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Antibiotic resistance is now a worldwide therapeutic problem. Since the beginning of anti-infectious treatment bacteria have rapidly shown an incredible ability to develop and transfer resistance mechanisms. In the last decades, the design variation of pioneer bioactive molecules has strongly improved their activity and the pharmaceutical companies partly won the race against the clock. Since the 1980s, the new classes of antibiotics that emerged were mainly directed to Gram-positive bacteria. Thus, we are now facing to multidrug-resistant Gram-negative bacteria, with no therapeutic options to deal with them. These bacteria are mainly resistant because of their double membrane that conjointly impairs antibiotic accumulation and extrudes these molecules when entered. The main challenge is to allow antibiotics to cross the impermeable envelope and reach their targets. One promising solution would be to associate, in a combination therapy, a usual antibiotic with a non-antibiotic chemosensitizer. Nevertheless, for effective drug discovery, there is a prominent lack of tools required to understand the rules of permeation and accumulation into Gram-negative bacteria. By the use of a multidrug-resistant enterobacteria, we introduce a high-content screening procedure for chemosensitizers discovery by quantitative assessment of drug accumulation, alteration of barriers, and deduction of their activity profile. We assembled and analyzed a control chemicals library to perform the proof of concept. The analysis was based on real-time monitoring of the efflux alteration and measure of the influx increase in the presence of studied compounds in an automatized bio-assay. Then, synergistic activity of compounds with an antibiotic was studied and kinetic data reduction was performed which led to the calculation of a score for each barrier to be altered.

抗生素耐药性已成为全球范围内的治疗难题。自抗感染治疗之初，细菌便迅速展现出令人瞩目的能力，以发展及转移耐药机制。在过去的几十年间，先驱生物活性分子的设计变异显著提升了其活性，制药公司部分地赢得了与时间赛跑的胜利。自1980年代起，新出现的抗生素类别主要针对革兰氏阳性菌。因此，我们现在面临着对抗多药耐药性革兰氏阴性菌的挑战，而对这些菌种的治疗手段几近匮乏。这些细菌的主要耐药性源于其双层细胞膜，该细胞膜共同阻碍了抗生素的积累，并在抗生素进入时将这些分子排出。主要挑战在于使抗生素穿越不可渗透的细胞膜并到达其作用靶点。一种有前景的解决方案是在联合治疗中，将常规抗生素与一种非抗生素性化疗增敏剂相结合。然而，为了实现有效的药物发现，对于理解革兰氏阴性菌渗透和积累规律的必要工具，存在显著缺口。通过使用多药耐药性肠杆菌，我们引入了一种高内涵筛选程序，用于化疗增敏剂的发现，该程序通过定量评估药物积累、屏障改变及其活性谱的推断来实施。我们组装并分析了控制化学品库以验证概念。分析基于对研究化合物存在下实时监测的泵出改变和流入增加的自动化生物试验。随后，研究了化合物与抗生素的协同活性，并进行了动力学数据分析，最终计算了每个需要改变的屏障的评分。