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Year : 2019  |  Volume : 30  |  Issue : 3  |  Page : 399-402
In vitro antimicrobial potential of infant mouthwashes against streptococcus mutans biofilm: A preliminary study

Health Sciences Center, School of Dentistry, Ceuma University, São Luís, Brazil

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Date of Web Publication9-Aug-2019


Background: Children and teenagers accumulate dental plaque easily due to immature motor coordination present at this specific age. Thus, chemical solutions such as mouthwashes are used for biofilm control. The widespread use of mouthwash could potentially change the oral environment though there is no evidence of its effects on the biofilm. Aim: The present study aimed to investigate the in vitro antimicrobial potential of infant mouthwashes on mature Streptococcus mutans biofilm. Methods: The susceptibility of S. mutans biofilm UA 159 (ATCC700610) to infant mouthwashes was tested with childrens mouthwashes containing the following active agents: G1-cetylpyridinium chloride, G2-xylitol and triclosan and G3-Malva sylvestris and xylitol. Phosphage-buffered saline (PBS) was used at the negative control (G4). In this study, cariogenic biofilm was exposed once a day for one minute to the mouthwashes over a period of five days. Following this, an aliquot of each mouthwash used was seeded in brain heart infusion (BHI) agar and then incubated at 37°C, 5% CO2 for 48 h. The results were expressed as colony-forming units (CFU) and converted into log10. The results were submitted to ANOVA and Tukey's test at 5%. Results: It was observed 7.75, 7.66, and 7.49 CFUlog10 values to G1, G2, and G3, respectively, with 9.53 CFUlog10 value to G4. Accordingly, all studied mouthwashes showed no significant statistical difference between them but with statistically significant bacterial reduction in comparison to control group. Conclusion: Infant mouthwashes presented a highly significant antimicrobial effect on cariogenic biofilm in an in vitro model, which raises concern when used by a young population.

Keywords: Biofilm, dental caries, mouthwashes

How to cite this article:
Sampaio GG, Leódido G, Gonçalves LM, Paschoal MA. In vitro antimicrobial potential of infant mouthwashes against streptococcus mutans biofilm: A preliminary study. Indian J Dent Res 2019;30:399-402

How to cite this URL:
Sampaio GG, Leódido G, Gonçalves LM, Paschoal MA. In vitro antimicrobial potential of infant mouthwashes against streptococcus mutans biofilm: A preliminary study. Indian J Dent Res [serial online] 2019 [cited 2023 Sep 28];30:399-402. Available from:

   Introduction Top

Dental caries is a type of disease that may begin in childhood and is present throughout the individuals' entire lifetime.[1] It is closely related to the accumulation of dental biofilm, known to be a dense, noncalcified, well-structured mass, made up exclusively of the bacteria involved, enmeshed in a sticky matrix that firmly adheres to the teeth.[1] High sucrose consumption and difficulty in removing the biofilm are factors that contribute to the prevalence of dental caries in socioeconomically underprivileged children and teenagers. This could be due to lack of knowledge on the part of the parents and the children themselves regarding healthy oral habits.[1],[2]

Different methods are used to control dental biofilm. They include mechanical resources (dental floss and toothbrushing), coadjuvant factors such as chemical substances (mouthwashes) or by food consumption with low carbohydrate concentrations and low cariogenic potential.[3]

Mouthwashes have been used for chemical control of bacterial plaque as they help reduce levels of pathogenic bacteria that cause tooth decay.[4],[5] In general, oral antiseptics do not have a complex composition (fluoride salts, cetylpyridinium chloride [CPC] and triclosan, xylitol).[4],[5] The antimicrobial actions of each mouthwash mainly differs due to the active ingredient present along with the presence or absence of fluoride.[6]

Sodium fluoride, CPC, triclosan, chlorhexidine (CHX), thymol and xylitol are the most common active components of mouthwashes due to their cariostatic effects on dental biofilms.[7]

The current recommendation is that they should be used once a day, preferably after brushing at night, and their use in children is recommended to be from 6 years of age onwards as children above this age know how to rinse their mouths without swallowing the solution.[6]

This might not be followed on a daily basis due to a lack of knowledge regarding the correct use/indications of mouthwashes by parents; lack of professional guidance regarding use of the product as it is available in the market without prescription. A combination of these factors results in widespread unsupervised use of this product, thus leading to adverse side effects in children and teenagers.

This study was performed to evaluate the antimicrobial efficacy of these products against Streptococcus mutans in biofilms due to the aformentioned factors.

