........

1. K.VINOTHINI
1 PG

2. CONTENTS

3. INTRODUCTION

4. Dental caries is defined as a
multifactorial ,transmissible ,infectious
disease caused primarily by the complex
interaction of cariogenic oral flora
(biofilm) with fermentable dietary
carbohydrates on the tooth surface over
time
-Sturdevant,5th edition

5. The microbiologic disease of the calcified
tissues of teeth,characterised by
demineralisation of the inorganic portion and
destruction of the organic substances of the
tooth –Shafers
Caries is defined as a localized post-
eruptive,pathological process of external origin
involving softening of the hard tooth tissue and
proceeding to the formation of a cavity -WHO

6. KOCH’ POSTULATE
a) The bacterium should be constantly
associated with lesions of the disease
b) It should be possible to isolate the
bacterium in pure culture from the lesion
c) Inoculation of such pure culture into
suitable laboratory animals should reproduce
the same lesion of the disease
d) It should be possible to re-isolate the
bacterium in pure culture from the lesions
produced in the experimental animals.

7. PLAQUE HYPOTHESIS
• SPECIFIC PLAQUE HYPOTHESIS
• NON SPECIFIC PLAQUE HYPOTHESIS
• ECOLOGICAL PLAQUE HYPOTHESIS

9. NON SPECIFIC PLAQUE HYPOTHESIS

10. ECOLOGICAL PLAQUE HYPOTHESIS
MARSH IN 1994

11. • Certain low abundance microbial pathways
can cause inflammatory disease by interfering
with the host immune system and remodelling
the microbiota.

12. THEORIES OF DENTAL CARIES
• The legend of worms living worms
• Humoral theory balance between humors
• Vital theory within the tooth
• Chemical theory(parmly-1819) chymal
agent
• Parasitic or septic theory(fincus)
micro-organism
• Chemicoparasitic theory- D.Miller
decalcification and dissolution

13. • Proteolytic theory(gottelib)-
microbes invade organic pathways
• Proteolysis chelation
theory(schwatz)-organic components
forms chelates
• Levine’s theory-chemical
relationship
• Sucrose chelation theory-Ca and P
release

15. Carounanidy usha,Dental caries- A complete changeover,Journal of conservative
dentistry,March 26’2009.

16. SUCROSE METABOLISM
Glucosyltransferase
-Dental caries in a biological context

17. Trait Property of mutant Effect on cariogenicity*
glucosyltransferase** decreased colonization and
plaque formation
reduced
fructosyltransferase loss of extracellular fructan none
fructanase no breakdown of
extracellular fructans
none
IPS production no intracellular glycogen reduced
antigen 1/11 decreased ability to adhere none
enzyme II (PTS) decreased sucrose
transport
none
lactic dehydrogenase no lactic acid production reduced
“aciduricity”*** reduced tolerance of low
pH
reduced

18. Sugar transport High and low affinity transport systems
operating over a wide range of conditions
to ensure substrate uptake, even under
extreme environments, e.g. low pH.
Acid production An efficient glycolytic pathway rapidly
producing low terminal pH values in
plaque.
Aciduricity Cells have specific biochemical attributes
enabling them to survive, metabolize and
grow at low pH values.
Extracellular polysaccharide
(EPS) production
EPS contributes to the plaque matrix,
consolidates attachment of cells, and
may localize acidic fermentation
products.
Intracellular polysaccharide
(IPS) production
IPS utilization allows acid production to
continue in the absence of dietary sugars.

20. ORAL BIOFILM

22. • Acquired pellicle formation
• Initial adhesion
• Maturation
• Dispersion of biofilm cells
• Cooperation in biofilm
• Metabolic communication
BIOFILM FORMATION

23. 1st step of biofilm formation is
the attachment of acquired
pellicle ,which is a thin protein –
containing film derived from
salivary glycoproteins to a clean
tooth surface
More energy will be released if glycoproteins are attached to the tooth
surface

24. Forces of interaction can be divided into 3 types
LONG-RANGE FORCES MEDIUM-RANGE FORCES SHORT RANGE FORCES
50-100 nm 10-50 nm <5nm
•Coulomb
interactions
•Van der Waals forces
•Dipole –dipole
interactions
•Hydrophobic
interactions
•Covalent bonds
•Electrostatic
Interactions
•Hydrogen bonds
•Ionic interactions
•Lewis acid-base
interactions
With these forces,proteins are adsorbed and re-arranged ,some conformational changes
are undertaken

