Quantitative and qualitative analysis of the antifungal activity of allicin alone and in combination with antifungal drugs

Abstract

The antifungal activity of allicin and its synergistic effects with the antifungal agents flucytosine and amphotericin B (AmB) were investigated in Candida albicans (C. albicans). C. albicans was treated with different conditions of compounds alone and in combination (allicin, AmB, flucytosine, allicin + AmB, allicin + flucytosine, allicin + AmB + flucytosine). After a 24-hour treatment, cells were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) to measure morphological and biophysical properties associated with cell death. The clearing assay was conducted to confirm the effects of allicin. The viability of C. albicans treated by allicin alone or with one antifungal drug (AmB, flucytosine) in addition was more than 40% after a 24-hr treatment, but the viability of groups treated with combinations of more than two drugs was less than 32%. When the cells were treated with allicin alone or one type of drug, the morphology of the cells did not change noticeably, but when cells were treated with combinations of drugs, there were noticeable morphological changes. In particular, cells treated with allicin + AmB had significant membrane damage (burst or collapsed membranes). Classification of cells according to their cell death phase (CDP) allowed us to determine the relationship between cell viability and treatment conditions in detail. The adhesive force was decreased by the treatment in all groups compare to the control. Cells treated with AmB + allicin had a greater adhesive force than cells treated with AmB alone because of the secretion of molecules due to collapsed membranes. All cells treated with allicin or drugs were softer than the control cells. These results suggest that allicin can reduce MIC of AmB while keeping the same efficacy.

Figure 1. Cell viability of C. albicans treated with allicin and antifungal drugs.

(a) The viability of C. albicans as a function of allicin concentration. The concentration of allicin was increased from 0 (control) to 5 µg/mL. (b) Cell viability as a function of treatment conditions. C. albicans cells were treated with allicin alone, one kind of drug (AmB, flucytosine), and the combinations of AmB + allicin, flucytosine + allicin, and AmB + flucytosine + allicin.

Figure 2. SEM images of C. albicans treated with allicin or antifungal drugs for 24 hours.

(a) Control cells. The images shown in (b)–(g) are of cells treated with allicin alone, AmB, flucytosine, AmB + allicin, flucytosine + allicin, and AmB + flucytosine + allicin, respectively.

Figure 3. AFM images of C. albicans treated with allicin or antifungal drugs for 24 hours.

(a) Control cells. The images shown in (b) – (g) are of cells treated with allicin alone, AmB, flucytosine, AmB + allicin, flucytosine + allicin, and AmB + flucytosine + allicin, respectively. The arrows indicate significant morphological changes.

Figure 4. Clearing assays with AmB (a), flucytosine (b), allicin (c), AmB + allicin (d), flucytosine + allicin (e), and AmB + flucytosine + allicin (f).

Spot number 1 indicates the control sample. The spots numbered 2, 3, and 4 correspond to antifungal treatments with the concentrations of MIC₁₀×10⁻¹, MIC₁₀, and MIC₉₀, respectively.

Figure 5. Changes in the biophysical properties of C. albicans according to treatment condition.

(a) Changes in the adhesive force between the cells surface and the AFM tip and (b) changes in the stiffness of the cell membrane.