Adhami et al, J Dent Res Dent Clin Dent Prospects, 2023, 17(3), 177-181

doi: 10.34172/joddd.2023.40461
TUOMS
https://joddd.tbzmed.ac.ir PRESS

Original Article

Effect of 20-ppm ozone and 1% chlorhexidine gels on plaque

index and Streptococcus mutans counts in the dental plaque
in 6–12-year-old children: A randomized, double-blind
clinical trial
ID ID
Ziya Ebrahim Adhami1 , Leila Erfanparast1, Zahra Molaei1* , Javid Sadeghi2, Azam Yazdanparas2
1
Department of Pediatric Dentistry, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran
2
Department of Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

ARTICLE INFO Abstract

Article History: Background. One of the methods to control dental caries is to use ozone. Since it is difficult
Received: June 25, 2023 for children to use mouthwashes, the present study aimed to evaluate 20-ppm zone and 1%
Accepted: September 2, 2023 chlorhexidine (CHX) gels’ effects on the plaque index and Streptococcus mutans counts in
ePublished: November 11, 2023 6–12-year-old children.
Methods. In the present double-blind clinical trial, 165 children, 6–12 years of age, referring
Keywords: to the Department of Pediatric Dentistry, Tabriz Faculty of Dentistry, were selected based on
Chlorhexidine gel, Dental inclusion and exclusion criteria and randomly assigned to three groups: ozone gel, CHX gel,
plaque, Ozone gel, and control. The subjects were instructed to place an adequate amount of the gels on all the
Streptococcus mutans surfaces of their teeth with one clean finger. The patients and evaluators were blinded to the
study groups. The plaque index and S. mutans counts in plaque samples were determined before
intervention and three weeks after intervention on the buccal surface of the most posterior
maxillary tooth (left or right). S. mutans counts were determined by culture. STATA software
version 14 was used for statistical analyses using Wilcoxon, Kruskal-Wallis, and post hoc Dum
tests. Statistical significance was defined at P < 0.05.
Results. The 20-ppm ozone and 1% CHX gels significantly decreased dental plaque compared
to the control group (P < 0.05), and their effects were similar (P > 0.05). These gels significantly
decreased the colonies and bacterial counts of S. mutans (P < 0.05).
Conclusion. The performance of 20-ppm ozone gel in decreasing the dental plaque and S.
mutans counts was similar to 1% CHX gel.

Introduction postoperative pain, plaque and biofilm, root canals, dentin

Dental caries is caused by the demineralization of the hypersensitivity, temporomandibular joint disorders, and
tooth structure by the organic acids produced by the tooth bleaching procedures.14-21 Ozone can be applied in
oral bacteria in the dental plaque through the anaerobic different forms, such as gas, ozone water, and ozone oil.
metabolism of dietary sugars.1-5 Streptococcus mutans Nagayoshi et al22 evaluated the effect of ozone water
and Lactobacillus acidophilus are found in the cariogenic on the survival of oral and plaque microorganisms and
biofilms and play a significant role in caries.5,6 Decreasing reported that it prevented dental plaque aggregation in
the counts of cariogenic bacteria in the dental plaque is vitro. Anumula et al23 reported that using water containing
one of the measures to prevent the initiation of dental ozone as a mouthwash resulted in a significant decrease in
caries and treat it.7,8 Significant advances have been made S. mutans counts compared to CHX after 7 and 14 days,
in preventing and treating dental caries during the past suggesting that it could be used as an alternative to CHX.
century. Chlorhexidine (CHX) is an antimicrobial agent It is important to prevent dental caries and improve
that can inhibit the growth and proliferation of S. mutans oral hygiene in 6–12-year-old children who are in the
and potentially prevent dental caries.9,10 Using CHX to mixed dentition period, with permanent teeth erupting.14
prevent dental caries is a nonsurgical caries management Previous studies have suggested different materials to
strategy.11 Another way to control the caries process is control dental plaque and decrease the load of cariogenic
by using ozone.12,13 Using ozone has been successful in bacteria. One of the methods to manage dental caries is the
managing wound healing, dental caries, oral lichen planus, use of ozone and CHX. In vitro studies have confirmed the
gingivitis and periodontitis, halitosis, jaw osteonecrosis, role of ozone in controlling dental plaque and decreasing

