Restor Dent Endod.  2014 Nov;39(4):288-295. 10.5395/rde.2014.39.4.288.

Synergistic effect of xylitol and ursolic acid combination on oral biofilms

• Zou Y
• Lee Y
• Huh J
• Park JW

• KMID: 2316910
• DOI: http://doi.org/10.5395/rde.2014.39.4.288

Abstract

OBJECTIVES
This study was designed to evaluate the synergistic antibacterial effect of xylitol and ursolic acid (UA) against oral biofilms in vitro.
MATERIALS AND METHODS
S. mutans UA 159 (wild type), S. mutans KCOM 1207, KCOM 1128 and S. sobrinus ATCC 33478 were used. The susceptibility of S. mutans to UA and xylitol was evaluated using a broth microdilution method. Based on the results, combined susceptibility was evaluated using optimal inhibitory combinations (OIC), optimal bactericidal combinations (OBC), and fractional inhibitory concentrations (FIC). The anti-biofilm activity of xylitol and UA on Streptococcus spp. was evaluated by growing cells in 24-well polystyrene microtiter plates for the biofilm assay. Significant mean differences among experimental groups were determined by Fisher's Least Significant Difference (p < 0.05).
RESULTS
The synergistic interactions between xylitol and UA were observed against all tested strains, showing the FICs < 1. The combined treatment of xylitol and UA inhibited the biofilm formation significantly and also prevented pH decline to critical value of 5.5 effectively. The biofilm disassembly was substantially influenced by different age of biofilm when exposed to the combined treatment of xylitol and UA. Comparing to the single strain, relatively higher concentration of xylitol and UA was needed for inhibiting and disassembling biofilm formed by a mixed culture of S. mutans 159 and S. sobrinus 33478.
CONCLUSIONS
This study demonstrated that xylitol and UA, synergistic inhibitors, can be a potential agent for enhancing the antimicrobial and anti-biofilm efficacy against S. mutans and S. sobrinus in the oral environment.

Keyword

Biofilm; Streptococcus mutans; Streptococcus sobrinus; Ursolic acid; Xylitol

MeSH Terms

Biofilms*
Hydrogen-Ion Concentration
Polystyrenes
Streptococcus
Streptococcus mutans
Streptococcus sobrinus
Xylitol*
Polystyrenes
Xylitol

Figure

• 

  Figure 1 Isobologram for the antimicrobial interaction between UA and xylitol against Streptococcous mutans KCOM 1207 (◊), S. mutans KCOM 1128 (□), S. mutans UA 159 (△) and S. sobrinus ATCC 33478 (×). Dashed line indicates additive interactions. FIC, fractional inhibitory concentrations; UA, ursolic acid.

• 

  Figure 2 Effect of xylitol or UA alone and xylitol-UA combined groups on the biofilm growth of S. mutans UA 159 (a) S. sobrinus 33478; (b) and mixture of S. mutans UA 159 with S. sobrinus 33478; (c) cultured in BHI containing 20 mM sucrose. UA, ursolic acid.

• 

  Figure 3 The number of biofilm cells in S. mutans UA 159 (a1), S. sobrinus 33478 (b1), and co-culture of S. mutans UA 159 with S. sobrinus 33478 (c1) after certain incubation times (3, 6, 12, and 24 hours). Relative number of biofilm of different age (3 hours ■, 6 hours ▓, 12 hours ▒, 24 hours ░) grown at various treatment and pH change (3 hours ●, 6 hours ○, 12 hours ▼, 24 hours △) in S. mutans UA 159 (a2), S. sobrinus 33478 (b2), and co-culture of S. mutans UA 159 with S. sobrinus 33478 (c2). UA, ursolic acid.

Reference

1. Seneviratne CJ, Zhang CF, Samaranayake LP. Dental plaque biofilm in oral health and disease. Chin J Dent Res. 2011; 14:87–94.

2. Rautemaa R, Ramage G. Oral candidosis-clinical challenges of a biofilm disease. Crit Rev Microbiol. 2011; 37:328–336.

3. Busscher HJ, Rinastiti M, Siswomihardjo W, van der Mei HC. Biofilm formation on dental restorative and implant materials. J Dent Res. 2010; 89:657–665.

4. Nicolas GG, Lavoie MC. Streptococcus mutans and oral streptococci in dental plaque. Can J Microbiol. 2011; 57:1–20.

5. Mosci F, Perito S, Bassa S, Capuano A, Marconi PF. The role of Streptococcus mutans in human caries. Minerva Stomatol. 1990; 39:413–429.

