Abstract
Objectives
The accumulation of oral bacterial biofilm is the main etiological factor of oral diseases. Recently, electrolyzed hydrogen-rich water (H-water) has been shown to act as an effective antioxidant by reducing oxidative stress. In addition to this general health benefit, H-water has antibacterial activity for disease-associated oral bacteria. However, little is known about the effect of H-water on oral bacterial biofilm. The objective of this study was to confirm the effect of H-water on streptococcal biofilm formation.
Methods
In vitro streptococcal biofilm was quantified using crystal violet staining after culture on a polystyrene plate. The effect of H-water on the expression of genes involved in insoluble glucan synthesis and glucan binding, which are critical steps for oral biofilm formation, was evaluated in MS. In addition, we compared the number of salivary streptococci after oral rinse with H-water and that with control tap water. Salivary streptococci were quantified by counting viable colonies on Mitis Salivarius agar-bacitracin.
Results
Our data showed that H-water caused a significant decrease in in vitro streptococcal biofilm formation. The expression level of the mRNA of glucosyltransferases ( gtfB, gtfc, and gtfI ) and glucan-binding proteins ( gbpC, dblB ) were decreased remarkably in MS after H-water exposure for 60 s. Furthermore, oral rinse with H-water for 1 week led to significantly fewer salivary streptococci than did that with control tap water.
Conclusions
Our data suggest that oral rinse with H-water would be helpful in treating dental biofilm-dependent diseases with ease and efficiency.
1
Introduction
Streptococcus mutans and Streptococcus sobrinu s are the major causative agents of human dental caries and are considered to be the principal cariogenic bacteria among the oral streptococci . Therefore, ecologically driven changes in oral biofilms caused by S. mutans and S. sobrinus are responsible for such diseases. The removal of oral biofilms has been an essential method for the prevention of dental caries . Hence, the importance of effective plaque control has been emphasized over the years. To date, mechanical plaque elimination with assorted devices, like the toothbrush, remains the primary and most widely accepted means of controlling plaque and maintaining good oral hygiene. However, it is difficult for most people to attain adequate plaque removal and control with such mechanical devices. As an adjunct to mechanical methods, oral hygiene products containing chemotherapeutic agents with a variety of antimicrobial mechanisms have been beneficial and desirable.
There are several gargling products that can be used as adjuvants to control dental plaque. The most frequently used agent is chlorhexidine (CHX). CHX mouth rinse is effective in preventing and controlling plaque formation, breaking up existing plaque, and inhibiting and reducing the development of gingivitis . Although the clinical effectiveness of CHX has been well documented, many adverse effects, such as swollen, red, and bleeding gums; desquamation or soreness of oral mucosa; altered taste sensation; staining of the teeth and the tongue; and gastritis, have also been reported . In addition, some oral bacteria develop resistance to the antibacterial activity of CHX . Another antimicrobial mouth rinse product containing triclosan, a chlorophenol, has been suspected to cause resistance in bacterial strains and allergic contact dermatitis . In addition, cetylpyridinium chloride mouth rinse has been found to cause tooth staining and burning sensation . These various limitations led to the development of safer alternative oral hygiene products.
Recently, electrolyzed hydrogen-rich water (H-water) has been shown to be a natural antibiotic material that overcomes such difficulties. Hydrogen (dihydrogen; H 2 ) acts as an effective antioxidant by reducing oxidative stress . In addition, H-water exhibits antibacterial activity for oral bacteria, such as Streptococcus mutans , Fusobacterium nucleatum , Porphyromonas gingivalis , and Tannerella forsythia , which are associated with oral diseases . Furthermore, H-water might be beneficial in suppressing the progression of periodontitis by decreasing gingival oxidative stress . These data suggest that H-water can be used as an adjuvant to prevent dental caries and periodontal disease.