   Methods Top

The susceptibility of biofilms of S. mutans UA 159 (ATCC700610) to the following infant mouthwashes was tested: G1-CPC (Cepacol Teen– Safoni Aventis Farmacêutica Ltda., Suzano, SP, Brazil), G2-xylitol and triclosan (Dentalclean Garfield-Rabbit Ind. Com de Prod. de Higiene Pessoal Ltda., Londrina, PR, Brazil) and G3-Malva sylvestris and xylitol (Malvatrikids Júnior-Daudt Oliveira Ltda., Rio de Janeiro, RJ, Brazil). Phosphate-buffered saline solution was used as a negative control.

Bacterial inoculum preparation

The S. mutans strain was placed in brain heart infusion (BHI) with 1% glucose for 18–24 h. After this period, the suspension was submitted to washing, and the turbidity of the resulting material was adjusted by means of spectrophotometer (BEL Engineering, SP, Brazil) until absorbancy similar to that of a suspension for stock solution at 1 × 106 bacteria/ml was attained.[8],[9]

Study design model

After reactivation in the previously described medium, aliquots of S. mutans (1 ml) were transferred to 24-well plates containing hydroxyapatite discs (Clarkson Chromatography Inc., South Williamsport, PA) simulating the tooth surfaces and kept in contact for 24 h to allow bacterial adhesion. After this period, the medium was replaced with nutrient-rich BHI solution containing 1% sucrose, changed daily, for the period of 5 days to allow cariogenic biofilm formation under 37°C, 5% CO2.[10] Before the daily change of culture medium, the discs were exposed to 1 ml of the mouthwashes for 1 min. The same procedure was performed for the negative control group.

Quantitative analysis

For quantitative analysis, the discs were transferred to tubes containing 5 ml PBS and submitted to sonication by using three pulses of 15 s at a power of 6 W (Branson Sonifier 150; Branson Ultrasonics, Danbury, CT) at intervals of 15 s each. A volume of 100uL of the homogenized suspension was used for serial decimal dilution, and afterwards seeded on BHI agar and incubated at 37°C, 5% CO2 for 48 h. The results were expressed as colony-forming units (CFU) and transformed into log10. The tests were performed in triplicate for each group and repeated twice (n = 6).

Statistical analysis

After checking assumptions of equality of variances and normal distribution data, Anova test was used to analyse the difference in CFU values of the mean value obtained in all groups. This was followed by the Tukey's test, taking the value of 5% as reference for statistically significant differences. All tests were performed using the software program SPSS for Windows, version 23.0 (IBM, Armonk, NY, USA).

   Results Top

According to the data analysis obtained, illustrated in [Figure 1], the authors observed that the biofilms formed by S. mutans exposed to the three children's mouthwashes tested on a daily basis for five days showed reduction in bacteria with statistically significant difference in comparison with the control group (P < 0.05), however, without statistical difference between them (P > 0.05).
Figure 1: Colony-forming unit/ml (Log10) values of Streptococcus mutans biofilms after treatment tested. Different letters indicate significant statistical differences (*ANOVA followed Tukey's test, P < 0.05). * Different lower case letters denote statistical difference at 5% of significance level (ANOVA test followed by Tukey's test)

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   Discussion Top

The aim of this study was to analyze the antimicrobial action of infant mouthwashes based on CPC (Cepacol Teen), triclosan/xylitol (Dentalclean Garfield) and Malva sylvestris/xylitol (Malvatrikids Júnior), and the use of PBS as negative control, against biofilms formed by S. mutans. As a result, it was verified that all mouthwashes tested presented antimicrobial action against the cariogenic biofilm tested.

Oral health improvement is an integral component of good general health. Many children and adolescents have inadequate oral and general health because of active and uncontrolled dental caries.[11] Oral microorganisms are considered crucial for the initiation and progression of dental caries. Among the various microorganisms studied, S. mutans have been regarded as one of the most virulent caries-producing organisms.[12] As S. mutans is an important cariogenic bacteria and reducing its counts is associated with a decrease in caries risk, it is worthwhile examining the comparative effects of commercial antiseptics on S. mutans to determine their potential as anticaries agents.

In the present study, it was used on a monospecies biofilm compounded by S. mutans. Other investigations have also used the same bacteria in a biofilm counterpart [13],[14] while some others used diffusion method [15],[16] with different microorganisms including S. sanguinis and Lactobacillus acidophilus, both involved with caries etiology and progression. There are advantages testing the current products on biofilm rather than planktonic cells or diffusion method as this provides better understanding on how they act in a way that is similar to what occurs inside the oral cavity and also closely simulates in vivo conditions.

