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ORIGINAL ARTICLE
Year : 2023 | Volume
: 22
| Issue : 3 | Page : 365-372
Effectiveness of three mouth rinsing agents against mutans Streptococcus and Lactobacillus species – A comparative study
Vijaya Lakshmi Bolla1, M Jyothi2, Shanthan Reddy Mettu2, Mallela G Manoj Kumar2, Kakumanu Nageswara Rao3, Munnangi Surendra Reddy4, Pradeep Koppolu5
1 Asst Professor, Department of Periodontics, Government Dental College and Hospital, Hyderabad, Telangana, India
2 Asst Professor, Professor and Head, Department of Pedodontics and Preventive Dentistry, Panineeya Institute of Dental Sciences, Hyderabad, Telangana, India
3 Associate Professor, Department of Pathology, ACSR, Government Medical College, Nellore, Andhra Pradesh, India
4 Professor, Department of Pedodontics and Preventive Dentistry, SVS Institute of Dental Sciences, Mahabubnagar, Telangana, India
5 Asst Professor, Department of Preventive Dental Sciences, Dar Al Uloom University, Riyadh, Saudi Arabia
Correspondence Address:
Vijaya Lakshmi Bolla
Department of Periodontics, Government Dental College and Hospital, Hyderabad, Telangana
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/aam.aam_95_22
Abstract
Background: Dental caries is one of the most common chronic childhood diseases and is a cause of continuous discomfort through impaired function and esthetics. To control dental caries, the removal of plaque is of utmost importance, and that further necessitates the use of chemotherapeutic agents. Several side effects associated with chlorhexidine have stimulated the search for an alternative chemotherapeutic agent. Aims: This study aims to compare the effectiveness of probiotic mouth rinse with kidodent mouth rinse, and placebo against mutans Streptococcus and Lactobacillus acidophilus (LA). Materials and Methods: The present study was a randomized parallel double-blinded clinical study which included 90 children of ages 6–15 years who were randomly allocated into three groups: Group 1 – placebo (n = 30), Group 2 – kidodent (n = 30), and Group 3 – probiotic (n = 30). Stimulated salivary samples were collected from all the children after rinsing with distilled water (first reading), and after rinsing with respective mouthwash (placebo/kidodent/probiotic mouth rinse) of their groups during the first visit (second readings). Samples were collected again after using mouth rinse for 14 days (third readings) and used for detection of pH, and levels of Streptococcus mutans (SM) and LA counts. Data were subjected to statistical analysis. Results: Statistically significant difference was seen between placebo versus kidodent, placebo versus probiotic but there was no statistically significant difference between kidodent versus probiotic after the immediate rinse and post 15 days of rinse. Conclusion: Kidodent and probiotic mouth rinse are more efficacious and have equivalent efficacy in reducing SM and LA.
Abstract in French Résumé
Contexte: Les caries dentaires sont l'une des maladies infantiles chroniques les plus courantes et sont la cause d'une gêne permanente due à une altération de la fonction et de l'esthétique. fonction et de l'esthétique. Pour lutter contre les caries dentaires, l'élimination de la plaque dentaire est de la plus haute importance, ce qui nécessite l'utilisation d'agents chimiothérapeutiques. agents chimiothérapeutiques. Plusieurs effets secondaires associés à la chlorhexidine ont stimulé la recherche d'un agent chimiothérapeutique alternatif. alternative. Objectifs: cette étude vise à comparer l'efficacité d'un bain de bouche probiotique à celle d'un bain de bouche kidodent, et d'un placebo contre les mutans Streptococcus et Lactobacillus acidophilus (LA). Matériaux et méthodes: La présente étude était une étude clinique randomisée en parallèle et en double aveugle. 90 enfants âgés de 6 à 15 ans ont été répartis au hasard en trois groupes : Groupe 1 - placebo (n = 30), Groupe 2 - kidodent (n = 30), et Groupe 3 - probiotique (n = 30). Des échantillons salivaires stimulés ont été prélevés chez tous les enfants après avoir été rincés à l'eau distillée (première lecture). après s'être rincés à l'eau distillée (première lecture), et après s'être rincés avec les bains de bouche respectifs (placebo/kidodent/rince-bouche probiotique) de leurs groupes lors de la première visite (deuxième lecture). la première visite (deuxième lecture). Les échantillons ont été collectés à nouveau après avoir utilisé le bain de bouche pendant 14 jours (troisième lecture) et ont été utilisés pour la détection du pH, et les niveaux de numération des Streptococcus mutans (SM) et des LA. Les données ont été soumises à une analyse statistique. Résultats: Une différence statistiquement significative statistiquement significative entre le placebo et le kidodent, le placebo et le probiotique, mais il n'y a pas de différence statistiquement significative entre le kidodent et le probiotique après le traitement. kidodent versus probiotique après le rinçage immédiat et après 15 jours de rinçage. Conclusion: Les bains de bouche kidodent et probiotiques sont plus efficaces et ont une efficacité équivalente dans la réduction de la SM et de la LA.
