Download Features of biofilms

Oral Biofilm
Contents
•Biofilm –Definition
•Studies on Biofilm
•Why make a biofilm?
•Stages of biofilm growth
•Structure Of Biofilm
•Features Of Biofilm
•Dental Plaque
•Classification Of Plaque
•Clinical appearance of plaque
•Composition of plaque
•Microbiota Of plaque
•Plaque Formation
•Growth Dynamics of plaque
• Microbial Interactions in dental Plaque
•Principle of bacterial transmission
•Microbial Homeostasis
•Plaque and Disease
•Microbial specificity of Periodontal
Diseases
•Denture Plaque
•Conclusion
Bio film
A biofilm is "a microbially derived sessile community
characterized by cells that are irreversibly attached to a
substratum or interface or to each other, are embedded
in a matrix of extracellular polymeric substances that
they have produced, and exhibit an altered phenotype
with respect to growth rate and gene transcription.”
(Donlan and Costerton, 2002)
Antonie Van Leeuwenhoek,first observed
microorganisms on tooth surfaces and can be
credited with the discovery of microbial biofilms.
95 % of bacteria existing in nature are in
biofilms
Studies on oral biofilms
- Plaque structure (Frank and Houver, 1970; Schroeder & de Boever, 1970;
Nyvad and Fejerskov, 1989)
-Bacterial composition (Bowden et al, 1975; Bowden, 1991; Moore and
Moore, 1994)
- Mechanisms involved in bacterial adherence (Gibbons, 1989;
Ofek and Doyle, 1994)
-Immune responses (Brandtzaeg, 1988; Ebersole, 1990; Michalek and Childers,
1990; Smith and Taubman, 1992),
-Concepts of competition and responses to the
environment among oral bacteria (Ritz, 1967, 1969; van der Hoeven and
de Jong, 1984; Hamilton and Buckley, 1991).
Ecological Advantages: Why Make A Biofilm?
1.Protection from the Environment
(Host defences,dessication,antimicrobial agents)
2.Nutrient Availability and Metabolic Cooperativity
3.Acquisition of New Genetic Traits
Stages of Biofilm Growth
A biofilm is organized to maximize energy, spatial
arrangements and movement of nutrients and byproducts
Physical composition, degree of organization and multispecies organization characterize the four stages of
biofilm growth
Stage I - Quiescent or least metabolically active state.
Conversion or transformation from Stage I to Stage II
requires significant genetic up-regulation.
Stage III - Maturity of the biomass. New antigens may
be expressed, genetic exchange enhanced & membrane
transport maximized.
Stage IV (apoptosis or death) signals detachment,
eroding or sloughing from the biofilm.
Structure Of Biofilm
Heterogeneous containing microcolonies of bacterial cells
encased in an EPS matrix and separated from other
microcolonies by interstitial voids (water channels)
Stoodley et al. (1997) defined certain criteria or characteristics
that could be considered descriptive of biofilms in general,
including a thin base film, ranging from a patchy monolayer of
cells to a film several layers thick containing water channels.
Basic structural unit of the biofilm is the microcolony.
Microcolonies - Single-species populations or multimember
communities of bacteria
Surface and interface properties, nutrient availability, the
composition of the microbial community, and hydrodynamics, can
affect biofilm structure
Under high shear stresses, such as on the surface of teeth during
chewing, the biofilm (dental plaque) is typically stratified and
compacted
Altered in response to flow conditions
Laminar flow- Patchy , rough round cell aggregates separated by
interstitial voids.
Turbulent flow - Patchy, but elongated "streamers" that oscillated
in the bulk fluid
Structure Of Biofilm
Fluid layer bordering the biofilm - Stationary sublayer
Fluid layer in motion. Nutrient components penetrate this fluid
medium by molecular diffusion.
Steep diffusion gradients, especially for oxygen, exist in the
more compact lower regions of biofilm, which explains changes
in microbial composition.
Interstitial voids or channels

Integral part of the biofilm structure.
Liquid flow occurs in water channels, allowing diffusion of
nutrients, oxygen, and antimicrobial agents.

Lifeline of the system, provide a means of circulating nutrients
as well as exchanging metabolic products with the bulk fluid
layer

Mechanisms for the formation & maintenance of these
structures.


