A Microfluidic Device for Simple and Rapid Evaluation of Multidrug Efflux Pump Inhibitors

Iino, Ryota; Nishino, Kunihiko; Noji, Hiroyuki; Yamaguchi, Akihito; Matsumoto, Yoshimi

doi:10.3389/fmicb.2012.00040

Cited by 23 publications

(26 citation statements)

References 48 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Drug Phenotypes Of Acrabmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

et al. 2015

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The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine ␤-naphthylamide (PA␤N) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PA␤N and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PA␤N, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PA␤N. Strong action of PA␤N in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PA␤N acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PA␤N acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PA␤N in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PA␤N on membrane integrity are compounded in cells unable to extrude PA␤N. IMPORTANCEThe increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds. Multidrug resistance among human bacterial pathogens remains a grave social concern. Numerous strategies have been proposed to curtail the rampant increase in multidrug resistance among human pathogens, ranging from the effective integration of pharmacokinetic and pharmacodynamic parameters and implementation of antimicrobial stewardship (1) to the development of novel antibiotics based on either an existing or a novel chemical scaffold, exploitation of new cellular targets, and directly tackling the cellular mechanisms that confer multidrug resistance (2). Efflux of antibiotics from the cell is one of the common mechanisms of antibiotic resistance in bacteria, with resistance developing when the rate of drug efflux across the membrane exceeds that of drug influx (3).Bacterial genomes encode several membrane-bound multidrug efflux systems (4, 5). These systems are usually under t...

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