Single-cell microfluidics facilitates the rapid quantification of antibiotic accumulation in Gram-negative bacteria.
Single-cell microfluidics facilitates the rapid quantification of antibiotic accumulation in Gram-negative bacteria.
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DOI：
10.1039/d0lc00242a
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发表时间：
2020-08-07
期刊：
影响因子：
6.1
通讯作者：
Pagliara S
中科院分区：
文献类型：
--
作者：
Cama J;Voliotis M;Metz J;Smith A;Iannucci J;Keyser UF;Tsaneva-Atanasova K;Pagliara S
一种用于定量革兰氏阴性菌中抗生素积累的新型快速单细胞测定法揭示了有关细菌药物积累的重要见解。革兰氏阴性菌的双膜细胞被膜是细胞内抗生素积累的强大屏障。对这些细胞中抗生素转运的定量理解对于药物开发至关重要，但由于缺乏合适的研究技术，这一点已被证明是难以捉摸的。在这里，我们结合联合收割机微流体和延时自体荧光显微镜，快速定量抗生素积累在数百个单独的大肠杆菌细胞。通过连续操纵微流体环境，我们证明了静止期大肠杆菌，传统上比生长细胞更难对付抗生素，与活跃生长的细胞相比，显示抗生素氧氟沙星的积累减少。我们的新的微流控方法有利于定量比较的作用，微环境与细胞药物积累的关键膜转运途径的情况下。与传统技术不同的是，我们的分析是快速的，研究随着细胞被给予药物的积累。该平台为研究抗生素在细菌中的积累提供了一个强大的新工具，这对于下一代抗生素的合理开发至关重要。
A novel, rapid single-cell assay for quantifying antibiotic accumulation in Gram-negative bacteria reveals important insights about bacterial drug accumulation. The double-membrane cell envelope of Gram-negative bacteria is a formidable barrier to intracellular antibiotic accumulation. A quantitative understanding of antibiotic transport in these cells is crucial for drug development, but this has proved elusive due to a dearth of suitable investigative techniques. Here we combine microfluidics and time-lapse auto-fluorescence microscopy to rapidly quantify antibiotic accumulation in hundreds of individual Escherichia coli cells. By serially manipulating the microfluidic environment, we demonstrated that stationary phase Escherichia coli, traditionally more refractory to antibiotics than growing cells, display reduced accumulation of the antibiotic ofloxacin compared to actively growing cells. Our novel microfluidic method facilitates the quantitative comparison of the role of the microenvironment versus that of the absence of key membrane transport pathways in cellular drug accumulation. Unlike traditional techniques, our assay is rapid, studying accumulation as the cells are dosed with the drug. This platform provides a powerful new tool for studying antibiotic accumulation in bacteria, which will be critical for the rational development of the next generation of antibiotics.
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DOI：
10.1016/j.bbapap.2008.11.005
发表时间：
2009-05
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Delcour AH
通讯作者：
Delcour AH
影响因子：
16.6
作者：
Modi, Souvik;Lopez-Domenech, Guillermo;Kittler, Josef T.
通讯作者：
Kittler, Josef T.
大肠杆菌K-12的构造框架，单基因敲除突变体：Keio Collection。
影响因子：
9.9
作者：
通讯作者：
--
影响因子：
13.6
作者：
Goormaghtigh, Frederic;Van Melderen, Laurence
通讯作者：
Van Melderen, Laurence
DOI：
10.1073/pnas.152206799
发表时间：
2002-07-23
影响因子：
11.1
作者：
Nestorovich, EM;Danelon, C;Bezrukov, SM
通讯作者：
Bezrukov, SM