Effects of Fresh Garlic Extract on Candida albicans Biofilms

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.

TABLE 1.

XTT assay results for adherent and biofilm-associated C. albicans treated with FGE

Phase	Mean absorbance at 492 nm ± SD at the following FGE concentration (μg/ml)
	0	0.5	1	2	4
Adherence phase	3.318 ± 0.107	0.079 ± 0.149	0.000 ± 0.000a
Mature biofilm phase
1 h treatment	3.049 ± 0.084			1.675 ± 0.231	1.690 ± 0.330b
48 h treatment	3.079 ± 0.063			2.714 ± 0.148	2.509 ± 0.243b

^aThe P value is <0.001 for the mean absorbance values across the concentration range of 0, 0.5, and 1 μg/ml, as determined by ANOVA.

^bThe P value is <0.001 for the mean absorbance values across the concentration range of 0, 2, and 4 μg/ml, as determined by ANOVA.

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.