Essential oils from edible plants especially those classified as GRAS are one of the safe alternatives to traditional antibiotics (Kalemba and Kunicka 2003). But the commercialization of these plant-based products is often hampered by the inability to purify bioactive compounds from these plants. Two such compounds, thymol, and carvacrol are the main constituents of O. vulgare L., both compounds are known to exhibit antimicrobial activity against a number of pathogens (Chorianopoulos et al. 2004; Thosar et al. 2013; Ultee et al. 2002). Interestingly, an in vitro study has demonstrated that thymol, and carvacrol show selectively higher antimicrobial activity against the tested pathogenic bacteria (Escherichia coli, Clostridium perfringens, and Salmonella) than the beneficial probiotic bacteria Lactobacillus (Du et al. 2015). When thymol and carvacrol were tested in vivo for their efficacy to inhibit C. perfringens in broilers it was found that although the population of the pathogen was not reduced significantly but the treatment alleviated intestinal lesions caused by these pathogens (Du et al. 2015). The same group (Du et al. 2016) in their in vivo study on broilers has further demonstrated many beneficial effects of thymol and carvacrol including an increase in feed conversion efficiency, increase in immunity against virus and tumor. It has been reviewed earlier also that herbs that serve as a primary source of these essential oils exhibit anticancer activities (Craig 1999). Hence, thymol and carvacrol in addition to possessing desired antimicrobial activity also have known health benefits making these essential oils a promising alternative antimicrobial agent.
Dental caries and periodontal diseases are most prevalent microbial diseases and S. mutans is one of the most important bacterium involved in dental caries (Loesche 1986; Selwitz et al. 2007). To keep a good oral hygiene, it is very important to check the growth of S. mutans. However, over and unwarranted use of antibiotics has also resulted in the development of antibiotic resistance in these pathogens (Leistevuo et al. 2000). Since, thymol and carvacrol exhibit good antimicrobial activity these essential oils were purified from O. vulgare L. and their antimicrobial activity against S. mutans was determined. Furthermore, the antimicrobial activity of thymol and carvacrol was compared with clove oil, a traditionally used essential oil in dentistry, and with chlorhexidine digluconate a widely used compound in mouthwashes (McBain et al. 2003; Thosar et al. 2013). To our knowledge, this is the first such detailed report on the antimicrobial and antibiofilm activity of thymol and carvacrol against S. mutans.
IC50 values of thymol, carvacrol, clove oil and chlorhexidine digluconate were found to be 54, 65, 306 and 4.9 µg/ml, respectively in this study. These results were further confirmed by MTT assay wherein 200 µg/ml of thymol and carvacrol reduced the cell viability by 87 ± 6 and 74 ± 8%, respectively. While, chlorhexidine digluconate resulted in the death of 37 ± 3% of the cells at a very low concentration of 2.5 µg/ml. In earlier studies, the essential oils from thyme and origanum exhibited high MIC values in a range of 256–512 ?g/ml against group A Streptococci (GAS) (Magi et al. 2015). While in the same study commercially available carvacrol exhibited a MIC value in the range of 64–256 µg/ml against GAS (Magi et al. 2015). Decontamination of lettuce using a solution of carvacrol and thymol was also reported since these essential oils are edible (Bagamboula et al. 2004). In another study, the antimicrobial activity of microencapsulated carvacrol and thymol was determined against many microorganisms including foodborne pathogens Escherichia coli O157:H7, Staphylococcus aureus, and Listeria innocua. The MIC of the microencapsulated carvacrol and thymol against these organisms were in the range of 225–375 ppm (Guarda et al. 2011). The positive control chlorhexidine digluconate suppressed the growth and viability of S. mutans at much lower concentrations as found in earlier studies also (McBain et al. 2003). While, the MIC values of eugenol a main constiuent of clove oil against S. mutans were reported to be 100 µg/ml (Freires et al. 2015).
As far as the mechanism of the antimicrobial activity is concerned, it is suggested that these compounds are involved in the permeabilization and depolarization of the cytoplasmic membrane. Which results in the reduction of the pH gradient across the cytoplasmic membrane. This lowering of pH gradient leads to the disturbance in proton motive force subsequently leading to the depletion of intracellular ATP level and cell death (Ultee et al. 2002). The increase in the number of dead cells following the treatment with thymol and carvacrol was also observed in this study (Figs. 3b, 4) through propidium iodide staining a dye that can only penetrate into the cells having compromised cell wall. Deformed and lysed cells as observed under scanning electron microscope further confirms that thymol and carvacrol results in the lysis of the cells. The overexpression of autolysin genes involved in the restructuring of the cell wall further confirms these findings. Our results also show the over expression sodA and ymcA genes, suggesting that treatment with thymol and carvacrol induces general and oxidative stress in the cells. Chlorhexidine digluconate also exhibit its antimicrobial activity primarily through membrane disruption (McBain et al. 2003). Inhibition of glycosidic and proteolytic enzymes by chlorhexidine digluconate is also reported (Hastings 2000). Although carvacrol and thymol also disrupt the cell membrane but these essential oils are not water-soluble which may adversely affect their penetration into the bacterial cells resulting in lower activity. This argument is also supported by the findings that nano form of thymol exhibit better antimicrobial activity than the native form because of the improved dispersibility (Shah et al. 2012).
Another important trait of pathogenicity is biofilm formation. Biofilms exhibit greater resistance to antimicrobial agents and are difficult to treat (Ahn et al. 2008; Curtis et al. 2011). Therefore, the antibiofilm activities of carvacrol (M-1) and thymol (M-2) against S. mutans was determined. The results of crystal violet assay and the qualitative examination of the biofilms under SEM (Fig. 6) suggest that the two compounds significantly reduce the biofilm formation by S. mutans. In gene expression studies also the downregulation of glycosyl transferase B (gtfB) gene suggests the inhibition of biofilm formation. Previously the antibiofilm activity of thymol and carvacrol against Pseudomonas aeruginosa has been reported (Ceylan and Ugur 2015). Chlorhexdine digluconate also reduced the biofilm formation at a concentration of 10 µg/ml. Inhibition of biofilm formation by chlorhexidine has been reported in in vivo studies also (Bailón-Sánchez et al. 2014). As far as the antimicrobial and antibiofilm activities of clove oil are concerned clove oil was found to be least effective.
Results presented in this study strongly suggest that thymol and carvacrol exhibit significant antimicrobial and antibiofilm activities against S. mutans. Since these compounds are derived from edible plants classified as GRAS, these essential oils can be used in mouthwashes or toothpastes for controlling oral bacteria and for maintaining good oral hygiene. Furthermore, unlike chlorhexidine digluconate, thymol and carvacrol have some health benefits in addition to the desired antimicrobial activity. Although, chlorhexidine digluconate is widely used and do not have any known health hazard but its effect on taste buds is well known (Helms et al. 1995).