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The Effects of Xylitol on Bacterial Growth Ryan Cusick Research and Thesis 493 Advisor: Dr. Douglas Oba Abstract The purpose of this work was to determine if xylitol inhibited the in vitro growth of the oral bacteria Streptococcus mutans, Lactobacillus lactis, Lactobacillus delbrunckii and Streptococcus lactis. Two 15% xylitol solutions were made by dissolving 0.15 g xylitol in 1 mL of filtered saliva or distilled water. Saturated disks were placed on the surface of Tryptic Soy Milk agar that had been confluently plated with four plates of each bacterium. The controls included distilled water, filtered saliva, and the commercial antibiotic, tetracycline. The cultures were incubated in a carbon dioxide environment. Zones of inhibition were observed only on the tetracycline control. Xylitol solutions of 15% did not inhibit growth in the bacteria. INTRODUCTION Dental hygiene and the prevention of cavities is a global concern. Tooth decay, which is the destruction of tooth enamel, is triggered when bacteria that live on teeth digest carbohydrates left on the surface of a tooth. Oral bacteria produce acids that destroy the enamel of teeth; over time this destruction of enamel results in caries, commonly know as cavities. A biofilm of different bacteria provide an environment that is conducive to caries formation (Briner et al. 1986). The prevention of dental decay includes fluoride therapy, fissure sealants, dietary counseling and oral hygiene measures such as brushing and flossing (Lewis 1995). Treatments involving remineralization of tooth enamel have been introduced. Since damaged tooth enamel facilitates caries formation, increasing remineralization may result in cavity prevention (Reynolds 1997). 2 Some manufacturers of chewing gum have promoted their products as a method to prevent tooth decay. Trident gum containing Recaldent is purported to deliver calcium beneath the surface of the tooth (Reynolds 1997). Chewing gum also enhances the amount of salivary flow in the mouth (Makinen 1972). Xylitol, a five-carbon molecule related to xylose is an alcohol found in certain gums such as Clen Dent. Xylitol induces synthesis of salivary enzymes (Knuuttila and Makinen 1975) such as lactoperoxidase, which breaks down plaque and reduces attachment ability of Streptococcus mutans and other oral bacteria (Makinen et al. 1976). Streptococcus mutans plays a crucial role in the initial attachment of the bacterial biofilm to the surface of the teeth. Most microorganisms, including S. mutans, cannot metabolize xylitol, however, a few microorganisms have been found to develop the ability to digest the substrate over a period of months (Knuuttila and Makinen 1975). Because S. mutans cannot ferment xylitol, acid production is reduced preventing oral pH levels from dropping (Trahan 1995). Chewing gum containing xylitol prevents caries formation (Hildebrant and Sparks 2000). Kandelman and Gagnon (1990) showed that increasing the percentage of xylitol in gum from 15% - 65% did not affect caries inhibition. Although the percent of xylitol in the gums did not affect the observed inhibition of caries, the frequency of the chewing did (Hildebrant and Sparks 2000). The mechanical process of mastication breaks down plaque, thus physically inhibiting caries formation (Kandelman and Gagnon 1990). A brief summary of established roles xylitol plays in inhibiting caries are the following: first, plaque is broken down because the pH level is maintained and S. mutans cannot thrive, because bacterial attachment to the tooth surface is decreased. Second, 3 xylitol causes a salivary response, rinsing teeth of bacteria and further reducing bacterial attachment. Third, it induces enzyme synthesis, increasing plaque breakdown. Most microorganisms cannot metabolize xylitol preventing the acidic end product that erodes tooth enamel. Finally, the bacterium grows in the presence of different carbohydrates, but growth is retarded by xylitol. As a main carbohydrate source, xylitol eliminates bacterial growth (Trahan 1995). Although xylitol eliminates bacterial growth it has not been investigated whether xylitol’s inhibition of dental bacteria is due to anti-microbial activity. When xylitol is the only