Abstract：
BACKGROUND: Polymyxin E is widely recognized as a last resort for treating multidrug-resistant gram-negative bacteria. Unfortunately, the effectiveness of polymyxin E is significantly reduced when treating life-threatening bacterial infections due to plasmid-mediated polymyxin E resistance. The synergistic effect of applying a polymyxin E adjuvant is a promising strategy for overcoming the growing threat of antibiotic-resistant pathogens.
OBJECTIVE: To evaluate the synergistic effect of fisetin and polymyxin E on S. typhimurium infections in vivo and further elucidate the underlying mechanism of this effect.
METHODS: The effect of combining fisetin and polymyxin E to treat mobilized colistin resistance-1-positive (MCR-1-positive) gram-negative bacteria in vitro was examined using various methods, such as checkerboard assays, growth curves and time‒kill curves. To elucidate the mechanism by which fisetin affects MCR-1, we employed ultraviolet (UV) absorption spectroscopy, thin layer chromatography (TLC), and western blot analysis to investigate its effect at the protein level. Subsequently, molecular dynamics simulations (MDS) and metabolomics analysis were utilized to determine the site of interaction between fisetin and MCR-1 as well as the potential pathways and mechanisms involved. A new nanoemulsion of fisetin was produced using high-pressure homogenization, and its stability was tested. Finally, two animal models of S. typhimurium HYM2 infection were established to evaluate the synergistic effect of polymyxin E and fisetin in vivo.
RESULTS: Our study revealed that fisetin exhibited a synergistic effect when combined with polymyxin E against MCR-1-positive S. typhimurium. The TLC results demonstrated that fisetin could inhibit the phosphoethanolamine (PEA) transfer of the MCR-1 protein, thereby restoring the activity of polymyxin E in strains against MCR-1. The MDS analysis indicated robust and immediate binding between fisetin and the MCR-1 protein, with both hydrophobic and polar effects playing significant roles in determining the binding energy of the former. Metabolomic studies demonstrated that the addition of fisetin significantly modulated bacterial metabolites. Moreover, it effectively inhibited the activity of ABC transporters in bacteria, thereby mitigating bacterial drug resistance and enhancing the therapeutic efficacy of polymyxin E. Furthermore, in mouse and chick models of infection, intragastric administration of the fisetin nanoemulsion together with polymyxin E resulted in significant therapeutic benefits, including increased survival rates, reduced bacterial colonization, and decreased levels of inflammatory factors.
CONCLUSIONS: Fisetin, an MCR-1 inhibitor and a promising synergistic partner of polymyxin E, has significant potential for clinical application in the treatment of S. typhimurium infections, particularly those resulting extensively from drug-resistant MCR-1-positive strains.
OBJECTIVE: To evaluate the synergistic effect of fisetin and polymyxin E on S. typhimurium infections in vivo and further elucidate the underlying mechanism of this effect.
METHODS: The effect of combining fisetin and polymyxin E to treat mobilized colistin resistance-1-positive (MCR-1-positive) gram-negative bacteria in vitro was examined using various methods, such as checkerboard assays, growth curves and time‒kill curves. To elucidate the mechanism by which fisetin affects MCR-1, we employed ultraviolet (UV) absorption spectroscopy, thin layer chromatography (TLC), and western blot analysis to investigate its effect at the protein level. Subsequently, molecular dynamics simulations (MDS) and metabolomics analysis were utilized to determine the site of interaction between fisetin and MCR-1 as well as the potential pathways and mechanisms involved. A new nanoemulsion of fisetin was produced using high-pressure homogenization, and its stability was tested. Finally, two animal models of S. typhimurium HYM2 infection were established to evaluate the synergistic effect of polymyxin E and fisetin in vivo.
RESULTS: Our study revealed that fisetin exhibited a synergistic effect when combined with polymyxin E against MCR-1-positive S. typhimurium. The TLC results demonstrated that fisetin could inhibit the phosphoethanolamine (PEA) transfer of the MCR-1 protein, thereby restoring the activity of polymyxin E in strains against MCR-1. The MDS analysis indicated robust and immediate binding between fisetin and the MCR-1 protein, with both hydrophobic and polar effects playing significant roles in determining the binding energy of the former. Metabolomic studies demonstrated that the addition of fisetin significantly modulated bacterial metabolites. Moreover, it effectively inhibited the activity of ABC transporters in bacteria, thereby mitigating bacterial drug resistance and enhancing the therapeutic efficacy of polymyxin E. Furthermore, in mouse and chick models of infection, intragastric administration of the fisetin nanoemulsion together with polymyxin E resulted in significant therapeutic benefits, including increased survival rates, reduced bacterial colonization, and decreased levels of inflammatory factors.
CONCLUSIONS: Fisetin, an MCR-1 inhibitor and a promising synergistic partner of polymyxin E, has significant potential for clinical application in the treatment of S. typhimurium infections, particularly those resulting extensively from drug-resistant MCR-1-positive strains.
