Toothbrush Bristle Wear and Adherence of Streptococcus mutans

1. Toothbrush Bristle Wear and Adherence of Streptococcus mutans Robert Goldsmith, DMD UMDNJ – Department of Pediatric Dentistry May 12, 2006

2. Purpose of the Study To determine if toothbrush bristle wear: •impacts the adherence of S. mutans •affects the extent of adherence at 0, 8 and 24 hours after air-drying

3. Review of Literature Microbial Contamination of Toothbrushes Svanberg M (1978).Contamination of toothpaste and toothbrush by Streptococcus mutans. Scand. J Dent. Res. 86:412-414. Purpose: Examined the contamination of toothbrushes by S. mutans Methods: Two Swedish adult subjects with a salivary S. mutans concentration of 106 colony forming units (CFU) were each given three new toothbrushes. After three consecutive days of use toothbrushes were air dried at room temperature for 15 minutes, 12 hours, and 24 hours and CFU were counted. Results: Greater than 106 CFU of S. mutans remained on toothbrush bristles up to 15 minutes after use and that approximately 104 CFU of S. mutans remained after 24 hours of air-drying.

4. Kozai K, Iwai T, Miura K. Residual contamination of toothbrushes by microorganisms. J Dent Child 56:201-204, 1989. Purpose: Investigated the microbial contamination of toothbrushes after regular use and rinsing by children. Methods: 150 Japanese children, 6 years of age, were given a new toothbrush without toothpaste and asked to brush and rinse the toothbrush three different ways - rinsed slightly in running tap-water, rinsed in running tap-water with a finger used to manipulate the bristles slightly, and rinsed well in running tap-water with vigorous manipulation of bristles. Toothbrushes were then collected and allowed to air-dry for 0, 6 and 24 hours. Results: Toothbrushes harbored approximately 4.47 x 104S. mutans CFU immediately after rinsing with the number decreasing to 2.55 x 104 CFU after 6 hours and to 1.35 x 103 CFU after 24 hours. Conclusions: The extent to which the toothbrush was rinsed affected the degree of contamination, with well-rinsed brushes containing fewer microorganisms.

5. Malmberg E, Birkhed D, Norvenious G, Noren JG, Dahlen G. Microorganism on toothbrushes from day-care centers. Acta Odontol Scand 52:93-98, 1994. Purpose: Examined microbial adherence on 44 toothbrushes used by children 4-to-6 years old at Swedish day-care centers. Methods: Unsupervised tooth brushing without toothpaste after breakfast and lunch using toothbrushes provided by their parents. Toothbrushes were air-dried for two hours after each use. Vigorous hand shaking was performed for two minutes to remove bacteria from toothbrushes. Bacteria were plated on agar media and CFU were counted Results: Over 50% of the time the most frequent bacteria found on the toothbrush two hours after use was Streptococci, predominantly S. salivarius, S. sanguis, and S. mitis. Forty-one percent of the toothbrushes contained Lactobacilli and none showed beta-hemolytic Streptococci. Conclusion: After two hours of air-drying toothbrushes become heavily contaminated with microorganisms and that the level and viability of the microorganisms vary based on the extent of dryness.

6. Wetzel WE, Schaumburg C, Ansari F, Kroeger T, Sziegoleit A. Microbial contamination of toothbrushes with different principles of filament anchoring. JADA, 136:June 2005:758-765. Purpose: Examined the microbial contamination of toothbrushes with different filament anchoring using 45 German children ages 6- to-13 years old. Methods: Toothbrushes were divided into three groups by type of anchoring construction: staple-set tufting, in-mold tufting, and individual in-mold placement of filaments. Subjects used two toothbrushes with a pea-sized amount of toothpaste, cleaning the maxillary and mandibular teeth on one side with one toothbrush and those on the opposite side with the other toothbrush. The brushes were examined immediately after brushing, two hours later and eight hours later. Results: Anchoring systems and the drying intervals both had a significant effect on the microbial contamination of the brushes with individual in-mold filament placement retaining the least amount of microorganisms compared with tufting.