available carbohydrate bacterial growth is not seen. However, when xylitol is one of several carbohydrates, the bacterium grows but the growth is retarded by xylitol (Trahan 1995). The purpose of this work was to determine if xylitol demonstrated anti-bacterial activity of the in vitro growth of the oral bacteria S. mutans, Lactobacillus lactus, Lactobacillus delbrunckii and Streptococcus lactis. Materials and Methods Two xylitol solutions were made by dissolving 0.15 g xylitol in 1 mL of filtered saliva or distilled water. These solutions were aseptically micropipetted onto and absorbed by a sterile paper disk. Each disk was placed on the surface of Tryptic Soy Milk agar, which had been confluently plated with either S. mutans, L. lactis, L. delbrunckii or S. lactis. Four plates of each bacterium were prepared. The controls included distilled water, filtered saliva, and the commercial antibiotic Tetracycline. The cultures were incubated for 36 hours at 37 º C in a carbon dioxide environment. of inhibition were measured. Results Zones 4 Solutions of 15% xylitol (w/v) in distilled water or filtered saliva did not cause zones of inhibition in any one of the four bacteria plated. The tetracycline control caused the only observed zones of inhibition. Each zone of inhibition plus or minus the standard deviation is given below in Table 1. Table 1. The growth inhibition, in mm, of bacteria by the presence of xylitol and controls, the mean +/- standard deviation. Bacteria plates Filtered Saliva Distilled Water 15% Xylitol in distilled water 15% Xylitol in filtered saliva S. mutans L. delbrunkii L. lactis S. lactis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Tetracycline 25 +/- 4.7 35+/-0.58 36 +/- 8.8 26 +/- 4.7 Conclusion The purpose of this study was to determine if xylitol inhibited the in vitro growth of various oral bacteria. The results indicate that xylitol did not inhibit the growth of bacteria in a carbon dioxide environment on Tryptic Soy Milk agar. Although previous studies have established that S. mutans will not grow in the presence of xylitol as a carbohydrate source, this study shows that inhibition is not due to anti-microbial activity. This study determined that xylitol solutions of 15% did not inhibit bacterial growth in a carbon dioxide environment on Tryptic Soy Milk agar. Therefore, the main mode of inhibition by xylitol appears to be the replacement of the carbohydrate source. Any further research with xylitol should focus on the replacement value of the crystalline alcohol as a carbohydrate source for oral bacteria. 5 Acknowledgements I would like to thank Dr. Oba for all his help, the biology faculty for their insights and corrections, Jennifer Barnes for help in the lab, and those who have helped revising the paper. 6 Sources Cited Brinier, WW, E Grossman, RY Buckner, GF Rebitksi, TE Sox, E Setser, and ML Ebert, 1986. Assessments of susceptibility of plaque bacteria to chlorhexidine after six months oral use. Journal of Periodontal Research 21S: 53-59. Hildebrant, GH and B Sparks. 2000. Maintaining mutans streptococci suppression with xylitol chewing gum. Journal of American Dental Association 131 (7): 909-916. Kandelman, D, and G Gagnon. 1990. A 24-month Clinical Study of the Incidence and Progression of Dental Caries in Relation to Consumption of Chewing Gum Containing Xylitol in School Preventative Programs. Journal of Dental Research 69: 1771-1775. Knuuttila, MLE and KK Makinen. 1975. Effect of Xylitol on the Growth and Metabolism of Streptococcus mutans. Caries Research 9: 177-189. Lewis, D 1995. Periodic health examination, 1995 update: 2. Prevention of dental caries. Canadian Medical Association Journal 152: 836-834. Makinen, KK. 1972. The role of sucrose and other sugars in the development of dental caries. International Dental Journal 22:363-368. Makinen, KK. Tenovuo, J and Scheinin, A. 1976. Xylitol-induced increase of lactoperoxidase activity. Journal of Dental Research 55:652-660. Reynolds, EC. 1997. Enamel Remineralization and Casein Peptides. School of Dental Science, Journal of Dental Research 76(9): 1587-95. Trahan, L. 1995. Xylitol: a review of its action on mutans streptococci and dental plaque- its clinical significance. International Dental Journal 45 (1): 77-92.