Candida auris, the youngest Candida species, is known to cause candidiasis and candidemia in humans and has been related to several hospital outbreaks. Moreover, Candida auris infections are largely resistant to the antifungal drugs currently in clinical use, necessitating the development of novel medications and approaches to treat such infections. Following up on our previous studies that demonstrated eugenol tosylate congeners (ETCs) to have antifungal activity, several ETCs (C1-C6) were synthesized to find a lead molecule with the requisite antifungal activity against C. auris. Preliminary tests, including broth microdilution and the MUSE cell viability assay, identified C5 as the most active derivative, with a MIC value of 0.98 g/mL against all strains tested. Cell count and viability assays further validated the fungicidal activity of C5. Apoptotic indicators, such as phosphatidylserine externalization, DNA fragmentation, mitochondrial depolarization, decreased cytochrome c and oxidase activity and cell death confirmed that C5 caused apoptosis in C. auris isolates. The low cytotoxicity of C5 further confirmed the safety of using this derivative in future studies. To support the conclusions drawn in this investigation, additional in vivo experiments demonstrating the antifungal activity of this lead compound in animal models will be needed.

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Background Candida species are opportunistic fungus that can cause serious infections, particularly in immunocompromised population. The number of fungal infections has increased steadily with Candida species being responsible for ˃ 70% of these instances, particularly in hospitalized patients with significant underlying conditions. Pharmacological resistance in Candida species and the advent of Candida auris have elevated candidiasis to a major public health concern. Candida auris is an emerging multidrug-resistant fungus that can cause catastrophic bloodstream infections and high fatality rates, particularly in hospitalized patients with major medical issues. Antifungal study of trimetallic nanoparticles (NPs) of various types have been studied as a therapy option for efficient and safe control of candidiasis. These NPs were highlighted for being environmentally friendly and sustainable synthetic preparative possibilities. Objective: This work aimed to synthesize and characterize novel Cu-Zn-Fe trimetallic NPs and determine their in vitro antifungal activity and mechanism of antifungal action against Candida auris isolates. Methods The synthesis and characterization of Cu-Zn-Fe trimetallic NPs was done by standard methods. The antifungal capability of these NPs were determined by calculating minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) following CLSI recommended guidelines. Susceptibility on planktonic cells and biofilms was further confirmed by MuseTM cell count and viability assay and scanning electron microscopy (SEM) respectively. For insight antifungal mechanisms, apoptosis and cell cycle arrest were studied by exploring different apoptotic markers and MuseTM cell analyzer. Results Characterizations by Fourier-transform infrared spectroscopy (FTIR), diffuse reflectance UV-visible spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) determine the successful biosynthesis of Cu-Zn-Fe trimetallic NPs. Susceptibility assay confirmed the fungicidal activity of Cu-Zn-Fe NPs with MIC and MFC values of 12.5 and 25 μg/ml respectively. These results were further confirmed by viability assay reporting the cell viability of 45.5%, 13.5%, and 1.8% when C. auris cells were treated with 1/2 MIC, MIC, and 2MIC respectively. Cell cycle analysis revealed that 91.2% of healthy developing untreated control cells were in G0/G1 phase, whereas 5.2% and 3.7% of cells were in the S phase and G2/M phase, respectively. In contrast, NP-treated cells were observed to be arrested in S phase with 49.3% cells at 2MIC. To study the physiology of cell death caused by NPs, we investigated mitochondrial membrane potential (∆ψm), with live cells having stable (∆ψm) whereas treated cells showed loss of (∆ψm). Another important parameter of apoptosis in yeast cells is the release of cytochrome C from the mitochondria to the cytosol and NP-treated cells resulting in decreased mitochondrial cytochrome C and elevated cytosolic cytochrome C levels. Both results confirmed the potential test NPs in causing apoptotic cell death in C. auris. Conclusion The trimetallic (Cu-Zn-Fe) nanoparticles displayed strong antifungal activity against C. auris, with a potential to arrest the cell cycle at S-phase, which could be linked to the DNA damage. Important yeast apoptotic markers suggested that the test NPs have a potential to cause apotosis in C. auris. All these findings suggest the potential of these trimetallic NPs to be taken to the next level of research in the development of novel antifungal medications.

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