It is generally known that two closely related species of mutans streptococci (MS), namely S. mutans and S. sobrinus , are the predominantly prevalent caries-associated species in humans. Among the physiological traits of MS that are most relevant to cariogenesis are their syntheses of extracellular polysaccharides from sucrose, which fosters their firm attachment to teeth and promotes tight cell clustering. The enzymes involved in the synthesis of extracellular polysaccharides, such as water-insoluble glucans, are extracellular glucosyltransferases (GTFs) . The GTF genes in S. mutans are classified in terms of the solubility of their gene products: insoluble glucan synthesis ( gtfB ), and insoluble/soluble glucan synthesis ( gtfC ) . Mutations of these GTF family genes lead to a reduction in the incidence of dental caries in rats exposed to MS carrying the mutant genes, indicating that these GTFs in S. mutans are responsible for the pathogenesis of dental caries . In S. sobrinus , gtfI plays an important role in water-insoluble glucan synthesis . Furthermore, binding with insoluble glucan through the glucan-binding protein (GBP) is crucial for the attachment of MS to solid surfaces in biofilm formation . Zhu et al. identified GbpC as the protein that is responsible for sucrose-dependent aggregation in S. mutans . Mutants lacking the gbpC gene formed biofilms that were structurally different and defective when compared to those of the wild type . On the other hand, the S. sobrinus dblB gene, which is one of the four gbpC protein gene homologs, is the primary gene responsible for aggregation in this species . Therefore, it is important to reveal the association between gbpC or dblB gene expression and biofilm formation in MS.
The salivary microbial species reflect the composition of the oral microbial community and could serve as a biomarker of the health and disease status of the oral cavity. Saliva allows dental plaque to flourish and also detaches layers of plaque . The level of certain bacterial species in saliva can reflect their presence in plaque . Previous studies have shown a significant correlation between the salivary concentration of MS and their proportions in plaque . Therefore, saliva would be an appropriate sample from which to estimate the quantity of S. mutans biofilm in oral cavity.
The object of this study was to evaluate the effect of H-water on oral streptococci. We first examined whether H-water affects in vitro streptococci biofilm formation. We also analyzed the expression of genes involved in glucan binding and insoluble glucan synthesis in S. mutans and S. sobrinus . Furthermore, we compared salivary streptococcal numbers after oral rinse with H-water and that with control tap water to estimate the effect of H-water on plaque MS levels.
2
Materials and methods
2.1
Bacterial culture
The bacteria tested in this study were Streptococcus mutans (ATCC 25175) and Streptococcus sobrinus (ATCC 27607). Streptococci were maintained on brain heart infusion (BHI) medium and grown under aerobic conditions.
2.2
Effect of H-water on streptococcal biofilm formation
An in vitro biofilm formation assay was performed in accordance with the protocol published by Toole .
H-water (dissolved hydrogen, 1500 ppb; oxidation-reduction potential, −600 mV to −700 mV) was purchased from Nanotec (nano H ® , South Korea). Streptococcal colonies were inoculated in BHI-0.3% sucrose broth and incubated for overnight. The culture broth was inoculated to 50 mL of the same liquid medium in the polystyrene tube to be 1% (V/V). Incubation for approximately 6 h leads to the exponential phase of bacteria (0.2–0.3 at OD 600, or 10 7 –10 8 cells/ml). The bacterial broth was centrifuged and the pellet was washed with phosphate-buffered saline (PBS). Streptococcal pellets were exposed to 5 mL of H-water or tap water for 0, 15, 30, and 60 s, respectively, after which were diluted with 10-fold volume of sterile PBS. After centrifugation, bacterial pellet was resuspended in BHI-0.3% sucrose medium and cultured for 24 h in 12-well polystyrene plates coated with sterile saliva to form bacterial biofilm. After discarding the supernatants, the attached bacteria were washed three times with PBS and stained with 0.2% crystal violet in 10% ethanol for 30 min to visualize the attached bacteria (biofilm). After washing five times, crystal violet was extracted with acid solvent, and absorbance was measured at 590 nm. The absorbance of test samples was normalized with that of tap-water. All experiments were repeated three times.