25. THICKNESS
In uncolonized areas, the pellicle reaches a
thickness of 0.01-1 micrometers within 24 hrs
COMPOSITION
Salivary
glycoproteins,phosphoproteins,lipids,gcf
,remnants of cell walls .
PERMEABILITY
Selectively permeable restricting the transport of
ions in and out of the hard tissues

26. •Microbial colonization of teeth requires that bacteria adhere
to the tooth surface.
•Planktonic bacteria recognize the protein binding sites in
acquired pellicle (alpha-amylase and proline rich
glycoprotein/proteins) and bind to the pellicle.
Attachment of single bacterial cell (0-24 hrs)
Growth of bacteria (4-24 hrs)

27. Initially they are non
specifically associated
-Vanderwaal’s force and
repulsive electrostatic
forces
Within short time,these
physiochemical forces
become stronger due to
adhesins on the microbial
surface-short range
interactions with
complementary receptors ,

28. Streptococcus
sanguinis
Actinomyces
species
Streptococcus
oralis
Haemophilus
species
Capnocytophaga
species
Neisseia
species
Streptococcus
mitis
Streptococcus
mutans

29. • As soon as the pioneer bacteria attach to the
pellicle,they secrete extra polysaccharides.
• The main surface attachment are fimbriae and
fibrils.

30. • Forces are hydrogen bonds,hydrophobic
interactions,calcium bridges,van der waals
forces,acid-base interactions and electrostatic
interactions

31. Streptococcus sanguinis
Streptococcus oralis
Actinomyces
naeslundi
Type 1-fimbriae
•Protein –protein
interactions.
Type 2-fimbriae
•Carbohydrate –
protein
interactions
Streptococcus oralis in addition has a galactose binding adhesin..
Actinomyces species can also bind to galactosyl residues in
glycoproteins

32. • Streptococcus mutans is much less than
streptococcus sanguinis in adhering to the
tooth surface.
• 10⁴-10⁵ cells of strep.mutans per mm of
saliva should be present before one cell can
be recovered.
• The equivalent conc of strep.sanguinis is 10^3
ml/cells. ( Van Houte and Green ,1974 )

33. • After the pioneer bacteria attach ,those early
colonizing bacteria provide specific binding
sites either directly or through salivary
glycoproteins binding to the pioneer
organisms for subsequent bacterial
colonization.
• Later colonizing bacteria recognize
polysaccharide or protein receptors on the
pioneer bacterial surface and attach to them.

34. Bacteria coaggregate, forming the typical corn cob forms, bristle
brush forms, or other forms in mature oral biofilm.

35. • Subsequent attached bacterial species include
Fusobacterium
nucleatum
Treponema
species
Tannerella
forsythensis
P.gingivalis
Aggregatibacter
actinomycetomco
mitans

36. Streptococcus
mutans
Fusobacterium nucleatum
P.gingivalis
•The fundamental mechanism of aggregation is
polysaccharide recognition between bacteria
•.The polysaccharide recognition sites vary from
one paired bacterial recognition to another
paired bacterial recognition.

37. CO-AGGREGATION
• Coaggregation bridges usually refer
to a structure of one bacterial
species with two or more different
receptors which can be recognized
by different adhesions of two or
more different bacterial species.
• F. nucleatum - coaggregation bridge
species
streptococcal
species
Obligate
anaerobes
Fusobacterium
nucleatum

38. DISPERSION
• In mature biofilm, bacteria leave the biofilm
by single cell detachment or a cluster of cells
detaching .
• The model of biofilm dispersion includes
erosion, sloughing and seeding.
Limited nutrients present at the
original site
host defense such as fluid shear
force of saliva, which tries to
limit biofilm development
ACTIVE
DISPERSION
PASSIVE
DISPERSION
1
2

39. • A proportional shift occurs during biofilm
development.
• The relative amount of
• Bacterial aggregation is a very complex process.
• Mature biofilms typically contain many porous
layers and water channels through the biofilm,
providing the bacteria essential nutrients.
Actinomyces,
Corynebacterium,
Fusobacterium and
Veillonella increase.
Streptococci and
Neisseria decrease

40. BACTERIAL COOPERATION

41. The foundation of biofilm metabolic communication is
coaggregation, which provides cells close access and makes
communication more convenient between bacterial species.
•EPS is the major component of biofilms.
• All biofilms contain EPS, although the type
of EPS in biofilms varies from one biofilm to
the other due to the bacterial growth status
and the substrates for bacterial metabolism.

