, especially in the pump deletion mutants, and caused fluorescein accumulation to disappear in ΔC. The checkerboard method revealed that D13-9001 acts synergistically with aztreonam, ciprofloxacin, and erythromycin only against the MexAB-OprM producer (ΔBC/pABM), and PAβN acts synergistically, especially with erythromycin, in all strains including the pump deletion mutants. The results obtained from PAβN 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 PAβN, which appeared to be a substrate of the pumps and permeabilized the membranes in E. coli.
authors.affiliation: Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan. yoshimi@sanken.osaka-u.ac.jp
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Kohei
authors.initials: K
authors.lastname: Hayama
authors.forename: Shouichi
authors.initials: S
authors.lastname: Sakakihara
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Nishino
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
authors.forename: Ryota
authors.initials: R
authors.lastname: Iino
authors.forename: Akihito
authors.initials: A
authors.lastname: Yamaguchi
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D005798:Genes, Bacterial; D026901:Membrane Transport Proteins / Q000187:drug effects*; D008826:Microbial Sensitivity Tests; D044085:Microfluidics / Q000295:instrumentation*; D008856:Microscopy, Fluorescence; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D026901:Membrane Transport Proteins