| Background:There is an increasing concern about non-albicans Candida due to its high isolation frequency in candidiasis recently and rapidly acquried resistance or nature resistance to fluconazole. Among clinically common non-albicans Candida species, C. glabrata has become the second most common pathogen isolated from patients and possesses rapidly acquired resistance; despite of a low detection frequency in candidiasis, C. krusei is intrinsically resistant to fluconazole. High drug resistance in C. glabrata and C. krusei brings challenges to clinically successful managenment for infections caused by C. glabrata and C. krusei. Comprehensively considering isolation frequency and drug resistance in clinically common non-albicans Candida, C. glabrata and C. krusei were selected to be the tested Candida species. The number of antifungal agents is limited and the discovery of new antifungal drugs takes years and years and costs a great deal of money. Accordingly, there is increasing interest in the development of the combination therapy of one antifungal drug and another non-antifungal drug, to counteract the increasing drug resistance in fungi. Tacrolimus is widely used to suppress the immune response in organ-transplantation patients and these patients are more vulnerable to suffer from fungal infections. Our previous research demonstrated that fluconazole combined with tacrolimus exerted strong synergistic effects against resistant C. albicans. It deserves investigation whether the combination could have synergistic effects on C. glabrata and C. krusei.Objective:This study aimed to evaluate the static and dynamic antifungal effects of fluconazole in combination with tacrolimus against C. glabrata and C. krusei with different susceptibilies, and to seek the potential mechanisms underlying the synergistic effects. Methods:The in vitro static and dynamic effects of fluconazole combined with tacrolimus against C. glabrata and C. krusei with different susceptibilities were investigated by checkerboard broth microdilution method and time-killing curve method, respectively. According to the antifungal susceptibility testing document M27-A3, MICs of tacrolimus and fluconazole alone and in combination were determined by XTT assay, with data analyzed by fractional inhibitory concentration index model. As to evaluation of the dynamic combined antifungal effects, colony counting was carried out to investigate the antifungal activity at0,6,12,24and48h after drug exposure, and the colony-forming-unit for each incubation time point per mL was plotted as the vertical ordinate of the time-kill curve. The antifungal mechanistic studies of the drug combination were performed from the following three aspects:visual evaluation of impacts of calcium channel inhibitor benidipine on the antifungal effects of the drug combination by plate-streaking method; quantification of expression levels of the fluconazole resistance genes (ERG11, CDR1, PDH1and SNQ2) after exposure to different drugs by real-time quantitative PCR; evaluation of tacrolimus on fluconazole efflux by flowcytometry using rhodamine6G as the fluorescent dye.Results:Tacrolimus enhanced the susceptibility of fluconazole against the dose-depending susceptible (MIC32μg/mL) and resistant C. glabrata strains, as well as all C. krusei strains, while degree of reduction in MICs varied with susceptibilities of strains. Addition of tacrolimus resulted in a four-fold and sixteen-fold downward shift in fluconazole MICs of the isolates with MIC of32μ.g/mL and with MICs≥256μg/mL, respectively, and the FICI values were approximately0.25and less than0.1. There was no reduction in MICs with regard to the C. glabrata isolates (MIC=8μg/mL). The synergy was further confirmed by the time-kill assay. When they were in combination against C. glabrata and C. krusei, there was a2.31and2.25log10CFU/mL decrease at24h compared with FLC alone, respectively. In addition, by observing the fungal growth after drug exposure using the plate-streaking method, the results indicated that addition of benidipine enhanced the antifungal effects of fluconazole/tacrolimus against C. glabrata and C. krusei. Further mechanism studies revealed that expression of ERG11and SNQ2genes in C. glabrata were significantly down-regulated (reduced by90%and66%, respectively) after exposure to drug combination, whereas that of CDR1gene was significantly up-regulated and no significant changes of expression of PDH1gene were observed. Flowcytometric assays of drug efflux pumps in C. glabrata manifested that tacrolimus inhibited fluconazole efflux, and especially at the time point of40min, the inhibition was the most obvious (the fluorescence intensity in the tacrolimus-containing group was2.76-fold that in the control group).Conclusion:Fluconazole in combination with tacrolimus exerts synergistic effects against C. glabrata and C. krusei. Inhibition of calcium channel could enhance the synergistic antifungal effects, indicating that the combined antifungal effects are related with calcium regulation. The potential mechanisms are related to decrease in ERG11and SNQ2gene expression and inhibition of fluconazole efflux in C. glabrata. Combination of tacrolimus and FLC may represent a promising approach of overcoming the resistance of C. glabrata and C. krusei to fluconazole and this study could provide insight into antifungal agent discovery. |
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