*Corresponding author: Zahra Molaei, Email: [email protected]

© 2023 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited.
Ebrahim Adhami et al

S. mutans counts.24,25 Ozone water is not very durable, To determine S. mutans counts in both the case group
and its preparation requires special tools.26 On the other and the control group, during the first session, dental
hand, it is difficult to use mouthwashes in this age group. plaque samples were taken before determining the plaque
Therefore, in the present study, the gel form of ozone index, and each sample was separately transferred to
was used in the younger age group, and unlike previous the microbiology laboratory in previously prepared
studies, samples were taken from the dental plaque itself. microtubes. In the laboratory, each sample was dissolved
In addition, a previous study used the gel form of ozone in 500 mL of normal saline and evaluated to determine the
by rubbing it on teeth and gingiva to treat gingivitis.27 colony counts in one of the microtubes in each group to
However, no study has evaluated the effect of ozone gel on determine the proper dilution. In three microtubes, 45 µL
S. mutans in children. of normal saline was placed, and 5 µL from the microtubes
containing the dissolved plaque was transferred into
Methods the first microtube; 5 µL of the first microtube was
In the present double-blind, randomized clinical transferred into the second, and 5 µL from the second
trial, sixteen 6–12-year-old children referring to the was transferred into the third microtube. This way, three
Department of Pediatric Dentistry, Tabriz Faculty of dilutions were prepared in microtubes, and 10 µL from
Dentistry, were evaluated. First, the parents signed an each dilution was separately transferred into MSB (mitis
informed consent form. The inclusion criteria consisted salivarius bacterium) solid agar medium as a selective
of an age range of 6–12 years, systemic health, no known bacterial culture medium and spread on the plate surface
allergy, no antibiotic therapy in the previous three weeks, homogeneously. The plates were incubated for 48 hours
patients with a high risk of caries (dmft > 3),28 and consent at 37 ºC under 95% nitrogen and 5% carbon dioxide,
to participate in the study. The exclusion criteria consisted and the formed colonies were counted. The presence of
of a lack of cooperation in the sampling procedure at the S. mutans was evaluated under a microscope. Of all the
specific time interval, the incidence of allergy symptoms plates, the plate with the lowest colony counts was selected
and signs to the materials used in the study, and not as the proper dilution, and the same concentration was
observing the instructions provided. used for the rest of the microtubes. After counting, the
The sample size was determined at n = 50 in each group number of colonies was multiplied by the reverse of the
using the plaque index value from a study by Indurkar and selected dilution. Since 10 µL of the solution was used for
Verm 27 by considering type I error at 0.05 and an 80% culturing on the plate surface for counting the colonies,
study power. To improve the study’s validity, the sample the achieved numeric value was multiplied by 100 to
size was increased by 10%, and finally, 55 samples were achieve the colony counts in 1 mL. Therefore, S. mutans
included in each group. The samples were assigned to counts were estimated in CFU/mL.30 STATA software
three groups: ozone gel, CHX gel, and control, using the version 14 was used for the statistical analyses of the data
random allocation rule. The patients and the examiner with Wilcoxon, Kruskal-Wallis, and post hoc Dunn tests.
were blinded to the study groups. All the subjects Statistical significance was defined at P < 0.05.
were instructed in oral hygiene measures, including
toothbrushing and flossing. The subjects in the 20-ppm Results
ozone and 1% CHX gel groups rubbed the gels on their The median bacterial counts (the number of colonies)
tooth surfaces twice daily for three weeks in addition to decreased in all the environments after the intervention;
routine oral hygiene measures.29 In the control group, the however, this decrease was significant in CHX and ozone
subjects rubbed one clean finger twice daily on all the tooth environments, with the least median counts in the CHX
surfaces. The subjects refrained from drinking and eating environment (Figure 1). The results showed that the
for half an hour after using the gels.29 The educational film bacterial counts and plaque sizes significantly decreased
was displayed in the parents’ presence and handed to them in all the groups after the intervention (P < 0.05) (Table 1).
in person. The intervention in both groups continued for Table 2 shows no significant differences in bacterial counts
three weeks. Baseline sampling (before intervention) and (P = 0.466) and plaque sizes (P = 0.365) between the groups
three weeks after the intervention were carried out in before the intervention. However, there were significant
the early morning hours when the children were fasting, differences in these two variables between the groups
and it was recommended that they not brush their teeth after the intervention (P = 0.001 for bacterial counts and
or use dental floss before the sampling procedure. After P = 0.003 for plaque sizes). Two-by-two comparisons of
sampling, the subjects brushed their teeth and continued the groups showed that the bacterial counts in the CHX
their routine oral hygiene procedures. In all the subjects, and ozone groups were lower than the control group
the baseline plaque index and S. mutans counts were (P = 0.001); however, there was no significant difference
determined in the samples collected from the buccal between the CHX and ozone groups (P = 0.380) (Table 3).
surface of the most posterior maxillary tooth (right or left In addition, plaque sizes in the CHX and ozone groups
side). The samples were taken from all the surfaces of the were significantly less than those in the control group
tooth in question with a toothpick and transferred into a (P = 0.001). However, there was no significant difference
microtube.29 between the CHX and ozone groups (P = 0.430) (Table 3).

