Carević Tamara, Kolarević Stoimir, Kolarević Margareta Kračun, Nestorović Nataša, Novović Katarina, Nikolić Biljana, Ivanov Marija
Institute for Biological Research "Siniša Stanković", National Institute of the Republic of Serbia, University of Belgrade, Bulevar despota Stefana 142, Belgrade 11108, Serbia.
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, Belgrade 11042, Serbia.
Biomed Pharmacother. 2024 Dec;181:117642. doi: 10.1016/j.biopha.2024.117642. Epub 2024 Oct 31.
Citrus flavonoids are group of bioactive polyphenols. Here, we investigated the potential of diosmin, myricetin and neohesperidin as possible inhibitors of Pseudomonas aeruginosa. This bacterium is a major clinical challenge due to its propensity to form resistant biofilm. The aims of this study were to examine flavonoids antibacterial activity using the microdilution method, assays intended to determine several antibiofilm mechanisms (crystal violet, congo red binding, extracellular DNA (eDNA) test and confocal laser scanning microscopy (CLSM) live/dead cell imaging), followed by virulence genes RT-qPCR analysis. Furthermore, we aimed to examine in vivo toxicity of the compounds as well as their efficacy in P. aeruginosa zebrafish embryo infection model. Minimal inhibitory concentrations of tested flavonoids towards P. aeruginosa were in range 0.05 - 0.4 mg/mL. A high potential of the compounds to disturb both the formation of the bacterial biofilm and its eradication was recorded, including significant reduction in biofilm biomass, exopolysaccharide and eDNA production. Biofilm treatment with diosmin resulted in the lowest percentage of live microbial cells as observed in the CLSM live/dead cell imaging. The lasI, pvdS, and rhlC genes were found to be downregulated in the presence of diosmin and myricetin. Only diosmin stood out as non-embryotoxic. Consequently, in vivo analysis using a zebrafish model of P. aeruginosa infection showed an antivirulence effect of diosmin. Our findings suggest that diosmin could be potential candidate for the development of new agent that target P. aeruginosa infections by reducing its virulence mechanisms.
柑橘类黄酮是一类具有生物活性的多酚。在此，我们研究了地奥司明、杨梅素和新橙皮苷作为铜绿假单胞菌潜在抑制剂的可能性。由于该细菌易于形成耐药生物膜，它是一个主要的临床挑战。本研究的目的是使用微量稀释法检测黄酮类化合物的抗菌活性，通过实验确定几种抗生物膜机制（结晶紫、刚果红结合、细胞外DNA（eDNA）检测和共聚焦激光扫描显微镜（CLSM）活/死细胞成像），随后进行毒力基因RT-qPCR分析。此外，我们旨在检测这些化合物的体内毒性以及它们在铜绿假单胞菌斑马鱼胚胎感染模型中的疗效。受试黄酮类化合物对铜绿假单胞菌的最低抑菌浓度范围为0.05 - 0.4mg/mL。记录到这些化合物具有干扰细菌生物膜形成及其根除的高潜力，包括生物膜生物量、胞外多糖和eDNA产量的显著降低。在地奥司明处理的生物膜中，共聚焦激光扫描显微镜活/死细胞成像显示存活微生物细胞的比例最低。发现在地奥司明和杨梅素存在的情况下，lasI、pvdS和rhlC基因下调。只有地奥司明表现出无胚胎毒性。因此，使用铜绿假单胞菌感染斑马鱼模型进行的体内分析显示地奥司明具有抗毒力作用。我们的研究结果表明，地奥司明可能是开发通过降低其毒力机制来靶向铜绿假单胞菌感染的新型药物的潜在候选物。
