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Microbiology of dental plaque differences between immature and
mature plaque.
Microbial metabolism and plaque
biofilm pathogenic potential.
Prof. d-r Kabaktchieva - 2014y.
The
dental professional comes
into contact with two of the
most widespread of all human
diseases - dental caries and
periodontal diseases
gingivitis
caries
caries
Unlike typical infectious diseases, dental caries and
periodontal diseases are not caused by
a single pathogenic microorganism.
 Dental
caries and periodontal
diseases result from the
accumulation of many different
species of bacteria that form
 dental plaque
a naturally acquired
bacterial biofilm that
develops on the teeth

Dental plaque
Dental plaque is a
multi-species
biofilm,
Some bacterial
species may be of
greater relevance in
the development of
caries and periodontal
diseases.
To understand the role of dental
plaque in caries
 must
first know how dental plaque
forms ?
 how changes in the proportions of
different plaque bacteria
can contribute to the development of this
oral disease?
Dental Plaque: A Microbial Biofilm
 Most
natural surfaces have their
own coating of microorganisms,
or biofilm, adapted to their
individual habitats.
 Bacterial
adhesion to surfaces
primarily involves two types of
reactions: physicochemical and
biochemical.
These same interactions
occur in the formation of
plaque and calculus on
oral structures.

All living cells in nature, including bacterial
plaque cells, have a net-negative surface charge.

The cells can, therefore, be attracted to
oppositely charged surfaces as skin.
in the case of bacterial plaque,
have attracting between the surfaces of
cells , teeth, and soft tissues of the oral
cavity.
The microorganisms within bacterial plaque
can produce :

extracellular coatings, such as
slime layers which prolong the existence of
biofilms ;

a variety of surface fibrils, or appendages,
that extend from their cell walls.

These mechanisms mediate attachment of bacteria to a
substrate by providing additional attachment structures
between the tooth surface and the plaque, thus allowing
the formation of adherent matrices.
Bacterial Colonization of the Mouth

Microorganisms found in the oral cavity are
naturally acquired from the environment.

Bacteria are acquired from the atmosphere,
food, human contact.

Bacteria form colonies between saliva
and hard tissues such as erupted teeth,
and exposed root cementum and dentin.

Prior to eruption, the external surface of
tooth enamel is lined by remnants of the
enamel-forming organ.

These tissue remnants are:
- the reduced enamel epithelium
and
- the basal lamina

The basal lamina connects the epithelium to the
enamel surface.

The basal lamina is also continuous with organic
material that fills the microscopic voids in the
superficial enamel.

This subsurface organic material appears as a
fringe-like structure attached to the basal lamina
and is composed of residual enamel matrix
proteins.
This material is referred to as a subsurface
pellicle.
The pellicle originates from local cells during
tooth formation; therefore, it is considered to
be of endogenous origin.
SSP
B
AP
ES
The transmission electron micrograph demonstrates :
- remnants of the subsurface pellicle (SSP)
- the acquired pellicle (AP)
- They are between the enamel surface (ES) and
RA
HD
EM
ES
BL
Figure . Junction of reduced enamel epithelium and enamel.
The reduced ameloblasts (RA) are attached to the enamel by
hemidesmosomes (HD) and a basal lamina (BL).
EM, enamel matrix remnants form a subsurface pellicle; ES,
 When
the tooth emerges into the oral
cavity, the remnants of the reduced
enamel epithelium are:
worn off
- digested by salivary and bacterial
enzymes
-

An erupted tooth immediately becomes
covered by a thin, microscopic coating of
saliva materials.

The salivary components become adsorbed
to the surface of the enamel within seconds.
This coating is also referred to as a
pellicle.

Because the pellicle is acquired after eruption
of teeth, it is said to have an exogenous
origin
the pellicle was formed by a substance
from outside the tooth, rather than during
the development of teeth.
The
oral bacteria can form
colonies in the acquired
pellicle.
The Acquired Pellicle
The coating of salivary origin that forms on
exposed tooth surfaces is called the
acquired pellicle.
It is acellular and
consists primarily of
glycoproteins
derived from saliva
B
АР
ЕS
A glycoprotein is a protein molecule
that includes an attached carbohydrate
component.

