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Brazilian Journal of Oral Sciences
Piracicaba Dental School - UNICAMP
EISSN: 1677-3225
Vol. 7, Num. 24, 2008, pp. 1489-1492

Brazilian Journal Oral Sciences, Vol. 7, No. 24, Jan/Mar 2008, pp. 1489-1492

Efficacy of mouthrinse spray in inhibiting cariogenic biofilm formation on toothbrush bristles

Andresa Piacezzi Nascimento1 Gisele Faria2 Evandro Watanabe3 Izabel Yoko Ito4

1MS, PhD student of Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto - SP, Brazil.
2
MS, PhD, Professor of Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto - SP, Brazil.
3
MS, PhD, Professor of Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina -MG, Brazil. 4PhD, Professor of Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto - SP, Brazil.
Correspondence to: Andresa Piacezzi Nascimento Rua Barão do Amazonas, nº 1167, ap. 123 Centro, Ribeirão Preto – SP, Brazil CEP: 14010-120 Phone: +55-16-3602-42-17 Fax: + 55-16-3602-4878 E-mail: piacezzi@gmail.com

Received for publication: October 24, 2007 Accepted: March 26, 2008

Code Number: os08004

Abstract

Aim: The aim of this study was to evaluate the efficacy of Periogard® (Colgate-Palmolive) and Plax® (Colgate-Palmolive) in inhibiting biofilm formation of mutans streptococci (MS) on toothbrush bristles used just once for 2 minutes with no dentifrice.
Methods: This study was performed into 3 phases in which mouthrinses and sterile distilled water (control group) were sprayed 6 times (to standardize the method) on toothbrush bristles used by 53 university students. Toothbrushes were kept at room temperature for 3 hours and put into test tubes each containing bacitracin sucrose broth (selective enrichment for MS). After incubation at 37ºC for 3 to 4 days, MS biofilm on the bristles were analyzed and colony-forming units (CFU) were counted by stereomicroscope under reflected light.
Results: 41 students completed the 3 phases of this study and 38 (92.7%) control group toothbrushes had CFU of MS that varied from 1 to uncountable. Periogard® and Plax® inhibited biofilm formation on the bristles of 97.4% and 84.2% toothbrushes, respectively.
Conclusion: Periogard® and Plax®, in spray, reduced/eliminated biofilm formation of MS on the toothbrush bristles. However, Periogard® was more efficacious than Plax® for this purpose.

Key words: chlorhexidine, triclosan, biofilm, mutans streptococci, toothbrush

Introduction

Toothbrushes become contaminated by microorganisms of the oral cavity after use1 working as a source of microbial transmission/dissemination. Therefore, patients re-using their toothbrushes may reinfect themselves with pathogenic microorganisms. As early as 1920, COBB2 cited the toothbrush as a cause of repeated infections of the mouth. Besides the reinfection risk there is a risk of cross-infection when microorganisms on one toothbrush are transferred to others kept in close proximity. Also, the cross-infection may occur when people share toothbrushes.

Svanberg3 showed that toothbrushes and toothpaste tubes can be contaminated by Streptococcus mutans after using. In an in vitro study Goldsmith4 et al. showed that bristle wear impacts the adherence of S. mutans on toothbrushes at 0, 8, and 24 hours after air-drying. The retention and survival of S. mutans on the toothbrushes vary according to the type of brush5. The number of microorganisms increases with the size of the exposed area5.

Nelson-Filho et al.6 demonstrated that toothbrushes used by children were heavily contaminated by mutans streptococci (MS), which is identified as the major etiological agent of human dental caries7. Therefore, the control of these microorganisms is relevant for the maintenance of oral health. Once dentistry emphasizes the prevention concepts, toothbrushes should be replaced and disinfected regularly, because reduction of toothbrush contamination may be important in the control of several oral diseases. However, only in the last two decades studies have related about in vivo microbial contamination of toothbrushes, suggesting methods for their disinfection6,8-10.

