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.

Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli

“…Highly sensitive fluorescent probes, including 8-anilino-1-naphthalenesulfonic acid (ANS), fluorescein-di-␤-D-galactopyranoside (FDG), and Sytox Green, have been used to gauge PA␤N=s membrane-destabilizing effects in E. coli and P. aeruginosa cells (33,34,35). In these studies, changes in fluorescence, which indicate internalization of these normally impervious fluorescent compounds, were measured 10 to 20 min (33,35) or over an hour (34) after incubation with various amounts of PA␤N.…”

“…In these studies, changes in fluorescence, which indicate internalization of these normally impervious fluorescent compounds, were measured 10 to 20 min (33,35) or over an hour (34) after incubation with various amounts of PA␤N. In contrast, preincubation of cells with PA␤N was avoided in this study to minimize any membrane damage prior to the start of real-time assays.…”

“…Moreover, we found that ANS is a substrate of the RNDtype efflux pumps (data not shown); therefore, PA␤N-mediated inhibition of the RND pumps is expected to reduce ANS efflux from the envelope, resulting in higher fluorescence intensities, thus complicating interpretation of the data. When FDG, a substrate of AcrB, was used to study the effects of PA␤N and other compounds in increasing membrane permeability, the authors noted that as little as 4 g/ml of PA␤N was enough to initiate the release of presumably intracellularly generated fluorescein, the cleaved product of FDG, from a strain lacking TolC (33,35). However, since efflux-deficient cells, such as those lacking TolC, cannot remove PA␤N from the cell, the secondary effects of PA␤N are expected to be greatly amplified.…”

“…However, addition of EDTA from 10 M to 5,000 M failed to increase NPN fluorescence significantly (data not shown), indicating that chelation of outer membrane-bound cations is not a critical factor in the PA␤N-mediated increase in NPN fluorescence. We then employed PMXBN, a compound previously used to provide evidence that PA␤N, like PMXBN, acts by destabilizing the outer membrane permeability barrier (33,35). As was done with PA␤N, PMXBN was added 200 s after the initiation of NPN efflux.…”

“…The authors showed that in strains expressing RND efflux pumps, PA␤N causes minimum damage to membrane integrity and potential, whereas in strains lacking a major efflux pump, it can impose some toxic effects (20,32). However, the conclusion that PA␤N acts principally as an efflux pump inhibitor was questioned by three recent publications (33,34,35). One of the main objections stems from the observation that PA␤N causes elevated toxicity, by damaging the membrane, in cells lacking major efflux pathways.…”