Verapamil inhibits efflux pumps in Candida albicans, exhibits synergism with fluconazole, and increases survival of Galleria mellonella

The emergence of resistance requires alternative methods to treat Candida albicans infections. We evaluated efficacy of the efflux pump inhibitor (EPI) verapamil (VER) with fluconazole (FLC) against FLC-resistant (CaR) and -susceptible C. albicans (CaS). The susceptibility of both strains to VER and FLC was determined, as well as the synergism of VER with FLC. Experiments were performed in vitro for planktonic cultures and biofilms and in vivo using Galleria mellonella. Larval survival and fungal recovery were evaluated after treatment with VER and FLC. Data were analyzed by analysis of variance and Kaplan-Meier tests. The combination of VER with FLC at sub-lethal concentrations reduced fungal growth. VER inhibited the efflux of rhodamine 123 and showed synergism with FLC against CaR. For biofilms, FLC and VER alone reduced fungal viability. The combination of VER with FLC at sub-lethal concentrations also reduced biofilm viability. In the in vivo assays, VER and FLC used alone or in combination increased the survival of larvae infected with CaR. Reduction of fungal recovery was observed only for larvae infected with CaR and treated with VER with FLC. VER reverted the FLC-resistance of C. albicans. Based on the results obtained, VER reverted the FLC-resistance of C. albicans and showed synergism with FLC against CaR. VER also increased the survival of G. mellonella infected with CaR and reduced the fungal recovery.

随着耐药性的出现，亟需开发治疗白色念珠菌（Candida albicans）感染的替代疗法。本研究评估了外排泵抑制剂（efflux pump inhibitor, EPI）维拉帕米（verapamil, VER）联合氟康唑（fluconazole, FLC）对氟康唑耐药型白色念珠菌（FLC-resistant C. albicans, CaR）以及氟康唑敏感型白色念珠菌（FLC-susceptible C. albicans, CaS）的抗菌效果。我们分别测定了两种菌株对VER及FLC的敏感性，同时验证了VER与FLC的协同作用。实验分别在体外开展了浮游菌培养（planktonic cultures）与生物膜（biofilms）相关测试，并利用大蜡螟（Galleria mellonella）开展体内实验。经VER与FLC处理后，我们对幼虫存活率及真菌回收量进行了评估。数据采用方差分析（analysis of variance）与Kaplan-Meier检验进行统计学分析。结果显示，亚致死浓度（sub-lethal concentrations）下VER与FLC联合使用可抑制真菌生长。VER可抑制罗丹明123（rhodamine 123）的外排，并对CaR菌株与FLC展现出协同抗菌活性。针对生物膜的实验表明，单独使用FLC或VER即可降低真菌活性；亚致死浓度下二者联合同样可降低生物膜的活性。在体内实验中，单独或联合使用VER与FLC均可提升CaR感染幼虫的存活率。仅在CaR感染且经VER与FLC联合处理的幼虫中，观察到真菌回收量降低的现象。VER可逆转白色念珠菌对FLC的耐药性。基于上述实验结果，VER可逆转白色念珠菌对FLC的耐药性，并对CaR菌株与FLC展现出协同抗菌活性；同时VER可提升CaR感染大蜡螟的存活率，并降低其体内真菌回收量。