Using nanoparticle optics assay for direct observation of the function of antimicrobial agents in single live bacterial cells
Using nanoparticle optics assay for direct observation of the function of antimicrobial agents in single live bacterial cells
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DOI：
10.1021/bi0351110
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发表时间：
2004-01-13
期刊：
影响因子：
2.9
通讯作者：
Xu, XHN
中科院分区：
文献类型：
--
作者：
Kyriacou, SV;Brownlow, WJ;Xu, XHN
原核生物和真核生物均报告了多药耐药性（MDR），这凸显了设计和筛选更有效的新药所面临的挑战。例如，铜绿假单胞菌（革兰氏阴性菌）的外排泵可以挤出多种结构和功能多样的底物，从而导致 MDR。在这项研究中，我们提出了一个新平台，利用纳米粒子光学和单活细胞成像，以纳米级和单细胞分辨率实时研究抗生素在活细菌细胞（铜绿假单胞菌）中的作用模式。以银（Ag）纳米颗粒（紫、蓝、绿、红）的颜色指数作为尺寸指数（30 +/- 10、50 +/- 10、70 +/- 10 和 90 +/- 10 nm），用于在纳米尺度上实时测量细胞壁的尺寸变化和膜渗透性。我们已经证明，随着氨曲南（AZT）浓度的增加和孵育时间的增加，细胞中积累的银纳米粒子的数量增加，表明AZT诱导膜通透性的大小变化和细胞壁的破坏。结果表明，纳米颗粒光学测定可以作为一种新的强大工具，用于在纳米尺度上实时表征活细胞中抗菌剂的作用模式。此外，对WT细菌突变体（nalB-1和DeltaABM）的研究表明，外排泵（MexA-MexB-OprM）有效地将底物（纳米颗粒）挤出细胞，表明MDR机制涉及诱导膜通透性和内在泵机制的变化。
Multidrug resistance (MDR) has been reported in both prokaryotes and eukaryotes, underscoring the challenge of design and screening of more efficacious new drugs. For instance, the efflux pump of Pseudomonas aeruginosa (gram-negative bacteria) can extrude a variety of structurally and functionally diverse substrates, which leads to MDR. In this study, we present a new platform that studies modes of action of antibiotics in living bacterial cells (P. aeruginosa), in real-time, at nanometer scale and single-cell resolution using nanoparticle optics and single living cell imaging. The color index of silver (Ag) nanoparticles (violet, blue, green, and red) is used as the sized index (30 +/- 10, 50 +/- 10, 70 +/- 10, and 90 +/- 10 nm) for real-time measurement of sized transformation of the cell wall and membrane permeability at the nanometer scale. We have demonstrated that the number of Ag nanoparticles accumulated in cells increases as the aztreonam (AZT) concentration increases and as incubation time increases, showing that AZT induces the sized transformation of membrane permeability and the disruption of the cell wall. The results demonstrate that nanoparticle optics assay can be used as a new powerful tool for real-time characterization of modes of action of antimicrobial agents in living cells at the nanometer scale. Furthermore, studies of mutants of WT bacteria (nalB-1 and DeltaABM), suggest that an efflux pump (MexA-MexB-OprM) effectively extrudes substrates (nanoparticles) out of the cells, indicating that the MDR mechanism involves the induction of changes in membrane permeability and the intrinsic pump machinery.