6. Bowden GH. Mutans streptococci caries and chlorhexidine. J Can Dent Assoc. 1996; 62:700703–707.

7. Gluzman R, Katz RV, Frey BJ, McGowan R. Prevention of root caries: a literature review of primary and secondary preventive agents. Spec Care Dentist. 2013; 33:133–140.

8. Shen Y, Stojicic S, Haapasalo M. Antimicrobial efficacy of chlorhexidine against bacteria in biofilms at different stages of development. J Endod. 2011; 37:657–661.

9. Ly KA, Milgrom P, Rothen M. Xylitol, sweeteners, and dental caries. Pediatr Dent. 2006; 28:154–163.

10. Trahan L, Bourgeau G, Breton R. Emergence of multiple xylitol-resistant (fructose PTS-) mutants from human isolates of mutans streptococci during growth on dietary sugars in the presence of xylitol. J Dent Res. 1996; 75:1892–1900.

11. Trahan L, Mouton C. Selection for Streptococcus mutans with an altered xylitol transport capacity in chronic xylitol consumers. J Dent Res. 1987; 66:982–988.

12. Assev S, Stig S, Scheie AA. Cariogenic traits in xylitol-resistant and xylitol-sensitive mutans streptococci. Oral Microbiol Immunol. 2002; 17:95–99.

13. Trahan L, Söderling E, Dréan MF, Chevrier MC, Isokangas P. Effect of xylitol consumption on the plaque-saliva distribution of mutans streptococci and the occurrence and long-term survival of xylitol-resistant strains. J Dent Res. 1992; 71:1785–1791.

14. Lee SH, Choi BK, Kim YJ. The cariogenic characters of xylitol-resistant and xylitol-sensitive Streptococcus mutans in biofilm formation with salivary bacteria. Arch Oral Biol. 2012; 57:697–703.

15. Leng S, Hao Y, Du D, Xie S, Hong L, Gu H, Zhu X, Zhang J, Fan D, Kung HF. Ursolic acid promotes cancer cell death by inducing Atg5-dependent autophagy. Int J Cancer. 2013; 133:2781–2790.

16. Zhou L, Ding Y, Chen W, Zhang P, Chen Y, Lv X. The in vitro study of ursolic acid and oleanolic acid inhibiting cariogenic microorganisms as well as biofilm. Oral Dis. 2013; 19:494–500.

17. Söderling EM, Ekman TC, Taipale TJ. Growth inhibition of Streptococcus mutans with low xylitol concentrations. Curr Microbiol. 2008; 56:382–385.

18. Assev S, Vegarud G, Rölla G. Growth inhibition of Streptococcus mutans strin OMZ 176 by xylitol. Acta Pathol Microbiol Scand B. 1980; 88:61–63.

19. do Nascimento PG, Lemos TL, Bizerra AM, Arriaga ÂM, Ferreira DA, Santiago GM, Braz-Filho R, Costa JG. Antibacterial and antioxidant activities of ursolic acid and derivatives. Molecules. 2014; 19:1317–1327.

20. Kim S, Song M, Roh BD, Park SH, Park JW. Inhibition of Streptococcus mutans biofilm formation on composite resins containing ursolic acid. Restor Dent Endod. 2013; 38:65–72.

21. Torella JP, Chait R, Kishony R. Optimal drug synergy in antimicrobial treatments. PLoS Comput Biol. 2010; 6:e1000796.

22. CLSI. M07-A9 Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Apprived standard. 9th ed. Wayne PA: Clinical and Laboratory Standards Institute;2012. p. 1–63.

23. Misra S, Raghuwanshi S, Gupta P, Saxena RK. Examine growth inhibition pattern and lactic acid production in Streptococcus mutans using different concentrations of xylitol produced from Candida tropicalis by fermentation. Anaerobe. 2012; 18:273–279.

24. Forssten SD, Björklund M, Ouwehand AC. Streptococcus mutans, caries and simulation models. Nutrients. 2010; 2:290–298.

25. Cura F, Palmieri A, Girardi A, Martinelli M, Scapoli L, Carinci F. Lab-Test(®) 4: dental caries and bacteriological analysis. Dent Res J (Isfahan). 2012; 9:S139–S141.

26. 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; 54:661–665.

27. de Soet JJ, van Loveren C, Lammens AJ, Pavicić MJ, Homburg CH, ten Cate JM, de Graaff J. Differences in cariogenicity between fresh isolates of Streptococcus sobrinus and Streptococcus mutans. Caries Res. 1991; 25:116–122.

28. Martinez AR, Abranches J, Kajfasz JK, Lemos JA. Characterization of the Streptococcus sobrinus acid-stress response by interspecies microarrays and proteomics. Mol Oral Microbiol. 2010; 25:331–342.