Primitive circulatory system.
Structure Of Biofilm
Extracellular Polymeric Substances
50% to 90% of the total organic carbon
Polysaccharides- Neutral or polyanionic (Gram – ve Bacteria)
Anionic property allows association of divalent cations such as
calcium and magnesium, which have been shown to cross-link
with the polymer strands and provide greater binding force in a
developed biofilm
Gram-positive bacteria - EPS cationic.
EPS highly hydrated . Prevents desiccation in some natural
biofilms.
Hydsrophobic/Hydrophillic
Structure Of Biofilm
EPS may vary in its solubility
EPS of biofilms not generally uniform but may vary spatially
and temporally.
Amount of EPS increases with age of the biofilm.
EPS may associate with metal ions, divalent cations, other
macromolecules
EPS production affected by nutrient status of the growth
medium
Slow bacterial growth will also enhance EPS production
EPS may also contribute to the antimicrobial resistance
properties of biofilms
Features of biofilms
Protection for resident bacteria by giving them a competitive
advantage over free floating bacteria.
Biofilms also offer a nutritional advantage to bacteria that reside
within them (Bowden 1997)
Antimicrobial Resistance in Biofilm

Resistance of bacteria to antimicrobial agents is increased in the
biofilm (Allison 1990,Costerton 1999)
1. Slower rate of growth of bacterial species in biofilm
(Ashby 1994,Brooun 2000)
2. Strongly charged or chemically highly reactive agents can fail to
reach deeper zones of the biofilm because the biofilm acts as an ion
exchange resin, removing such molecules from solution (Gilbert 1999)
3. Extracellular enzymes such as Beta lactamase, formaldehyde
lyase, formaldehyde dehydrogenase may become trapped and
concentrated in the ECM thus inactivating some antibiotics.
Features of biofilms
4. Multidrug resistance pumps that can extrude
antimicrobial agents from the cell (Brooun 2000)
5. Quorum sensing seems to play a role in antibiotic
resistance
6. Conjugation, transformation plasmid transfer and
transposon transfer have occur more easily in a biofilm.
Features of biofilms
Quorum Sensing
 Mechanism of gene regulation in which bacteria coordinate the
expression of certain genes in response to the presence or absence of
small signal molecules
 The QS bacteria release, detect and respond to accumulation of
small signal molecules, in a cell density-dependent manner, thereby
regulating the expression a set of target genes.
 The term "quorum" is used to describe this kind of signal system,
since a certain number of micro-organisms must be present for the
signal to be sensed and for the population to respond to the signal.
Features of biofilms
Activities under
quorum-sensing control include
1.Secondary metabolite production
2.Motility
3.Symbiosis
4.Nodulation
5.Conjugal plasmid transfer
6.Biofilm maturation and virulence
Features of biofilms
Autoinducers
 The
stimuli of quorum- sensing systems are signal molecules,
called autoinducers.

Produced at a basal constant level, and the concentration thus is a
function of microbial density.