7. Review of Literature Toothbrush Bristle Wear McKendrick AJW, McHugh WD, Barbenel LMH. Toothbrush age and wear: An analysis. Br Dent J 130:66-68, 1971. Purpose: Examined toothbrush wear over two years using 103 adults with a mean age of 20.7 years. Methods: 50 subjects were issued electric toothbrushes and 53 subjects were issued hand-held toothbrushes. They were instructed to return their used toothbrushes when they thought the brush was worn out. Results: The average brush age at replacement was 10.5 weeks. Conclusions: The researchers concluded that the way in which a toothbrush is used for cleaning teeth is more important than length of time in use.

8. Glaze PM, Wade AB. Toothbrush age and wear as it relates to plaque control. J Clin Periodontol 13:52-56, 1986. Purpose: Examined toothbrush wear and plaque control. Methods: 40 British dental students, ages 19-to-26 years old. One group used a single toothbrush for 10 weeks while the other group were given new toothbrushes every two weeks for ten weeks. At biweekly visits, plaque and calculus were measured. The degree of toothbrush wear was assessed subjectively and placed into one of three categories: good, fair, and poor condition. Brush head surface area was assessed using calipers at different locations on the trim of the toothbrushes and by multiplying the greatest measurements in each direction. Results: Subjects using the same toothbrush for the ten week period had significantly more plaque on their teeth than subjects who replaced their brushes. However, it was only when the mean brush-head surface area increased to 68% above that of unused brushes that plaque scores significantly increased.

9. Rawls HR, Mkwayi-Tulloch NJ, Casella R, Cosgrove R. The measurement of toothbrush wear. J Dent Research 68(12):1781-1785, 1989. Purpose: Develop a quantitative measure of toothbrush wear based on bristle splaying Methods: Toothbrushes were damaged using a toothbrush wear machine. Bristle wear was measured subjectively and scored as follows: “0” A brush that a person could not be sure if it had been used or not (0-25% wear) “1” bristles that spread apart in many of the tufts (25- 49% wear) “2” all tufts were spread apart and many bristles were curled and/or matted (50-75% wear) “3” most tufts overlap and were matted and many curled and bent bristles were seen (76-100% wear) Results: Bristle splaying was strongly influenced by the length of time a toothbrush was used and that wear rating provided a quick and effective way of determining bristle deterioration.

10. Summary Microbial Contamination: • Toothbrushes become heavily contaminated with many microorganisms after regular use. • Different amounts of bacteria adhere to toothbrushes at different time points. • Level and viability of the microorganisms varies based on the amount of toothbrush bristle rinsing and dryness after use. Toothbrush Bristle Wear: • Bristles wear out over time and can affect plaque removal • Bristle wearcan be created andassessed through different means

11. Hypotheses 1.      Toothbrush group will affect adherence of S. mutans to new and worn toothbrushes as measured by the number of recoverable microorganisms. 2. Worn toothbrush bristles will harbor more S. mutans than new toothbrush bristles immediately after contamination with bacteria as measured by the number of recoverable microorganisms. 3. After 8 hours of air-drying, worn toothbrush bristles will harbor more S. mutans than new toothbrush bristles as measured by the number of recoverable microorganisms. 4 After 24 hours of air-drying, worn toothbrush bristles will harbor more S. mutans than new toothbrush bristles as measured by the number of recoverable microorganisms.

12. MethodsCreating Toothbrush Bristle Wear An orthodontic typodont from a front and side viewwith metal bands and brackets on the teeth with four rubber bands placed around the typodont to hold it closed and to assure constant pressure of the toothbrushes against the bracketed teeth.

13. Standardization of Toothbrush Bristle Wear Goal: To identify worn toothbrushes that meet the criteria for a category “3” toothbrush classification as specified by Rawls et al (1989): most tufts overlap and are matted together or many bristles are bent and curled. Method: •4 independent observers were given a verbal description and visual representations of new and worn toothbrushes by group (labeled A, B and C). They were then given 30 worn toothbrushes, 10 from each group and asked to rate whether the worn toothbrushes met the appropriate criteria for a category “3” toothbrush. •Two training sessions produced 100% reliability.