Results of various studies have pointed out the efficacy of mouthwashes against different bacteria present in the oral cavity. Corroborating with the present investigation, Savas et al.[13] demonstrated the efficacy of different antibacterial agents against S. mutans monospecies biofilm. The results showed that biofilms treated with cetylpyridinium chloride achieved a substantial bacterial reduction in comparison to other studied substances. Added to these results, Sentila et al.[14] investigated the antimicrobial effects of mouthwashes and toothpastes containing triclosan on S. mutans biofilms. In conclusion, the oral hygiene products containing triclosan, independent of product (mouthwash X toothpaste), proved to be the most effective followed by those containing fluoride and herbal products only. In addition, the effects of antiseptic agents have been tested on periodontal tissues as well [17],[18],[19] achieving good results regarding gingival bleeding. However, since the principal issue in children is still dental caries, the focus of the present study was based on the effects of mouthwashes against cariogenic bacteria.

Regarding the results, it could be observed that the CPC mouthwash had the greatest bacterial reduction (2.04 log10 reduction), with no statistical difference with the other studied mouthwashes against S. mutans biofilms. CPC is an antiseptic quaternary ammonium compound with a high affinity for Gram-positive bacteria such as Streptococcus mutans.[20] As with CHX, CPC has a high binding affinity for negatively charged bacterial cell walls. It causes membrane disruption, leakage of cytoplasmic components, and inhibition of metabolism and proliferation. In dental biofilms, it prevents cellular aggregation and thus plaque maturation.[20],[21] Currently, there is minimal evidence for the use of CPC in children as an anticaries agent although studies have demonstrated that presurgical 0.05% CPC mouthrinses by teenagers aged 10–15 years are efficacious at reducing both aerobic and anaerobic microorganisms.[22],[23] Cepacol Teen, tested in the present study, containing 0.05% CPC, presented additional inhibitory actions possibly related to other components such as surfactants. As is already known, 0.12% CHX is considered as the gold standard oral antiseptic. Since mouthrinses may not be suitable for children younger than 6 years and due to CHX side effects, other antiseptics are also being currently investigated. The advantages of CPC can thus be summarized as follows: same efficacy of CHX regimen, lower costs and reduced chance of patient intolerance.[22],[23] Although the use of these substances is not recommended by pediatric dentists, many children and teenagers are often using this without proper guidance, which demonstrates the importance of studies of this nature aiming to provide clear and current information to this population and their parents as well.

Apart from the CPC results, other active agents tested were also effective against cariogenic bacteria. The triclosan-based mouthwash (Dentalclean Garfield) contained coadjuvants such as xylitol and sodium fluoride that acted together. Triclosan alone had only a moderate effect on plaque formation, whereas in addition to another substance, this effect could be greater. Corroborating with these results, an in vivo investigation demonstrated that this substance associated with a copolymer (that was not tested here) was capable of diminishing the population of the species Veillonella and Fusobacteria, by 89.8% and 91.2%, respectively.[24] In addition, the fluoride present in the tested mouthwashes also helped remineralize tooth enamel, interfering in the metabolism and growth of acid producing bacteria in bacterial plaque, and inhibiting the formation of polysaccharides that promote the adhesion of bacteria to the enamel surface, thus resulting in bacterial reduction.[25] Yet, M. sylvestris, same active agent found in G3, has proved to be an efficient antiseptic, chemopreventive/chemotherapeutic plant extract on different pathogens and bacteria, as verified in the current investigation.[26]

Irrespective of how promising the results were shown to be, there are limitations to this type of study. These include in vitro study, using hydroxyapatite discs instead of human and/or bovine dental substrates and the absence of saliva, which would all be vital factors for verifying the potential of mouthwashes.

In relation to antibacterial activity of the mouthwashes tested in vitro, there was also inhibition of S. mutans growth in the biofilms. Therefore, the oral antiseptic products presented in this study might be complementary options for controlling dental biofilm where necessary. There could also be related side effects to unsupervised use of these products, especially among children. Adequate information should be provided to the parents to warn them of the side effects from indiscriminate use of these substances at an early age.


Since the use of these mouthwashes deemonstrated a definite reduction in the number of cariogenic bacteria, the chance of development of oral microbiota modification with resultant antimicrobial resistance should be studied, especially in children. Hence, efforts of dental professionals and advertising sector must propagate the indications and side effects of the unrestricted use of those substances.

Financial support and sponsorship

Publication support was sponsored by Fundação de Amparo à Pesquisa e Desenvolvimento do Estado do Maranhão (FAPEMA).

Conflicts of interest

There are no conflicts of interest.

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Correspondence Address:
Prof. Marco Aurélio Benini Paschoal
Post-Graduate Program of Integrated Dentistry, Ceuma University, Rua Josué Montelo, No 1, Renascença II, São Luís
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijdr.IJDR_500_17

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