1.
Cariology IV
Microbiology of dental caries
Zdenek Broukal, Erika Lencova
Institute of Dental Research

2.
• Ontogenesis of oral microbial ecosystem
• Cariogenic microorganisms
- Streptococcus mutans
- Heterofermentative lactobacilli
- Other oral microorganisms
• Dental caries as an infectious disease
- Concept of the window of infectivity
- Mechanisms of oral infection
- Contributing factors

3.
Ontogenesis of oral microbial
ecosystem I.
• Several hrs after delivery
– Transient flora of the birth canal and St. epidermidis from skin surface
• Up to 5-6 months
– S. viridans, S. oralis, S. sanguinis, lactobacilli, diphteroids
• Teeth eruption
– 1. Infectious window period for S. mutans, G- cocci (Veilonella,
Neisseria), anaerobic streptococci, gramm negative rods
• Beginning of physiological dentition exchange
– Infectious window period for S. mutans, G+ anaerobic rods
(actinomycetes), G+ aerobic rods (Nocardia), G+ Rothia dentocariosa
• Adolescence
– G- anaerobes (Prevotella), Porphyromonas spp. , Capnocytophaga spp.,
anaerobic Vibrio spp., spirillae

4.
Ontogenesis of oral microbial
ecosystem II.
• In edentulous oral cavity of a newborn/toddler
microorganisms adhere partially to mucous
membranes, partially form planktonic microflora in
the saliva and in salivary biofilm covering mucous
membranes
• After the teeth eruption 90-95 % of microbial
population forms plaque, the rest covers mucous
membranes and form planktonic microflora

5.
Ontogenesis of oral microbial
ecosystem III.
0%
20%
40%
60%
80%
100%
0 1 2 3 4 5 7 9 11 13 15 1 2
G+ G+ cocci and
rods
G- cocci and rods
filaments and
fusobacteria
spirili and vibrio
spp.
Toothbrushing No oral hygiene Toothbrushing
days

6.
Investigating association of
microbial flora with dental caries
• 1878 J. Tomes – microorganisms in dentin tubules
• 1892 W.D. Miller – chemo-parasite theory of dental caries aetiology
• 1922 J. Clark – streptococci in dentin tubules – S. mutans
• 1938 G. Soggnaes – experimental dental caries in rodent model
– Physiological breed of laboratory rodents
– Cariogenic diet
– Choosing suitable animal models for experimental dental caries –
hamster, rat, (mice, gerbil, …)
– Selecting cell lines (rat Sprague-Dawley)
• 1963 R.W. Schaedler – gnotobiotic and germ-free animal models in
research

7.
Investigating association of microbial
flora with dental caries - evidence
• 1960 P.H. Keyes, R.J. Fitzgerald
– Caries transmitted experimentally in hamster and rat models
with cariogenic diet that were kept together via bedding,
excrements etc.
– germ-free animals with cariogenic diet had no dental caries
– Mono-contamination of germ-free animals with individual oral
microbial strains of rodent and human origin
– 1964 – “cariogenic“ streptococci, actinomycetes
• 1965 J.van Houte, J. Carlsson – “cariogenic“ streptococci =
S. mutans
• 1967 B. Mejàre and others – levels of S. mutans in saliva correlate
with caries experience in humans
• 1978 B. Köhler – S. mutans is transmitted from mother to child –
window of infectivity

8.
Taxonomy I.
(Bergey´s Manual of Determinative Bacteriology 2nd Edition, 1992)
Domain (Bacteria)
Kingdom (Procaryotae /Monera/)
Phylum (Firmicutes)
Class (Bacilli)
Order (Lactobacillales)
Family (Lactobacillaceae)
• Genus (Streptococcus)
Species (S. mutans)

9.
Oral lactobacilli
Heterofermentative
• main substrate: starch (glucose,
maltose, dextrins)
• slow metabolism
• produce only 50% lactic acid and
considerable amounts of ethanol,
acetic acid and carbon dioxide
• not so low pH
– L. casei
– L. fermentum
– L. salivarius
– L. plantarum
– L. brevis
– L. cellobiosus
– L. buchneri
Homofermentative
• main substrate: glucose,
sucrose
• rapid metabolism
• produce more than 85% lactic
acid from glucose
• low pH
– L. acidophillus
– L. acidophillus sensu
stricto
– L. crispatus
– L. gasseri
– L. rhamnosus