Oral fluids and small molecules can slowly
diffuse through the acquired pellicle into the
superficial enamel;

If the pellicle is displaced, ( by a prophylaxis),
the pellicle begins to reform immediately;

It takes about a week for the pellicle to develop
its condensed, mature structure, which may
also incorporate bacterial products.


Colonization of the acquired pellicle can be
beneficial for the bacteria, because the
pellicle components can serve as nutrients.
For example:
proline-rich salivary proteins
may be degraded by bacterial
collagenases.
This action releases
peptides, free amino acids, +
salivary mucins may enhance
the growth of dental plaque
organisms, such as
actinomycetes and
spirochetes.
The
carbohydrate components
of certain pellicle glycoproteins
may serve as receptors for
proteins that bind bacteria to
surfaces - e.g., adhesins,
Тhereby the adhesins contributing to
bacterial adhesion to the tooth.
The
-
binding sites on the pellicle,
are also host proteins, including:
immunoglobulins (i.e., antibodies),
the enzyme lysozyme, and
proteins of the complement system.
These host proteins originate from saliva and
gingival sulcus fluid.
 Аn
antagonistic relationship often exists
between different types of bacteria
competing for the binding sites.
 For
example:
it has been shown that
some streptococci
synthesize and release
proteins called
bacteriocins, which can
inhibit some strains of
Actinomyces and
Actinobacillus species.
Dental Plaque Formation

All bacteria that initiate plaque formation
come in contact with the organically coated
tooth surface by chance.

Forces exist that tend either to allow bacteria
to accumulate on teeth or to remove them.

Shifts in these forces determine whether
more or less plaque accumulates at a given
site on a tooth.

Bacteria tend to be removed from the teeth
during mastication of foods, by the tongue,
and by toothbrushing and other oral hygiene
activities.

For this reason, bacteria tend to accumulate
on teeth in sheltered, undisturbed
environments, which basically are sites at
risk.

These sites include the occlusal fissures,
the surfaces apical to the contact between
adjacent teeth, and in the gingival sulcus.
 Therefore,
it is no coincidence
that the major plaque-based
diseases - caries and
inflammatory periodontal
diseases arise at these sites
where plaque is most abundant
and stagnant.
 Initial
plaque formation may take
as long as 2 hours.
 Colonization
begins as a series
of isolated colonies, often
confined to microscopic tooth
surface irregularities.
 With
the aid of nutrients from saliva
and host food, the colonizing bacteria
begin to multiply.
 About
2 days are required for the
plaque to double in mass,
 During
which time the bacterial
colonies have been growing together.
 The
most dramatic change in
bacterial numbers occurs during
the first 4 or 5 days of plaque
formation.
 After
approximately 21 days,
bacterial replication slows, and
plaque accumulation becomes
relatively stable.
 The
increasing thickness of the plaque
limits the diffusion of oxygen to the
entrapped original, oxygen-tolerant
populations of bacteria.
 As
a result, the organisms that survive
in the deeper aspects of the
developing plaque are either
facultative or obligate anaerobes.
 The
forming bacterial colonies are
rapidly covered by saliva.
 When
seen with the scanning electron
microscope, growing colonies protrude
from the surface of the plaque as
domes .
“Domes” have
appearance
of a cluster of
igloos
beneath
newly fallen
snow
Scanning electron micrograph
of dome formation in the plaque.
In individuals with poor oral
hygiene, superficial dental
plaque may incorporate :
- food debris ,
- human cells such as epithelial
cells (desquamated cells)
- leukocytes.


This debris is called materia
alba, which literally means
"white matter.“

Unlike plaque, it is usually
removed easily by rinsing with
water.
cloud
 At
times, the
plaque
demonstrates
staining, which is
caused by
chromogenic
bacteria, which
produce a brown
pigment.
Molecular Mechanisms of
Bacterial Adhesion

The initial bacterial attachment to the acquired
pellicle ( A) is thought to involve physicochemical
interactions (e.g., electrostatic forces and
hydrophobic bonding) between molecules of the
amino acids phenylalanine and leucine.

details A. A side chain of a phenylalanine
component of a bacterial protein interacts via
hydrophobic bonding with a side chain of a leucine
component of a salivary glycoprotein in the
acquired pellicle.

The hydrophobicity of some streptococci, is caused
by cell wall-associated molecules including
glucosyltransferase, an enzyme that converts the
glucose portion of the sugar, sucrose, into extracellular
polysaccharide.

