Ⅰ. Introduction
The characteristics of Streptococcus mutans and S. sobrinus are acid production and aciduricity [1,2]. Since this characteristics, they play a central role in the induction of dental caries [3], and S. mutans and S. sobrinus have been called cariogenic bacteria. Especially, compared to planktonic bacteria, their biofilm is associated more with dental caries due to acid production in limited space [4]. Also, the biofilm of S. mutans and S. sobrinus is more resistant for antimicrobial agents than planktonic bacteria because of glucan [5]. Glucan is an exopolysaccharide of S. mutans and S. sobrinus and plays a barrier role in cariogenic biofilm [6]. Therefore, glucan inhibits the penetration of antimicrobial agents into biofilm. Chemical gargle solutions are used to prevent infectious diseases such as caries and periodontitis in oral cavity [7,8]. Since some gargle solutions contain alcohol, their use in oral health has been controversial [9-11]. Therefore, dental gargle solution for the prevention of caries is not recommended for children.
Electrolyzed water (EW) is generated by the decomposition of water (H2O) into oxygen (O2) and hydrogen gas (H2) by an electric current being passed through the water. The characteristics of EW was differ according to the type of electrodes, such as silver and platinum, and the type of molecules added such as citric acid and sodium chloride. Also, the physical properties of EW vary according to the presence or absence of a membrane in the chamber [12]. Several types of EW such as acidic, basic, and neutral water have been used in many fields such as industry, agriculture, and food because of its disinfecting effects [12,13]. Recently, neutral electrolyzed water (NEW) was tried to apply to the medical and dental areas, as it is harmless to biological tissues [14-16]. Also, NEW has anti-cancer, anti-oxidative and anti-bacterial effects [17-19]. Acidic electrolyzed water has bactericidal effect on Listeria monocytogenes and Escherichia coli [13]. Basic electrolyzed water affects oral hygiene including bactericidal activity, biofilm removal and bacterial growth inhibition [19]. However, acidic and basic electrolyzed water were not sure of harmfulness due to their acidic and basic condition.
The purpose of this study was to investigate the antimicrobial effects of NEW on cariogenic bacteria and their biofilm, and to compare the antimicrobial activity of NEW and commercial gargle solutions.
Ⅱ. Materials and Methods
1. Bacterial species and cultivation
S. mutans ATCC 25175 and S. sobrinus ATCC 33478 were used in this study and cultivated by brain heart infusion broth (BHI)(BD bioscience, Sparks, MD, USA). In order to form biofilm, S. mutans and S. sobrinus were cultivated in BHI broth containing 2% sucrose according to the methods of Lee et al. [3].
2. Production of electrolyzed water
Two types of EW were used in this study and in order to investigate its antibacterial activity. EW was generated from an electrolyzing distilled water containing 0.05% and 0.15% sodium chloride in an undivided anode chamber and cathode chamber. Also, the platinum electrodes were placed in the chamber horizontally. The pH and oxidation reduction potential (ORP) level of the EW was measured by a pH meter (Thermo Fisher Scientific, Waltham, MA, USA). The free available chlorine species (HClO and ClO-) were determined using N,N-diethyl-p-phenylenediamine (Sigma-Aldrich Co. St. Louis, MO, USA) [20]. The free chlorine, pH and ORP of the EW were analyzed within 10 min after electrolysis.
3. Antimicrobial efficacy of the EW against cariogenic bacteria
S. mutans and S. sobrinus were cultivated in BHI broth for 36 h and counted by a bacterial counting chamber (Marienfeld, Lauda-Konigshofen, Germany). The bacterial density was adjusted to 1 × 107 cells/mL with BHI broth. Each bacterial suspensions (1 mL) was harvested by centrifugation at 4,000 × g for 10 min and then washed by phosphate buffered saline (PBS). The bacterial pellets were treated with 1 mL of the EW and commercial gargle solutions (alcohol- or fluoride-containing gargle) for 30 sec or 1 min, and then immediately added to 1 mL of BHI broth. The samples were diluted serially 10 fold to 105, each diluted suspension was spread on an agar plate and incubated at 37℃ until the colonies could be counted.
4. Comparison of biofilm biomass and viable bacteria
For biofilm formation, a plastic coverslip (SPL bioscience, Gyoengi-do) was coated with saliva according to the methods of Lee et al. [21]. S. mutans and S. sobrinus were inoculated into BHI broth including 2% sucrose and dispensed on the saliva-coated cover slip in a 12-well polystyrene plate (SPL bioscience). The plates were incubated at 37℃ for 72 h, and the media were changed with fresh BHI broth including 2% sucrose each day. The biofilms were washed twice with PBS to remove planktonic bacteria and treated with the EW or commercial gargle solutions for various durations. The EW and commercial gargle solution were immediately aspirated by suction pump. Each biofilm was stained with 0.5% crystal violet and washed three times with PBS. Absolute alcohol was added to dissolve the crystal violet on the bacteria in biofilm, and the supernatant was transferred into a 96-well polystyrene plate (SPL bioscience). The optical density of the supernatant was measured at a wavelength of 590 nm by a microplate reader (Biotek. Winooski, VT, USA). In order to analyze the viable bacteria in the biofilm, electrolyzed water-treated biofilms were disrupted mechanically, and weak sonication was performed (output 3W) to homogenize the bacteria. The bacterial suspensions were serially diluted from 103 to 106 with BHI broth, and the diluted suspensions (50 μL) were plated on Mitis-salivarius agar (BD bioscience). The plates were incubated at 37℃ for 48 h, and the colony forming units of each bacteria on each plate were counted.
