1. Get the Key for Tomorrow’s Dentistry a symposium organized by the Danisco Cultor and Lotte Korea and sponsored by KOREAN CARIES PREVENTION ASSOCIATION Seoul April 22, 2001

2. The Influence and Mechanism of Xylitol on Mutans Streptococci Luc Trahan, PhD Groupe de Recherche en Écologie Buccale Faculté de médecine dentaire Université Laval Québec, Canada

3. Principal Proposed Mechanisms of Action of Xylitol n Lack of acid production by plaque bacteria n Increased saliva secretion and salivary buffer capacity n Inhibition of demineralization of sound enamel n Remineralization of decalcified sites n Inhibition of acid production by MS from dietary sugars n Inhibition of MS growth n Selection of a new less virulent MS population n Reduction of the transmission / implantation of Ms

4. Aim and Content Aim: To survey and summarize the influence and the mechanisms of action of xylitol on Mutans Streptococci n Background information on the dental plaque ecosystem and the utilisation of sugars by MS n Details of the effects of xylitol on MS n Recent progresses on the mechanisms of action n Summary and take home messages

5. ContentContent n Background information on the dental plaque ecosystem and the utilisation of sugars by MS n Details of the effects of xylitol on MS u Inhibition of growth and acid production from sugars u Selection of a new MS population n Recent progresses on the mechanisms of action u Disturbed protein synthesis and reduced survival u Virulence properties n Summary and take home messages

6. The Caries Etiological Factors Constitute a Fragile Ecosystem Xylitol PlaqueTooth Sugar CARIES Saliva Fluoride Multiple Factors Sealant Antiseptics

7. Modification of the Plaque Ecosystem by Sugar Consumption S M SUGARS ACIDS ACIDS SUGARS(sucrose)

8. Ecological Plaque Hypothesis and Prevention of Caries EnvironmentalchangesEcologicalshift FermentableSugarAcidNeutralpHLowpH S. oralis S. sanguis MSLactobacilliRemineralizationDemineralization Marsh, P., 1991

9. Dietary Sugars: Source of Energy and Carbon for the Growth of Plaque Bacteria

10. Acidic End-products Generation from Dietary Sugars by Plaque Bacteria Demineralization Remineralization Sugars Xylitol Sorbitol Critical pH

11. Xylitol: a Caries Preventive 5C- Sugar Alcohol CH 2 OH H-C-OH HO-C-H H-C-OH CH 2 OH XYLITOL C 5 H 12 O 5 CH 2 OH H-C-OH HO-C-H H-C-OH CH 2 OH XYLITOL C 5 H 12 O 5 CH 2 OH H-C-OH HO-C-H H-C-OH CH 2 OH SORBITOL C 6 H 14 O 6 CH 2 OH H-C-OH HO-C-H H-C-OH CH 2 OH SORBITOL C 6 H 14 O 6 CH 2 OH C=O HO-C-H H-C-OH CH 2 OH FRUCTOSE C 6 H 12 O 6 CH 2 OH C=O HO-C-H H-C-OH CH 2 OH FRUCTOSE C 6 H 12 O 6 A NATURAL 5 CARBON SUGAR ALCOHOL (PENTITOL) ALCOHOL (PENTITOL)

12. Dietary Sugar Utilization by Oral Streptococci ADP ATP Sugar~P Glucose~P PEP Pyruvate Lactate ADP ATP Lactic acid Sugar ADP ATP Sugar ADP+PPi Polysaccharides reserves ATP Glucose GlucanFructan Sugar PTS P Sucrose Fructose ++ E

13. 5-C alcohol (xylitol) PTS PERMEASE ± acidic end-products Lactic acid Acetic acid Ethanol etc... 5-C alcohol 5-C alcohol~P 2-Keto pentose 2-Keto pentose~P Carbohydrate pathways DH Kinase Epimerisation  Five-carbon Sugar Alcohols Utilization by Non-oral Bacteria ADP ATP

14. 5-C alcohol (xylitol) PTS PERMEASE ± acidic end-products Lactic acid Acetic acid Ethanol etc... 5-C alcohol 5-C alcohol~P 2-Keto pentose 2-Keto pentose~P Carbohydrate pathways DH Kinase Epimerisation  Incapacity for Oral Bacteria to 5-Carbon Sugar Alcohols Incapacity for Oral Bacteria to Utilize 5-Carbon Sugar Alcohols ADP ATP    

15. Inducible Six-carbon Sugar Alcohols Utilization by Mutans Streptococci ADP ATP Mannitol~P Sorbitol~P Glucose~P PEP Pyruvate Lactate ADP ATP Lactic acid ADP+PPi Polysaccharides reserves ATP Sorbitol PTS mtl Mannitol PTS sor

16. ContentContent n Background information on the dental plaque ecosystem and the utilisation of sugars by MS n Details of the effects of xylitol on MS u Inhibition of growth and acid production from sugars u Selection of a new MS population n Recent progresses on the mechanisms of action u Disturbed protein synthesis and reduced survival u Virulence properties n Summary and take home messages