42. • Since almost all bacterial species in biofilm can
undergo biosynthesis and degrade EPS, EPS
becomes the communication medium
between bacterial species.
• Provide shelter to the bacteria
• Helps biofilm maintain biofilm structures
• influences ion exchange within biofilms and controls
the hydrophilic or hydrophobic characteristics

43. OXYGEN METABOLISM AND
EXCHANGE
• Oxygen metabolism and exchange within the
biofilm between different aerobic and obligate
anaerobic species has a special significant role
for the survival of obligate anaerobes.
AEROBIC
ORGANISM
LOW REDOX
POTENTIAL
SURVIVAL OF
OBLIGATE
ANAROBES

44. • F. nucleatum is an important bridge bacterium, which
can aggregate with both aerobic and obligate
anaerobic species, allowing the two species to live
together.
• F. nucleatum and Prevotella intermedia can grow in a
wide pH range from 5.0 to 7.0, but P. gingivalis is very
susceptible to a pH lower than 6.5.
However, P. intermedia and F. nucleatum can
produce ammonia and organic acids by fermenting
glutamate and aspartate,providing a more neutral
environment for P. gingivalis and other acid-sensitive
bacterial species.
Other communications
Short-chain fatty acids, exogenous quinines and
vitamin K.
- Hojo and colleagues.

45. • Streptococci possess the strongest ability of
producing bacteriocins among all oral bacteria.
• Bacteriocins produced by S. mutans can be
differentiated into two types termed mutacins.
• Bacteriocins have a broad prospect for application
in the field of medicine. A new class of drugs can be
developed which is targeted and contains only minor
side effects, using its unique narrow- spectrum
antibacterial properties, to avoid microbial flora
and bacterial resistance and achieve the desired
clinical effect under the condition of ensuring the
balance of normal flora in the human body.

46. • In biofilm, the process of bacteria producing signal
molecules, transporting, sensing and controlling a
series of acts is called a quorum sensing system.
• QS systems control a wide range of responses,
including bacterial surface adhesion, extracellular
matrix production, synthesis of biosurfactants, spore
formation, competency, bioluminescence and virulence
factor expression, etc.
• QS systems are highly specific and accurate, which are
the basis of precise regulations of the different
bacterial phenotypes

47. • There are three types of recognized QS
systems in bacteria
GRAM POSITIVE-OLIGOPEPTIDES
GRAM NEGATIVE-ACYL-HOMOSERINE LACTONE
BOTH-Al-1 & 2

48.  Gram-positive bacteria synthesize oligopeptides (red wavy
lines) that are typically modified at specific amino acids and
are actively secreted.
Detection occurs via a two-component signal transduction
circuit, leading to the phosphorylation of a response regulator
protein, which can bind promoter DNA and regulate
transcription of target genes.

49. • In Gram-negative bacteria,
AHLs (red triangles) are
produced by LuxI-like
proteins and are detected by
LuxR-type proteins.
• AHLs freely diffuse across the
cell membrane and increase
in concentration in the
environment in proportion to
cell growth.
• LuxR-type proteins, when
bound to cognate
autoinducers, bind specific
promoter DNA elements and
activate transcription of
target genes

50. • Quorum sensing in Vibrio harveyi.
Two parallel two-component
systems detect AI-1 (blue
triangles), an AHL synthesized by
LuxLM, and AI-2 (red circles), a
furanosyl borate diester, which is
synthesized by LuxS.
• Binding of the autoinducers by
LuxN and LuxPQ leads to the
dephosphorylation of LuxU and
LuxO. Dephosphorylation of LuxO
relieves repression of luxCDABE

51. • Recent evidence has shown that competence-
stimulating peptide (CSP) can induce alarmones which
are intracellular signal molecules and are produced due
to harsh environmental factors, and can convey
sophisticated messages in a population including the
induction of altruistic cellular suicide under stressful
conditions
• S. mutans, the QS systems consist of approximately five
proteins, including a 21-amino-acid CSP.
• CSP signaling molecules are highly species-specific
• Eckert et al
STAMP STREPTOCOCCUS
MUTANS

