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Over the last few decades, candidiasis has exhibited an increasing incidence worldwide, causing high mortality in immunocompromised patients. Candida albicans is one of the leading opportunistic fungal pathogens. However, due to the increased use of antifungal agents, resistance of C. albicans to conventional agents, especially fluconazole, has frequently emerged. Therefore, research on the use of combinations of current drugs to sensitize antifungal agents and overcome fungal resistance has attracted considerable attention. This study demonstrated for the first time that D-penicillamine (PCA) combined with fluconazole showed a synergistic effect against C. albicans. PCA combined with fluconazole not only showed synergistic effects against planktonic cells of C. albicans, but also showed synergistic effects against C. albicans biofilms formed within 12 h in vitro. In addition, a Galleria mellonella infection model was used to evaluate the in vivo effects of this drug combination. The results showed that the combination of the two drugs could improve the survival rate, decrease the fungal burden, and reduce the tissue invasion of G. mellonella larvae. Finally, we explored the potential synergistic mechanisms of the drug combination, mainly including inhibition of the morphological transformation, reduction of the intracellular calcium concentration, and the activation of metacaspase, which is closely related to cell apoptosis. These findings might provide novel insights into antifungal drug discovery and the treatment of candidiasis caused by C. albicans.

Candida albicans is a common opportunistic fungal pathogen that may cause nosocomial fungal infections. The resistance of Candida albicans to traditional antifungal drugs has been increasing rapidly in recent years, and it brings a great challenge in clinical treatment. N-butylphthalide is originally extracted from the seed of Apium graveolens and is currently used for the treatment of ischemic stroke in the clinic. This study demonstrated that n-butylphthalide exhibited antifungal activity against Candida albicans with minimum inhibitory concentrations of 128 μg/ml; moreover, n-butylphthalide combined with fluconazole showed synergistic antifungal effects against resistant Candida albicans, resulting in a decrease in the minimum inhibitory concentrations of fluconazole from >512 to 0.25–1 μg/ml. Time-killing curves verified the antifungal activity in dynamic. Besides, n-butylphthalide exhibited anti-biofilm activity against Candida albicans, biofilms preformed 1,024 to 0.5–8 μg/ml. Furthermore, in vitro antifungal effects of n-butylphthalide were confirmed in vivo. N-butylphthalide prolonged survival rate of larvae infected by Candida albicans, reduced the fungal burden in larvae and caused less damage to larval tissues. Notably, n-butylphthalide inhibited hyphal growth and induced intracellular reactive oxygen species accumulation and a loss in mitochondrial membrane potential, which was a potential antifungal mechanism. Besides, the synergistic effects between n-butylphthalide and fluconazole potentially relied on the mechanism that n-butylphthalide significantly promoted drug uptake, and suppressed drug efflux via down-regulating the drug transporter encoding genes CDR1 and CDR2. These findings demonstrated the antifungal effects and mechanisms of n-butylphthalide against Candida albicans for the first time, which might provide broad prospects for the identification of new potential antifungal targets.

The incidence of resistant Candida isolates has increased continuously in recent decades, especially Candida albicans. To overcome this resistance, research on antifungal sensitizers has attracted considerable attention. Linezolid was found to inhibit the growth of Pythium insidiosum and synergize with amphotericin B against Cryptococcus neoformans. The objective of this study was to determine the interactions of linezolid and azoles against C. albicans in vitro and in vivo. In vitro, linezolid combined with azoles induced synergistic effects not only against some susceptible C. albicans isolates, but also against all tested resistant C. albicans isolates. For all resistant isolates, exposure to the combination of linezolid with azoles induced a significant decrease in the minimum inhibitory concentrations (MIC) of azoles, from >512 to 0.5–1 μg/mL for fluconazole, from >16 to 0.25–1 μg/mL for itraconazole, and from >16 to 0.03–0.25 μg/mL for voriconazole. Additionally, linezolid synergized with fluconazole against biofilms that were preformed for ≤ 12 h from both susceptible and resistant C. albicans, and the sessile MIC of fluconazole decreased from >1024 to 1–4 μg/mL. In vivo, linezolid plus azoles prolonged the survival rate of infected Galleria mellonella larvae twofold compared with the azole monotherapy group, significantly decreased the fungal burden of the infected larvae, and reduced the damage of resistant C. albicans to the larval tissue. These findings will contribute to antifungal agent discovery and new approaches for the treatment of candidiasis caused by C. albicans.