17. Growth Inhibition of S. mutans Strains by Xylitol Strains% inhibition ATCC 27352 GS5-2 Ingbritt NTCC 10449 6715 8E3 (freshisolate) 67.3 (freshisolate) 82 0 76 85 84 68 33

18. Effect of Xylitol on Acid Production Rate by Resting Cells of S. mutans Vadeboncoeur, C., 1983 MINUTES 0102030 40 µ m o l e H + / 1 6 m g c e l l s ( d r y w e i g h t ) 0 10 20 30 40 Strain % inhibition R 27352 X S 6715 Ingbritt 0 49 68 38 from xylitol from glucose + xylitol

19. Definition of MS Sensitivity to Xylitol Xs wild type strains xylitol-sensitive Xs wild type strains : xylitol-sensitive Strains that are inhibited by xylitol Xr natural mutants xylitol-resistant Xr natural mutants : xylitol-resistant Strains that are not inhibited by xylitol None of these strains ferment xylitol and none produce acid from it.

20. Effect of the Addition of Xylitol to Growing X S and X R S. mutans Trahan, L., 1985 X R (± xyl) X S (+ H 2 O) X S (+ xyl) Time (hours) 0123456 O D ( 6 6 0 n m ) 0,20 0,30 0,40 0,50 0,60 0,70 0,10 1 4 C - x y l i t o l a c c u m u l a t e d 0 20 40 60 80 100 120 XRXR XSXS + xylitol

21. FRUCTOSEXYLITOL FRUCTOSE-6~PXYLITOL~P IIC Enzyme I Enzyme I~P HPr(His~P) HPr PEP Pyruvate IIB ~ IIC IIA P ~ P IIB ~ IIA ~ PP FRUCTOSE FRUCTOSE-1-~P Constitutivefructose-PTS Induciblefructose-PTS S. mutans Constitutive Fructose-PTS also Transports Xylitol Benchabane, H., 1997

22. Xylitol Transport and Expulsion in S. mutans FRUCTOSE PTS Sugar Xylitol Sugar~P PEP Pyruvate ADP ATP ADP ATP Xylitol~P EXPULSION Xylitol Pi Lactate Lactic acid Trahan L. 1996

23. Establishment and Consequences of a Xylitol Futile Cycle FRUCTOSE PTS Sugar Sugar~P PEP Pyruvate ADP ATP ADP ATP Xylitol~P EXPULSION Xylitol Pi Lactate E Lactic acid Xylitol

24. PTS Sugar Sugar~P PEP Pyruvate ADP ATP ADP ATP Lactate FRUCTOSE PTS Xylitol Lactic acid Absence of Constitutive Fructose PTS Activity in Xr Mutants

25. Effect of Xylitol and Xylitol-5-phosphate on Selected Glycolytic Enzymes in Vitro Enzyme % inhibition by xylitol xylitol 5-P PGI0>90 6-PFK 0 50 50 1-PFK 0 10 10 PK0>90 LDH0 0

26. Proposed Model of the Effect of Xylitol on S. mutans Glucose-6-P Fructose-6-P Fructose-1,6-di-P GA3P 1,3-DPG DHAP 2-PG PEPPyruvateLactate Xylitol-P Glucose PGI 6-PFK PKLDH Xylitol 3-PG - - + Xylitol Glucose PTS man PTS fru PMF Glycogen Lactic acid Trahan L., 1996

27. ContentContent n Background information on the dental plaque ecosystem and the utilisation of sugars by MS n Details of the effects of xylitol on MS u Inhibition of growth and acid production from sugars u Selection of a new MS population n Recent progresses on the mechanisms of action u Disturbed protein synthesis and reduced survival u Virulence properties n Summary and take home messages

28. In vitro Selection of X R Natural Mutants Following Growth in the Presence of Xylitol XYLITOLXYLITOLXYLITOLXYLITOL

29. In vivo Selection of X R Natural Mutants in Saliva and Plaque of Xylitol Consumers XYLITOLXYLITOL

30. Proportion of salivary natural X R MS strains in patients Patients Number of strains %XR%XR%XR%XR Total XRXRXRXR XSXSXSXS Controls2021810 Xylitol-consumers 54 47 787

31. Occurrence in 1988 of X R Salivary Strains in Patients from the Ylivieska Xylitol Study (1982-1984) Group % X R Controls 35.0 ± 6.4 Active consumers 82.5 ± 5.7 Former consumers N 16 14 13 74.6 ± 8.6

32. Effect of Xylitol Consumption on the Plaque Ecosystem S M XYLITOL

33. ContentContent n Background information on the dental plaque ecosystem and the utilisation of sugars by MS n Details of the effects of xylitol on MS u Inhibition of growth and acid production from sugars u Selection of a new MS population n Recent progresses on the mechanisms of action u Disturbed protein synthesis and reduced survival u Virulence properties n Summary and take home messages