10.
S. mutans – ecological
requirements
• Presence of solid surfaces in the oral cavity
– hard dental tissues, dentures, infant obturator in cleft
children
• Repeated infection
• Increased frequency of sucrose intake
• Ecological niche
– mode of existence that a species has within an ecosystem –
smaller proportion of other viridans streptococci (showing
alpha haemolysis on blood agar - S. viridans, S. oralis, S.
sanguinis)

11.
S. mutans – topography
• Pits and fissures
• Interdental spaces
• Carious defects
• SM is not necessarily present on all teeth
• SM can be transmitted iatrogenically
• Rare on mucous membranes
• Proportion in salivary planktonic bacteria
reflects the proportion in plaque – saliva as
material for microbial examination
• Treatment of carious defects does not
decrease the proportion of SM in plaque

12.
S. mutans – colonies morphology
Colonies of S. mutans on
the spoon contaminated
with saliva and dipped for
36 hrs in BHI broth + 5%
sucrose
Colonies of S. mutans on
MSA broth, incubated for
24 hrs, GasPak

13.
Lactobacilli - ecological requirements
• Presence of solid surfaces in the oral cavity
– hard dental tissues, dentures, infant obturator in
cleft children
• Increased proportion of aciduric flora
• Increased frequency of sucrose intake
• Carious defects
• Increased amount of plaque

14.
Lactobacilli – topography
• Carious defects
• Proportion in salivary planktonic bacteria
partially reflects the proportion in plaque –
saliva as material for microbial examination
• Treatment of carious defects does decrease the
proportion of SM in plaque

15.
Actinomycetes - ecological
requirements
• Presence of solid surfaces in the oral cavity
– hard dental tissues, dentures, infant obturator in
cleft children
• Wide range of plaque flora
• Increased frequency of sucrose intake
• Carious defects
• Increased amount of plaque

16.
Actinomycetes – topography
• Interdental spaces
• Subgingival plaque
• Carious defects
• Proportion in salivary planktonic bacteria does
not reflect the proportion in plaque
• Treatment of carious defects does not decrease
the proportion of A. in plaque
• Abundant in the human mouth and induce root
surface caries in hamsters and gnotobiotic
animals

17.
Virulence factors – cariogenicity I.
• S. mutans
– Energy utilisation - α,(1-2) glycosidic bonds in sucrose
– Intensive glycolysis under wide range of pH
– Production of EPS – intermicrobial matrix in plaque
– Production of IPS – intracellular glycogen (ADPglucose
pyrophosphorylase (GlgC) and glycogen synthase (GlgA)
– Specific adherence to acquired pellicle
– Tolerance towards high sucrose concentration - up to 40 %
– Microaerofilic
• Heterofermentative lactobacilli
– Optimum pH for metabolism 3.5 – 5.5
– Enzymatic set for intensive glycolysis
– Amylase

18.
Virulence factors – cariogenicity II.
• A. odontolyticus, A. naeslundii
– Glycolysis under wide range of pH
– Production of IPS – intracellular glycogen
– Specific adherence to acquired pellicle
– Wide range of proteases
– Microaerophilic

19.
Relationship of other
microorganisms to dental caries
• Mixed flora of dental plaque
– Microaerophilic conditions for cariogenic
microorganisms
– Aciduricity (tolerance of, and growth at low pH)
– Co-aggregation – increased cohesion of plaque
– proteases – destruction of protein matrix of
enamel/dentin
– amylase – hydrolysis of starch to oligosaccharides

20.
Koch’s postulates
• Suspected pathogenic microorganism should be
present in all cases of the disease and absent from
healthy animals
• The microorganism should be grown in pure
culture
• Cells from pure culture should cause disease in a
healthy animal
• The microorganism should be re-isolated and
shown to be the same as the original

21.
Metabolic activity of cariogenic
microorganisms – pH of the
environment
hetero-
fermenta-
tive
lactobacilli
S. mutans
0
20
40
60
80
100
7 6 5 4 3 2
pH
%

22.
Production of polysaccharides by SM
– Extracellular polysaccharides (α 1-3, 1-4, 1-6
glucane) – adhesion factor
• glucosyltranspherase (GTF)
– Intracellular polysaccharides (intracellular
glycogen-like) – energy source
• ADPglucose pyrophosphorylase (GlgC
• glycogen synthase (GlgA)

23.
Extracellular polysaccharides
Microorganism Extracellular polysaccharides
S. mutans
α1-4, α1-3, glucane
α1-6, glucane
mutane
dextrane
lactobacilli β2-6, fructane levane
actinomycetes
β2-6, fructane
α1-6, glucane
levane
dextrane

24.
Density of plaque
Fermentable
polysaccharides
Cariogenic
plaque
Non-cariogenic
plaque
EPS

25.
Chemo-parasite theory
W.D.Miller 1892
Dental caries occurs as a result
of hard dental tissues
demineralization with organic
acids – end products of sucrose
metabolism of oral
microorganisms.

