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Acta Odontológica Latinoamericana

versión On-line ISSN 1852-4834

Acta odontol. latinoam. vol.28 no.2 Buenos Aires ago. 2015



Antiseptic mouthwashes: in vitro antibacterial activity


Evandro Watanabe1, Andresa P. Nascimento2, Juliane M. Guerreiro-Tanomaru3, Ana M. Razaboni1, Denise de Andrade4, Mário Tanomaru-Filho3

1 Department of Restorative Dentistry. University of São Paulo (USP), School of Dentistry of Ribeirão Preto. Ribeirão Preto, SP, Brazil.
2 Apis Flora Industrial e Comercial, Ribeirão Preto, SP, Brazil.
3 Department of Restorative Dentistry. São Paulo State University (UNESP), Araraquara School of Dentistry. Araraquara, SP, Brazil.
4 Department of General and Specialized Nursing, University of São Paulo (USP), Ribeirão Preto Nursing School, Ribeirão Preto, SP, Brazil.

CORRESPONDENCE Prof. Dr. Evandro Watanabe Faculdade de Odontologia de Ribeirao Preto Universidade de Sao Paulo. Departamento de Odontologia Restauradora. Avenida do Cafe s/ no, Monte Alegre, Ribeirao Preto, SP, Brasil. CEP: 14.040-904 E-mail:


Mouthwashes are used as an adjunct to tooth brushing for improving breath and preventing oral diseases. The aim of this study was to compare the in vitro Maximum Inhibitory Dilution (MID) of 3 mouthwashes with different active ingredients against mutans streptococci (MS). The products analyzed were PeriogardR, CepacolR and PlaxR Fresh Mint. Their antibacterial activity was assessed in duplicate in 96-well microtiter plates against 36 clinical isolates of MS. Each mouthwash was submitted to a serial two-fold dilution (1/2.5 to 1/5120) using double concentration of Tryptose Soy Broth with 1.0% yeast extract. The final volume in each well was 100 mL plus 5 mL of a bacterial suspension, equivalent to 107 CFU/mL. They were incubated microaerobically at 37oC for 48 hours and the MIDs determined. MID was 1/320 for PeriogardR and CepacolR, and 1/20 for PlaxR. Statistical analysis revealed that the MID of PeriogardR MID did not differ from that of CepacolR (p>0.05), and was higher than that of PlaxR (p<0.05). In conclusion, the antiseptic mouthwashes containing chlorhexidine (PeriogardR) and cetylpyridinium chloride (CepacolR) had higher in vitroantibacterial activity (MID) against MS than the antiseptic mouthwash containing triclosan (PlaxR), according to microbiological method employed.

Keywords: Microbial Sensitivity Tests; Mouthwashes; Streptococcus.


Antissépticos bucais: atividade antibacteriana

Os antissepticos bucais sao utilizados mundialmente como adjuvantes da escovacao para melhoria do halito e prevencao de doencas bucais infeciosas. O objetivo deste estudo foi comparar in vitro a Diluicao Inibitoria Maxima (DIM) de 3 antissepticos bucais com diferentes principios ativos contra estreptococos do grupo mutans (EGM). Os produtos analisados foram PeriogardR, CepacolR e PlaxR FreshMint. A atividade antibacteriana foi avaliada em duplicata em placas de microtilulacao de 96 pocos contra 36 isolados clinicos de EGM. Cada antisseptico bucal foi submetido a diluicao dupla seriada (1/2,5 a 1/5120) com o emprego de concentracao dupla de TryptoseSoyBrothwith adicionado de 1,0% de extrato de levedura. O volume final em cada poco foi de 100 mL mais 5 mL da suspensao bacteriana equivalente a 107 UFC/mL. A incubacao foi realizada em microaerofilia a 37oC por 48 horas e a DIM deteminada. PeriogardR e CepacolR apresentaram DIM de 1/320, e PlaxR de 1/20. Os resultados submetidos asanalises estatisticas revelaram que a DIM do PeriogardR nao foi diferente do CepacolR (p>0,05) sendo maior que do PlaxR (p<0,05). Em conclusao, os antissepticos bucais contendo clorexidina (PeriogardR) e cloreto de cetilpiridinio (CepacolR) demonstraram maior atividade antibacteriana in vitro (DIM) contra os EGM do que o antisseptico bucal contendo triclosan (PlaxR) de acordo com o metodo microbiologico utilizado.