Mots-clés: Bacillus clausii UBBC-07 bain de bouche, kidodent bain de bouche, lactobacilles, bain de bouche probiotique, Streptococcus mutans
Keywords: Bacillus clausii UBBC-07 mouth rinse, kidodent mouth rinse, lactobacilli, probiotic mouth rinse, Streptococcus mutans
Introduction 
Streptococcus mutans (SM) and Lactobacillus acidophilus (LA) are two of the most important microbial factors in dental caries.[1] Dental caries results in both impaired function and may raise concerns of esthetics. The treatment of caries can also be a financial burden for individuals. Thus, there is a need to identify individuals at risk for the development of caries and to target preventive measures along with developing an active treatment for them. In third-world countries, especially, there is a lack of awareness that caries can be prevented by simple hygiene practices and the use of cost-effective, easily available products, which can further help to reduce the global burden in developing countries.[2] The use of chemotherapeutic agents is especially essential for children, as the effectiveness of their tooth brushing, particularly at interproximal sites may be limited due to factors such as lack of dexterity, motivation, or proper supervision.[3]
Kidodent mouth rinse is a combination of mouth rinse containing xylitol (5%), sodium fluoride (0.05%), and triclosan (0.03%). A noncariogenic sugar substitute that occurs naturally, xylitol, cannot be metabolized by oral bacteria. Sodium fluoride inhibits carbohydrate utilization of oral microorganisms by blocking enzymes involved in the bacterial glycolytic pathway and is effective in reducing caries. Triclosan is a nonionic phenolic anti-plaque agent with broad-spectrum antimicrobial activity. There is limited data on triclosan in spite of being incorporated in certain toothpastes and mouth rinses.[4]
The WHO defines probiotics as “living microorganisms that, when administered in proper amounts, provide beneficial effects to the host;” thus, they are often used to treat or prevent different conditions and diseases in humans. They play a role in the prevention of dental caries by their capacity to compete, antagonize and prevent the proliferation of cariogenic bacteria.[5]
Most enormously examined Bacillus species include Bacillus subtilis, Bacillus clausii, Bacillus cereus, Bacillus coagulans, and Bacillus licheniformis. An advantage of including spores such as Bacilli is that they are heat stable and can therefore be stored at room temperature in a desiccated form without affecting the viability. They are also capable of surviving at low pH which other nonspore formers such as Lactobacillus spp cannot. B. clausii UBBC-07 suspension is registered as an over-the-counter medicinal supplement. B. clausii strains also carry resistance to a number of antibiotics including erythromycin, cephalosporins and cycloserine, kanamycin, tobramycin, and amikacin. Thus, probiotic B. clausii with its ability to survive in acidic pH and various mechanisms of action can be used to prevent dental caries.[6]
Materials and Methods The present study was a randomized parallel double-blinded clinical study. All children in the age group of 6–15 years who attended the Outpatient Department of Pedodontics And Preventive Dentistry, Panineeya Mahavidyalaya Institute of Dental Sciences and Research Centre, Hyderabad, India were screened for participation in the study. The ethical committee number is PMVIDSandRC/IEC/PEDO/DN/0106-16.
Selection of the subjects
Children were selected based on the criteria mentioned below.
Inclusion criteria
Children in the age group of 6–15 yearsChildren with <4 carious teeth and poor oral hygieneHad not taken antibiotic or probiotic in any form within 2 weeks before the study.Exclusion criteria
Children with systemic diseases, immune-compromising conditions, and special children were excluded from the study.Study procedure
Clinical examination
The examination was done by a single examiner [Figure 1], [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]b, [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e, [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d, [Figure 4]e, [Figure 4]f, [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]d. The study procedure was explained in detail to the parents of the children selected for the study. Written informed consent was obtained from parents or guardians of the children before the commencement of the study. The research protocol described here was approved by the institutional ethical committee.
