Thymol and carvacrol induce autolysis, stress, growth inhibition and reduce the biofilm formation by Streptococcus mutans

Abstract

Organic compounds from plants are an attractive alternative to conventional antimicrobial agents. Therefore, two compounds namely M-1 and M-2 were purified from Origanum vulgare L. and were identified as carvacrol and thymol, respectively. Antimicrobial and antibiofilm activities of these compounds along with chlorhexidine digluconate using various assays was determined against dental caries causing bacteria Streptococcus mutans. The IC₅₀ values of carvacrol (M-1) and thymol (M-2) against S. mutans were 65 and 54 µg/ml, respectively. Live and dead staining and the MTT assays reveal that a concentration of 100 µg/ml of these compounds reduced the viability and the metabolic activity of S. mutans by more than 50%. Biofilm formation on the surface of polystyrene plates was significantly reduced by M-1 and M-2 at 100 µg/ml as observed under scanning electron microscope and by colorimetric assay. These results were in agreement with RT-PCR studies. Wherein exposure to 25 µg/ml of M-1 and M-2 showed a 2.2 and 2.4-fold increase in Autolysin gene (AtlE) expression level, respectively. While an increase of 1.3 and 1.4 fold was observed in the super oxide dismutase gene (sodA) activity with the same concentrations of M-1 and M-2, respectively. An increase in the ymcA gene and a decrease in the gtfB gene expression levels was observed following the treatment with M-1 and M-2. These results strongly suggest that carvacrol and thymol isolated from O. vulgare L. exhibit good bactericidal and antibiofilm activity against S. mutans and can be used as a green alternative to control dental caries.

Keywords: Alternative antimicrobials; Carvacrol; Oral hygiene; S. mutans; Thymol.

Fig. 1

Chemical structure of carvacrol (M-1) and thymol (M-2) (refer to Additional file 1 for raw data)

Fig. 2

Growth inhibition of S. mutans in the presence of clove oil (CO), carvacrol (M-1; CAR), thymol (M-2; THY), and chlorhexidine digluconate (CHD). The experiment was done in triplicate and values are presented as mean ± S.D error. Asterisk represents the p value <0.05 (refer to Additional file 1 for raw data)

Fig. 3

The change in the viability of S. mutans when grown with clove oil (CO), carvacrol (M-1; CAR), thymol (M-2; THY), and chlorhexidine digluconate (CHD). a Shows percent change in the metabolic activity of S. mutans using MTT assay with various concentrations of test compounds. While b shows the percent population of dead and live cells as determined by Live/dead staining when grown with test compounds. The experiment was done in triplicate and values are presented as mean ± S.D error. Asterisk represents the p value <0.05. Dagger the concentration of CHD is 2.5, 5, 10 and 20 µg/ml (refer to Additional file 1 for raw data)

Fig. 4

The change in the viability of S. mutans as determined by Live/dead staining when untreated (a, CONT), treated with 25 µg/ml of clove oil (b, CO), carvacrol (d, M-1) and thymol (e, M-2) and 2.5 µg/ml of chlorhexidine digluconate (c, CHD). Orange or red cells represent dead cells while green cells represent live cells (refer to Additional file 1 for raw data)

Fig. 5

Percent inhibition of the biofilm formation by S. mutans in the presence of test compounds. Values are presented are mean ± S.D error of three independent experiments. Dagger chlorhexidine digluconate was tested at a concentration of 2.5, 5, 10 and 20 µg/ml. Asterisk represents the p value <0.05 (refer to Additional file 1 for raw data)

Fig. 6

Qualitative assessment of S. mutans biofilms grown in the presence of test compounds. Control biofilms show high density of cells with homogeneous shape (A). While, cells treated with chlorhexidine digluconate (B), carvacrol (C) and thymol (D) show short chains of cells with many deformed and lysed cells marked by arrows (refer to Additional file 1 for raw data)

Fig. 7

RT-PCR analysis of S. mutans grown with the test compounds targeting Autolysin AtlE, gtfB, sodA and ymcA genes. The expression of AtlE, sodA and ymcA genes was induced by the test compounds while, gtfB gene was downregulated (refer to Additional file 1 for raw data)