Palavras-chave: Testes de Sensibilidade Microbiana; Antissepticos Bucais; Streptococcus.



Mouthwashes have been widely employed in the fields of Preventive Dentistry and Periodontics1-3. The use of antimicrobial mouthwashes has been proposed as a means for reducing the levels of oral bacteria, specifically Streptococcus mutans. A wide range of mouthwashes containing different active ingredients is available in the market. PeriogardR is a mouthwash which contains 0.12% chlorhexidine gluconate and other ingredients (Table 1). Chlorhexidine is a cationic biguanide with broad-spectrum antimicrobial action, effective against dental biofilm and gingivitis3-6. Chlorhexidine has an important feature, substantivity, due to its ability to bind to oral tissues and act for extended periods after application4. CepacolR is a cetylpyridinium chloride-based (CPC) mouthwash (Table 1).

Table 1: Chemical composition of the mouthwashes.

CPC is a quaternary ammonium compound included in the group of the cationic surface-active agents7. It acts primarily by penetrating the cell membrane, causing leakage of cell components, disruption of the bacterial metabolism, inhibition of cell growth, and finally, cell death8. PlaxR contains 0.03% triclosan (2,4,4'-trichloro-2'- hydroxydiphenyl ether), 0.20% polyvinyl-methyl ether/maleic acid (PVM/MA) copolymer which is used jointly with triclosan to increase its antimicrobial activity,9 and other ingredients (Table 1). Due to their ability to form biofilms on teeth, mutans streptococci (MS) are considered major etiological agents of human dental caries10. Thus, mouthwashes containing active ingredients against them may help prevent caries. Mouthwashes are expected to maintain their antimicrobial activity even after dilution in oral fluids, and their antimicrobial activity against different strains of S. mutans can be evaluated in vitro, The aim of this study was to compare in vitro Maximum Inhibitory Dilution (MID) of 3 mouthwashes containing different active ingredients against 36 clinical isolates of MS.


The following products were evaluated: PeriogardR (Colgate-Palmolive, Sao Bernardo do Campo, SP, BR), CepacolR (Aventis Pharma, Suzano, SP, BR) and PlaxR Fresh Mint (Colgate-Palmolive, Sao Bernardo do Campo, SP, BR) as shown in Table 1.Antibacterial activity was assessed in duplicate in 96-well microtiter plates against 36 clinical isolates of mutans streptococci (MS). Each mouthwash was submitted to a serial two-fold dilution (1/2.5 to 1/5120) using double concentration of Tryptose Soy Broth with 1.0% yeast extract. The final volume in each well was 100 mL plus 5 mL of a bacterial suspension equivalent to 107 CFU/mL. They were incubated microaerobically at 37oC for 48 hours and the MIDs determined (the highest dilution of each product that inhibited the bacterial growth).

Statistical analysis
Results were expressed as scores determined from MID. Groups were compared using the Kruskal- Wallis nonparametric test. When this test showed significant difference between groups, Dunn's multiple comparison test, which allows two-by-two comparison between groups, was applied. The significance level in the statistical testing was 5% (p<0.05).


The mouthwashes had different MIDs. Statistical analysis revealed that the MID of PeriogardR did not differ from that of CepacolR (p>0.05) but was higher than that of PlaxR (p<0.05) against 36 clinical isolates of MS (Table 2).

Table 2: Data of 36 clinical isolates of mutans streptococci inhibited by each dilution of the mouthwashes.


