Effects of Fresh Garlic Extract on Candida albicans Biofilms
Jennifer A. Shuford, James M. Steckelberg and Robin Patel*
+ Author Affiliations

Mayo Clinic College of Medicine
Department of Medicine
Division of Infectious Diseases
200 First St. SW
Rochester, MN 55905
The effects of fresh garlic extract (FGE) against planktonic Candida spp. have been demonstrated in vitro and have been attributed to the action of allicin (3), a sulfur-containing compound that is formed at levels of approximately 3 to 5 mg/g of fresh clove (8). Biofilm-associated, or sessile, Candida albicans organisms demonstrate increased resistance to traditional antifungal agents that have activity against their planktonic counterparts (5). Therefore, we investigated the activity of FGE against both planktonic and sessile forms of C. albicans.

(This data was presented in part at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., 30 October to 2 November 2004.)

FGE was prepared as previously described by Lemar and colleagues (7). Briefly, fresh garlic cloves were crushed in sterile saline at a concentration of 40 mg/ml. FGE was filter sterilized and stored at −70°C until use. We utilized C. albicans GDH 2346, a strain isolated from a denture stomatitis patient that is known to produce an extracellular matrix containing a mannoprotein adhesin, with a previously described method of C. albicans biofilm formation (2, 4). Sterile silicone elastomer disks were immersed in 1 × 107 CFU of C. albicans, incubated at 37°C on an orbital shaker for 90 min, and then rinsed. One-third of the disks were placed in 0, 0.5, or 1.0 mg of FGE/ml in yeast nitrogen base and incubated at 37°C for 48 h prior to evaluation. The other disks were incubated in yeast nitrogen base for 48 h to form mature biofilms. The untreated, mature biofilms were incubated with 0, 2, or 4 mg of FGE/ml in saline for 1 or 48 h prior to evaluation. All biofilms were evaluated with XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay, a semiquantitative, colorimetric assay of metabolic activity (2). MICs for planktonic organisms were measured using the NCCLS macrodilution method. All experiments were performed in triplicate on three different days. Comparisons between groups were performed by analysis of variance (ANOVA). P values of <0.05 were considered significant.

The mean absorbances at 492 nm for disks treated immediately after adherence, mature biofilms treated for 1 h, and mature biofilms treated for 48 h are presented in Table 1. MICs for planktonic yeast were 0.0625 to 0.125 mg/ml.

These data demonstrate activity of FGE against C. albicans in its planktonic, adherent, and sessile phases. The in vitro activity decreases as the biofilm phenotype develops, as noted previously with traditional antifungal drugs (1). The superior activity at 1 versus 48 h of treatment probably relates to the half-life of FGE at 37°C and would be an important consideration in the development of in vivo uses (6). Although only one strain of C. albicans was used, the same MIC range and metabolic assay results were demonstrated with a clinical candidemia isolate (data not shown). These results appear promising and merit further investigation for determination of the antifungal activity of FGE against C. albicans biofilms.

Comparison between allicin and fluconazole in Candida albicans biofilm inhibition and in suppression of HWP1 gene expression.
Khodavandi A1, Harmal NS, Alizadeh F, Scully OJ, Sidik SM, Othman F, Sekawi Z, Ng KP, Chong PP.
Author information
Abstract
Candida albicans is an opportunistic human pathogen with the ability to differentiate and grow in filamentous forms and exist as biofilms. The biofilms are a barrier to treatment as they are often resistant to the antifungal drugs. In this study, we investigated the antifungal activity of allicin, an active compound of garlic on various isolates of C. albicans. The effect of allicin on biofilm production in C. albicans as compared to fluconazole, an antifungal drug, was investigated using the tetrazolium (XTT) reduction-dependent growth and crystal violet assays as well as scanning electron microscopy (SEM). Allicin-treated cells exhibited significant reduction in biofilm growth (p<0.05) compared to fluconazole-treated and also growth control cells. Moreover, observation by SEM of allicin and fluconazole-treated cells confirmed a dose-dependent membrane disruption and decreased production of organisms. Finally, the expression of selected genes involved in biofilm formation such as HWP1 was evaluated by semi-quantitative RT-PCR and relative real time RT-PCR. Allicin was shown to down-regulate the expression of HWP1.

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.

Abstract

Allicin was effective in vitro against Candida, Cryptococcus, Trichophyton, Epidermophyton, and Microsporum. The minimal inhibitory concentrations (MICs) of allicin against these organisms were 3.13 to 6.25 μg/ml by the agar dilution method and 1.57 to 6.25 μg/ml by the broth dilution method, using Sabouraud glucose (SG) medium. However decreased activity was demonstrated against Aspergillus. The MIC of allicin against various pathogenic fungi was affected considerably by differences in the experimental conditions, e.g., incubation time, inoculum size, type of medium, and medium pH. The MIC of allicin against Candida, Cryptococcus, and Aspergillus remained constant after more than 3 days of incubation, and that against Dermatophytes remained constant after more than 10 days of incubation. Decreasing the inoculum size increased the susceptibility to allicin. The antifungal activity of allicin was stronger on SG agar medium with a pH of 5.6 than on the same medium with a pH of 6.0 or higher. By microscopical observation, allicin induced morphological abnormalities in hyphae of Trichophyton mentagrophytes Morita. Percent germination of spores of the Morita strain at 24 h in SG agar medium was greatly decreased with an allicin concentration of 3.13 μg/ml, and the lethal dose for the spores was about four times higher than the fungistatic concentration. These results suggest that allicin inhibits both germination of spores and growth of hyphae.

Abstract
A checkerboard microdilution method, performed according to the recommendations of the National Committee for Clinical Laboratory Standards, was used to study the in vitro interaction of fluconazole and allicin in 24 fluconazole-resistant clinical isolates of Candida albicans, one experimentally induced strain S-1, and one ATCC type strain 10231. The interaction intensity was determined by spectrophotometric methods and visual reading of the checkerboard assay, and the nature of the interactions was assessed using two nonparametric approaches [fractional inhibitory concentration index (FICI) and ΔE models]. Synergism was observed in 23 strains using FICI, and in 22 strains using ΔE. The ΔE model gave results consistent with FICI, but no antagonistic action was observed. The positive interactions were also confirmed by the time-killing test and agar diffusion in the selected strains. Moreover, the in vivo experiment showed that a combination of fluconazole and allicin exhibited a good synergism against C. albicans.