52. MICROBIOLOGY OF CARIES
AT SPECIFIC SITES

54. ENAMEL CARIES
•Fissures are the most caries prone sites of the dentition and the strongest corelation
between the plaque levels of mutans streptococci and caries have been
found at these sites
In a cross sectional study,71% of
carious fissures had viable
counts of mutans streptococcus.
70 % of fissures that were caries
free had no detectable mutans
streptococci (Loesche et al,1975)
In a longitudinal study of
fissures,the proportion of
mutans streptococcus increased
significantly at the time of lesion
diagnosis (Loesche
&straffon,1979)
A subsequent longitudinal study
confirmed these findings and
demonstrated even stronger
relationship between mutans
streptococci and caries initiation
-Loesche et al,1984

56. • Early signs of fissure
caries develops at the
fissure entrance than
fissure proper
• % of streptococci higher
at fissure entrance than
with the fissure proper
(Meiers &
Schachtele,1984)
• S.mutans (serotype c)
• Caries free and active
tooth
• S.sobrinus (serotype d)
• Caries active recruits

57. • Vellionella species reduce the impact of acid
production by utilizing the lactate produced
from sugar metabolism
• S.salivarius,S.sanguinis and A.naeslundi
produce alkali from salivary components.

58. ROOT CARIES
• Early studies –Actinomyces species in root
caries.
In a cross sectional study of plaque
overlying root surfaces,mutans
streptococci alone or in
combination with lactobacilli is
isolated more frequently
(Billings et al,1985)

59. GRAM POSITIVE
Actinomyces
Streptococcus
Bifidobacterium
Cornybacterium
Lactobacillus
Peptostreptococcus
propionibacterium
GRAM NEGATIVE
Prevotella
Campylobacter
Capnocytophaga
Fusobacterium
Leptotrichia
Selenomonas
Veillonella

60. •This diversity is also observed in active non cavitated
root surface lesions.
Substantial increase in
A.naeslundi
Gram negative species
like prevotella
intermedia and
Capnocytophaga
species

61. High proportions
of lactobacilli and
gram positive rods
like Actinomyces
Eubacterium
saburrem –
considerable
proportion
Actinomyces israelii
Actinomyces
gerencseriae

63. Initial occupants-extension of plaque
community to immediately adjacent tooth
Metabolites from plaque-penetrate thin
epithelial lining of sulcus-inducing strong
inflammatory reaction.
Capillaries dilate become permeable –leakage

64. Some metabolites have chemotactic
properties that induce infiltration of white
blood cells into this region
Sulcular tissue release of plasma like
fluid containing
immunoglobulins,polymorphonuclear
leukocytes,albumins and hemins
NEW
NICHES
COCCI TO FILAMENTOUS
BACTERIA AND
SPIROCHETES

65. Bacteriodes
melongenicus
Proteins and iron
containing
compounds are
available
Very pathogenic plaque
because this produces
several enzymes that
destroy gingival epithelium

66. Bacterial invasion of dentine and root canals
Dentine can be invaded by
• By direct progression of an enamel caries lesion
• From caries of the root surface
• From a periodontal pocket via lateral or accessory canals
• As a result of secondary or recurrent caries
• As a result of fracture or trauma during operative
procedures

68. ADVANCING FRONT OF THE DENTINAL LESION
•The microbial community from the advancing front of a dentinal
lesion is diverse and contains many facultatively- and obligately-
anaerobic
•Gram positive bacteria belonging to the genera Actinomyces,
Bifidobacterium,Eubacterium,Lactobacillus,Parvimonas (formerly Pe
ptostreptococcus),Propionibacterium .
•Streptococci are recovered less frequently,Gram negative bacteria
such as Prevotella, Porphyromonas, and Fusobacterium spp. can also
be isolated but they are generally present only in low numbers.

69. •The microflora found in the dentine and pulp of periodontally-diseased
human teeth is also diverse and may be derived predominantly from
the subgingival area.
•Numerous Gram positive and Gram negative species have been
identified; some are more prevalent in the dentine (e.g. A.
odontolyticus, Bifidobacterium spp.), some predominate in the pulp
(e.g. black-pigmented anaerobes), while others are found equally at
both sites (e.g. A. naeslundii, Veillonella spp., F. nucleatum).
•Dentine collagen is denatured and modified during the caries process,
and becomes more susceptible to breakdown by non-specific proteases,
and this explains the presence of both acidogenic and proteolytic
bacteria.

