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. 2018 Feb 28:2018:4508709.
doi: 10.1155/2018/4508709. eCollection 2018.

Curcumin as a Promising Antibacterial Agent: Effects on Metabolism and Biofilm Formation in S. mutans

Bingchun Li  1   2 , Xinlong Li  1   2 , Huancai Lin  1   2 , Yan Zhou  1   2
Affiliations

Curcumin as a Promising Antibacterial Agent: Effects on Metabolism and Biofilm Formation in S. mutans

Bingchun Li et al. Biomed Res Int. .

Abstract

Streptococcus mutans (S. mutans) has been proved to be the main aetiological factor in dental caries. Curcumin, a natural product, has been shown to exhibit therapeutic antibacterial activity, suggesting that curcumin may be of clinical interest. The objective of this study is to evaluate the inhibitory effects of curcumin on metabolism and biofilm formation in S. mutans using a vitro biofilm model in an artificial oral environment. S. mutans biofilms were treated with varying concentrations of curcumin. The biofilm metabolism and biofilm biomass were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and the crystal violet assay. Confocal laser scanning microscopy was used to analyse the composition and extracellular polysaccharide content of S. mutans biofilm after curcumin treatment. The biofilm structure was evaluated using a scanning electron microscope. The gene expression of virulence-related factors was assessed by real-time PCR. The antibiofilm effect of curcumin was compared with that of chlorhexidine. The sessile minimum inhibitory concentration (SMIC50%) of curcumin against S. mutans biofilm was 500 μM. Curcumin reduced the biofilm metabolism from 5 min to 24 h. Curcumin inhibited the quantity of live bacteria and total bacteria in both the short term (5 min) and the long term. Moreover, curcumin decreased the production of extracellular polysaccharide in the short term. The expression of genes related to extracellular polysaccharide synthesis, carbohydrate metabolism, adherence, and the two-component transduction system decreased after curcumin treatment. The chlorhexidine-treated group showed similar results. We speculate that curcumin has the capacity to be developed as an alternative agent with the potential to reduce the pathogenic traits of S. mutans biofilm.

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Figures

Figure 1
Figure 1
(a) Antibiofilm effects of different concentrations of curcumin and the correspondent concentrations of DMSO on S. mutans biofilm. The bacteria were inoculated in a 96-well microtiter plate containing 1% BHIS medium to form a 24 h biofilm, then washed with PBS, and incubated in 1% BHIS with different concentrations of curcumin (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001). After 24 h of incubation, the Colony-Forming Units (CFU) of lived bacteria in biofilm were evaluated by the MTT assay. (b) Effect of SMIC50 curcumin on the growth of S. mutans.
Figure 2
Figure 2
Antibiofilm effect of different curcumin exposure times on S. mutans biofilm. A 24 h biofilm was incubated in curcumin at the SMIC50 for different times. The Colony-Forming Units (CFU) of lived bacteria in biofilm were evaluated by the MTT assay (a), and the percentage of biofilm biomass was evaluated by the crystal violet (CV) assay (b). The data represent the mean ± SD of three independent tests. The asterisks (∗) indicate significant differences (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).
Figure 3
Figure 3
CLSM imaging of S. mutans biofilm grown in 1% BHIS. After 24 h of growth, the biofilm was treated with 1% BHIS (control), 500 μM curcumin, or 0.12% chlorhexidine for 5 min (a) and 24 h (b). Each micrograph represents 4 optical sections: green representing live bacteria, red representing dead bacteria, combined green and red from two channel images, and three-dimensional reconstructions of the control biofilm without any treatment, the 500 μM curcumin-treated biofilm, and the 0.12% chlorhexidine-treated biofilm. The total bacteria biomass and the Colony-Forming Units (CFU) of lived bacteria are quantified in (c) and (d). The data represent the mean ± SD. The asterisks (∗) indicate significant differences (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).
Figure 4
Figure 4
CLSM images of S. mutans biofilm. (a, b) Three-dimensional reconstructions of the untreated (control) biofilm, the 500 μM curcumin-treated biofilm, and the 0.12% chlorhexidine-treated biofilm at 5 min (a) and 24 h (b). EPS was labelled in red (Alexa Fluor 647), bacterial cells were labelled in green (SYTO9), and red and green superimposed appear as yellow. Images were obtained at 20x magnification. (c) Image representing the volume of EPS, calculated according to 5 random sites of each sample, repeated three times. (d) Change in biofilm thickness at 5 min and 24 h, respectively, calculated from data obtained from fluorescence CLSM. Data are presented as the mean ± SD. The asterisks (∗) indicate significant differences (∗∗P < 0.01, ∗∗∗P < 0.001).
Figure 5
Figure 5
Morphological characteristics of S. mutans biofilm treated with or without drugs. Representative SEM images of 24 h S. mutans biofilm grown in curcumin for 5 min (b) and 24 h (e) and grown in chlorhexidine for 5 min (c) and 24 h (f). The image of 24 h S. mutans biofilm grown in 1% BHIS for 5 min (a) and 24 h (d) as a control. Magnifications of 2000x, 5000x, and 10,000x are shown for each condition. The black arrows highlight the EPS of S. mutans, which was markedly reduced.
Figure 6
Figure 6
Results of qRT-PCR to examine the gene expression of different virulence systems in S. mutans UA159. (a) EPS synthesis system; (b) carbohydrate metabolism system; (c) quorum-sensing system; (d) two-component transduction system. All _targets were amplified using primers. Different gene expression levels were normalized to the level of 16sRNA gene transcripts. Data are presented as the mean ± SD. The asterisks (∗) indicate significant differences (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).
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