摘要：
背景：多粘菌素E被广泛认为是治疗多药耐药革兰氏阴性菌的最后手段。不幸的是,在治疗由于质粒介导的多粘菌素E耐药性而危及生命的细菌感染时,多粘菌素E的有效性显著降低。应用多粘菌素E佐剂的协同作用是克服抗生素抗性病原体日益增长的威胁的有希望的策略。
目的：评价非塞素和多粘菌素E对体内鼠伤寒沙门氏菌感染的协同作用，并进一步阐明其作用机制。
方法：使用各种方法检查了非塞素和多粘菌素E联合治疗体外动员的粘菌素抗性-1阳性（MCR-1阳性）革兰氏阴性菌的效果，比如棋盘化验，生长曲线和时间杀死曲线。为了阐明fisetin影响MCR-1的机制，我们采用了紫外（UV）吸收光谱，薄层色谱（TLC），和蛋白质印迹分析以研究其在蛋白质水平上的作用。随后，利用分子动力学模拟（MDS）和代谢组学分析来确定非瑟酮和MCR-1之间的相互作用位点以及所涉及的潜在途径和机制。采用高压均质化技术制备了一种新的非塞丁纳米乳液,并对其稳定性进行了测试。最后,建立了两种鼠伤寒沙门氏菌HYM2感染的动物模型，以评估多粘菌素E和非塞素在体内的协同作用。
结果：我们的研究表明，当与多粘菌素E组合时，非塞素对MCR-1阳性鼠伤寒沙门氏菌表现出协同作用。薄层色谱(TLC)实验结果表明,非塞素能抑制MCR-1蛋白的磷酸乙醇胺(PEA)转移,从而恢复菌株中多粘菌素E对MCR-1的活性。MDS分析显示Fisetin和MCR-1蛋白之间的牢固和直接结合，疏水和极性效应在确定前者的结合能中起着重要作用。代谢组学研究表明，非瑟酮的添加显着调节细菌代谢产物。此外，有效抑制细菌中ABC转运蛋白的活性,从而减轻细菌耐药性并提高多粘菌素E的治疗效果。在小鼠和小鸡感染模型中，非塞素纳米乳剂与多粘菌素E一起胃内给药可带来显着的治疗效果，包括提高生存率，减少细菌定植，炎症因子水平降低。
结论：Fisetin，MCR-1抑制剂和多粘菌素E的有希望的协同伴侣，在鼠伤寒沙门氏菌感染的治疗中具有巨大的临床应用潜力，特别是由耐药MCR-1阳性菌株广泛产生的那些。
目的：评价非塞素和多粘菌素E对体内鼠伤寒沙门氏菌感染的协同作用，并进一步阐明其作用机制。
方法：使用各种方法检查了非塞素和多粘菌素E联合治疗体外动员的粘菌素抗性-1阳性（MCR-1阳性）革兰氏阴性菌的效果，比如棋盘化验，生长曲线和时间杀死曲线。为了阐明fisetin影响MCR-1的机制，我们采用了紫外（UV）吸收光谱，薄层色谱（TLC），和蛋白质印迹分析以研究其在蛋白质水平上的作用。随后，利用分子动力学模拟（MDS）和代谢组学分析来确定非瑟酮和MCR-1之间的相互作用位点以及所涉及的潜在途径和机制。采用高压均质化技术制备了一种新的非塞丁纳米乳液,并对其稳定性进行了测试。最后,建立了两种鼠伤寒沙门氏菌HYM2感染的动物模型，以评估多粘菌素E和非塞素在体内的协同作用。
结果：我们的研究表明，当与多粘菌素E组合时，非塞素对MCR-1阳性鼠伤寒沙门氏菌表现出协同作用。薄层色谱(TLC)实验结果表明,非塞素能抑制MCR-1蛋白的磷酸乙醇胺(PEA)转移,从而恢复菌株中多粘菌素E对MCR-1的活性。MDS分析显示Fisetin和MCR-1蛋白之间的牢固和直接结合，疏水和极性效应在确定前者的结合能中起着重要作用。代谢组学研究表明，非瑟酮的添加显着调节细菌代谢产物。此外，有效抑制细菌中ABC转运蛋白的活性,从而减轻细菌耐药性并提高多粘菌素E的治疗效果。在小鼠和小鸡感染模型中，非塞素纳米乳剂与多粘菌素E一起胃内给药可带来显着的治疗效果，包括提高生存率，减少细菌定植，炎症因子水平降低。
结论：Fisetin，MCR-1抑制剂和多粘菌素E的有希望的协同伴侣，在鼠伤寒沙门氏菌感染的治疗中具有巨大的临床应用潜力，特别是由耐药MCR-1阳性菌株广泛产生的那些。