34. Effect of a Heat or Xylitol Stress on 14 C-labelled Protein Profile of S. mutans

35. Densitometric Analysis of Protein Profiles of Xr and Xs MS Strains After a Xylitol Exposure Xs strain Xr strain Protein ID ControlXylitolexposure ControlXylitolexposure HSP-701.001.081.000.53HSP-601.000.821.000.54 40 kDa 1.001.001.091.53 71 kDa 1.001.161.000.58 63 kDa 1.001.111.000.57 57 kDa 1.00 1.031.001.37 53 kDa 1.000.951.000.53 50 kDa 1.000.951.001.48

36. Effect of a Xylitol Shock on the Survival Capacity of S. mutans 123.1 Number of surviving cells (CFU /mL) S. mutans 123.1 X S S. mutans 123.1 X R Control Xylitol X/C Control Xylitol X/C Control Xylitol X/C Control Xylitol X/C 3.51±0.13 x10 10 1.51±0.47 x10 10 0.43 1 2.62±0.68 x10 9 2.78±0.38 x10 9 1.06 4 1.45±0.38 x10 9 0.654±0.09 x10 9 0.45 2 8.15±0.34 x10 8 8.44±0.42 x10 8 1.03 5 3.07±0.60 x10 9 2.19±0.05 x10 9 0.71 3 1.60±0.39 x10 9 1.91±0.43 x10 9 1.19 6 1, 2, 3 p<0.0003 4, 5, 6 p>0.11

37. ContentContent n Background information on the dental plaque ecosystem and the utilisation of sugars by MS n Details of the effects of xylitol on MS u Inhibition of growth and acid production from sugars u Selection of a new MS population n Recent progresses on the mechanisms of action u Disturbed protein synthesis and reduced survival u Virulence properties n Summary and take home messages

38. Preliminary Results on the Attenuated Virulence Properties of X R MS Strains Selected by Xylitol... u a reduced acid production from sugars u a modified extracellular polysaccharides synthesis u a modified bacterial agglutination/aggregation capacity u an elevated glucan-binding protein C elevated 20 folds When X R strains are compared with corresponding X S strains the accumulated evidence suggest:

39. u an increased dextran-dependent aggregation capacity u a reduced adherence to saliva coated hydroxyapatite and glass surfaces u a reduced cariogenicity in a rat model u an absence of DnaK and GroEL stress proteins response to a xylitol exposure u a larger bacterial cells shedding from plaque to saliva Preliminary Results on the Attenuated Virulence Properties of X R MS Strains Selected by Xylitol

40. ContentContent n Background information on the dental plaque ecosystem and the utilisation of sugars by MS n Details of the effects of xylitol on MS u Inhibition of growth and acid production from sugars u Selection of a new MS population n Recent progresses on the mechanisms of action u Disturbed protein synthesis and reduced survival u Virulence properties n Summary and take home messages

41. Summary of the Effects of Xylitol on Mutans Streptococci... n A variable intensity inhibition of growth on most fermentable sugars resulting from: u the establishment of an energy spending futile cycle u the strong inhibition of glycolytic pathway enzymes n An inhibition of acid production from dietary sugars also resulting from the above mentioned mechanisms

42. Summary of the Effects of Xylitol on Mutans Streptococci n A selection of X R MS strains with apparently less virulent properties n A disturbance of protein synthesis, including stress proteins DnaK and GroEL, resulting in a reduced capacity to survive further exposure to xylitol n A reduced capacity to be transmitted from mother to child

43. Clinical Benefits for Patients Chewing Xylitol Gum n a reduced caries incidence via: u an increased saliva secretion and salivary buffer capacity u an inhibition of demineralization of sound enamel u a remineralization of decalcified sites u an inhibition of acid production from dietary sugars n a less acidogenic and less aciduric plaque ecosystem The accumulated evidence suggests :

44. Additional Clinical Benefits from the Inhibitory Action of Xylitol on MS n a reduction of: u plaque indices u the amount of plaque accumulated u the number of MS in plaque and saliva u the capacity to transmit MS strains n a selection of Xr MS strains with: u apparently less virulent properties u a loss of MS strains from plaque to saliva The accumulated evidence suggests:

45. Take Home Message # 1 n Xylitol influences positively the plaque microflora in establishing ecological conditions less appropriate for the low-pH bacteria producing acidic end-products and thus beneficial for the patients n Long and/or frequent exposure to xylitol does not result, for oral bacteria, in adaptation to metabolize the sugar-alcohol

46. Take Home Message # 2 n Xylitol is not only unfermentable by oral bacteria but it also inhibits most mutans streptococci n Biochemical mechanisms explaining the inhibition of the growth of, and acid production by, mutans streptococci have been well known for a few years n Knowledge on other mechanisms involving disturbed proteins synthesis by xylitol and reduced survival of MS to repeated exposures to xylitol is progressing

47. Take Home Message # 3 n The occurrence of xylitol-resistant strains in xylitol consumers is explained by a simple selection of natural mutants n Xylitol-resistance of MS does not impair its use in caries prevention and is most probably beneficial for the patients n Knowledge of the hypothesized less virulent properties of xylitol-resistant strains is progressing

48. This presentation is the result of various studies conducted with numerous graduate students and colleagues from Québec and Finland THANK YOU

49. n A campus with more than 35 000 students n A Faculté de médecine dentaire since 1969 A french speaking university founded in 1683