Use of resazurin to detect mefloquine as an efflux-pump inhibitor in Pseudomonas aeruginosa and Escherichia coli

Vidal-Aroca, Faustino; Meng, Alexandra; Minz, Tanja; Page, Malcolm G. P.; Dreier, Jürg

doi:10.1016/j.mimet.2009.09.021

Cited by 39 publications

(29 citation statements)

References 29 publications

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“…Permeabilizing activity of PAβN on outer membrane of E. coli by the assay using nitrocefin (Figure 10) was comparable to the activity obtained from the FDG assay and was higher than that on outer membrane of P. aeruginosa [15]. Outer membrane permeabilizing activity of PAβN have also been published by other researchers using resazurin as a substrate [43]. In our FDG assay, the effect of PAβN on the outer membrane of E. coli was visible in 4 µg/ml (Figure 6).…”

Section: Discussionsupporting

confidence: 83%

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

et al. 2011

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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.

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Section: Discussionsupporting

confidence: 83%

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

et al. 2011

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“…The permeabilizing activity of PAβN on the outer membrane of E. coli by nitrocefin hydrolysis assay was comparable to the activity obtained in the FDG assay (Matsumoto et al, 2011) and was higher than that reported previously for the outer membrane of P. aeruginosa (Lomovskaya et al, 2001). The outer membrane-permeabilizing activity of PAβN has also been published by other researchers using resazurin as a substrate (Vidal-Aroca et al, 2009). In the FDG assay, the effect of PAβN on the outer membrane of E. coli was visible at 4 μg/ml (Figure 1D).…”

Section: Discussionmentioning

confidence: 62%

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

Iino¹,

Nishino²,

Noji³

et al. 2012

Front. Microbio.

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Recently, multidrug-resistant pathogens have disseminated widely owing essentially to their increased multidrug efflux pump activity. Presently, there is a scarcity of new antibacterial agents, and hence, inhibitors of multidrug efflux pumps belonging to the resistance–nodulation–cell division (RND) family appear useful in the treatment of infections by multidrug-resistant pathogens. Moreover, recent progress in microfabrication technologies has expanded the application of nano/micro-devices to the field of human healthcare, such as the detection of infections and diagnosis of diseases. We developed a microfluidic channel device for a simple and rapid evaluation of bacterial drug efflux activity. By combining the microfluidic device with a fluorogenic compound, fluorescein-di-β-D-galactopyranoside, which is hydrolyzed to a fluorescent dye in the cytoplasm of Escherichia coli, we successfully evaluated the effects of inhibitors on the RND-type multidrug efflux pumps MexAB–OprM and MexXY–OprM from Pseudomonas aeruginosa in E. coli. Our new method successfully detected the MexB-specific inhibitory effect of D13-9001 and revealed an unexpected membrane-permeabilizing effect of Phe-Arg-β-naphthylamide, which has long been used as an efflux pump inhibitor.

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“…6). This ruled out the possibility that the changes in formazan reduction rates are caused by MexEF-OprN-mediated efflux of TV as observed for other redox probes (68). In addition, TV sensitivity levels were comparable between PA14, PA14::mexF, PA14::mexT, and PA14nfxC, which further suggests that TV is not a MexEF-OprN substrate (data not shown).…”

Section: Figmentioning

confidence: 75%

MexT Functions as a Redox-Responsive Regulator Modulating Disulfide Stress Resistance in Pseudomonas aeruginosa

et al. 2012

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MexT is a global LysR transcriptional regulator known to modulate antibiotic resistance and virulence in Pseudomonas aeruginosa. In this study, a novel role for MexT in mediating intrinsic disulfide stress resistance was demonstrated, representing the first identified phenotype associated with inactivation of this regulator in wild-type cells. Disruption of mexT resulted in increased susceptibility to the disulfide stress elicitor diamide [diazenedicarboxylic acid bis(N,N,-di-methylamide)]. This compound is known to elicit a specific stress response via depletion of reduced glutathione and alteration of the cellular redox environment, implicating MexT in redox control. In support of this, MexT-regulated targets, including the MexEF-OprN multidrug efflux system, were induced by subinhibitory concentrations of diamide. A mexF insertion mutant also exhibited increased diamide susceptibility, implicating the MexEF-OprN efflux system in MexT-associated disulfide stress resistance. Purified MexT protein was observed to form an oligomeric complex in the presence of oxidized glutathione, with a calculated redox potential of ؊189 mV. This value far exceeds the thiol-disulfide redox potential of the bacterial cytoplasm, ensuring that MexT remains reduced under normal physiological conditions. MexT is activated by mutational disruption of the predicted quinone oxidoreductase encoded by mexS. Alterations in the cellular redox state were observed in a mexS mutant (PA14nfxC), supporting a model whereby the perception of MexS-associated redox signals by MexT leads to the induction of the MexEF-OprN efflux system, which, in turn, may mediate disulfide stress resistance via efflux of electrophilic compounds.

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Use of resazurin to detect mefloquine as an efflux-pump inhibitor in Pseudomonas aeruginosa and Escherichia coli

Cited by 39 publications

References 29 publications

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

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

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

MexT Functions as a Redox-Responsive Regulator Modulating Disulfide Stress Resistance in Pseudomonas aeruginosa

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