14. Visual Representation of a New and Worn Toothbrush New Worn

15. Examining New and Worn toothbrush bristles under a dissecting microscope New toothbrush bristles are tightly packed Worn toothbrush bristles are splayed

16. Examining New and Worn toothbrush bristles under a scanning electron microscope New Bristle (360X) •Round •Smooth Worn Bristle (370X) •Jagged •Irregular

17. Measurement of Toothbrush Bristle Splaying Goal: •To compare bristle splaying between new and worn toothbrushes by group Method: •10 toothbrushes from each group, 5 new and 5 worn were used. •Three randomly selected tufts, magnifying loops of 2X magnification, a millimeter caliper and an adequate light source. •The degree of bristle splaying was measured within a tuft from one edge to the most splayed bristle at the other edge of the tuft.

18. Toothbrush bristle splaying for all toothbrush groups both new and worn N=45 N=45 N=45  A statistically significant difference in mean bristle splaying of 1.7 mm +/- 0.74 mm was seen between all toothbrushes, both new and worn (t = 172.7; P = 0.0001).

19. Toothbrush bristle splaying between new and worn toothbrushes by group    • Toothbrush group A had a mean difference in bristle splaying of 2.21 mm +/- 0.99 (t = 67.8; P = 0.0001). Toothbrush group B had a mean difference in bristle splaying of 0.68 mm +/- 0.13 (t = 271.0; P = 0.0001). Toothbrush group C had a mean difference in bristle splaying of 2.10 mm +/- 0.27 (t = 270.1; P = 0.0001).

20. Methodology Flow Chart for Bacteria 30 adult toothbrushes Group A (10) Group B (10) Group C (10) Not submitted to bristle wear (15 - 5 per group) Submitted to bristle wear (15 - 5 per group) Toothbrushes dipped into a test tube containing S. mutans Four random tufts were removed from the toothbrush heads with a hemostat and placed into a test tube containing 3ml of phosphate buffered saline Serial dilutions 10-1 to 10-5 were performed and plated on Mitis Salivarius Agar 100 microliters of this bacterial solution (sample) and 100 microliters of Brian Heart Infusion media (control) were pipetted into two separate wells of a 96-well microplate Rinsed for five seconds by dipping into non-sterile tap water Vortexed for 30 seconds to remove bacteria Plates were grown aerobically in a humidity chamber at 37°C for two days until colony forming units were large enough to be visually counted Serial dilutions (10-1 to 10-3) and 100 microliters of each was plated on Mitis Salivarius Agar and incubated at 37° C for two days Optical reading at 620nm was used to measure the amount of S. mutans in the sample compared to the control Colony forming units of S. mutans were visually counted

21. Statistical Analysis • Dependent Variable • • Bacterial adherence to toothbrush bristles • Independent Variables • • Toothbrush status (new vs. worn) and toothbrush group (A, B and C) • Stat View program; Super ANOVA program • • Means and Standard Deviations - Adherence of S. mutans to new and worn toothbrushes at different time points • • Bacterial data was transformed to log10. • • Independent t-tests were used to compare (new vs. worn) • • A one-factor ANOVA was used to compare (toothbrush groups A, B and C). Post Hoc Scheffe’s test for significance • •  = 0.05, Power = 80%

22. Adherence of S. mutans at different time points by toothbrush group     At 0 hours, the results of the analysis of variance (F = 8.2; df = 2) comparing the three toothbrush groups indicated that there were significant differences (P = 0.0008). At 8 hours, the results of the analysis of variance (F = 28.2; df = 2) comparing the three toothbrush groups indicated that there were significant differences (P = 0.0001). At 24 hours, the results of the analysis of variance (F = 15.46; df = 2) comparing the three toothbrush groups indicated that there were significant differences (P = 0.0001).  

23. Adherence of S. mutans at different time points by toothbrush status  At 0 hours, the results of the t-test comparison (t = 4.21; df = 1) indicated that S. mutans adherence to new toothbrushes were significantly (P = 0.0448) more than S. mutans adherence to worn toothbrushes. At 8 and 24 hours respectively, the results of the t-test comparison (t = 1.44 and t = 2.13 respectively) indicated that S. mutans adherence to new and worn toothbrushes were not significantly different (P = 0.2358 and P = 0.1496 respectively).