2.3
Quantitative Real-time PCR (RT-qPCR)
To determine the effects of H-water on the genes expression of glucosyltransferases and glucan-binding proteins, streptococci were cultured in BHI-0.3% sucrose broth for 8 h with pre-exposure to H-water or sterile tap water for 60 s. We analyzed mRNA expression of the gtfB, gtfC, and gtfI , dblB genes in S. mutans and S. sobrinus, respectively, by real-time PCR. Total RNA was extracted from the whole bacterial culture, including the planktonic and attached bacteria, using QIAzol solution (QIAGEN, CA, USA). After DNase treatment, isolated RNA (1 μg each) was reverse transcribed using the Omniscript RT kit in the presence of random primers (QIAGEN, CA, USA). The resultant cDNA was amplified on a real-time PCR instrument (ABI Prism 7500, Applied Biosystems, CA, USA) using gene-specific primer pairs and SYBR Premix Ex Taq (TaKaRa, WI, USA) as described by the manufacturer. The primers were added to a final concentration of 10 μM. In addition, 10 μL of 2X SYBR Premix Ex Taq (TaKaRa, WI, USA) was used, and the reactions were carried out in a total volume of 20 μL as recommended by the manufacturer. The primers for 16S rRNA, gtf , gbpC , and dblB were used, as described previously . Changes in the levels of gene expression were also calculated using the “delta-delta threshold cycle” method described by Niu et al. . The relative gene expression levels were normalized to those determined for 16S rRNA in the same samples. All experiments were repeated two times.
2.4
Effect of oral rinse with H-water on salivary streptococci
Informed consent was obtained from all volunteers before starting the in vivo oral rinse experiments. The study was approved by the Ethics Committee of the Kyungpook National University. A double-blinded study design was used to evaluate the effect of oral rinse with H-water or control tap water on salivary streptococcal number. The subjects were healthy adults with a minimum of 20 natural teeth including at least 4 molars. Subjects were excluded for the following reasons: a medical condition requiring premedication; antibiotic use within 2 weeks of the first treatment period; inability to comply with protocol; orthodontic appliances interfering with obtaining 20 gradable teeth; and/or rampant caries, open or untreated caries, severe gingivitis, or advanced periodontitis requiring prompt treatment. Subjects agreed not to receive dental prophylaxis and to refrain from using antibiotics, any non-study dentifrice, or other oral hygiene products ( e.g. , floss, chewing gum) for the study duration. A total of six subjects participated, and each tried three cycles of oral rinse with H-water or tap-water. In other words, a total of 18 trials were performed to compare salivary streptococcal number after oral rinse with tap water or H-water. The participants were told to abstain from all mechanical plaque control measures except the toothbrush but to rinse 3 times a day with 30 mL of the assigned solution each for 60 s. During the first week of the 2-week cycle, H-water had been used as a test solution, and saliva was collected at the end of the first week. Then, tap water had been used as control solution, and saliva was collected at the end of the second week. After a one-week interval, another 2-week cycle trial was started at the same condition.
Whole saliva was collected between 1 and 2 pm after rinsing shortly with tap water. Salivary samples were processed immediately for the quantification of streptococci. Saliva sample (1 mL) was diluted 10-fold serially to 10 −5 with PBS, after which 100 μL was spread on Mitis Salivarius agar-bacitracin (MSB). After culture for two days under aerobic condition, the number of colony-forming units (CFU) was measured
2.5
Statistical analysis
Significant variation analysis was used to calculate the parametric, two-tailed, non-paired t -test, and analysis of variance, and the non-parametric Mann–Whitney U test was used to compare two populations. All analyses were performed using Origin 8.0 (OriginLab, Northampton, MA, USA), and P-values of ≤0.05 were considered statistically significant.
2
Materials and methods
2.1
Bacterial culture
The bacteria tested in this study were Streptococcus mutans (ATCC 25175) and Streptococcus sobrinus (ATCC 27607). Streptococci were maintained on brain heart infusion (BHI) medium and grown under aerobic conditions.
2.2
Effect of H-water on streptococcal biofilm formation
An in vitro biofilm formation assay was performed in accordance with the protocol published by Toole .
H-water (dissolved hydrogen, 1500 ppb; oxidation-reduction potential, −600 mV to −700 mV) was purchased from Nanotec (nano H ® , South Korea). Streptococcal colonies were inoculated in BHI-0.3% sucrose broth and incubated for overnight. The culture broth was inoculated to 50 mL of the same liquid medium in the polystyrene tube to be 1% (V/V). Incubation for approximately 6 h leads to the exponential phase of bacteria (0.2–0.3 at OD 600, or 10 7 –10 8 cells/ml). The bacterial broth was centrifuged and the pellet was washed with phosphate-buffered saline (PBS). Streptococcal pellets were exposed to 5 mL of H-water or tap water for 0, 15, 30, and 60 s, respectively, after which were diluted with 10-fold volume of sterile PBS. After centrifugation, bacterial pellet was resuspended in BHI-0.3% sucrose medium and cultured for 24 h in 12-well polystyrene plates coated with sterile saliva to form bacterial biofilm. After discarding the supernatants, the attached bacteria were washed three times with PBS and stained with 0.2% crystal violet in 10% ethanol for 30 min to visualize the attached bacteria (biofilm). After washing five times, crystal violet was extracted with acid solvent, and absorbance was measured at 590 nm. The absorbance of test samples was normalized with that of tap-water. All experiments were repeated three times.