Figure 3: Armamentarium. (a) Armamentarium for srceening. (b) Armamentarium for collection of stimulated saliva (c) Armamentarium for estimation of pH. (d) Armamentarium for determination of bacterial counts, (e) Incubator
Figure 4: Clinical procedure. (a) Examination of child. (b) Study groups (mouth rinses). (c) Collection of stimulates saliva sample. (d) Rinsing with Placebo mouth rinse. (e) Rinsing with Kidodent mouth rinse. (f) Rinsing with probiotic mouth rinse
Figure 5: Estimation of pH and determination of bacterial counts. (a) Estimation of pH. (b) Determination of bacterial counts. (c) Grading of streptococcus Mutans. (d) Grading of Lactobacillus acidophilusThe two mouth rinses used in the study were Kidodent mouth rinse from Dr Reddy's Laborotory Ltd, Hyderabad, India and Bacillus clausii UBBC07 and placebo suspensions from Unique Biotech Ltd, Hyderabad, India.
Distribution of subjects [Figure 2]a and [Figure 2]b
Selected 90 children were randomly allocated into three groups using a lottery system as follows [Figure 4]b:
Group 1 – placebo group (n = 30) (control group)Group 2 – kidodent group (n = 30) (experimental group)Group 3 – probiotic group (n = 30) (experimental group).First visit (first readings): the children were made to sit on the dental chair in an upright position and were asked to rinse with distilled water for 1 min (n = 90). Stimulated saliva samples were collected from all the children. The subjects were asked not to eat or drink 30 min before the collection of saliva, and not perform any physical exercises for at least an hour.
Method of collection of saliva sample (stimulated whole saliva) [Figure 3]b and [Figure 4]c
The children were instructed to chew the paraffin tablet for 5 min to stimulate salivation and facilitate the transfer of bacteria from the dental surfaces to the saliva. Saliva samples were collected in the collection cups at the interval of 15–20 s within the stipulated 5 min of time. The collected saliva samples were used for the detection of pH and determination of SM and LA counts.
Detection of pH of saliva [Figure 3]c and [Figure 5]a
Determination of the salivary pH for each sample was performed, using a special test paper (CRT buffer) and readings were recorded.
Microbiological examination [Figure 3]d and [Figure 5]b
Semiquantitative determination of SM and Lactobacillus was carried out using the CRT® Bacteria Test (Ivoclar Vivadent). The blue Mitis Salivarius agar with bacitracin was used to detect mutans streptococci, whereas the light culture medium with Rogosa agar was used to evaluate lactobacilli. The agar carrier was removed from the test vial and NaHCO3 tablet was placed at the bottom of the vial. After the removal of protective foils carefully, both the agar surfaces were coated with the saliva collected using a pipette and excess saliva was allowed to drip off. The agar carrier was slid back into the vial and closed tightly. The test vial was placed upright in an incubator and incubated at 37°C/99°F for 48 h [Figure 3]e. After the removal of the vial from the incubator, the density of the SM and lactobacilli colonies were compared with the corresponding evaluation pictures in the enclosed model chart and readings were recorded as first (baseline) readings.
Grading of Streptococcus mutans and Lactobacillus acidophilus colony-forming units [Figure 5]c and [Figure 5]d
Grade 1, Grade 2 = Inactive lesions (IA) – <105 CFU/mlGrade 3, Grade 4 = Active lesions (A) – > or = 105 CFU/ml.First visit (second reading): the mouth rinses were dispensed by the other examiner to eliminate the bias as it was a double-blind design. The children were asked to rinse with the respective mouthwash (placebo/kidodent/probiotic mouth rinse) that was randomly allocated in their groups.
Distribution of samples
Group 1 (n = 30) – rinse with 10 ml of placebo for 1 min [Figure 4]dGroup 2 (n = 30) – rinse with 10 ml of kidodent mouthwash for 1 min [Figure 4]eGroup 3 (n = 30) – rinse with 10 ml of probiotic mouthwash for 1 min [Figure 4]f.Stimulated saliva samples were collected from the children after rinsing with allotted mouthwash and used for detection of pH and determination of SM and LA counts. The readings thus obtained were recorded.
Intervention period of 14 days
The children were prescribed to use the allocated mouthwash twice daily for 1 min in the interval of 12 h for 14 days, 30 min after tooth brushing, and were instructed not to drink or eat for 30 min after usage of mouthwash. They were encouraged to maintain routine oral hygiene practices throughout the intervention period. The parents were asked to supervise the child during the use of mouthwash. No treatment was performed in the intervention period.
