Assessment of antibacterial effect of hydrogen water on plaque from patients with chronic periodontitis.

BACKGROUND: Periodontitis is an inflammatory disease causing destruction of tissues surrounding the teeth. The primary etiological factor for periodontitis is plaque. An inference could be drawn that an overall reduction in microorganisms halts disease progression. It is desirable to have natural agents with minimal side effects to reduce the microbial load. AIM: The aim of the study is to assess the effect of hydrogen water on microbial count in plaque obtained from chronic periodontitis patients and to determine the antibacterial activity of hydrogen water at various time intervals.
MATERIALS AND METHODS: A total of twenty chronic periodontitis patients were included after obtaining approval from the institutional ethical committee. Written informed consent was obtained from all the twenty participants. Plaque samples were collected and exposed to hydrogen water at baseline, 1 min, 2 min 30 s, and 5 min. Samples were then cultured on blood agar and incubated in aerobic and anaerobic conditions. The colony forming units and total bacterial count were recorded after 24-48 h. STATISTICAL ANALYSIS: Intragroup pair-wise comparison was done using Wilcoxon sign-ranked test.
RESULTS: Hydrogen water showed antibacterial activity against aerobic and anaerobic organisms associated with chronic periodontitis. There was a statistically significant difference in the number of colony forming units from baseline to 1 and 2.5 min for the aerobic culture and also for baseline to 1, 2.5, and 5 min for the anaerobic culture.
CONCLUSION: The data of the present study indicate that hydrogen water has an antibacterial effect on microorganisms associated with chronic periodontitis. Copyright:

INTRODUCTION

Dental plaque is a highly complex biofilm comprising various species and strains of microbiota, adhering to tooth surfaces, other oral surfaces, and restorations in the oral cavity. Plaque associated with a tooth surface is characterized by Gram-positive rods and cocci including Streptococcus mitis, Streptococcus sanguis, Actinomyces viscosus, Actinomyces naselundii, and Eubacterium species. Tissue-associated plaque shows a predominance of Streptococcus oralis, Streptococcus intermedius, Peptostreptococcus micros, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythus, and Fusobacterium nucleatum.[1]

Periodontitis is an inflammatory disease of the supporting tissues of teeth initiated by microorganisms, resulting in progressive destruction of the tissues surrounding a tooth, leading to the eventual loss of the tooth. Hence, overall reduction of the bacteria present in the dental plaque may help prevent periodontal disease and limit its progression.[2]

Various agents are used to combat the microbial load in the oral cavity. The use of chemical agents for this purpose is proven to be effective, but the duration of action of these agents is short lived and they cannot be consumed lifelong. Therefore, the use of natural agents, which can be consumed on a daily basis and also have antibacterial effects on oral bacteria, is desirable. One such agent is “hydrogen water.”

Hydrogen water has shown antibacterial activities on standard strains of Actinobacillus actinomycetemcomitans, F. nucleatum, and Treponema denticola.[3] It has also been proven to be an antifungal agent by showing its activity against Candida albicans biofilm.[4] Studies conducted have confirmed its efficacy in suppressing the progression of periodontitis by reducing gingival oxidative stress, due to its antioxidative activity.[5]

Taking into account the above applications and advantages of hydrogen water, we conducted a study on plaque samples obtained from patients with chronic periodontitis. The bacteria present in the plaque were exposed to hydrogen water at various intervals of time, and the antibacterial effect of hydrogen water was assessed by direct contact test.

MATERIALS AND METHODS

Production of hydrogen water

Generation of hydrogen water was done using a commercially available hydrogen water bottle. The base of the bottle has electrodes, which helps in the production of hydrogen water. Distilled water was infused in the hydrogen water bottle. The process of electrolysis is initiated in 3 s after the power of the bottle is in an ON mode. The electrolysis of water generated hydrogen (H2) at the cathode, thus producing hydrogen water in the bottle at the end of 5 min.

Sample collection and procedure

Subjects for the study were selected from patients visiting the Department of Periodontology, in the Institute. Plaque samples were collected from 20 patients with chronic periodontitis aged 30–50 years after obtaining approval from the institutional ethical committee. Written informed consent was obtained from all the 20 participants. Patients with the presence of bleeding on probing, probing depth (PD) >3 mm, or clinical attachment loss (CAL) ≥5 mm were included in the study. Pregnant and lactating women, patients who received any medications 3 months before sampling, patients with cancerous or precancerous lesion, patients who underwent periodontal therapy in the last 6 months, patients with medically compromised conditions, and patients using mouth rinses and tobacco habitués were excluded from the study. The clinical examination included Loe and Silness gingival index, PD, and CAL using the Williams probe.

The plaque samples were collected in reduced transport fluid (RTF) and transported to the laboratory for analysis. It was vortexed for 30 s to allow equal dispersion of the plaque in the RTF.

A pilot study was conducted earlier to standardize the dilution of the plaque sample, and it was finalized that the plaque sample would be diluted with thioglycollate broth with hemin and Vitamin K in the ratio of 1:25.

