Rapid accumulation of fluorophores and fast kill identify drugs with bactericidal effects against Gram-negative bacteria

Supplementary material for 'Rapid accumulation of fluorophores and fast kill identify drugs with bactericidal effects against Gram-negative bacteria'.Antimicrobial resistance is a massive threat, but developing a new antibiotic can take decades. That time could be drastically reduced if we were able to anticipate desirable properties of a chemical, such as its potential to target specific bacterial compartments. This would provide the opportunity to prioritise the development of molecules that target, for instance, the cell membrane, as this does not rely on transporters and usually results in a fast-acting bactericidal effect. We used flow cytometry and a set of fluorophores together with a group of antibiotics to discriminate between antimicrobials acting on cell membrane versus intracellularly against two Gram-negative bacteria, E. coli and A. baylyi. We then chose Rhodamine 123 as a fluorescent marker to screen a commercial library of chemical compounds. Using flow cytometry, several drugs present in the Prestwick library were observed to have cytotoxic effects at 1 µM final concentration towards E. coli. This was confirmed with growth inhibitory assays in both E. coli and A. baylyi for Pantoprazole, Theophylline and Zoledronic acid. This represents an approach to the large-scale screening of small molecules with the potential to deliver fast-acting molecules that target cell membranes in Gram-negative bacteria.

《荧光探针快速富集与快速杀菌效应可识别针对革兰氏阴性菌（Gram-negative bacteria）的杀菌药物》补充材料。抗菌药物耐药性（antimicrobial resistance）是一项重大威胁，而研发一款新型抗生素往往需要数十年之久。若能预先预判化合物的理想特性——例如其靶向特定细菌区域的潜力——则可大幅缩短这一研发周期。这将使我们能够优先开发靶向细胞膜的分子，因为这类分子无需依赖转运蛋白，通常可快速发挥杀菌效应。本研究采用流式细胞术（flow cytometry）、一组荧光探针（fluorophores）以及多款抗生素，针对两种革兰氏阴性菌——大肠杆菌（E. coli）与A. baylyi——区分靶向细胞膜与胞内作用的抗菌剂。随后我们选择罗丹明123（Rhodamine 123）作为荧光标记物，对商用化合物库进行筛选。通过流式细胞术分析，我们观察到Prestwick化合物库中的多款药物在终浓度1 µM时，对大肠杆菌具有细胞毒性效应。针对泮托拉唑（Pantoprazole）、茶碱（Theophylline）与唑来膦酸（Zoledronic acid）的大肠杆菌及A. baylyi生长抑制实验，验证了上述结论。本研究为大规模筛选小分子化合物提供了一种可行方案，有望获得靶向革兰氏阴性菌细胞膜的速效抗菌分子。