2.3
Quantitative Real-time PCR (RT-qPCR)
To determine the effects of H-water on the genes expression of glucosyltransferases and glucan-binding proteins, streptococci were cultured in BHI-0.3% sucrose broth for 8 h with pre-exposure to H-water or sterile tap water for 60 s. We analyzed mRNA expression of the gtfB, gtfC, and gtfI , dblB genes in S. mutans and S. sobrinus, respectively, by real-time PCR. Total RNA was extracted from the whole bacterial culture, including the planktonic and attached bacteria, using QIAzol solution (QIAGEN, CA, USA). After DNase treatment, isolated RNA (1 μg each) was reverse transcribed using the Omniscript RT kit in the presence of random primers (QIAGEN, CA, USA). The resultant cDNA was amplified on a real-time PCR instrument (ABI Prism 7500, Applied Biosystems, CA, USA) using gene-specific primer pairs and SYBR Premix Ex Taq (TaKaRa, WI, USA) as described by the manufacturer. The primers were added to a final concentration of 10 μM. In addition, 10 μL of 2X SYBR Premix Ex Taq (TaKaRa, WI, USA) was used, and the reactions were carried out in a total volume of 20 μL as recommended by the manufacturer. The primers for 16S rRNA, gtf , gbpC , and dblB were used, as described previously . Changes in the levels of gene expression were also calculated using the “delta-delta threshold cycle” method described by Niu et al. . The relative gene expression levels were normalized to those determined for 16S rRNA in the same samples. All experiments were repeated two times.
2.4
Effect of oral rinse with H-water on salivary streptococci
Informed consent was obtained from all volunteers before starting the in vivo oral rinse experiments. The study was approved by the Ethics Committee of the Kyungpook National University. A double-blinded study design was used to evaluate the effect of oral rinse with H-water or control tap water on salivary streptococcal number. The subjects were healthy adults with a minimum of 20 natural teeth including at least 4 molars. Subjects were excluded for the following reasons: a medical condition requiring premedication; antibiotic use within 2 weeks of the first treatment period; inability to comply with protocol; orthodontic appliances interfering with obtaining 20 gradable teeth; and/or rampant caries, open or untreated caries, severe gingivitis, or advanced periodontitis requiring prompt treatment. Subjects agreed not to receive dental prophylaxis and to refrain from using antibiotics, any non-study dentifrice, or other oral hygiene products ( e.g. , floss, chewing gum) for the study duration. A total of six subjects participated, and each tried three cycles of oral rinse with H-water or tap-water. In other words, a total of 18 trials were performed to compare salivary streptococcal number after oral rinse with tap water or H-water. The participants were told to abstain from all mechanical plaque control measures except the toothbrush but to rinse 3 times a day with 30 mL of the assigned solution each for 60 s. During the first week of the 2-week cycle, H-water had been used as a test solution, and saliva was collected at the end of the first week. Then, tap water had been used as control solution, and saliva was collected at the end of the second week. After a one-week interval, another 2-week cycle trial was started at the same condition.
Whole saliva was collected between 1 and 2 pm after rinsing shortly with tap water. Salivary samples were processed immediately for the quantification of streptococci. Saliva sample (1 mL) was diluted 10-fold serially to 10 −5 with PBS, after which 100 μL was spread on Mitis Salivarius agar-bacitracin (MSB). After culture for two days under aerobic condition, the number of colony-forming units (CFU) was measured
2.5
Statistical analysis
Significant variation analysis was used to calculate the parametric, two-tailed, non-paired t -test, and analysis of variance, and the non-parametric Mann–Whitney U test was used to compare two populations. All analyses were performed using Origin 8.0 (OriginLab, Northampton, MA, USA), and P-values of ≤0.05 were considered statistically significant.
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