The diluted sample was then centrifuged at 1000 rpm for 2 min. Postcentrifugation, the supernatant was discarded. For the growth of bacteria, blood agar media with hemin and Vitamin K was used. The culture plate was divided into four quadrants. The diluted sample was inoculated in one quadrant to facilitate readings at baseline (negative control) in two different culture plates of blood agar using streak method of culturing. 1 ml of hydrogen water was then added to the pellet, and further, inoculation was done at 1, 2.5, and 5 min, respectively, in the culture plates.

One plate with the above culture was studied under aerobic conditions, which is in 5% CO2 and incubated for 24 h at 37°C in an incubator. The second plate was placed in an anaerobic jar with modified gas pack and incubated for 72 h at 37°C. After the respective incubation time period, the colony forming units and total aerobic and anaerobic bacterial count of both the plates were recorded using a digital colony counter.

The same procedure and interpretation were followed for all the 20 samples with hydrogen water (test group) and chlorhexidine (positive control).

Statistical procedure

Data collected were subjected to statistical analysis using Statistical Package for the Social Sciences (SPSS v 21.0, IBM, Armonk, NY). Normality of numerical data was checked using Shapiro–Wilk test. It was observed that the data did not follow a normal curve; hence, nonparametric tests have been used for comparisons. Intragroup comparison was done by Friedman's test while pair-wise comparison was done using Wilcoxon signed-rank test. A P < 0.05 was considered to be statistically significant, keeping α error at 5% and β error at 20%, thus giving a power to the study as 80%.

RESULTS

The effect of hydrogen water on the microbial count in the plaque obtained from patients with chronic periodontitis is presented in the following results.

Intragroup comparison was done using Friedman's test, for both aerobic and anaerobic cultures [Tables 1 and 2, respectively]. A statistically significant difference (P < 0.01) was seen for the values between the time intervals. This was followed by pair-wise comparison to determine the antibacterial activity of hydrogen water at exposure of 1, 2.5, and 5 min on the bacteria present in the plaque in patients with chronic periodontitis.

Table 1

Intra group comparison of hydrogen water group for aerobic culture

	n	Mean (CFU/ml)	SD	Minimum	Maximum	Median	Mean rank	χ2	P of Friedman’s test
Baseline	20	81.72×104	55.63×104	21×104	242×104	79.5×104	3.58
1 min	20	59.55×104	47.35×104	8×104	195×104	39.5×104	2.25	18.763	0.000**
2.5 min	20	45.72×104	30.51×104	5×104	130×104	46×104	1.83
5 min	20	53.55×104	38.71×104	11×104	130×104	43.5×104	2.33

**P<0.01 Statistically highly significant difference. n – Number of samples; P – Level/test of significance; CFU/ml – Colony forming units per milliliter; SD – Standard deviation; χ– Chi square value

Table 2

Intragroup comparison of hydrogen water group for anaerobic culture

	n	Mean (CFU/ml)	SD	Minimum	Maximum	Median	Mean rank	χ2	P of Friedman’s test
Baseline	20	115.2×104	74.11×104	25×104	252×104	100×104	3.61
1 min	20	75.2×104	44.92×104	20×104	190×104	72×104	2.28	18.400	0.000**
2.5 min	20	70.3×104	37.53×104	17×104	150×104	67×104	2.17
5 min	20	61.0×104	38.29×104	10×104	140×104	63.5×104	1.94

**P<0.01 Statistically highly significant difference. n – Number of samples; P – Level/test of significance; CFU/ml – Colony forming units per milliliter; SD – Standard deviation; χ– Chi square value

Table 3 shows the pair-wise comparison of the hydrogen water group using Wilcoxon sign-ranked test for aerobic culture. There was a statistically significant difference (P < 0.01 implies highly significant difference, and P < 0.05 implies significant difference) seen for the values between the time intervals baseline–1 min, baseline–2.5 min, and 1–2.5 min, indicating that there were a lesser number of colony forming units at 1 and 2.5 min when compared to baseline and at 2.5 min when compared 1 min. However, statistically insignificant difference was seen for the values between baseline–5 min (P = 0.067) and 1–5 min (P = 0.756).

Table 3

Pairwise comparison using Wilcoxon sign-ranked test of hydrogen water group for aerobic culture

Time pairs	Z	P
Baseline 0 min-1 min	−2.696	0.007**
Baseline 0 min-2.5 min	−3.099	0.002**
Baseline 0 min-5 min	−1.831	0.067#
1 min-2.5 min	−2.418	0.016*
1 min-5 min	−0.310	0.756#

*P<0.05 statistically significant difference, **P<0.01 Statistically highly significant difference, #P>0.05 nonsignificant difference. P – Level/test of significance; Z – Number of standard deviations from mean

Pair-wise comparison test of the hydrogen water group for anaerobic culture is depicted in Table 4. A statistically significant difference (P < 0.01) was seen, suggesting that the number of colony forming units was reduced at 1, 2.5, and 5 min when compared to baseline. However, there was no statistically significant difference seen for the values between the time intervals – 1–2.5 min (P = 0.179), 1–5 min (P = 0.327), and 2.5–5 min (P = 0.231).