Periogard® (Colgate-Palmolive - São Bernardo do Campo – SP -Brazil) is a mouthrinse which contains 0.12% chlorhexidine gluconate - the most effective antiplaque agent available11. Plax® (Colgate-Palmolive - São Bernardo do Campo – SP - Brazil), in its turn, is a mouthrinse which contains 0.03% triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) and 0.20% polyvinylmethyl ether/maleic acid (PVM/MA) copolymer which is used jointly with triclosan to increase its antimicrobial activity12.

The aim of this study was to evaluate the efficacy of Periogard® and Plax®, in spray, in inhibiting biofilm formation of MS on toothbrush bristles used just once by university students for 2 minutes with no dentifrice.

Material and Methods

This study was approved by Ethics Committee at the Faculdade de Ciências Farmacêuticas de Ribeirão Preto – Universidade de São Paulo (Process # 33). In this study participated 53 students of both genders (18 to 24 years old), matriculated at the Pharmacy course of the same institution. The inclusion criterion was not being using antibiotics for a period of 30 days.

Two mouthrinses were evaluated: Periogard® and Plax®. Sterile distilled water was used as control group. The 3 solutions were placed into individual plastic spray bottles.

The study was performed into 3 phases with a one-week interval between them. The study was randomized in which the 3 solutions were used in all phases by different groups of people to avoid interference of confusion factors in the results. At the end of the 3 phases, all participants had used the 3 solutions. In each phase, all volunteers received new toothbrushes (Contente® Soft, Suavetex - Uberlândia - MG - Brazil) and performed toothbrushing with no dentifrice, during 2 minutes. After toothbrushing, each participant rinsed the toothbrush with tap water as usually. Then, the toothbrushes were collected, numbered and the correspondent solution was sprayed 6 times (to standardize the method) on the bristles to a distance from 5 to 10cm between the toothbrush and the bottle. During this procedure, the toothbrushes were maintained in vertical head up position. Excess solution was removed by shaking the toothbrush. After that, the toothbrushes were kept on a rack to avoid contact among the bristles and put into a container at room temperature for 3 hours simulating the period between toothbrushing.

Microbiological procedures

After 3 hours at room temperature, each toothbrush was introduced aseptically in a test tube (25x150mm) with 10.0ml of bacitracin sucrose broth (CaSaB) avoiding contact of the bristles with the wall and bottom of the test tube. CaSaB was prepared as described by Jensen and Bratthall13 without trypan blue, according to Cesco et al.14. This culture medium is selective enrichment for MS and makes possible the biofilm formation on the toothbrush bristles. The toothbrushes were incubated at 37oC for 3 to 4 days. Then, they were removed from the test tubes. The toothbrush bristles were observed carefully from all sides and angles for the MS biofilm analysis and the colony-forming units (CFU) count, based on colony morphology, using a stereomicroscope under reflected light. The number of colonies was expressed according to the following parameters:

  • 0*: absence of cariogenic biofilm adhered on the bristles; culture medium was limpid, indicating absence of microorganisms;
  • 0: absence of cariogenic biofilm adhered on the bristles, but culture medium was turbid, indicating presence of othermicroorganisms;
  • 1 to 100: number of CFU were countable;
  • +100: colonies were not confluent and had values greater than 100;
  • Uncountable: bacterial development was intense, with confluent colonies, not allowing exact CFU count.

Statistical analysis

The data of the CFU count of MS were submitted to the nonparametric Friedman test. The established significance level was 5%. When the Friedman test showed differences among the 3 solutions, the Dunn’s multiple comparison procedure was employed to compare which pairs of solutions were different at the 5% significance level.

Results

From the initial 53 subjects, 41 completed the 3 phases of this study, but only 38 (92.7%) cases which formed the MS biofilm on the control group (CFU count varied from 1 to uncountable showed in Table 1) were considered to compare the efficacy of Periogard® and Plax®, in spray, in inhibiting biofilm formation of MS on toothbrush bristles. Figures 1a and 1b show MS biofilm on toothbrush bristles of the control group.