Bacterial autoinducers can be classified into three major chemical
groups
i)N-acyl homoserine lactones (AHLs) 70 species of Gram-negative
bacteria
ii) Oligopeptides, Gram- +ve bacteria
iii) Ribose-like S-4,5-dihydroxy-2,3-pentanedione
(DPD)/autoinducer-2 (AI-2), by both Gram-ve and + ve bacteria interspecies QS signaling molecule
Features of biofilms
At low population densities,
basal-level production of
autoinducer molecules results in
the rapid dilution of the
autoinducer signals in the
surrounding environment.
At high population densities, an
increase in bacterial number
results in accumulation of
autoinducers beyond a threshold
concentration, leading to the
activation of the response
regulator proteins, which in turn
initiate the quorum-sensing
cascade.
 These
molecules usually diffuse into the cells and bind directly to
a response regulator (Bassler, 1999; de Kievit and Iglewski, 2000).
 As
micro-organisms release the signal molecules when
approaching a surface, the signal molecule concentration increases
in the area between the micro-organism and the surface, due to
limited diffusion.
 The
micro-organisms will perceive this concentration increase
and thus sense the presence of the surface they are colonizing
(Costerton, 1999).
 The
signal molecules of quorum-sensing systems are often highly
specific. Quorum-sensing signaling thus serves intra species
communication purposes.
Features of biofilms
Quorum Quenching
Inhibition of quorum sensing (signaling molecules) by
degradation enzymes
Quorum-sensing-interfering compounds either produced
naturally by certain eukaryotic hosts, synthesized by chemical
methods, or produced by creating transgenic plants all have
either a positive or a negative effect on the expression of
bacterial phenotypes regulated by quorum sensing.
It is also possible that quorum quenching is used as a
defense mechanism against antibiotic-producing bacteria in
the ecological niche.
Quorum sensing disrupting compounds attenuate virulence of
bacteria
Features of biofilms
Gene Transfer
Biofilms - Ideal niche for the exchange of extrachromosomal
DNA
Conjugation - Greater rate between cells in biofilms than
between planktonic cells
Enhanced conjugation - Biofilm environment provides minimal
shear and closer cell-to-cell contact.
Since plasmids may encode for resistance to multiple
antimicrobial agents, biofilm association also provides a
mechanism for selecting for, and promoting the spread of, bacterial
resistance to antimicrobial agents
Features of biofilms
Dispersal
Shedding of daughter cells from actively growing cells
Detachment as a result of nutrient levels or quorum sensing, or
shearing of biofilm aggregates (continuous removal of small
portions of the biofilm) because of flow effects
Brading et al.1995 Three main processes for detachment are
erosion or shearing (continuous removal of small portions of the
biofilm), sloughing (rapid and massive removal), and abrasion
(detachment due to collision of particles from the bulk fluid with
the biofilm).
Eroded or sloughed aggregates from the biofilm retain certain
biofilm characteristics, whereas cells that have been shed as a result
of growth may revert quickly to the planktonic phenotype
Features of biofilms
Adaptation To Life In A Biofilm
Different gradients of chemicals, nutrients, and oxygen
create micro-environments to which micro-organisms
must adapt to survive.

Regulation of a vast set of genes, and the microorganisms are thus able to optimize phenotypic properties
for the particular environment.

Biofilm micro-organisms differ phenotypically
from their planktonic counterparts.

Features of biofilms
Dental Plaque
Dental
Plaque is defined clinically as a structured,
resilient, yellow grayish substance that adheres
tenaciously to the intraoral hard surfaces, including
removable and fixed restorations (Bowen et al, 1976)
Clinical importance - Primary etiologic agents in the
development of dental caries and periodontal diseases

(Socransky 1998,Haffajee 1994)
Dynamic and complex microbial community adherent to
the tooth surface

Classification of Plaque
Based on position on the tooth surface toward the gingival
margin, as follows:
 Supragingival plaque - At or above the gingival
margin; when in direct contact with the gingival margin, it
is referred to as marginal plaque
 Subgingival plaque - Below the gingival margin,
between the tooth & gingival pocket epithelium
Clinical appearance of Plaque
Supragingival plaque - Nearly invisible thin film on the
tooth surface to thick mats of material that completely
obscure the tooth surface and cover the gingival margin.
Sub gingival plaque - Difficult to visualize because it is
hidden from view within the gingival crevice or
periodontal pocket, composed of highly organized masses
of bacteria.
Composition Of plaque
Microorganisms
Non bacterial microorganisms
Intercellular matrix :Organic and inorganic
Organic constituents- Polysaccharides, proteins,
glycoproteins, and lipid material.
Inorganic components –Ca,P,Na,K,F
Microbiota of Plaque
Supragingival plaque –
Gram positive cocci and short rods - Tooth surface
Gram negative rods and filaments, spirochetes Outer surface
Sub gingival Plaque Tooth associated plaque - Filamentous microorganisms,gram
positive rods & cocci including Streptococcus mitis,S.sanguis,
Actinomyces viscosus, A. naesulundii, Eubacterium species
Deeper parts of the pocket- Filamentous organisms fewer in
number, in apical portion -absent.
Apical border of the plaque mass separated from the Junctional
epithelium by a layer of host leukocytes
Apical tooth associated region -  concentration of gram -ve
rods.
Tissue associated plaque
Gram negative rods and cocci ,filaments, flagellated rods, and
spirochetes.
Streptococcus oralis, S.intermedius, P.micros,P.gingivalis
P.Intermedia,T.forsythua,F.nucleatum (Dbart 1998,Dzink 1989)
Biofilms In the subgingival environment
• Bathed in GCF
• Faster formation of plaque.
• Sub gingival accumulation of toxic substances
• The pH increases in GCF where dental plaque has grown (6.9 to
8.6)
•Redox potential.
Biofilm associated with Crevicular epithelium
• S sanguis has multiple adhesions that allow it to adhere to oral
epithelium
Oral treponemes bind to fibronectin
P gingivalis binds to RBCs,macrophages,fibrinogen.
• A.A comitans and P.gingivalis once attached to oral epithelium
can become internalized by fusion and endocytosis. Thus
protected from immune system and antibiotics &have a most
nutritious environment.
• Crevicular epithelium is frequently ulcerated in disease, this
gives bacteria like P.gingivalis access to bind to type IV collagen
in basement membranes which could facilitate its potential
invasion of gingival connective tissue.
Formation of plaque
Dental plaque forms via an ordered sequence of events, resulting in
a structurally- and functionally-organized, species-rich microbial
community
Distinct stages in plaque formation include:
 Acquired pellicle formation
 Reversible adhesion
 Co-adhesion resulting in attachment of secondary colonizers to
already attached cells
 Multiplication and biofilm formation
 Detachment.
Pellicle Formation
 Pellicle forms by selective adsorption of the environment
macromolecules (Scannapieco1990)