Table 4

Pairwise comparison using Wilcoxon sign-ranked test of the hydrogen water group for anaerobic culture

Time pairs	Z	P
Baseline 0 min-1 min	−3.324	0.001**
Baseline 0 min-2.5 min	−3.025	0.002**
Baseline 0 min-5 min	−2.744	0.006**
1 min-2.5 min	−1.345	0.179#
1 min-5 min	−.980	0.327#
2.5 min-5 min	−1.198	0.231#

**P<0.01 Statistically highly significant difference, #P>0.05 nonsignificant difference. P – Level/test of significance; Z – Number of standard deviations from mean

The above results indicate that hydrogen water does act as an antibacterial agent against pathogens associated with chronic periodontitis.

DISCUSSION

Mechanical plaque control is an effective strategy for preventing the progression of oral diseases. A daily use of an effective mouth rinse as well can be considered as an adjuvant strategy to tooth brushing. Chlorhexidine, as a mouth rinse, has been considered as a gold standard for many decades now, but long-term use of chlorhexidine is known to cause side effects.[6] Hence, discovery of potential natural agents exhibiting antibacterial effect on oral bacteria is desirable. Hydrogen water is one such natural agent with an antibacterial potential.[378]

In a study carried out by Lee et al.,[3] antibacterial effect of hydrogen water was checked in vitro and in vivo. When compared to the effect of tap water, samples treated with hydrogen water showed a significant reduction in the growth of all periodontopathogens (standard strains) in culture, toothbrushes, and saliva. The results of our study were similar to the above study. In our present study, there was a statistically significant reduction in the colony forming units of aerobic and anaerobic bacteria when they were treated with hydrogen water as compared to baseline.

Kim et al.[7] studied the effect of hydrogen water on streptococcal biofilm formation. The results showed that there was a significant decrease in in vitro biofilm formation. Furthermore, oral rinse with hydrogen water for 1 week led to significantly fewer salivary streptococci than did that with control tap water. Hence, the authors concluded that oral rinse with hydrogen water would be helpful in treating dental biofilm-dependent diseases with ease and efficiency. In our present study, we found that the aerobic and anaerobic bacteria which constitute the biofilm were significantly reduced by hydrogen water.

The results of the present study were also in accordance with the study done by Baek et al.,[8] wherein the antibacterial activity of hydrogen-rich water was tested against bacteria associated with oral disease. Bacterial strains of Streptococcus mutans, F. nucleatum, and Tannerella forsythia were cultured and treated with hydrogen water and tap water, and the number of bacterial colonies was measured. Hydrogen water showed antibacterial effect against all four pathogens in comparison to tap water.

Zeng et al.,[9] through their research on the antibacterial mechanism of electrolysed water on Escherichia coli and Staphylococcus aureus, suggested that the products obtained after electrolysis of water increased the permeability of the bacterial cell membrane and led to the leakage of cellular components, thus killing the bacteria. However, the exact mechanism by which the hydrogen water reacts with bacterial membrane is beyond the scope of this study.

Studies on rats by Yoneda et al.[10] and Kasuyama et al.[11] have shown that consumption of hydrogen-rich water was beneficial in suppressing periodontitis progression and limiting the alveolar bone resorption. The results of our present study suggest that hydrogen water has an antibacterial effect on aerobic and anaerobic bacteria in the plaque obtained from patients with chronic periodontitis. Considering that the plaque microbiota is the initiating etiological agent for chronic periodontitis, we can assume that the antibacterial potential of hydrogen water will help halt the disease progression.

Azuma et al.[12] studied the effects of drinking hydrogen water on nonsurgical periodontal treatment in 13 patients. The participants in the hydrogen water group showed greater improvements in probing pocket depth and clinical attachment level as compared to the control group at 2, 4, and 8 weeks.

To the best of our knowledge, no studies have reported any unwanted/adverse effects of consumption of hydrogen water.

The results of our in vitro study have been very encouraging to say the least. They indicate an antibacterial potential for hydrogen water against clinical plaque samples in patients with chronic periodontitis. The adjunctive use of hydrogen water as an irrigant, mouth rinse, especially in patients with chronic periodontitis, needs to be further explored.

CONCLUSION

Chronic periodontitis is a multifactorial disease although bacterial onslaught through accumulation of plaque is responsible for initiating and increasing the severity of disease.

The analysis of data from the present study is suggestive that hydrogen water has an antibacterial activity against aerobic and anaerobic bacteria present in the plaque obtained from the patients with chronic periodontitis. This remarkable finding of something as natural as water, but having an antibacterial potential, has certainly opened our minds to further research on hydrogen water and its applications.

Presence of bacterial biofilm causes a low-grade inflammation, which leads to persistent activation of inflammatory cells, resulting in a high degree of reactive oxidative species (ROS) release.Over produced ROS deplete the endogenous anti-oxidants thereby leading to increased oxidative damage. Recent studies also indicate that treatment with hydrogen water has an antioxidative effect on cells, tissues, and organs as well as a beneficial role in the prevention against oxidative stress-related disorders.

In continuation to this study, we are now inspired to take this forward and test hydrogen water with respect to its antioxidant potential in patients with chronic periodontitis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.