After using Periogard® spray, only 1 (2.6%) toothbrush presented 1 CFU of MS on toothbrush bristles (subject # 1) and 37 (97.4%) toothbrushes had no biofilm formation of MS (Table 1). From the 37 toothbrushes without biofilm formation on bristles, 9 (24.3%) presented other microorganisms that were indicated by the turbid culture medium (CaSaB) and 28 (75.7%) showed absence of any microorganism indicated by limpid CaSaB.

After using Plax® spray, 6 (15.8%) toothbrushes presented MS on toothbrush bristles varied from 1 to 2 CFU (Table 1) and 32 (84.2%) toothbrushes showed no biofilm formation of MS. From the 32 toothbrushes without biofilm formation on bristles, 16 (50.0%) presented development of other microorganisms and 16 (50.0%) were free of microbial growth. The statistical analysis among the 3 spray solutions (sterile distillated water, Periogard® and Plax®) showed significant difference in inhibition of cariogenic biofilm formation on toothbrush bristles (p<0.01). Both mouthrinses (Periogard® and Plax®) were statistically more efficacious in reduction/ inhibition of biofilm formation of MS than sterile distilled water (p<0.001). However, Periogard® was more efficacious than Plax® (p<0.01) for this purpose.

Discussion

The use of dentifrices during toothbrushing may reduce toothbrush microbial contamination15 because they may contain antimicrobial agents. In this study, toothbrushing was performed with no dentifrice, in order to avoid interferences on toothbrush contamination by MS and evaluation the efficacy of mouthrinses used for toothbrush disinfection. Some researchers evaluated toothbrush disinfection by the immersion of them into antimicrobial solutions6,8,9. In this study, antimicrobial solutions in spray were employed as described by some authors10,16,17. The advantage of this method is that it is easy and quick to be applied. Furthermore, it is economic because avoid solution waste and only one spray bottle may be used by several family members.

The evaluation of toothbrush contamination was performed by toothbrush immersion method into liquid culture medium, in order to detect cariogenic biofilm formation on the bristles. This method allows the detection of a greater MS number than the desorption method in which toothbrushes are submitted to mechanic agitation or sonication for microorganisms desorption, and the resultant suspensions are diluted and plated in solid culture media10.

CaSaB has the antibiotic bacitracin and a high concentration of sucrose which inhibit the growth of other microorganisms. The increased concentration of sucrose also promotes adhesion of MS on toothbrush bristles and it may cause the biofilm formation as well as attainment of distinct colonies. The growth of MS occurs on bristles, not in the culture medium. When the microbial growth in the culture medium is found is due to some microorganisms which can resist to the concentrations of bacitracin and sucrose, but do not have the ability to adhere13.

Thirty-eight control group toothbrushes showed growth of MS. These data indicate that they constitute resident flora of the human mouth, and the artifacts (toothbrushes) used to infection control must be disinfected. That also shows that the drying period of the bristles do not eliminate the microorganisms.

After using Periogard® spray, only 1 toothbrush (case # 1) was colonized by 1 CFU of MS (Table 1). Similar results were obtained by Nelson-Filho et al.6, who demonstrated that the immersion of toothbrushes used by children in a solution containing 0.12% chlorhexidine gluconate inhibited biofilm formation of MS in 100% from the toothbrushes. Similar results also were obtained in a study with adults’ toothbrushes by Sato et al.10, who demonstrated that a solution containing 0.12% chlorhexidine digluconate in spray inhibited the biofilm formation of MS in 100% from the toothbrushes.

In another study by Bhat, Hegde and George9, toothbrushes used by children were placed into a solution containing 0.2% chlorhexidine gluconate for 24 hrs. After this period, there was no growth of MS.

Plax® spray reduced/eliminated significantly the cariogenic biofilm formation on the toothbrushes. However, it was less effective than Periogard®. There is not another study evaluating the effect of Plax® on cariogenic biofilm formation on toothbrush bristles so it was not possible to compare the results of this study with others from literature.

Some toothbrushes disinfected with Periogard® or Plax® presented turbid culture medium. That may be due to the growth of microorganisms which resisted to the concentrations of bacitracin and sucrose present in the CaSaB and to the concentrations of antimicrobial agents present in the mouthrinses.