Some glucosyltransferases have been designated as hydrophobins.

These polysaccharides include "sticky" glucans that,
through hydrogen bonding, are thought to contribute to
the mediation of bacterial adhesion (Fig. C).

Once the bacteria adhere, they are often "entombed" as additional glucan is produced.

C. The host's dietary sucrose is converted by
the bacterial enzyme, glucosyltransferase, to
the extracellular polysaccharide,
glucan, which has many
hydrophobic groups and can
interact with amino acid side-chain
groups, such as serine, tyrosine,
and threonine. in the acquired
pellicle.
Molecular Mechanisms of Bacterial Adhesion

B. The negatively charged carboxyl group of a
bacterial protein is attracted to a positively
charged calcium ion (i.e., electrostatic attraction),
which in turn is attracted to a negatively charged
phosphate group of a salivary phosphoprotein in
the acquired pellicle.

Bacteria also have external cell-surface
proteins termed adhesins, which have
lectin-like activity, because they can bind
to carbohydrate components of
glycoproteins.
Тhe adhesins may be located on bacterial
surface appendages, such as fimbriae
(Figure D).
 Fimbria-associated adhesins probably
mediate bacterial adhesion via ionic or
hydrogen-bonding interactions.


D. The fimbrial surface appendage extends
from the bacterial cell to permit the terminal
adhesin portion to bind to a sugar component
of a salivary glycoprotein

Facultative anaerobes can exist in an
environment with or without oxygen;

obligate anaerobes cannot exist in an
environment with oxygen.

Lectins are plant proteins with receptor sites
that bind specific sugars.
Another molecular mechanism of bacterial
adhesion is calcium bridging. (Figure B).

In this process positively charged,
divalent calcium ions in the saliva help to link
the negatively charged cell surfaces of
bacteria to the negatively charged acquired
pellicle
Bacteria in the Dental Plaque

The bacteria colonize the teeth in a
reasonably predictable sequence.

The first to adhere are primary colonizers,
sometimes referred to as "pioneer species”.

These are microorganisms that are able to
stick directly to the acquired pellicle.
 Those
that arrive later are secondary
colonizers.
 They
may be able to colonize an
existing bacterial layer, but they are
unable to act as primary colonizers.
 Generally
speaking, the primary
colonizers are not pathogenic.
If the plaque is allowed to remain undisturbed,
it eventually becomes populated with secondary
colonizers that are the likely etiologic agents of
dental caries and periodontal diseases.



The earliest colonizers are cocci (spherical
bacteria), especially streptococci, which
constitute 47% to 85% of the cells found during
the first 4 hours after professional tooth cleaning.
These organisms tend to be followed by
short rods and filamentous bacteria.
 Тhe
most abundant colonization is on
the proximal surfaces, in the fissures
of teeth, and in the gingival sulcus
region.
 Cocci
are probably the first to adhere
because they are small and round and,
therefore, have a smaller energy barrier
to overcome than other bacterial forms.

The first colonizers tend to be aerobic
(oxygen-tolerant) bacteria including Neisseria and
Rothia;

The streptococci, the gram-positive facultative
rods, and the actinomycetes are the main
organisms in plaque found in early fissures and
approximal plaque.

As plaque oxygen levels fall, the proportions of
gram-negative rods (e.g., fusobacteria) and
gram-negative cocci such as Veillonella
tend to increase.
Of the early colonizers:
- Streptococcus sanguis often appears first,
- followed by Streptococcus mutans

Both depend on a sheltered environment for
growth and the presence of extracellular
carbohydrate (e.g., sucrose).
Sucrose is used to synthesize intracellular
polysaccharides that serve as an internal
source of energy, as well as external
polysaccharide coats.
 The
polysaccharide coating helps
protect the cell from the osmotic
effects of sucrose.
 In
addition, the coating reduces the
inhibitory effect of toxic metabolic end
products, such as lactic acid, on
bacterial survival.
 Тhe
non-motile cells, including
streptococci and actinomycetes, come into
contact with the tooth randomly,
 motile
cells such as the spirochetes are
likely to be attracted by chemotactic factors
(e.g., nutrients).
 Surface
receptors probably provide a
means of attachment for secondary
colonizers onto the initial bacterial layer.
 Bacteria
that cannot adhere easily
to the tooth initially via organic
coatings can probably attach by
strong lectin-like, cell-to-cell
interactions with similar or
dissimilar bacteria that are already
attached (i.e., the primary
colonizers).
 Gram-negative,
anaerobic species such
as Treponema, Porphyromonas,
Prevotella, and Fusobacterium species
predominate in the subgingival plaque
during the later phases of plaque
development, but they may also be
present in early plaque.

