| Abstract|| |
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 2020 Nov 28];30:399-402. Available from: https://www.ijdr.in/text.asp?2019/30/3/399/264120
| Introduction|| |
Dental caries is a type of disease that may begin in childhood and is present throughout the individuals' entire lifetime. 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. 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.,
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.
Mouthwashes have been used for chemical control of bacterial plaque as they help reduce levels of pathogenic bacteria that cause tooth decay., In general, oral antiseptics do not have a complex composition (fluoride salts, cetylpyridinium chloride [CPC] and triclosan, xylitol)., The antimicrobial actions of each mouthwash mainly differs due to the active ingredient present along with the presence or absence of fluoride.
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.
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.
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|| |
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.,
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. 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.
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).
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|| |
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)|
Click here to view
| Discussion|| |
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. 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. 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 , while some others used diffusion method , 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. 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. 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 ,, 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. 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., 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., 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., 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. 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. 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.
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.
| References|| |
Maske TT, van de Sande FH, Arthur RA, Huysmans MCDNJM, Cenci MS.In vitro
biofilm models to study dental caries: A systematic review. Biofouling 2017;9:1-15.
Rossi GN, Sorazabal AL, Salgado PA, Squassi AF, Klemonskis GL. Toothbrushing procedure in schoolchildren with no previous formal instruction: Variables associated to dental biofilm removal. Acta Odontol Latinoam 2016;29:82-9.
Anil S, Anand PS. Early childhood caries: Prevalence, risk factors, and prevention. Front Pediatr 2017;5:157.
Uzer Celik E, Tunac AT, Ates M, Sen BH. Antimicrobial activity of different disinfectants against cariogenic microorganisms. Braz Oral Res 2016;30:e125.
Marinho VC, Chong LY, Worthington HV, Walsh T. Fluoride mouthrinses for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2016;7:CD002284.
Zero DT. Dentifrices, mouthwashes, and remineralization/caries arrestment strategies. BMC Oral Health 2006;6 Suppl 1:S9.
Osso D, Kanani N. Antiseptic mouth rinses: An update on comparative effectiveness, risks and recommendations. J Dent Hyg 2013;87:10-8.
Leite HL, Cavalcante SI, de Sousa EM, Gonçalves LM, Paschoal MA. Streptococcus mutans
photoinactivation using a combination of a high potency photopolymerizer and rose Bengal. Photodiagnosis Photodyn Ther 2016;15:11-2.
de Freitas MT, Soares TT, Aragão MG, Lima RA, Duarte S, Zanin IC, et al.
Effect of photodynamic antimicrobial chemotherapy on mono- and multi-species cariogenic biofilms: A literature review. Photomed Laser Surg 2017;35:239-45.
de Sousa DL, Lima RA, Zanin IC, Klein MI, Janal MN, Duarte S, et al.
Effect of twice-daily blue light treatment on matrix-rich biofilm development. PLoS One 2015;10:e0131941.
McDonalds RE, Avery DR, Dean JA. Dentistry for Child and Adolescent. 7th
ed. St. Louis: CV Mosby; 2000. p. 415-24.
Berkowitz RJ. Mutans streptococci: Acquisition and transmission. Pediatr Dent 2006;28:106-9.
Savas S, Kucukyılmaz E, Celik EU, Ates M. Effects of different antibacterial agents on enamel in a biofilm caries model. J Oral Sci 2015;57:367-72.
Sentila R, Gandhimathi A, Karthika S, Suryalakshmi R, Michael A. In-vitro
evaluation and comparison of the anti-microbial potency of commercially available oral hygiene products against Streptococcus mutans
. Indian J Med Sci 2011;65:250-9.
] [Full text]
LuIs HS, Luis LS, Bernardo M.In vitro
study of the effect of an essential oil and a delmopinol mouth rinse on dental plaque bacteria. Indian J Dent Res 2016;27:648-51.
] [Full text]
Evans A, Leishman SJ, Walsh LJ, Seow WK. Inhibitory effects of antiseptic mouthrinses on Streptococcus mutans
, Streptococcus sanguinis
and Lactobacillus acidophilus
. Aust Dent J 2015;60:247-54.
Slot DE, Berchier CE, Addy M, Van der Velden U, Van der Weijden GA. The efficacy of chlorhexidine dentifrice or gel on plaque, clinical parameters of gingival inflammation and tooth discoloration: A systematic review. Int J Dent Hyg 2014;12:25-35.
Haps S, Slot DE, Berchier CE, Van der Weijden GA. The effect of cetylpyridinium chloride-containing mouth rinses as adjuncts to toothbrushing on plaque and parameters of gingival inflammation: A systematic review. Int J Dent Hyg 2008;6:290-303.
Gunsolley JC. Clinical efficacy of antimicrobial mouthrinses. J Dent 2010;38 Suppl 1:S6-10.
Ayad F, Prado R, Mateo LR, Stewart B, Szewczyk G, Arvanitidou E, et al.
A comparative investigation to evaluate the clinical efficacy of an alcohol-free CPC-containing mouthwash as compared to a control mouthwash in controlling dental plaque and gingivitis: A six-month clinical study on adults in San Jose, Costa Rica. J Clin Dent 2011;22:204-12.
Williams MI. The antibacterial and antiplaque effectiveness of mouthwashes containing cetylpyridinium chloride with and without alcohol in improving gingival health. J Clin Dent 2011;22:179-82.
Slots J. Low-cost periodontal therapy. Periodontol 2000 2012;60:110-37.
Keni NN, Aras MA, Chitre V. Chlorhexidine allergy due to topical application. Indian J Dent Res 2012;23:674-6.
] [Full text]
Fine DH, Furgang D, Markowitz K, Sreenivasan PK, Klimpel K, De Vizio W, et al.
The antimicrobial effect of a triclosan/copolymer dentifrice on oral microorganisms in vivo
. J Am Dent Assoc 2006;137:1406-13.
Shahid M. Regular supervised fluoride mouthrinse use by children and adolescents associated with caries reduction. Evid Based Dent 2017;18:11-2.
Razavi SM, Zarrini G, Molavi G, Ghasemi G. Bioactivity of Malva sylvestris
L. a medicinal plant from Iran. Iran J Basic Med Sci 2011;14:574-9.
Prof. Marco Aurélio Benini Paschoal
Post-Graduate Program of Integrated Dentistry, Ceuma University, Rua Josué Montelo, No 1, Renascença II, São Luís
Source of Support: None, Conflict of Interest: None