抗葡萄球菌的夫西地酸作为外排泵抑制剂与氟康唑联合治疗阴道念珠菌病的小鼠模型。

BACKGROUND: Candida albicans is the most common fungus that causes vaginal candidiasis in immunocompetent women and catastrophic infections in immunocompromised patients. The treatment of such infections is hindered due to the increasing emergence of resistance to azoles in C. albicans. New treatment approaches are needed to combat candidiasis especially in the dwindled supply of new effective and safe antifungals. The resistance to azoles is mainly attributed to export of azoles outside the cells by means of the efflux pump that confers cross resistance to all azoles including fluconazole (FLC). OBJECTIVES: This study aimed to investigate the possible efflux pump inhibiting activity of fusidic acid (FA) in C. albicans resistant isolates and the potential use of Fusidic acid in combination with fluconazole to potentiate the antifungal activity of fluconazole to restore its activity in the resistant C. albicans isolates. METHODS: The resistance of C. albicans isolates was assessed by determination of minimum inhibitory concentration. The effect of Fusidic acid at sub-inhibitory concentration on efflux activity was assayed by rhodamine 6G efflux assay and intracellular accumulation. Mice model studies were conducted to evaluate the anti-efflux activity of Fusidic acid and its synergistic effects in combination with fluconazole. Impact of Fusidic acid on ergosterol biosynthesis was quantified. The synergy of fluconazole when combined with Fusidic acid was investigated by determination of minimum inhibitory concentration. The cytotoxicity of Fusidic acid was tested against erythrocytes. The effect of Fusidic acid on efflux pumps was tested at the molecular level by real-time PCR and in silico study. In vivo vulvovaginitis mice model was used to confirm the activity of the combination in treating vulvovaginal candidiasis. RESULTS: Fusidic acid showed efflux inhibiting activity as it increased the accumulation of rhodamine 6G, a substrate for ABC-efflux transporter, and decreased its efflux in C. albicans cells. The antifungal activity of fluconazole was synergized when combined with Fusidic acid. Fusidic acid exerted only minimal cytotoxicity on human erythrocytes indicating its safety. The FA efflux inhibitory activity could be owed to its ability to interfere with efflux protein transporters as revealed by docking studies and downregulation of the efflux-encoding genes of both ABC transporters and MFS superfamily. Moreover, in vivo mice model showed that using fluconazole-fusidic acid combination by vaginal route enhanced fluconazole antifungal activity as shown by lowered fungal burden and a negligible histopathological change in vaginal tissue. CONCLUSION: The current findings highlight FA's potential as a potential adjuvant to FLC in the treatment of vulvovaginal candidiasis.

背景：白色念珠菌是最常见的真菌，它会引起免疫功能正常的女性阴道念珠菌病，并导致免疫功能低下的患者发生灾难性感染。由于白色念珠菌对唑类药物的耐药性不断增加，此类感染的治疗受到了阻碍。需要新的治疗方法来对抗念珠菌病，尤其是在新的有效和安全的抗真菌药物供应减少的情况下。唑类药物的耐药性主要归因于细胞外的唑类药物通过外排泵的输出，这导致所有唑类药物（包括氟康唑（FLC））的交叉耐药。

目的：本研究旨在研究夫西地酸（FA）对白色念珠菌耐药株的外排泵抑制活性，并探讨其在联合氟康唑时的潜在用途，以增强氟康唑的抗真菌活性，恢复其对耐药白色念珠菌的活性。

方法：通过测定最小抑菌浓度评估白色念珠菌分离株的耐药性。通过荧光素 6G 外排试验和细胞内积累来检测夫西地酸在亚抑菌浓度下对流出活性的影响。进行小鼠模型研究以评估夫西地酸的抗外排活性及其与氟康唑联合使用的协同作用。通过定量测定麦角固醇生物合成来评估夫西地酸的作用。通过测定最小抑菌浓度来研究氟康唑与夫西地酸联合使用的协同作用。用红细胞检测夫西地酸的细胞毒性。通过实时 PCR 和计算机模拟研究检测夫西地酸对流出泵的作用。采用体内阴道阴道炎小鼠模型证实该组合治疗阴道念珠菌病的活性。

结果：夫西地酸显示出流出抑制活性，因为它增加了细胞内荧光素 6G 的积累，而荧光素 6G 是 ABC 外排转运蛋白的底物，并减少了其在白色念珠菌细胞中的外排。氟康唑与夫西地酸联合使用具有协同作用。夫西地酸对人红细胞的细胞毒性最小，表明其安全性。FA 的外排抑制活性可能归因于其干扰外排蛋白转运体的能力，这一点通过对接研究和 ABC 转运体和 MFS 超家族的外排编码基因的下调得到了证实。此外，体内小鼠模型表明，通过阴道途径使用氟康唑-夫西地酸联合治疗可增强氟康唑的抗真菌活性，表现为真菌负荷降低，阴道组织的组织病理学变化可忽略不计。

结论：目前的研究结果强调了 FA 作为氟康唑治疗阴道念珠菌病的潜在辅助药物的潜力。