Further studies should be done to evaluate the efficacy of Plax® spray in inhibiting biofilm formation of MS on toothbrush bristles used by people of different age groups, schooling and socioeconomic level. Other mouthrinses with different antimicrobial agents also must be evaluated. In conclusion, Periogard® and Plax®, in spray, reduced/eliminated biofilm formation of MS on toothbrush bristles. However, Periogard® was more efficacious than Plax® for this purpose.

References

  1. Glass RT, Lare MM. Toothbrush contamination: a potential health risk? Quintessence Int. 1986; 17: 39-42.
  2. Cobb CM. Toothbrush as a cause of repeated infections of the mouth. Boston Med Surg J. 1920; 183: 263-4.
  3. Svanberg M. Contamination of toothpaste and toothbrush by Streptococcus mutans. Scand J Dent Res. 1978; 86: 412-4.
  4. Goldsmith RN, Shey Z, Houpt MI, Fine D, Schreiner H, Greenberg B. Toothbrush bristle wear and adherence of Streptococcus mutans. Pediatr Dent. 2007; 29: 243-7.
  5. Bunetel L, Tricot-Doleux S, Agnani G, Bonnaure-Mallet M. In vitro evaluation of the retention of three species of pathogenic microorganisms by three different types of toothbrush. Oral Microbiol Immunol. 2000; 15: 313-6.
  6. Nelson-Filho P, Macari S, Faria G, Assed S, Ito IY. Microbial contamination of toothbrushes and their decontamination. Pediatr Dent. 2000; 22: 381-4.
  7. Okada M, Soda Y, Hayashi F, Doi T, Suzuki J, Miura K, Kozai K. Longitudinal study of dental caries incidence associated with Streptococcus mutans and Streptococcus sobrinus in pre-school children. J Med Microbiol. 2005; 54: 661-5.
  8. Caudry SD, Klitorinos A, Chan ECS. Contaminated toothbrushes and their disinfection. J Can Dent Assoc. 1995; 61: 511-6.
  9. Bhat SS, Hegde KS, George RM. Microbial contamination of tooth brushes and their decontamination. J Indian Soc Pedod Prev Dent. 2003; 21: 108-12.
  10. Sato S, Pedrazzi V, Lara EHG, Panzeri H, Albuquerque Júnior RF, Ito IY. Antimicrobial spray for toothbrush disinfection: an in vivo evaluation. Quintessence Int. 2005; 36: 812-6.
  11. Addy M. Chlorhexidine compared with other locally delivered antimicrobials: a short review. J Clin Periodontol. 1986; 13: 957-64.
  12. Nabi N, Mukerjee C, Schmid R, Gaffar A. In vitro and in vivo studies on triclosan/PVM/MA copolymer/NaF combination as an anti-plaque agent. Am J Dent. 1989; 2: 197-206.
  13. Jensen B, Bratthall D. A new method for the estimation of mutans streptococci in human saliva. J Dent Res. 1989; 68: 468-71.
  14. Cesco RT, Bignelli P, Santos CP, Ito IY. Toothbrushes: evaluation of contamination level by streptococci of mutans group. In: 5th World Congress on Preventive Dentistry; São Paulo, 1995. São Paulo: Brazilian Association for Preventive Dentistry, 1995. p.103.
  15. Quirynen M, De Soete M, Pauwels M, Gizani S, Van Meerbeek B, Van Steenberghe D. Can toothpaste or a toothbrush with antibacterial tufts prevent toothbrush contamination? J Periodontol. 2003; 74: 312-22.
  16. Meier S, Collier C, Scaletta MG, Stephens J, Kimbrough R, Kettering JD. An in vitro investigation of the efficacy of CPC for use in toothbrush decontamination. J Dent Hyg. 1996; 70: 161-5.
  17. Neal PR, Rippin JW. The efficacy of a toothbrush disinfectant spray – an in vitro study. J Dent. 2003; 31: 153-7.

© Copyright 2008 - Piracicaba Dental School - UNICAMP São Paulo - Brazil


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