24. Adherence of S. mutans to new toothbrushes by group at different time points    At 0 hours, the results of the analysis of variance (F = 8.81; df = 2) comparing the three toothbrush groups indicated that there were significant differences (P = 0.0011).  At 8 hours, the results of the analysis of variance (F = 19.48; df = 2) comparing the three toothbrush groups indicated that there were significant differences (P = 0.0001).  At 24 hours, the results of the analysis of variance (F = 16.88; df = 2) comparing the three toothbrush groups indicated that there were significant differences (P = 0.0001).

25. Adherence of S. mutans on worn toothbrushes by group at different time points   At 0 hours, the results of the analysis of variance (F = 2.99; df = 2) comparing the three toothbrush groups indicated no significant differences (P = 0.0670).  At 8 hours, the results of the analysis of variance (F = 12.88; df = 2) comparing the three toothbrush groups indicated that there were significant differences (P = 0.0001).  At 24 hours, the results of the analysis of variance (F = 4.44; df = 2) comparing the three toothbrush groups indicated that there were significant differences (P = 0.0215).

26. Adherence of S. mutans on New and Worn Group C Toothbrushes at different time points  No significant differences were seen between adherence of S. mutans on new and worn group C toothbrushes at 0 hours (t = 3.43; df = 1; P = 0.0805) and 8 hours (t = 3.68; df = 1; P = 0.0711).  At 24 hours (t = 21.58; df = 1; P = 0.0002), a significant difference in mean bacterial adherence was seen between new and worn group C brushes.

27. Conclusions • Toothbrush group affects adherence of S. mutans to both new and worn toothbrushes. • New toothbrushes tend to harbor more S. mutans than worn toothbrushes at 0, 8 and 24 hours after air-drying, but was only significant at 0 hours. • S. mutans adheres to toothbrushes immediately after inoculation and can be recovered on toothbrushes 8 and 24 hours later.

28. Explanation of Results •Capillary action of the liquid bacterium on the toothbrush bristles • Different toothbrush head shapes/bristle surface area • Different lengths of toothbrush bristles • Different number of bristles per tuft • Different means of tufting

29. Limitations of the Study 1. The use of a one-time application of a liquid bacterial culture of S. mutans to measure adherence does not replicate the oral environment in the mouth. Toothbrushes were only inoculated once and then measured over time. 2. Times points chosen were 0, 8 and 24 hours, to facilitate the conduct of the study when the laboratory was available. Even though this was practical, it may have simulated life more closely if 0, 12 and 24 hours where chosen because people brush their teeth in the morning and then 12 hours later when they go to bed.

30. Future Studies 1) Repeated inoculations of toothbrushes with S. mutans over time 2) Another study could look at specific sites on the bristles where bacteria adhere and colonize over time 3) Different bacteria

31. Acknowledgements 1) Ms. Marie McKiernan for her help, time, knowledge and support in the laboratory. Without her, this project could not have been completed.  2) Dr. David Furgang for his help and guidance with all aspects of this project, especially the statistical analysis.  3) Dr. Kabilan Velliyagounder for his help with the use of the scanning electron microscope.  4) Ms. Weijuan Han for her help with the statistical analyses.  5) My fellow post-graduate students, especially Dr. Natalie Sanche and Dr. Shylon Mathew for their help in the laboratory.  6) The third year dental students, Ms. Janna Kohout, Ms. Heather Wolen, Ms. Maryam Shariff, Mr. Mark Danbe and Mr. Reza Movahed who helped in evaluating toothbrushes.  7) The Pediatric Dental faculty for sharing their knowledge of pediatric dentistry: Dr. Jack Budnick, Dr. Mary Burke, Dr. Jorge Caceda, Dr. Jerry Guzzy, Dr. Mahdu Mohan, Dr. Melvyn Oppenheim, Dr. R. Glenn Rosivack and Dr. Nanci Tofsky.  8) Ms. Debra Goldsmith and Ms. Maria Navarro for supplying toothbrushes for this study.  9) Ms. Carmen Logatto and Ms. Debra Bereski for their help in scheduling committee meetings.  10) My family, especially my mother Dr. Rachelle Goldsmith for her support and guidance throughout this project. 11)  My thesis advisory committee: Dr. Zia Shey, Dr. Daniel Fine, Dr. Helen Schreiner, Dr. Barbara Greenberg, and Dr. Milton Houpt for their advice and encouragement throughout this project.