Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan.
PLoS One. 2011 Apr 12;6(4):e18547. doi: 10.1371/journal.pone.0018547.
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.
荧光素二-β-D-半乳糖苷（FDG）是一种荧光化合物，在大肠杆菌的细胞质中被β-半乳糖苷酶水解，生成荧光染料荧光素。我们发现 FDG 和荧光素都是外排泵的底物，因此开发了一种使用 FDG 和微流控通道装置评估大肠杆菌中外排泵抑制活性的新方法。我们使用了大肠杆菌 MG1655 野生型、ΔacrB（ΔB）、ΔtolC（ΔC）和同时缺失 acrB 和 tolC（ΔBC）的菌株，这些菌株都带有编码铜绿假单胞菌 MexAB-OprM（pABM）或 MexXY-OprM（pXYM）的质粒。我们评估了两种抑制剂，MexB 特异性的吡啶并嘧啶（D13-9001）和非特异性的苯丙氨酸-精氨酸-β-萘酰胺（PAβN）。使用荧光显微镜下的微流控通道装置观察抑制剂对泵的影响。AcrAB-TolC 和类似的泵有效地阻止了 FDG 进入野生型细胞，因此没有荧光。相反，ΔB 或 ΔC 容易将 FDG 内流并水解为荧光素，而在 ΔB 中，剩余的泵将荧光素外排。因此，在微流控通道中观察到 ΔB 中的荧光介质和 ΔC 及 ΔBC 中的荧光细胞。D13-9001 显著增加了ΔBC/pABM 中的荧光细胞数量，但对ΔBC/pXYM 没有影响。PAβN 增加了所有菌株的培养基荧光，特别是在泵缺失突变体中，并且导致 ΔC 中荧光素的积累消失。棋盘法显示，D13-9001 仅对 MexAB-OprM 产生菌（ΔBC/pABM）与氨曲南、环丙沙星和红霉素表现出协同作用，而 PAβN 与所有菌株（包括泵缺失突变体），尤其是与红霉素，表现出协同作用。PAβN 的结果与膜通透剂多粘菌素 B 或多粘菌素 B 非肽的结果相似。该新方法阐明了 D13-9001 特异性抑制 MexAB-OprM，而 PAβN 似乎是泵的底物，并使大肠杆菌的膜通透性增加。