Pellicle consists of glycoproteins (mucins) ,proline rich proteins,
phosphoproteins, histidine rich proteins, enzymes, that function as
adhesion sites for bacteria

Electrostatic,vanderwaals and hydrophobic forces.
Characteristics
of the underlying hard surface are transferred
through the pellicle layers and can still influence initial bacterial
adhesion (Pratt terpestra1991)
observed a clear relationship between the type of
proteins adsorbed in the pellicle and the free energy of the
substratum surface.
Absolem
et al 1987
The
adsorption of saliva molecules onto solid surfaces is
dependent on the surface, and on composition of saliva and
complex intermolecular relationships.
The
mean pellicle thickness ranges from about 100nm at 2
hours to 500 to 1000nm at 24 to 48 hours.
Mucins
• Multifunctional molecules involved in the formation of acquired
pellicle, lubrication of tooth surfaces to reduce wear, and protection
of oral surfaces from dehydration (Levine 1987,Tabak 1982)
• HMW mucin (>1000 kD) called MUC 5 B and a LMW MUC 7
• MUC 5 B selectively forms heterotropic complexes with many
salivary proteins and incorporates them into the acquired pellicle
(Iontcheva 2000) . It attracts S.sanguis, S.gordonii, E.corrodens,
S.aureus, P.aeruginosa. (Cohen 1989)
• MUC 7 has a long protein core with sugar containing short side
chains of varying lengths. At the terminal ends of the sugars there
are often sialic residues that can bind to receptors on pellicle or
epithelial cells.
• S sanguis and S. mitis (some of the first bacteria to colonise
pellicles ),possess adhesions for sialic acid. Other bacteria such as
A.viscosus, A.naselundii, Prevotella intermedia, E.corrodens
Fusobacterium species have galactosyl adhesions (Kollenbrander
1992)
Proline rich proteins
• Heterogenous family of salivary proteins that contain abundant
amounts of proline, glutamic acid/glutamine and glycine.
• Three groups of PRPs:acidic(16kD),basic(6-9kD) and
glycosylated(36kD) (Bennick 1982)
• Acidic and glycosylated PRPs are found in newly formed
acquired pellicles (Bennick 1983)
Cystatins
•Physiologic inhibitors of cystine proteases such as cathepsins
B,H,L
•Cystatin SA play a role in regulating the activity of cysteine
proteases, role in pellicle formation and remineralisation (Johnson
1991)
Statherin
•LMW salivary acidic phosphoprotein
•Promotes bacterial adhesion to tooth surfaces (Amino 1994,Gibbons 1988)
•Four statherin variants present in saliva (Jensen 1991)
Histatins
•12 histidine rich small proteins.
•Possess antifungal properties.
•Histatin 5 effective against Candida albicans
•Acts by binding to specific protein receptors on the cell membrane of
the organism.
•Inhibitory effects of Histatin on aggregation,adherence,and protease
activity of P.gingivalis reported (Murakami1991)
Initial adhesion and attachment of bacteria
I stage -Initial transport of the bacterium to the tooth
surface. Through
Brownian
motion
Sedimentation of microorganisms
Liquid flow
Active bacterial movement
II- Initial adhesion
Initial, reversible adhesion of the bacterium, initiated by
the interaction between bacterium and surface, from a
certain distance (50nm) through long range and short
range forces, including van der waals attractive forces and
electrostatic repulsive forces.
Derjaguin,Landau,Verwey
and Overbeek (DLVO)
have postulated that ,above a separation distance of 1nm,the
summation of the electrostatic and van der waals forces
descibes the total long range interaction. (Rutter 1984)

