Abstract
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.
Highlights
Increase in multidrug resistance among clinical isolates is a major problem in infection control
We focused on a new technique for a simple and rapid measurement of the activities of bacterial drug efflux pumps and inhibitors by using a microfluidic device recently reported by Matsumoto et al (2011)
EVALUATION OF INHIBITORS BY THE CLASSIC CHECKERBOARD METHOD Checkerboard minimum inhibitory concentrations (MICs) determination is a simple method to evaluate the effects of inhibitors on antibacterial activity
Summary
Increase in multidrug resistance among clinical isolates is a major problem in infection control. The so-called “multidrug-resistant Pseudomonas aeruginosa (MDRP)” which is resistant to major antipseudomonal agents such as carbapenems, quinolones, and aminoglycosides (Sekiguchi et al, 2007b; Kirikae et al, 2008), has been isolated and identified as a cause of nosocomial outbreaks in Japan (Sekiguchi et al, 2007a; Satoh et al, 2008). The RND-type multidrug efflux systems have extremely broad substrate specificities and protect the cells from the actions of antibiotics. They usually function as three-component assemblies spanning the outer and cytoplasmic membranes and the periplasmic space of Gram-negative bacteria. The RND efflux system consists of three different proteins: a cytoplasmic membrane protein (such as MexB), a membrane fusion protein (MexA), and an outer membrane channel (OprM)
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