Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiotics to reverse drug resistance. Expression of ramA by Salmonella enterica serovar Typhimurium increases in response to efflux inhibition, irrespective of the mode of inhibition. We hypothesized that measuring ramA promoter activity could act as a reporter of efflux inhibition. A rapid, inexpensive, and high-throughput green fluorescent protein (GFP) screen to identify efflux inhibitors was developed, validated, and implemented. Two chemical compound libraries were screened for compounds that increased GFP production. Fifty of the compounds in the 1,200-compound Prestwick chemical library were identified as potential efflux inhibitors, including the previously characterized efflux inhibitors mefloquine and thioridazine. There were 107 hits from a library of 47,168 proprietary compounds from L. Hoffmann La Roche; 45 were confirmed hits, and a dose response was determined. Dye efflux and accumulation assays showed that 40 Roche and three Prestwick chemical library compounds were efflux inhibitors. Most compounds had specific efflux-inhibitor-antibiotic combinations and/or species-specific synergy in antibiotic disc diffusion and checkerboard assays performed with Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella Typhimurium. These data indicate that both narrow-spectrum and broad-spectrum combinations of efflux inhibitors with antibiotics can be found. Eleven novel efflux inhibitor compounds potentiated antibiotic activities against at least one species of Gram-negative bacteria, and data revealing an E. coli mutant with loss of AcrB function suggested that these are AcrB inhibitors.

中文翻译：

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革兰氏阴性菌的新型多药外排抑制剂。

抗生素的主动流出可防止其积累到有毒的细胞内浓度，这有助于临床相关的多药耐药性。抑制主动外排可增强抗生素活性，表明外排抑制剂可与抗生素联合使用以逆转耐药性。肠道沙门氏菌鼠伤寒沙门氏菌的ramA表达响应于流出抑制而增加，无论抑制方式如何。我们假设测量ramA启动子活性可以作为外排抑制的报告者。开发、验证和实施了一种快速、廉价和高通量的绿色荧光蛋白 (GFP) 屏幕来识别外排抑制剂。筛选了两个化合物库以寻找增加 GFP 产量的化合物。在 1,200 种化合物的 Prestwick 化学文库中，有 50 种化合物被确定为潜在的外排抑制剂，包括先前表征的外排抑制剂甲氟喹和硫利达嗪。来自 L. Hoffmann La Roche 的 47,168 种专有化合物库中有 107 条命中；45 个被确认命中，并确定了剂量反应。染料流出和积累测定表明，40 种 Roche 和三种 Prestwick 化学文库化合物是流出抑制剂。大肠杆菌、绿脓杆菌、鲍曼不动杆菌和鼠伤寒沙门氏菌。这些数据表明，可以发现外排抑制剂与抗生素的窄谱和广谱组合。11 种新型外排抑制剂化合物增强了对至少一种革兰氏阴性菌的抗生素活性，并且数据显示失去 AcrB 功能的大肠杆菌突变体表明这些是 AcrB 抑制剂。