70. BACTERIAS IN PULP
• Once bacteria are in the pulp, inflammation can occur which
may result eventually in necrosis of the root canal.
• A further consequence is that microorganisms can invade and
destroy tissue surrounding the apex of the root, producing a
spreading or localized infection .
• Diverse mixed culture of bacteria are cultured, including
black-pigmented anaerobes (Prevotella intermedia,
Prevotella melaninogenica, Porphyromonas endodontalis, P.
gingivalis), and Prevotella dentalis, Campylobacter
sputorum,Eubacterium spp.and Parvimonas (formerly Peptos
treptococcus) spp..

71. NECROTIC PULPS
INFECTED ROOT
CANALS AND
ABSCESSES
PAIN
• Propionibacterium
Eubacterium
• Fusobacterium
• Porphyromonas
endodontalis
• P. dentalis
• Parvimonas micra
• Porphyromonas
and Fusobacterium

72. MICROBIAL TESTS
•Lactobacillus colony count test
•Calorimetric snyder test
•Swab test
•Salivary buffer capacity
•Enamel solubility test
•Albans test
•Fosdick calcium dissolution test
•Dewar test
•Cariostat test

73. BACTERIAL INTERACTIONS

74. STREPTOCOCCUS MUTANS
Kreth et al reported dual species inhibitory
activities of S. mutans on S. gordonii, S.
pyogenes, Streptococcus mitis ATCC 903,
Streptococcus pneumoniae, Streptococcus
cristatus and S. sanguinis were significant;
the inhibitions on S. oralis, S. mitis ATCC
33399 and S. parasanguinis were medium;
and the inhibition on S. sobrinus was mild.

75. The numbers of S. mutans and S. sanguinis are negatively
associated.
If the level of S. mutans is high, the level of S.
sanguinis is low
LARGE AMOUNT OF
ORGANIC ACID PRODUCED
BY S.MUTANS THAN
S.SANGUINIS
•When S. mutans and S. sanguinis were cultivated under acidic
conditions (pH 5.5) at the same time, S. mutans grew better
than S. sanguinis.
• Acidic conditions can repress or damage the ATP-glucose
phosphotransferase activity of both strains, but the repression
on S. mutans was less than on S. sanguinis
1.

76. 2.•The mutacin inhibition order of S. mutans on S.
sanguinis from the smallest to the largest was double-
mutant strain (I− IV−), mutacin I-defective strain (I− IV+),
mutacin IV-defective strain (I+ IV−) and wild-type strain
(I+ IV+).
•The presence or absence of oxygen affects mutacin
expression. Under aerobic conditions the mutacin IV
structural gene was upregulated 5-fold compared with a
non-anaerobic condition
A second mechanism
of S. mutans inhibition
on S. sanguinis is the
mutacin
S. mutans excretes

77. .
S. mutans inhibits
the ability of S.
sanguinis to produce
hydrogen peroxide
(H2O2).
The H2O2 production of S. sanguinis was 66%
reduced when S. sanguinis was cultivated
with S. mutans compared with cultivated alone

78. STREPTOCOCCUS SANGUINIS

79. Lactobacillus casei This strain can produce
abundant lactic acid and
has a wide pH range.
Lactobacillus paracasei L. paracasei produces
bacteriocins, which make
pores in the cytoplasmic
membranes
Actinomyces naeslundii A. naeslundii consumes
H2O2 by protein oxidation
Veillonella Produce vitamin
K,consumes organic
acids.

80. Streptococcus
gorgondii
Pyruvate oxidase is affected and
the amount of H2O2 is reduced
in S.mutans.
Streptococcus oralis Cell division is faster than
S.mutans
Streptococcus
oligofermentans.
Produces hydrogen peroxide
a cooperative synergistic growth
relationship may exist between P.
gingivalis and T. denticola.
Fusobacterium
nucleatum.
Supports P. gingivalis growth by
providing a capnophilic
environment.
Porphyromonas
gingivalis

81. ANTIBIOTIC RESISTANCE

82. RESISTANCE
• Antibiotic resistance has become a worldwide problem in public
health.
• The resistance of Streptococci to penicillin, amoxicillin,
trimethoprimsulfamethoxazole and erythromycin was observed
in children treated with antibiotics.
• Biofilms can protect bacteria from a challenging environment
with several host defense mechanisms directed towards bacteria
or protect from applied antibiotics.
• The antigens of biofilm bacteria are hidden in the biofilm matrix
and become less suspectible to the host immune system.
• Physical injury of the biofilm can also be reduced by the biofilm
matrix.