Second visit (third readings): the children were recalled after 14 days. Stimulated saliva samples were collected as described in the first visit and used for detection of pH and determination of SM and LA counts. Thus third readings were obtained.
Statistical analysis
Data obtained were recorded in a tabular form and were subjected to statistical analysis. The statistician was kept blinded with regard to the allocation of subjects to the three groups. Data were analyzed using the Statistical Package for the Social Sciences version 23 (SPSS Inc., Chicago, Illinois, USA). Statistical significance was set at 0.05 (P < 0.05 was considered statistically significant). Overall intra and intergroup comparisons were carried out using the Friedman test and Kruskal–Wallis H test, respectively, and individual pairwise intra and intergroup comparisons were carried out using Wilcoxon signed-rank test and Mann–Whitney U test, respectively.
Results Intragroup comparison of SM colony-forming units (CFU) at various durations [Table 1] showed statistically significant difference at baseline, immediate, and post 15 days intervention in Group 1 (P = 0.018), Group 2 (P < 0.001), and Group 3 (P = 0.027).
Table 1: Intragroup comparison of Streptococcus mutans colony-forming unitIntergroup comparison of SM CFU [Table 2] showed a statistically significant difference between Group 1 versus Group 2 and Group 1 versus Group 3 but no statistically significant difference between Group 2 versus Group 3 after the immediate rinse and post 15 days of rinse.
Table 2: Intergroup comparison of Streptococcus mutans colony-forming unitIntragroup comparison LA CFU at various durations [Table 3] showed a statistically significant difference in comparison between baseline, immediate, and post 15 days intervention in Group 2 (P < 0.001) and Group 3 (P < 0.001) but no statistical significance in Group 1 (P = 0.846.)
Table 3: Intra group comparison of Lactobacillus acidophilus colony-forming unitIntergroup comparison of LA CFU [Table 4] revealed that there was no statistically significant difference between any comparisons at baseline. A statistically significant difference was present between Group 1 versus Group 2 (P = 0.030) and Group 1 versus Group 3 (P = 0.030) but no statistically significant difference between Group 2 versus Group 3 (P = 1) after immediate rinse. There was a statistically significant difference between Group 1 versus Group 2 (P < 0.001) and Group 1 versus Group 3 (P < 0.001) but no statistically significant difference between Group 2 versus Group 3 (P = 0.787) at post 14 days.
Table 4: Intergroup comparison of Lactobacillus acidophilus colony-forming unit Discussion The oral cavity harbors many microorganisms some being detrimental to the tooth structure. SM plays a major role in the initiation of caries, a common problem observed in all age groups. Pellicle a salivary coating when habituated by bacteria is termed plaque. The plaque provides the domain for the activities of lactobacilli to produce lactic acid, which dissolves the enamel by decalcifying it with extracellular dextran-based polysaccharide that allows them to cohere forming plaque on tooth surfaces.[7] Thus, the present study uses SM and LA which play a vital role in caries formation and progression, respectively.
The oral cavity is a habitat for temporary and permanent microorganisms. Some of these microorganisms play a major role in the occurrence of dental caries. The relationship between Streptococcus species and dental caries has been well documented with SM playing a major role in the initiation of caries. Sucrose is used by SM to produce a sticky, extracellular dextran-based polysaccharide that allows them to cohere forming plaque on tooth surfaces. The plaque provides the domain for the activities of lactobacilli to produce lactic acid, which dissolves the enamel by decalcifying it.[7] Thus, the present study uses SM and LA which play a vital role in caries formation and progression, respectively (can delete if the reframed is fine).
Mechanical plaque control is an effective method for the prevention of oral diseases, but it requires patient cooperation and motivation; therefore, chemical plaque control can be used as an adjunct for achieving the desired results.[8] Mouth rinses should be effective and safe for regular use in children. They are recommended only for those who have the ability to swish and expectorate without swallowing.[4] Hence, the present study included children >6 years of age.