A wide range of mouthwashes containing different active ingredients is available on the market. It is important to know their antimicrobial activity because they are mainly employed to control microorganisms. This study conducted a comparative analysis among three different mouthwashes - chlorhexidine gluconate, cetylpyridinium chloride and triclosanbased solutions - which were effective against MS from the initial dilution of 1/2.5. Chlorhexidine gluconate mouthwashes have been available on the market for a long time with concentrations ranging from 0.12% to 0.2%. The chlorhexidine-based mouthwash evaluated in this study (PeriogardR) contains 0.12% chlorhexidine gluconate. In this study, PeriogardR had the best in vitro antibacterial activity against MS, because it inhibited all the clinical isolates of MS at the 1/320 dilution. However, our results disagree with MIDs found in other studies, of 1/1611, 1/8012 and 1/16013. Other studies found that CepacolR was able to inhibit MS at 1/2012 and 1/4013 dilutions. In contrast, our study found that all clinical isolates of MS were inhibited by CepacolR at 1/320 dilution. In our study, PlaxR had lower MID than PeriogardR and CepacolR (p<0.05) and inhibited the growth of all MS at the 1/20 dilution, in agreement with Andre et al. 13. Thus, triclosan used with PVM/MA copolymer had in vitro antibacterial activity against the different clinical isolates of MS.
McMurry, Oethinger and Levy14 demonstrated in a study with Escherichia coli that the antibacterial activity of triclosan is due to its ability to block the synthesis of fatty acids by inhibiting the enoyl-acyl carrier protein reductase enzyme. This specific action affects several lipid synthesis-dependent processes, resulting in secondary effects on the cytoplasmic membrane15. Giertsen16 analyzed mouthwashes containing triclosan (3.5mM) associated with sodium lauryl sulphate (17.4mM), which were found to have a synergic effect against S. mutans(NTCC 10449). The MIC of triclosan and sodium lauryl sulphate against S. mutanswas 13.49μM. Moreover, sodium lauryl sulphate is an anionic surfactant that has often been used in mouthwashes and toothpastes and it is included in the PlaxR formula (Table 1). In addition, PlaxR contains a sodium fluoride
concentration of 270 ppm (270 μg/mL), which is less than that required for the MIC of 600 μg/mL against S. mutans17.
Herrera et al. 18 evaluated the antimicrobial activity of four commercially available mouthwashes, three containing 0.12% chlorhexidine (one containing alcohol and two alcohol-free) and one containing 0.12% chlorhexidine and 0.05% CPC (alcoholfree). They observed that the product containing alcohol was more effective than the alcoholfree rinses, except for the formulation including chlorhexidine and CPC, which had greater antimicrobial activity. The difference in the MID results of these studies may be related to the different sources of the MS (ATCC strain11, saliva13 and dentures12). According to Carlinet al. 19 PeriogardR and Plax WhiteningR can induce genetic damage. DNA damage is considered to be the prime mechanism during chemical carcinogenesis, and these data may be relevant in risk assessment for protecting human health and preventing carcinogenesis. Moreover, the ethanol concentration present in Cepacol induced mitotic recombination between homologous chromosomes in the Drosophila SMART assay involved in the genesis of numerous diseases, including cancer20. In conclusion, the mouthwashes containing chlorhexidine (PeriogardR) and cetylpyridinium chloride (CepacolR) had higher in vitro antibacterial activity (MID) against MS than the mouthwash containing triclosan (PlaxR), according to microbiol - ogical method employed. Further studies should be conducted to evaluate the in vitro and in vivo antimicrobial activities of PeriogardR, CepacolR, PlaxR and other mouthwashes against MS and a wide range of oral microorganisms, as well as their cytotoxicity.


The authors thank Izabel Yoko Ito, Ph.D., Full Professor of Microbiology (in memoriam) of University of Sao Paulo, School of Pharmaceutical Sciences of RibeiraoPreto, SP, Brazil for her microbiological knowledge applied to this research.


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