Effects of fresh garlic extract on Candida albicans biofilms.
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ABSTRACT: Biofilms are a protected niche for microorganisms, where they are safe from antibiotic treatment and can create a source of persistent infection. Using two clinically relevant Candida albicans biofilm models formed on bioprosthetic materials, we demonstrated that biofilm formation proceeds through three distinct developmental phases. These growth phases transform adherent blastospores to well-defined cellular communities encased in a polysaccharide matrix. Fluorescence and confocal scanning laser microscopy revealed that C. albicans biofilms have a highly heterogeneous architecture composed of cellular and noncellular elements. In both models, antifungal resistance of biofilm-grown cells increased in conjunction with biofilm formation. The expression of agglutinin-like (ALS) genes, which encode a family of proteins implicated in adhesion to host surfaces, was differentially regulated between planktonic and biofilm-grown cells. The ability of C. albicans to form biofilms contrasts sharply with that of Saccharomyces cerevisiae, which adhered to bioprosthetic surfaces but failed to form a mature biofilm. The studies described here form the basis for investigations into the molecular mechanisms of Candida biofilm biology and antifungal resistance and provide the means to design novel therapies for biofilm-based infections.Journal of Bacteriology 10/2001; 183(18):5385-94. · 3.19 Impact Factor
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ABSTRACT: A quantitative method is described for the determination of allicin (2-propene-1-sulfinothioic acid S-2-propenyl ester) in garlic, using standard additions of alliin (l-(+)-S-allylcysteine sulfoxide) in conjunction with supercritical fluid extraction (SFE) and high performance liquid chromatography analysis with UV-vis absorbance detection. Optimum CO(2)-SFE conditions provided 96% recovery for allicin with precision of 3% (RSD) for repeat samples. The incorporation of an internal standard (allyl phenyl sulfone) in the SFE step resulted in a modest improvement in recovery (99%) and precision (2% RSD). Standard additions of alliin were converted to allicin in situ by endogenous alliinase (l-(+)-S-alk(en)ylcysteine sulfoxide lyase, EC 188.8.131.52). Complete conversion of the spiked alliin to allicin was achieved by making additions after homogenization-induced conversion of the naturally occurring cysteine sulfoxides to thiosulfinates had taken place, thus eliminating the likelihood of competing reactions. Concentration values for allicin determined in samples of fresh garlic (Allium sativum L. and Allium ampeloprasum) and commercially available garlic powders (Allium sativum L.) by standard addition of alliin were found in all cases to be in statistical agreement (95% confidence interval) with values determined using a secondary allicin standard (concentration determined using published extinction coefficients). This method provides a convenient alternative for assessing the amount of allicin present in fresh and powdered garlic, as alliin is a far more stable and commercially prevalent compound than allicin and is thus more amenable for use as a standard for routine analysis.Journal of Agricultural and Food Chemistry 03/2004; 52(4):682-7. · 2.91 Impact Factor
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ABSTRACT: Biofilms formed by Candida albicans on small discs of catheter material were resistant to the action of five clinically important antifungal agents as determined by [3H]leucine incorporation and tetrazolium reduction assays. Fluconazole showed the greatest activity, and amphotericin B showed the least activity against biofilm cells. These findings were confirmed by scanning electron microscopy of the biofilms.Antimicrobial Agents and Chemotherapy 10/1995; 39(9):2128-31. · 4.57 Impact Factor
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2005, p. 473
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 1
Letters to the Editor
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 com-
pound 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 an-
tifungal agents that have activity against their planktonic coun-
terparts (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 ster-
ile saline at a concentration of 40 mg/ml. FGE was filter ster-
ilized 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 ? 107CFU 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 semiquan-
titative, colorimetric assay of metabolic activity (2). MICs for
planktonic organisms were measured using the NCCLS mac-
rodilution method. All experiments were performed in tripli-
cate 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 immedi-
ately 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 consider-
ation in the development of in vivo uses (6). Although only one
strain of C. albicans was used, the same MIC range and met-
abolic assay results were demonstrated with a clinical candi-
demia isolate (data not shown). These results appear promis-
ing and merit further investigation for determination of the
antifungal activity of FGE against C. albicans biofilms.
We thank J. Chandra, M. Ghannoum, and J. Douglas for use of the
GDH 2346 isolate.
1. Chandra, J., D. M. Kuhn, P. K. Mukherjee, L. L. Hoyer, T. McCormick, and
M. A. Ghannoum. 2001. Biofilm formation by the fungal pathogen Candida
albicans: development, architecture, and drug resistance. J. Bacteriol. 183:
2. Chandra, J., P. K. Mukherjee, S. D. Leidich, F. F. Faddoul, L. L. Hoyer, L. J.
Douglas, and M. A. Ghannoum. 2001. Antifungal resistance of candidal bio-
films formed on denture acrylic in vitro. J. Dent. Res. 80:903–908.
3. Ghannoum, M. A. 1988. Studies on the anticandidal mode of action of Allium
sativum (garlic). J. Gen. Microbiol. 134:2917–2924.
4. Hawser, S. P., and L. J. Douglas. 1994. Biofilm formation by Candida species
on the surface of catheter materials in vitro. Infect. Immun. 62:915–921.
5. Hawser, S. P., and L. J. Douglas. 1995. Resistance of Candida albicans bio-
films to antifungal agents in vitro. Antimicrob. Agents Chemother. 39:2128–
6. Koch, H. P., and L. C. Lawson. 1996. Knoblauch. Garlic: the science and
therapeutic application of Allium sativum L. and related species, 2nd ed.
Williams & Wilkins, Baltimore, Md.
7. Lemar, K. M., M. P. Turner, and D. Lloyd. 2002. Garlic (Allium sativum) as
an anti-Candida agent: a comparison of the efficacy of fresh garlic and freeze-
dried extracts. J. Appl. Microbiol. 93:398–405.
8. Rybak, M. E., E. M. Calvey, and J. M. Harnly. 2004. Quantitative determi-
nation of allicin in garlic: supercritical fluid extraction and standard addition
of alliin. J. Agric. Food Chem. 52:682–687.
Jennifer A. Shuford
James M. Steckelberg
Mayo Clinic College of Medicine
Department of Medicine
Division of Infectious Diseases
200 First St. SW
Rochester, MN 55905
*Phone: (507) 255–6482
Fax: (507) 255–7762
TABLE 1. XTT assay results for adherent and biofilm-associated C. albicans treated with FGE
Mean absorbance at 492 nm ? SD at the following FGE concentration (?g/ml)
Mature biofilm phase
1 h treatment
48 h treatment
3.318 ? 0.1070.079 ? 0.1490.000 ? 0.000a
3.049 ? 0.084
3.079 ? 0.063
1.675 ? 0.231
2.714 ? 0.148
1.690 ? 0.330b
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 ?g/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 ?g/ml, as determined by ANOVA.