Institute of Physical Chemistry of the Polish Academy of Sciences, ul. Kasprzaka 44/52, 01-224 Warszawa, Poland.
Lab Chip. 2018 Dec 7;18(23):3668-3677. doi: 10.1039/c8lc00916c. Epub 2018 Oct 30.
Measurement of antibiotic susceptibility at the level of single cells is important as it reveals the concentration of an antibiotic that leads to drug resistance in bacterial strains. To date, no solution for large-scale studies of antibiotic susceptibility at the single-cell level has been shown. Here, we present a method for production and separation of emulsions consisting of subnanoliter droplets that allows us to identify each emulsion by their spatial position in the train of emulsions without chemical barcoding. The emulsions of droplets are separated by a third immiscible phase, thus forming large compartments-tankers-each filled with an emulsion of droplet reactors. Each tanker in a train can be set under different reaction conditions for hundreds or thousands of replications of the same reaction. The tankers allow for long term incubation - needed to check for growth of bacteria under a screen of conditions. We use microfluidic tankers to analyze susceptibility to cefotaxime in ca. 1900 replications for each concentration of the antibiotic in one experiment. We test cefotaxime susceptibility for different initial concentrations of bacteria, showing the inoculum effect down to the level of single cells for more than a hundred single-cell events per tanker. Lastly, we use tankers to observe the formation of aggregates of bacteria in the presence of cefotaxime in the increasing concentration of the antibiotic. The microfluidic tankers allow for facile studies of the inoculum effect and antibiotic susceptibility, and constitute an attractive, label-free screening method for a variety of other experiments in chemistry and biology.
单细胞水平抗生素药敏性的测量非常重要，因为它揭示了导致细菌菌株产生耐药性的抗生素浓度。迄今为止，还没有针对单细胞水平抗生素药敏性的大规模研究的解决方案。在这里，我们提出了一种生产和分离亚纳升级液滴乳液的方法，该方法允许我们通过乳液列车中的空间位置来识别每个乳液，而无需化学条形码。液滴乳液通过第三不相容相分离，从而形成大的隔室-油轮-每个隔室都充满了液滴反应器的乳液。列车中的每个油轮都可以设置在不同的反应条件下，以进行相同反应的数百或数千次重复。油轮允许进行长期孵育-在条件筛选下检查细菌的生长情况所需的时间。我们使用微流控油轮在一个实验中分析约 1900 个抗生素浓度下头孢噻肟的药敏性，每个油轮中每个浓度的药敏性重复分析约 1900 次。我们测试了不同初始细菌浓度下的头孢噻肟药敏性，显示出接种物效应，每个油轮中有超过一百个单细胞事件可达到单细胞水平。最后，我们使用油轮观察在头孢噻肟存在下细菌聚集体的形成，随着抗生素浓度的增加。微流控油轮允许轻松研究接种物效应和抗生素药敏性，并构成一种有吸引力的、无标记的筛选方法，可用于化学和生物学的各种其他实验。