Total Interaction energy/Total Gibbs Energy GTOT= GA+GE
This is a function of the separation distance between a
negatively charged particle and a negatively charged surface in
a medium ionic strength suspension medium (Eg. Saliva).

For most bacteria, GTOT consists of a secondary minimum,
where a reversible binding takes place,5-20nm from the
surface, a positive maximum (an energy barrier B) to adhesion,
and a steep primary minimum (located at <2 nm from surface)
where an irreversible adhesion is established.

III- Attachment
Firm anchorage between bacterium and surface
Specific interactions (covalent, ionic or hydrogen bonding)
 Bonding between the bacteria and pellicle is mediated by
specific extracellular proteinaceous components (adhesions) of the
organism and complementary receptors (proteins,glycoprotweins
or polysaccharides) on the surface of the pellicle and is species
specific.
 Each Streptococcus and Actinomyces strain binds specific
salivary molecules.
Molecular interactions between adhesins and receptors
Short range forces involve specific sterochemical interactions
between components
On the microbial cell surface(adhesins) and receptors in the
acquired pellicle; these type of interactions contribute to the
tropism of an organism with a particular surface or habitat.
Streptococci ( especially, S Sanguis), the principal early
colonizers, bind to acidic proline rich proteins and other receptors
in the pellicle, such as alpha amylase and sialic acid (Hsu 1994)
Actinomyces species can also function as primary colonizers,
for example, A viscosus possesses fimbriae that contain adhesions
that specifically bind to proline rich proteins of the dental pellicle
(Gibbons 1988)
This firm attachment is followed by surface colonization
and biofilm formation, which eventually reaches the "climax
community" of dental plaque. (Stage IV)
Colonisation and Plaque maturation
 Co aggregation refers to the adhesion of two distinct bacterial
cell types to form visible aggregates
 18 genera from the oral cavity have shown some form of co
aggregation (Kolenbrander 1993)
 Cell cell interaction occurs primarily through the highly
specific sterochemical interactions of protein and carbohydrate
molecules located on the bacterial cell surfaces, in addition to the
less specific interactions resulting from hydrophobic, electrostatic
and van der waals forces.
Fusobacteria co aggregate with all other human oral bacteria,
whereas Veillonella, Capnocytophage,and Prevotellae bind to
streptococci and actinomycetes (Kollenbrander 1993,Whittaker 1996)
Mediated by lectin like adhesions and can be inhibited by lactose
and other galactosides.
Well characterized interactions of secondary colonizers with
early colonizers include the aggregation of Fusobacterium
nucleatum with Streptococcus sanguis, Prevotella loeschii with
A.viscosus and Capnocytophaga ochraceus with A.viscosus.
Streptococci show intrageneric co aggregation allowing them
to bind to the nascent monolayer of already bound streptococci.
 Early colonizers are either independent of defined complexes
(A.naseulundii ,A.viscosus) or members of the yellow
(streptococcus) or purple complexes (A.odontolyticus)
 The microorganisms primarily considered secondary colonizers
fell into the green orange or red complexes
 Each strain of an early colonizer is coated with distinct
molecules.Identical cells coated with a specific salivary molecule
may agglutinate which would lead to a micro concentration and
juxtapositioning of a particular strain.
Both streptococci and actinomycetes are facultative anaerobes,
They are taught to prepare a favorable environment for later
colonizers, which have more fastidious growth requirements.
In the latter stages of plaque formation, co aggregation between
different gram – species is likely to predominate. E.g.. F nucleatum
with P.gingivalis or T.denticola.
Bridging
Two non co aggregating strains may participate together in a
multigeneric aggregate if they recognize a common partner by
distinct mechanisms
For example A. Israeli does not co aggregate with S.oralis
P loescheii co aggregated with both strains by means of different
adhesins (Weiss et al 1988).
When the three types were mixed all three cell types were found
in the aggregate formed (Kollenbrander et al 1985)
Corn Cobs - A Model For Oral Microbial Biofilms
 Jones (1971-72)
 First described by Vincentini 1897 - Structure was composed of a
single microbial species and named them leptotrix racemosa.
 Central filamentous bacterium covered by coccal species.
 Attachment of the Cocci to the filament occurred via hair like
appendages (fimbriae) found on some species of oral streptococci
(Listgardten et al 1973).
 The fimbriae associated with corncob formation were found
to be located on one pole rather than being uniformly
distributed over the cell surface as found in other oral
streptococci.
 Another feature of the corncob is the firmness of the
attachment between the component microorganisms
 Test tube brush
Composed of filamentous bacteria to which gram negative rods
adhere.
F. nucleatum
Most numerous gram -ve species in healthy sites, numbers increase
markedly in periodontally diseased sites (Moore 1994)

