氟尼辛葡甲胺逆转奶牛乳房炎克柔念珠菌对伊曲康唑耐药性分子机制

念珠菌通过污染的乳导管、注射器等进入奶牛乳区，引起奶牛真菌性乳腺炎。克柔念珠菌毒力比白色念珠菌弱，但细胞表面疏水性强，对物质粘附力大更易侵入奶牛乳腺。分离的克柔念珠菌对氟康唑天然耐药，对伊曲康唑耐药性呈上升趋势。非甾体类消炎药对抗真菌药物具有增效作用并能降低药物的使用剂量。本研究评价氟尼辛葡甲胺（FM）与唑类药物联合对克柔念珠菌及其生物被膜的药效作用；采用流式细胞术、荧光定量PCR、激光共聚焦等方法，分析药物对菌膜通透性、ROS积累、线粒体膜电位影响；测定FM对药物转运泵活性、菌丝生长、胞外磷脂酶活性、生物被膜形成的影响；检测药物靶酶基因、麦角甾醇合成基因和粘附因子表达、HSP90和钙调磷酸酶活性；FM和伊曲康唑对克柔念珠菌粘附奶牛乳腺上皮细胞的干预作用；确证FM和伊曲康唑调节克柔念珠菌感染模式生物-大蜡螟的存活率、真菌消除率和组织病理学特征；阐明FM逆转克柔念珠菌对伊曲康唑耐药分子机制。

Candida enters the milk region of cows through contaminated milk ducts and syringes causing fungal mastitis. The virulence of Candida krusei is weaker than Candida albicans, but its cell surface is highly hydrophobic, and it has a strong adhesion to substances and is more likely to invade dairy cows' glands. In particular, Candida krusei is naturally resistant to fluconazole, and the resistance to itraconazole（ITC） is on the rise. Non-steroidal anti-inflammatory drugs have a synergistic effect on antifungal drugs and can reduce the dosage of the drugs. This study evaluated the efficacy of combination of flunixin meglumine (FM) and azoles on Candida cresolus and its biofilm. Membrane Permeability, ROS accumulation and mitochondrial membrane potential of Candida krusei treating FM and azoles alone and combined were analyzed using flow cytometry, fluorescence quantitative PCR and laser confocal microscopy. To evaluate FM effects on transport pump activity, hyphae growth extracellular phospholipase activity and biofilm formation. To analyze FM effects on drug target enzyme genes, ergosterol synthesis genes and Adhesion factor expression and HSP90 and calcineurin activity. To study FM effects on Candida krusei adhered to mammary epithelial cells. To confirm FM and ITC effects of on the survival rate of Candida krusei infection model organism-Galleria mellonella, fungal elimination and histopathological characteristics. The molecular mechanism by which FM reverses the resistance of Candida krusei to ITC was elucidated.