Iino Ryota, Nishino Kunihiko, Noji Hiroyuki, Yamaguchi Akihito, Matsumoto Yoshimi
Department of Applied Chemistry, Graduate School of Engineering, University of Tokyo Bunkyo-ku, Tokyo, Japan.
Front Microbiol. 2012 Feb 8;3:40. doi: 10.3389/fmicb.2012.00040. eCollection 2012.
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.
近年来，多药耐药病原体广泛传播，这主要归因于它们增强的多药外排泵活性。目前，新型抗菌药物匮乏，因此，属于耐药-固氮-细胞分裂（RND）家族的多药外排泵抑制剂似乎在治疗多药耐药病原体引起的感染中很有用。此外，微制造技术的最新进展已将纳米/微器件的应用扩展到人类医疗保健领域，如感染检测和疾病诊断。我们开发了一种微流控通道装置，用于简单快速地评估细菌的药物外排活性。通过将微流控装置与一种荧光化合物（荧光素-二-β-D-吡喃半乳糖苷，在大肠杆菌细胞质中水解为荧光染料）相结合，我们成功评估了抑制剂对大肠杆菌中铜绿假单胞菌的RND型多药外排泵MexAB-OprM和MexXY-OprM的影响。我们的新方法成功检测到了D13-9001对MexB的特异性抑制作用，并揭示了长期用作外排泵抑制剂的苯丙氨酸-精氨酸-β-萘酰胺出人意料的膜通透作用。