83. • The short distance between bacterial cells makes
cell-to-cell metabolic communications more
frequent.
• Scientists postulated a conception of “insurance
hypothesis,” which stated a single species was
more vulnerable by the environment than
multiple species
• The antibiotic resistance of bacterial cells in
biofilm was reported to be 1,000 to 1,500 times
greater than the resistance of planktonic cellsand
has become a rising problem in recent years.

84. • Antibiotic resistance genes can be transferred
between bacterial cells within biofilm resulting in
a biofilm-wide resistance to the antibiotics.
• The common carrier of resistance genes are
plasmids, which are circular double-stranded
DNA species presenting mainly in bacteria.
• The replication of plasmids is independent of
chromosomal DNA replication and the number of
plasmids in a cell varies widely.
• Plasmids with antibiotic resistance genes are
gained by bacterial conjugation.

85. Almost all biofilm cells displayed a higher minimum inhibitory
concentration (MIC) compared to their relative planktonic
cells. A. naeslundii, Campylobacter species, F. nucleatum, P.
intermedia/nigrescens, P. loescheii, S. intermedius, S.
parasanguis, S. sanguinis, V. atypical and V.
parvula demonstrated very significant (4–250-fold) antibiotic
resistance increases between biofilm and planktonic cells.
Sedlacek and Walker compared the antibiotic effect between biofilm and
planktonic cells.
There were 19 subgingival species investigated:
Actinomyces naeslundii, Actinomyces meyeri, Bacteroides species,
Bifidiobacterium species, Campylobacter species, Eubacterium species,
Fusobacterium alocis, F. nucleatum, Faecalibacterium prausnitzii, P. intermedia/nigrescens,
Prevotella loescheii, Streptococcus intermedius, Streptococcus parasanguis, Streptococcus
sanguinis (earlier named Streptococcus sanguis), Veillonella atypical and Veillonella parvula

86. CARIES VACCINE

87. • 1944 williams –homologous lactobacilli
vaccine
• Streptococcus mutans
•Interfering with adherence,colonization and
dissemination.
•Altering the ability to produce acid
•Reducing its stickiness by altering polysaccharide
mechanism
ACTIVE
IMMUNISATION
PASSIVE
IMMUNISATION

88. MOLECULAR MICROBIOLOGICAL
PERSPECTIVE

89. TRADITIONAL AND NEXT GENERATION
SEQUENCING
• DENATURING GRADIENT GEL
ELECTROPHOROSIS
• PCR BASED METHODS
• 16S Rrna GENE MICROARRAYS
• CHECKERBOARD HYBRIDIZATION
• OPEN ENDED APPROACHES AND NEXT
GENERATION SEQUENCING

90. OTHER MOLECULAR APPROACHES
• METAGENOMIC AND METATRANSCRIPTOMIC
APPROACHES
• METABOMOLIC APPROACH
-Dental caries from a molecular perspective-
caries research-2013

91. DESCRIPTIVE
APPROACH
FUNCTIONAL
APPROACH
What is the
microbial
colonization?
DNA
What is their
Genetic
potential?
DNA
What are the microbial communties doing?
Which genes
are expressed ?
RNA
What is the
protein
Content ?
PROTEINS
What is the
metabolic
output ?
METABOLITES
16S rDNA
approaches
Metageno
mics
Meta
transcriptomics
Meta-
proteomics
Meta -
biomics

92. REFERENCES
• STURDEVANT’S Art and science of Operative dentistry
• Shafer’s textbook of Oral Pathology
• Textbook of operative dentistry-vimal k sikri
• Dental caries the disease and its clinical management,Wiley
blackwell
• Rujee huang, Mingyun li and Richard L Gregory , Bacterial
interactions in dental biofilm, Virulence, 2011 Sep-Oct; 2(5): 435–
444.
• Nyvad,Dental Caries from a Molecular Microbiological Perspective
,Caries Research,2013;47:89-102
• Michael J,Federle ,Interspecies communication in bacteria,Journal
of Clinical Investigation,2003.
• Carounanidy usha,Dental caries- A complete changeover,Journal
of conservative dentistry,March 26’2009.
• Dental caries in a biological context

93. Thank you