There is evidence that oxygen does not penetrate more than 0.1 mm into the
dental plaque, a fact that may explain the presence of anaerobic bacteria in
early plaque.
Dental Plaque Matrix

The organisms are positioned
perpendicular to the tooth surface as a
result of competitive colonization.

The bacterial cells in the biofilm are
surrounded by an intercellular plaque
matrix (Figure – see next).
Figure . An electron
micrograph showing
palisades (P) of
bacteria
perpendicular to the
enamel surface
(ES),
bacterial cells that
are probably
secondary
colonizers (SC),
 the intercellular
plaque matrix (IPM),
 the acquired
pellicle (AP).
 The matrix is composed of both organic and
inorganic components that originate primarily
from the bacteria.

Polysaccharides derived from bacterial
metabolism of carbohydrates are a major
constituent of the matrix,
 salivary and serum proteins/
glycoproteins represent minor components.

Some bacteria on the surface of the biofilm
aggregate into distinctive structures that
include arrangements of cocci ("corn-cob"
configurations) and rods ("test-tube brush"
configurations)
radially arranged around a central filament
(Figure see next).
Figure : A. Cross section of "corn cob" from 2-month-old plaque.
A coarse fibrillar material attaches the cocci (C) to the central filament
(CF)..
B. Coarse "test-tube brush" formations consisting of
central filament (CF) surrounded by large, filamentous bacteria with
flagella uniformly distributed over its body (LF). Background consists of a
spirochete-rich microbiota (S).
Dental Plaque Metabolism
 For
metabolism to occur,
a source of energy is required.
For the caries-related S. mutans and
many other acid-forming organisms,
this energy source can be sucrose.
Almost immediately following exposure of
these microorganisms to sucrose, they
produce:

acid, intracellular polysaccharides, which
provide a reserve source of energy for each
bacterium

extracellular polysaccharides including
glucans (dextran) and fructans (levan).
 Glucans
can be viscid substances
that help anchor the bacteria to
the pellicle, as well as stabilize
the plaque mass.

Fructans can act as an energy
source for any bacteria having the
enzyme levanase.
 The
glucans constitute up to
approximately 20% of plaque dry
weight,
 Levans - about 10%, and
 Bacteria the remaining 70% to
80%.
 The
glucans and fructans are major
contributors to the intercellular
plaque matrix.
 Plaque
organisms grow under adverse
environmental conditions, which include:
- varying pH,
- temperature,
- ionic strength,
- oxygen tension,
- nutrient levels,
- antagonistic elements, such as competing
organisms ,
- the host inflammatory-immune response.

Variations of the aforementioned conditions
can affect the primary and extended
adherence of microorganisms to the surface,

as well the diffusion of essential elements
(i.e., oxygen and nutrients),

all of which are required for the prolonged
existence of bacterial biofilm.
 The
plaque organisms must find a
safe haven in relation to their
neighbors and the oral
environment.
 Such
a favorable location is
termed an ecological niche.
 With
dietary sugars entering the plaque,
anaerobic glycolysis results in
acidogenesis (acid production) and
accumulation of acid in the plaque.

If no acid-consuming organisms (e.g.,
Veillonella) are available to use the acids,
the plaque pH drops rapidly from 7.0 to
below 4.5.
 This
drop is important because enamel
begins to demineralize between pH 5.0 and
5.5.
 One
possible outcome of the drop in
pH may be the dissolution of the
mineralized tooth surface adjacent to
the plaque, resulting in carious
cavitation of the tooth.

This process provides the bacteria
access to the inorganic elements
(e.g., calcium and phosphate) needed
for their nutritional requirements.
 Until
supragingival plaque
mineralizes as dental calculus, it
can be removed by mechanical
debridement (e.g., toothbrushing,
flossing, or use of interdental
aids).
coloring

Dental plaque cannot be removed by
rinsing alone.

As the plaque matures, it becomes more
resistant to removal with a toothbrush.

almost 3 times as much pressure to
remove it on the third day as on the first.
END