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

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

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

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

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

The Caries Etiological Factors Constitute a Fragile Ecosystem Xylitol Plaque Tooth Sugar CARIES Saliva Fluoride Multiple Factors Sealant Antiseptics

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

Ecological Plaque Hypothesis and Prevention of Caries Environmental changes Ecological shift Fermentable Sugar Acid Neutral pH Low S. oralis S. sanguis MS Lactobacilli Remineralization Demineralization Marsh, P., 1991

Dietary Sugars: Source of Energy and Carbon for the Growth of Plaque Bacteria OD (660 mm) 4 8 1 2 , 3 5 Sugar could be: sucrose glucose lactose mannitol sorbitol etc.. Xylitol Time (hours)

Acidic End-products Generation from Dietary Sugars by Plaque Bacteria Demineralization Remineralization Sugars Xylitol Sorbitol Critical pH 19

Xylitol: a Caries Preventive 5C- Sugar Alcohol CH2OH H-C-OH HO-C-H SORBITOL C6H14O6 CH2OH H-C-OH HO-C-H XYLITOL C5H12O5 CH2OH C=O HO-C-H H-C-OH FRUCTOSE C6H12O6 A NATURAL 5 CARBON SUGAR ALCOHOL (PENTITOL)

Dietary Sugar Utilization by Oral Streptococci ATP Glucan Fructan Sugar ADP + + Sugar~P Fructose Glucose Sugar ADP+PPi ATP Polysaccharides reserves PTS Glucose~P ADP ATP Sucrose PEP ADP Pyruvate ATP Lactate Lactic acid

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

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

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

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

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

Effect of Xylitol on Acid Production Rate by Resting Cells of S. mutans 4 µ m o l e H + / 1 6 g c s ( d r y w i h t ) from glucose 3 Strain % inhibition R 27352 X S 6715 Ingbritt 49 68 38 2 1 + xylitol from xylitol 1 2 3 4 M I N U T E S Vadeboncoeur, C., 1983

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

Effect of the Addition of Xylitol to Growing XS and XR S. mutans XR (± xyl) + xylitol XS (+ H2O) , 7 , 6 XS (+ xyl) , 5 , 4 ) m n , 3 1 2 6 XS 6 ( 1 d D e t O a 8 , 2 l u m u 6 c c a l 4 o l i t y 2 x XR - C 4 1 , 1 1 2 3 4 5 6 T i m e ( h o u r s ) Trahan, L., 1985

S. mutans Constitutive Fructose-PTS also Transports Xylitol XYLITOL~P IIC FRUCTOSE XYLITOL Constitutive fructose-PTS IIB ~ IIA P PEP Enzyme I HPr(His~P) HPr FRUCTOSE IIC IIA IIB Enzyme I~P ~ ~ Pyruvate P P Inducible fructose-PTS FRUCTOSE-1-~P Benchabane, H., 1997

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

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

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

Effect of Xylitol and Xylitol-5-phosphate on Selected Glycolytic Enzymes in Vitro % inhibition by Enzyme xylitol xylitol 5-P PGI >90 6-PFK 50 1-PFK 10 PK >90 LDH

Proposed Model of the Effect of Xylitol on S. mutans Glycogen PMF Fructose-6-P 6-PFK Glucose PGI Fructose-1,6-di-P Glucose Glucose-6-P - DHAP PTSman GA3P + PK LDH Lactic acid PEP Pyruvate Lactate 1,3-DPG PTSfru 2-PG - 3-PG Xylitol Xylitol-P Xylitol Trahan L., 1996

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

In vitro Selection of XR Natural Mutants Following Growth in the Presence of Xylitol

In vivo Selection of XR Natural Mutants in Saliva and Plaque of Xylitol Consumers

Proportion of salivary natural XR MS strains in patients Number of strains % XR Patients Total XR XS Controls 20 2 18 10 54 47 Xylitol-consumers 7 87

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

Effect of Xylitol Consumption on the Plaque Ecosystem S M XYLITOL

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

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

Densitometric Analysis of Protein Profiles of Xr and Xs MS Strains After a Xylitol Exposure Xs strain Xr strain Protein ID Control Xylitol exposure Control Xylitol exposure HSP-70 1.00 1.08 0.53 HSP-60 0.82 0.54 71 kDa 1.00 0.58 1.00 1.16 63 kDa 1.00 0.57 1.00 1.11 57 kDa 1.00 1.37 1.00 1.03 53 kDa 1.00 0.53 1.00 0.95 50 kDa 1.00 1.48 1.00 0.95 40 kDa 1.00 1.09 1.53

Effect of a Xylitol Shock on the Survival Capacity of S. mutans 123.1 Number of surviving cells (CFU /mL) S. mutans 123.1 XS S. mutans 123.1 XR Control Xylitol X/C Control Xylitol X/C 3.51±0.13 x1010 1.51±0.47 x1010 0.43 1 2 .62±0.68 x109 2.78±0.38 x109 1.06 4 1.45±0.38 x109 0.654±0.09 x109 0.45 2 8.15±0.34 x108 8.44±0.42 x108 1.03 5 3.07±0.60 x109 2.19±0.05 x109 0.71 3 1.60±0.39 x109 1.91±0.43 x109 1.19 6 1, 2, 3 p<0.0003 4, 5, 6 p>0.11

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

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

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

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

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

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

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

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

Take Home Message # 1 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 Long and/or frequent exposure to xylitol does not result, for oral bacteria, in adaptation to metabolize the sugar-alcohol

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

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

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

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