Analyzing the antibacterial effects of food ingredients: model experiments with allicin and garlic extracts on biofilm formation and viability of Staphylococcus epidermidis

Abstract

To demonstrate different effects of garlic extracts and their main antibiotic substance allicin, as a template for investigations on the antibacterial activity of food ingredients. Staphylococcus epidermidis ATCC 12228 and the isogenic biofilm-forming strain ATCC 35984 were used to compare the activity of allicin against planktonic bacteria and bacterial biofilms. The minimal inhibitory concentration (MIC) and the minimum biofilm inhibitory concentration (MBIC) for pure allicin were identical and reached at a concentration of 12.5 μg/mL. MBICs for standardized garlic extracts were significantly lower, with 1.56 and 0.78 μg/mL allicin for garlic water and ethanol extract, respectively. Biofilm density was impaired significantly at a concentration of 0.78 μg/mL allicin. Viability staining followed by confocal laser scanning microscopy showed, however, a 100% bactericidal effect on biofilm-embedded bacteria at a concentration of 3.13 μg/mL allicin. qRT-PCR analysis provided no convincing evidence for specific effects of allicin on biofilm-associated genes. Extracts of fresh garlic are more potent inhibitors of Staphylococcus epidermidis biofilms than pure allicin, but allicin exerts a unique bactericidal effect on biofilm-embedded bacteria. The current experimental protocol has proven to be a valid approach to characterize the antimicrobial activity of traditional food ingredients.

Keywords: ATCC 12228; ATCC 35984; Allicin; Staphylococcus epidermidis; bacterial biofilm.

Figure 1

Minimum inhibitory concentration (mean ± SEM) of allicin determined in Staphylococcus epidermidis ATCC 12228 growing in planktonic cultures. (A) Results obtained with the standard CLSI broth dilution protocol; (B) Results (mean ± SEM) obtained with a live/dead staining and quantification of dead and live bacteria with confocal microscopy. Different lower-case letters (a, b) indicate significant differences (P < 0.001) between allicin concentrations.

Figure 2

Staphylococcus epidermidis (ATCC 35984) biofilm formation within 48 h and after exposure to different concentrations (0.098–25 μg/mL) of allicin. Different upper-case letters (A, B) indicate significant (P < 0.001) differences between time points within the same allicin concentration. Different lower-case letters (a–d) indicate significant (P < 0.0001) differences between allicin-treated groups within the same time point.

Figure 3

Minimum biofilm inhibitory concentration (MBIC) and viability of Staphylococcus epidermidis (ATCC 35984) within a biofilm exposed to different concentrations of allicin. (A) Results of the safranin staining method (% OD value at 540 nm wavelength, mean ± SEM). (B) Results of live/dead staining with SYTO® green and propidium iodide and quantitative evaluation with CLSM (mean ± SEM). Different letters (a–d) indicate significant (P < 0.0001) differences.

Figure 4

Comparison of the effect of aqueous (gWE) and ethanol (gEE) extracts of fresh garlic standardized for their allicin concentration on Staphylococcus epidermidis biofilm formation and bacterial viability in the biofilm. The left panel (A1, B1) show the results of the safranin staining methods as mean percentage (±SEM) of biofilm formation. The middle panel (A2, B2) give examples of this viability staining of the two extracts with the same allicin concentration (1.56 μg/mL allicin). The right panel (A3, B3) show the result (mean ± SEM) obtained with quantitative evaluation of bacterial viability determined by differential viability staining with SYTO® green and propidium iodide and confocal microscopy.

Figure 5

Staphylococcus epidermidis (ATCC 35984) biofilm architecture after exposure to increasing concentrations of allicin. CSLM images of S. epidermidis biofilm surface and smooth surface from (xz) (A, B); biofilm 3D and thickness images (C, D) after exposure to different allicin concentrations (0.098–1.56 μg/mL). The biofilm thickness decreased from 10.7 (controls) concentration-dependently to 8.6, 7.7, 8.0, 7.9, and 4.7 μm (1.56 μg/mL allicin) with increasing allicin concentrations. Cells were distinguished by staining total bacterial cells with SYTO® green (green) and nonviable bacterial cells with propidium iodide (red).

Figure 6

Relative mRNA expression (mean ± SEM) of selected biofilm-related genes in biofilm bacteria (A) and persister cells in suspension (B). Down- and upregulation of gene expression (marked by dashed lines) were considered significant when the relative expression was decreased or increased ≥4 folds.