Chemical agents act as an adjunct to mechanical control. They cannot replace mechanical plaque control. Although chlorhexidine is considered a gold standard chemical agent it has been reported to have a number of local side effects such as brown discoloration of the teeth and tongue, oral mucosal erosion, dry mouth, and unpleasant taste, necessitating the need to search for alternative chemical agents.[9]
Kidodent mouth rinse containing xylitol, sodium fluoride, and triclosan was used in this study. Xylitol disrupts the energy production process of SM, leading to a futile energy consumption cycle and cell death, thereby exhibiting effectiveness in the prevention of caries. It is conjectured that fluoride affects streptococcal cells by inhibiting one of the series of glycolytic enzymes, namely, enolase. Triclosan is said to increase the ability of mouthwash to bind to the oral mucosa, and thus, be available for longer periods of time.[4]
The probiotics approach, on the other hand, has been credited as a breakthrough in maintaining oral health. They use the beneficial bacteria commonly found in healthy mouths to fight against pathogenic bacteria. They also lower the pH, thus disabling microorganisms from producing the dental plaque and calculus that cause oral inflammation. They produce antioxidants that prevent stain and plaque formation by neutralizing the free electrons that are needed for mineral formation.[10] As the idea of replacing harmful microorganisms with nonharmful, inactivated, or genetically modified bacteria is attractive, the present study includes a probiotic mouth rinse (B. clausii UBBC-07). It is an established probiotic for intestinal health but no study until now had reported its relation to oral health. B. clausii is an aerobic, spore-forming bacterium that is able to survive transit through the acidic environment of the stomach and colonize the intestine even in the presence of antibiotics (Urdaci MC et al. 2004). B. clausii UBBC-07 is available for oral use as a suspension of 2 billion spores/5 mL. The immunomodulatory capacity of these B. clausii strains could be the result of the expression of some extracellular and/or cell wall-associated compounds involved in immuno-stimulation.[11]
A parallel-group study design was chosen in this study and three different groups of subjects received three different mouth rinses under similar conditions. The drawback of a crossover study is contamination or carryover. There is a possibility that some effect of the treatment used in one period might persist and alter the response observed in later treatment periods.[12] Stimulated whole saliva samples according to Krasse were the most convenient way to determine the level of SM and Lactobacillus spp. infection.[13] The possibility of semiquantitative determination of the salivary level of SM and Lactobacillus represents major progress in the current dental practice. CRT bacteria enable the simultaneous determination of the mutans streptococci and lactobacilli counts in saliva by means of selective agars. Juri and UkoviBagi concluded that the application of CRT Bacteria Tests represents a simple clinical procedure, which enables the detection of a patient's high risk of caries and at the same time provides some guidelines for the preventive procedure that can ensure appropriate disease prevention.[14]
To the best of our knowledge, there are no studies to date comparing kidodent mouth rinse with probiotic mouth rinse in the literature. Thus, the present study was conducted comparing these two mouth rinses against SM and LA.
Results in [Table 1] showed that Group 2 (kidodent) and Group 3 (probiotic) exhibited a reduction of active carious lesions and a statistically significant difference. Our results are in accordance with a study conducted by Arunakul et al.[15] but contradict the findings of Gonçalves et al.[16][Table 2] showed that kidodent and probiotic mouth rinses have statistically equivalent efficacy in reducing SM. This was in accordance with a study conducted by Subramaniam and Nandan[4][Table 3] showed no statistical significance in Group 1 (placebo), whereas Group 2 (kidodent) showed a reduction of active lesions. Hegde and Kamath[17] in a study found that a combination mouth rinse led to a statistically significant reduction (P < 0.001) of salivary SM and lactobacilli counts. Group 3 (probiotic) showed a reduction of active lesions and a statistically significant difference was noticed. [Table 4] showed a statistically significant difference between placebo versus kidodent and placebo versus kidodent but no statistically significant difference between kidodent versus probiotic after the immediate rinse and post 15 days of rinse.
Overall, the results of the present study showed that kidodent and probiotic mouth rinse are more efficacious in reducing SM and LA than placebo. Moreover, kidodent and probiotic mouth rinses have no statistical difference in reducing SM and LA.
With an increasing global problem of antibiotic resistance, probiotics by virtue of their natural therapy and potential therapeutic value can be used as an effective alternative to other traditional mouth rinses as a novel approach for reducing the incidence of caries.
The limitations of this study were as follows:
The two-week period may be insufficient to evaluate the substantivity of mouth rinsesA wash-out period would have been more authenticating.Further long-term research may be proposed for determining the minimum duration of probiotic administration required to produce an optimal effect and numerous randomized clinical studies will be required to clearly establish the potential of probiotics in preventing and treating oral infections and to get information on safety, side effects, and carryover effect of such bacteriotherapy.
Conclusion From the present study, it can be concluded as follows:
Kidodent mouth rinse and probiotic mouth rinse are more efficacious in reducing SM and LA than the placebo groupKidodent and probiotic mouth rinses have equivalent efficacy in reducing SM and LA.Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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