1. The effect of increased ventilation of toothbrush covers on retention of Streptococcus mutans biofilms INTRODUCTION The sale and use of toothbrush covers for storage purposes continues to be prolific, despite disapproval from the dental community. Covers offer protection from external contamination from coliforms, yeasts, molds, hairsprays and other products used around the sink. This makes them attractive merchandise for the uninformed consumer. Covers have minimal ventilation. They act as reservoirs for oral pathogens that remain on the brush following use and thrive in moist environments. Contaminated toothbrushes can re-infect users with oral pathogens. Streptococcus mutans, which causes dental caries, is the most commonly studied oral pathogen. The retention of S. mutans on toothbrushes has been studied as relating to many factors, including the use of toothbrush covers. S. mutans forms biofilms naturally. Early studies examined only the loosely adhered/easily removed S. mutans colonies on brushes. Only a few studies have been conducted regarding the retention of biofilms on toothbrush heads. No studies have yet quantified the effect of toothbrush covers on biofilm retention. Increasing the ventilation of toothbrush covers may reduce the retention of biofilms, such as S. mutans, on toothbrushes. http://www.linktotheweb.com/trpcapabrush.jpg http://textbookofbacteriology.net/nfStreptococcus_mutans.jpeg Joanna King HYPOTHESIS H 0 : There is no difference in the quantity of biofilms retained on toothbrushes stored in unmodified (4.2% ventilation) and modified (20% ventilation) toothbrush covers. REVIEW OF LITERATURE METHODS PROJECT SUMMARY Oral pathogens such as Streptococcus mutans are retained on toothbrushes following use, and can form biofilms which may reinfect users. Dental professionals discourage use of toothbrush covers which offer protection from external contaminants but are reservoirs for oral pathogens due to minimal ventilation. This proposal suggests that increasing the ventilation of toothbrush covers from 4.2% to 20% will reduce the retention of S. mutans. Toothbrushes will be vortexed in a bacterial suspension. Toothbrushes will be stored for 24 hours at room temperature; 4 with modified toothbrush covers, 4 with unmodified covers, and 4 uncovered. The toothbrushes will be incubated in BHI broth at 37  C for 48 hours. Biofilms will be dyed with crystal violet and counted under a stereoscopic microscope to determine differences between treatments. Increased ventilation, and thus decreased retention, would result in a safer dental product. PRELIMINARY WORK Calculated area of toothbrush covers and space devoted to ventilation. Strips were cut into toothbrush covers to bring ventilation from 4.2% up to 20% Quality control: Sterilized brushes with 90% ethyl alcohol for 5 minutes, 5 hours, and 24 hours. After 48 hours incubation, bacterial growth was noted on the brush sterilized for 5 minutes. No growth was noted at 5 and 24 hours. Dye: To determine if colonies could be dyed without background staining interfering with counts, a toothbrush head was stained with crystal violet and rinsed with alcohol. Bristle staining was light enough to detect biofilms. Dilution and Time: Serial dilution of bacterial suspension. Bacterial concentration was counted with hemacytometer. Brushes vortexed in 1:10, 1:100, 1:1000, and 1:10,000 dilutions in 5mL of PBS buffer. Brushes incubated and checked at 6 and 24 hours, no visible growth. At 48 hours biofilms were counted under stereoscopic microscope. It was determined that a 1:100 dilution (1.75x10 cells/mL) yielded the most suitable biofilm counts. 5 Figure 1. Streptococcus mutans biofilms adhered on toothbrush bristles following toothbrushing with fluoridated dentifrice (Nelson-Filho et al. 2004). EXPECTED RESULTS Figure 2. Mean S. mutans biofilms retained on toothbrushes. ANOVA test,P <0.005. Lower numbers of biofilms retained on brushes stored in modified covers. Control (uncovered brushes) have lowest number of biofilms retained FUTURE STUDIES Does this increase in ventilation significantly increase the contamination from external sources? Does the placement of the ventilation holes have any effect on external bacterial contamination? On oral pathogen retention? LITERATURE CITED Borso, H., Crump, R. and Schelling, M. 2004. The effect of toothbrush covers on bacterial retention. Journal of Dental Hygiene 78(4):1. Bunetel, L. et al.. 2000. In vitro evaluation of the retention of three species of pathogenic microorganisms by three different types of toothbrush. Oral Microbiology and Immunology 15:313-316. Nelson-Filho, P. et al. 2004. Effect of triclosan dentifrice on toothbrush contamination. Pediatric Dentistry 26:11-16. Sato, S. et al. 2004. Bacterial survival rate on toothbrushes and their decontamination with antimicrobial solutions. Journal of Applied Oral Science 12(2):99-103 Taji, SS. And Rogers, AH. 1998. The microbial contamination of toothbrushes. A pilot study. Australian Dental Journal 43(2)128-130. Acknowledgements Dr. Carolyn Mathur, Research Mentor Barbara Taylor Dr. John Spitznagel Dr. Karl Kleiner Toothbrushes are extensively contaminated by a variety of sources, including microorganisms resulting from the storage area (Taji and Rogers, 1998). Toothbrushes become contaminated by microorganisms with use and the contaminated toothbrush can be the cause of reinfection by oral pathogens (Sato et al. 2004). Toothbrush covers negatively affect retention of unadhered bacteria on toothbrushes (Borso et al. 2004). Survival rates of S. mutans decreases with exposure to air (Bunetel et al, 2000). S. mutans biofilms adhere tightly to toothbrushes and form countable colony forming units (Figure 1). Toothbrush bristles are contaminated following a single use (Nelson- Filho et al. 2004). Department of Biological Sciences, York College of Pennsylvania Sterilize toothbrushes and covers for 5 hours in 90% ethyl alcohol Vortex brushes for 3 minutes in a 1:100 dilution of bacterial suspension in PBS buffer 4 brushes, uncovered 4 brushes, unmodified covers Store at room temperature for 24 hours Dye with crystal violet Count biofilms under stereoscopic microscope Incubate at 37 degrees Celsius in BHI broth for 48 hours Analyze with ANOVA 4 brushes modified covers