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 Ebrahim Adhami et al

Table 1. Intragroup comparisons (before and after the intervention) of

bacterial counts and plaques between the study groups

Before After
Study groups P value*
Mean SD Mean SD

Bacteria 28750.5 15176 6946.9 10370 0.001

CHX
Plaque 88.32 10.40 30.75 6.70 0.001

Bacteria 29469 16781 8930.9 9225 0.001

Ozone
Plaque 84.07 16.20 29.75 5.92 0.001

Bacteria 35525.8 23850 20619.6 16735 0.001

Control
Plaque 86.14 10.76 34.71 6.35 0.001**
*Wilcoxon signed-rank test.
**Paired t-test.

Table 2. Intergroup comparisons of bacterial counts (colonies) and plaque

Figure 1. The frequency distributions of colony counts in the study groups
between the three groups before and after intervention
before and after the intervention.The means of bacterial counts after
intervention: control > ozone > CHX. The rectangle in the middle of each box
Before After
Study groups P value* P value* plot shows the 25, 50, and 75 percentiles, respectively. The 50 percentile is
(Sum ranks) (Sum ranks)
equal to the median of colony counts
Control 4919.5 6027.5
Bacterial
CHX 4351 0.466 3451.5 0.001 with the present study, the study above showed similar
count
Ozone 4424.5 4216
results concerning the effects of CHX and ozone on
decreasing plaque.
CHX 4398 5207
Patel et al33 reported that ozone gel with an olive oil base
Plaque size Ozone 4972.5 0.365 3862.5 0.003
as a sole treatment for periodontitis significantly improved
Control 4324.5 3491.5
clinical and microbiological parameters over time with no
*Kruskal-Wallis test. complications. Nagayoshi et al22 evaluated the effect of
Table 3. Two-by-two comparisons of the group in terms of bacterial counts ozone water on the survival of oral microorganisms and
and plaque size after intervention dental plaque, concluding that ozone water prevented
Groups Control CHX
dental plaque accumulation in vitro.
Ozone forms oxidized radicals in aqueous environments,
Bacterial counts after the CHX 0.001
intervention
disrupting cellular osmotic equilibrium by penetrating the
Ozone 0.001 0.380
cytoplasmic membrane. It also oxidizes amino acids and
Plaque size after the CHX 0.007 nucleic acid, finally lysing the cell.34
intervention Ozone 0.001 0.430 In the present study, S. mutans counts were evaluated
P values are based on Dunn’s test by culturing in the MSB agar medium. Three weeks after
the intervention, in all three groups, S. mutans colony
counts decreased significantly in the CHX and ozone
Discussion environments, with the least mean colony counts in the
In the present study, 20-ppm ozone and 1% CHX gels were CHX group. Evaluation of S. mutans counts in the study
applied to tooth surfaces twice daily for three weeks. The groups showed that both gels significantly decreased
plaque accumulation was similar between the three groups bacterial counts compared to the control group. In
before the intervention; however, there were significant addition, the effect of CHX and ozone in decreasing
differences between the groups after the intervention, i.e., bacterial counts was similar.
both gels significantly decreased plaque accumulation Unlike the present study, Mon et al35 reported a better
compared to the control group. In addition, the effect of performance of CHX than ozone in decreasing S. mutans
CHX and ozone gels on decreasing plaque was similar. counts at 15- and 30-day intervals in 10–12-year-old
Consistent with the present study, Bulani et al31 reported children. These researchers evaluated the salivary samples
similar effects of ozone oil and CHX gel on decreasing and reported the lowest debris and Oral Hygiene Index-
plaque and gingival indexes as clinical parameters. Simplified Score (OHI-S1) in the ozone water group.
These researchers showed the antimicrobial and anti- Therefore, they recommended ozone-containing water
inflammatory effects of ozone on gingival tissue with no instead of chemical mouthwashes in children.
complications. When ozone is disintegrated into oxygen, an oxygen-
Gandhi et al32 reported no significant differences in PI, rich environment is created, disrupting the plaque’s
GI, PD, CAL, and Pg and Aa counts between the CHX and natural ecosystem. The cellular enzymatic control
ozone groups. Since CHX is the gold standard as an anti- system is inhibited because ozone inhibits glycoproteins,
plaque and anti-gingivitis agent, ozone-containing olive glycolipids, and other amino acids, resulting in the
oil might be used as an auxiliary subgingival irrigation cessation of functions and microorganism death.36
solution in patients with chronic periodontitis. Consistent Unlike the present study and the studies mentioned

