Evaluation of Multidrug Efflux Pump Inhibitors by a New Method Using Microfluidic Channels
作者： Yoshimi Matsumoto , Kohei Hayama , Shouichi Sakakihara , Kunihiko Nishino , Hiroyuki Noji
DOI: 10.1371/JOURNAL.PONE.0018547
关键词: Biochemistry 、 Membrane transport protein 、 Fluorescence microscope 、 Escherichia coli 、 Polymyxin B 、 Chemistry 、 Fluorescence 、 Fluorescein 、 Efflux 、 Fluorescence-lifetime imaging microscopy 、 General Biochemistry, Genetics and Molecular Biology 、 General Agricultural and Biological Sciences 、 General Medicine
摘要: Fluorescein-di-β-d-galactopyranoside (FDG), a fluorogenic compound, is hydrolyzed by β-galactosidase in the cytoplasm of Escherichia coli to produce fluorescent dye, fluorescein. We found that both FDG and fluorescein were substrates efflux pumps, have developed new method evaluate efflux-inhibitory activities E. using microfluidic channel device. used MG1655 wild-type, ΔacrB (ΔB), ΔtolC (ΔC) ΔacrBΔtolC (ΔBC) harboring plasmids carrying mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes Pseudomonas aeruginosa. Two inhibitors, MexB-specific pyridopyrimidine (D13-9001) non-specific Phe-Arg-β-naphthylamide (PAβN) evaluated. The effects inhibitors on pumps observed device under fluorescence microscope. AcrAB-TolC analogous effectively prevented influx wild-type cells, resulting no fluorescence. In contrast, ΔB ΔC easily imported fluorescein, which was exported residual ΔB. Consequently, medium cells ΔBC channels. D13-9001 substantially increased cell number ΔBC/pABM but not ΔBC/pXYM. PAβN all strains, especially pump deletion mutants, caused accumulation disappear ΔC. checkerboard revealed acts synergistically with aztreonam, ciprofloxacin, erythromycin only against MexAB-OprM producer (ΔBC/pABM), synergistically, erythromycin, strains including mutants. results obtained from similar membrane permeabilizer, polymyxin B nonapeptide concentration. clarified specifically inhibited contrast PAβN, appeared be substrate permeabilized membranes coli.
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