Ⅲ. Results
1. Physical property of the electrolyzed water
When distilled water in the presence or absence of sodium chloride at the concentration of 0.05% and 0.15% was by an passed electric current through the platinum electrode, the level of pH was neutral, and did not change. However, the concentration of free chlorine was increased in a dose-dependent manner (Table 1). The concentration of free chlorine indicated hypochlorous acid (HOCl) and hypochlorite ion (OCl-).
2. Antimicrobial activity of the electrolyzed water
The antimicrobial activity of the generated EW was compared with two commercial gargle solutions, including an alcohol-containing gargle for adults and a fluoride-containing gargle for children. The EW from the water including sodium chloride showed significant antimicrobial activity against S. mutans and S. sobrinus (Fig. 1). Furthermore, the sodium chloride-containing EW has more antimicrobial activity compared to the gargle solution for children.
3. Effect of the electrolyzed water on biofilm of S. mutans and S. sobrinus
Mature biofilm of S. mutans and S. sobrinus was formed and treated with the EW and commercial gargle solutions. Fluoride-containing gargle as a gargle for children did not affect the biofilm of S. mutans and S. sobrinus. However, the EW from the water including sodium chloride and the alcohol-containing gargle significantly disrupted the biofilm (Fig. 2). Furthermore, the count of S. mutans and S. sobrinus in the biofilm was decreased by both the EW and the alcohol-containing gargle (Fig. 3). Interestingly, the EW disrupted more the biofilm and killed more the cariogenic bacteria in the biofilm than the alcohol-containing gargle.
Ⅳ. Discussion
Dental caries as tooth decay is the most prevalent infectious disease in children and is associated with S. mutans and S. sobrinus [22]. Gargle solutions have been used to prevent caries in daily life. However, general gargle solutions contain chemicals including alcohol to kill oral bacteria. Therefore, the use of gargle solutions for oral health has been controversial because of their harmful side effects [23,24]. Eventually, the gargle solution is reluctant to use in children. In this study, EW was investigated antimicrobial activity against S. mutans and S. sobrinus and compared with the conventional gargle solutions. Also, the EW was evaluated for its potential to replace conventional gargle solutions.
The EW was generated by an electric current by passing through it platinum electrodes in the presence of sodium chloride. This EW showed neutral pH and contained free chlorines such as hypochlorous acid (HOCl) and hypochlorite ion (OCl-). When the EW was generated using silver electrodes, the level of pH was neutral. However, the level of free chlorines was barely detected. The free chlorines in aqueous solution have antimicrobial activity [25]. The bactericidal mechanisms of free chlorines are to react with the bacteria surface components and to impair intracellular components. In current study, these characteristics of the EW from NaCl-containing water was examined antimicrobial and anti-biofilm activity against S. mutans and S. sobrinus. Although the antimicrobial activity of the EW was weaker than a commercial gargle solution with alcohol, it showed stronger antimicrobial activity compared to the gargle solution for children.
Interestingly, although the EW exhibited weaker antimicrobial effect on planktonic S. mutans and S. sobrinus than the commercial gargle solution, it was more effective in destroying the biofilm of S. mutans and S. sobrinus than the commercial gargle solution. On the basis of biofilm disruption, the EW destroys the surface barrier of the biofilm, penetrates into the biofilm, and then reacts with each bacterium. Since the ability of commercial gargle solution to destroy the biofilm is weak, the solution might not penetrate deeply into the biofilm. S. mutans and S. sobrinus produce abundant glucan from sucrose, and the glucan is a barrier of biofilm, protecting the bacteria in the biofilm and forming limited space [26,27]. Finally, the level of pH was rapidly decreased in the biofilm because S. mutans and S. sobrinus can produce lactic acid in a limited space and under conventional conditions. Therefore, dental caries is related more to the biofilm of cariogenic bacteria than to planktonic bacteria [21].
The NEW is effective in removing cariogenic biofilm as thoroughly as the commercial gargle solutions and showed antimicrobial activity against S. mutans and S. sobrinus. Furthermore, no harmful effects have been reported with the use of neutral EW. Therefore, the neutral EW from NaCl-containing water may be a candidate for the prevention of dental caries.