Combination Effect of Novel Bimetallic Ag-Ni Nanoparticles with Fluconazole against Candida albicans

Abstract

The increasing frequency of antifungal drug resistance among pathogenic yeast "Candida" has posed an immense global threat to the public healthcare sector. The most notable species of Candida causing most fungal infections is Candida albicans. Furthermore, recent research has revealed that transition and noble metal combinations can have synergistic antimicrobial effects. Therefore, a one-pot seedless biogenic synthesis of Ag-Ni bimetallic nanoparticles (Ag-Ni NPs) using Salvia officinalis aqueous leaf extract is described. Various techniques, such as UV-vis, FTIR, XRD, SEM, EDX, and TGA, were used to validate the production of Ag-Ni NPs. The antifungal susceptibility of Ag-Ni NPs alone and in combination with fluconazole (FLZ) was tested against FLZ-resistant C. albicans isolate. Furthermore, the impacts of these NPs on membrane integrity, drug efflux pumps, and biofilms formation were evaluated. The MIC (1.56 μg/mL) and MFC (3.12 μg/mL) results indicated potent antifungal activity of Ag-Ni NPs against FLZ-resistant C. albicans. Upon combination, synergistic interaction was observed between Ag-Ni NPs and FLZ against C. albicans 5112 with a fractional inhibitory concentration index (FICI) value of 0.31. In-depth studies revealed that Ag-Ni NPs at higher concentrations (3.12 μg/mL) have anti-biofilm properties and disrupt membrane integrity, as demonstrated by scanning electron microscopy results. In comparison, morphological transition was halted at lower concentrations (0.78 μg/mL). From the results of efflux pump assay using rhodamine 6G (R6G), it was evident that Ag-Ni NPs blocks the efflux pumps in the FLZ-resistant C. albicans 5112. Targeting biofilms and efflux pumps using novel drugs will be an alternate approach for combatting the threat of multi-drug resistant (MDR) stains of C. albicans. Therefore, this study supports the usage of Ag-Ni NPs to avert infections caused by drug resistant strains of C. albicans.

Keywords: Candida albicans; biofilms; efflux pumps; fuconazole resistance; synergistic effect.

Figure 1

Schematic illustration of the formation of Ag-Ni NPs using Salvia officinalis aqueous extract as a stabilizing and reducing agent.

Figure 2

(a) UV–vis spectra of Salvia officinalis Ag NPs, Ni NPs and Ag-Ni NPs at 30 °C (inset optical images), and (b) Fourier transform infrared (FTIR) spectra of Salvia officinalis extract and Ag-Ni NPs.

Figure 3

(a) Scanning electron microscopy (SEM) image, (b) energy-dispersive X-ray spectroscopy (EDX), (c) X-ray diffraction (XRD) patterns and (d) thermal gravimetric analysis-differential thermal analysis (TGA-DTA) curves of bimetallic Ag-Ni nanoparticles.

Figure 4

Viability profile of Candida albicans 5112 untreated cells (negative control), heat killed cells (positive control) and cells treated with 0.78 µg/mL (0.5 × MIC) and 1.56 µg/mL (MIC) of the bimetallic Ag-Ni NPs.

Figure 5

(a) Inhibition of morphogenesis in C. albicans 5112. Hyphae formation was monitored at different concentrations of Ag-Ni NPs (0.78 μg/mL, 0.5 × MIC; 1.56 μg/mL MIC). Untreated C. albicans 5112 was used as a negative control. (b) Anti-biofilm activity of Ag-Ni NPs. The picture shows the effect of Ag-Ni NPs on biofilm formation in C. albicans 5112. The biofilm matrix fluoresces green (Con A), whereas metabolically inactive cells fluoresce yellow-green (FUN-1). (c) Percent biofilm inhibition and cell viability within the biofilms with regard to different concentrations of Ag-Ni NPs.

Figure 6

Intracellular accumulation of R6G in FLZ-resistant Candida cells. Fluorescence microscopy of R6G-stained FLZ-resistant Candida 5112. In the negative control, there was no accumulation of the dye after addition of glucose, whereas exposure to 0.5 × MIC (0.78 μg/mL) and MIC (1.56 μg/mL) of Ag-Ni NPs resulted in accumulation of R6G inside the C. albicans 5112 cells.

Figure 7

Incorporation of PI by FLZ-resistant C. albicans 5112. The figure shows the membrane disruption ability of Ag-Ni NPs against C. albicans cells. The negative control had cells with intact cell membrane.

Figure 8

SEM images of FLZ-resistant C. albicans 5112. (A) Untreated control; yeast cells exposed to Ag-Ni NPs at different contractions: (B) 0.5 × MIC (0.78 μg/mL) and (C) MIC (1.56 μg/mL). Blue arrows show wrinkles, rupture, and distortion of C. albicans surfaces, whereas yellow arrows show discharge of intracellular material.