26.
Ecologic factors in dental caries
S. Socransky 1992
S. mutans S. sanguis
Selective ecological pressure (nutrition)
lactobacilli actinomycetes
Shortage of
sucrose
neutral pH
Enough of
sucrose acidic
pH
Healthy tooth caries

27.
Microbial
agent
Defending
mechanisms
Genotype hypothesis of SM cariogenicity
M. W. Russell 1994
Defending
mechanisms
Microbial agent
Defending
mechanisms
specific
pathogen
or virulent
genotype

28.
Hypotheses integration
Non-cariogenic
spectrum of
plaque
Selective
ecological
pressure
aciduric flora
(S. mutans)
Selective physical-
chemical pressure
acidophilic
flora
(lactobacilli)
Selective genetic
pressure
Cariogenic
spectrum of
plaque
virulent SM and
lactobacilli
genotypes

29.
Window of infectivity for cariogenic
microorganisms
• Conditions suitable for transmission of microorganisms
to planktonic flora
• Conditions suitable for adhesion of microorganisms to
dental surface/obturator/dentures
• Conditions suitable for repeated infection
• Contributing factors
– Frequent presence of sucrose in the oral cavity
– Decreased salivary secretion (during sleep)
– Quantity of infection

30.
0 6 12 18 24 30 36 5 6 7
age months years
Beginning of the
eruption of
permanent teeth
Window of
infectivity
Beginning of the
eruption of
temporary teeth
Succedaneous
window of
infectivity?
Window of infectivity
C.W. Caulfield 1993
Repeated
infection
Repeated
infection

31.
Mechanism of oral infection
Infection source
Transmission vector
Infection entry
window of infectivity
Contributing factors
Persistent infection
Repeated transmission
Transmission way

32.
Contributing factors of oral infection
• Risk behaviour of mother (siblings, father,
grandparents, carers)
• Untreated carious dentition of mother/others
• High levels of cariogenic microorganisms in saliva
• High frequency of sucrose intake especially before
sleeping (decreased salivary flow)
• Later start of tooth brushing
• Administration of sweetened medications
• Prolonged nursing/nursing as tranquilizing practice

33.
Evidence of SM transpher from
mother to child
Li and Caufield - 1995: 34 pairs mother - child;
70.6 % children had SM genotype identical with
mother
De Soet et al. - 1998, 21 pairs mother – child (with
lip or palate cleft); 38 % had SM genotype
identical with mother
Kozai et al. - 1999: 20 families, 51 % had SM
genotype identical with mother, 31 % with
father, 19 % other type of SM genotype
Dušková a Broukal – 1997: 40 families, 60 % had
SM genotype identical with mother, 8 % with
father, 32 % other type of SM genotype

34.
Proportions of SM and lactobacillli
in fissure plaque
Caries affected % z TVC (total viable count)
Tooth
Caries
experience
S. mutans S. sanguinis lactobacilli
With caries low 2.0 5 1
With caries high 25 8 4
Caries free
intact
dentition
< 1 13 0
Caries free low 2.0 21 < 1
Caries free high 8.0 6 < 1

35.
Hereditary fructose intolerance
• autosomal recessive disorder of fructose
metabolism first described in 1956
• incidence 1:22 000 of live births
• deficiency of fructose-1-phosphate aldolase (EC
2.1.2.13) activity
• accumulated fructose-1-phosphate inhibits
glycogen breakdown and glucose synthesis,
thereby causing severe hypoglycaemia
following ingestion of fructose
• patients develop a strong distaste for sweet
food
• almost all affected persons have intact dentition
and do not have S. mutans present in the mouth

36.
E-learning resources
www.textbookofbacteriology.net
www.db.od.mah.se/car/carhome.html
www.uic.edu/classes/peri/peri343/
http://www.dent.ucla.edu/ce/caries/index.html
http://www.dentistry.leeds.ac.uk/OROFACE/PAGE
S/micro/micro.html
http://dentistry.ouhsc.edu/intranet-
web/Courses/DSA8212/CarHomePage.html
http://hebw.cf.ac.uk/oralhealth/index.html