J Dent Res Dent Clin Dent Prospects, 2023, Volume 17, Issue 3 179
Ebrahim Adhami et al

in this study, Anumula et al23 reported that the effect of Formal analysis: Leila Erfanparast, Zahra Molaei, Javid Sadeghi,
ozone-containing water on decreasing S. mutans counts Azam Yazdanparast.
Funding acquisition: Ziya Ebrahim Adhami.
was higher than that of CHX; therefore, they suggested
Investigation: Leila Erfanparast, Zahra Molaei, Javid Sadeghi, Azam
continuous use of ozone-containing water as a mouthwash Yazdanparast.
to replace CHX. These researchers evaluated patients with Methodology: Leila Erfanparast, Zahra Molaei, Javid Sadeghi, Azam
a higher rate of dental caries, with an MS rate > 105 CFU. Yazdanparast.
CHX is one of the most commonly used oral Project Administration: Ziya Ebrahim Adhami.
Resources: Javid Sadeghi, Azam Yazdanparast.
antimicrobial agents in different formulations.37 CHX
Supervision: Ziya Ebrahim Adhami.
exerts lethal effects on bacterial membranes and is Validation: Ziya Ebrahim Adhami.
active on gram-negative and gram-positive bacteria. Writing–original draft : Ziya Ebrahim Adhami, Leila Erfanparast,
It lyses bacterial cell wall and precipitates bacterial Zahra Molaei, Javid Sadeghi, Azam Yazdanparast.
cytoplasmic contents. In addition, it destroys bacterial Writing–review & editing: Ziya Ebrahim Adhami, Leila Erfanparast,
Zahra Molaei, Javid Sadeghi, Azam Yazdanparast.
phosphoenolpyruvate and inhibits its metabolic activity.38
Moreover, CHX indirectly affects the enzymatic function Competing Interests
of dehydrogenase and adenosine triphosphates in the The authors deny any conflict of interest.
bacterial cell wall, disrupting the cellular membrane.39
Several other studies have shown the effects of ozone Ethical Approval
on decreasing S. mutans counts. For example, Polydoru et This study was approved by the Ethics Committee of (Ethics code:
IR.TBZMED.REC.1401.304) and registered at the Iranian Registry of
al40 showed the bactericidal effects of ozone on S. mutans Clinical Trials (Identifier: IRCT20220727055562N1; https://www.
after applying it for 80 seconds. In another study, these irct.ir/trial/65219).
researchers showed complete inhibition of S. mutans
growth after applying it for one minute after eight weeks Funding
of follow-up.41 The study was financially supported by Tabriz University of Medical
Sciences.
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