 Always
present whenever Treponema denticola and
Porphyromonas gingivalis are also present, - its presence predates
that of the other two species and may be required for their
colonization (Socransky 1998)
F.
nucleatum coaggregates with all of the early colonizers and the
late colonizers , acts as a bridge between early and late colonizers.
F.
nucleatum interacts with and binds host-derived molecules, such
as plasminogen.F. nucleatum is generally non proteolytic, but
organisms that coexist with it, such as P. gingivalis, are highly
proteolytic and can activate fusobacterium- bound plasminogen to
form fusobacterium-bound plasmin, a plasma serine protease
 Acquisition
of proteolytic ability on its cell surface confers on
the fusobacteria a new metabolic property, the ability to process
potential peptide signals in the community. These peptides may be
used as nutrients by fusobacteria or by other biofilm residents.
F. nucleatum also induces expression of - defensin 2, a small
cationic peptide produced by mucosal epithelial cells

 Although
F. nucleatum is often considered a periodontal
pathogen, it may instead contribute to maintaining homeostasis
and improving host defense against true pathogens
Role of surface characteristics in Adhesion
Macroscopic Surface Characteristics
1.Surface Energy
Interfacial surface free-energy (Busscher and Weerkamp, 1987) has been
postulated as a driving force for initial adhesion of microorganisms to solid surfaces.
Low surface free-energy micro-organisms adhere to low surface
free-energy substrata, and high surface free energy microorganisms to high surface free-energy substrata
(Busscher and Weerkamp, 1987)
2.Zeta Potential
Determined by the nature and number of ionogenic groups on the
cell surface
Depends on the pH and ionic strength of the suspending medium.
A low negative charge expectedly favors adhesion.
3.Hydrophobicity
Hydrophobic
micro-organisms are attracted to solid surfaces by
rejection from the aqueous phase.
Ionic,
ion-dipole, or hydrogen bond interactions may be stabilized
by hydrophobic bonds (Nesbitt et al., 1982).
Fimbriae
which may bridge gaps between micro-organisms and
tooth surfaces have been associated with hydrophobicity.
Lipoteichoic
acid may confer hydrophobicity to the microbial
surface.
Hydrophobic
streptococci adhere better to hydroxyapatite in vitro
than do less hydrophobic strains.
4.Topography of supragingival plaque
Initial growth along the gingival margin and from the interdental
space (Areas protected against shear forces)
Tooth surface irregularities offer a favorable growth path
5.Surface micro roughness
Rough intraoral surfaces accumulate and retain more plaque
Threshold level for surface micro roughness-about
0.2micrometres,above which bacterial adhesion will be facilitated
(Bollen 1998)
Microscopic Surface characteristics
1.Salivary Components
Salivary oligosaccharide-containing glycoproteins -Receptors
for oral streptococci in the salivary pellicle (Gibbons and Qureshi, 1978)

Salivary proline-rich protein 1 and statherin have been
implicated as receptors for type 1 fimbriae of A. viscosus (Gibbons et

al., 1988)
“Cryptitopes" may be revealed by the adsorption of the
molecule to the tooth surface, or by the enzymatic cleavage of
terminal residues.

2.Microbial Adhesins
 Bind stereochemically to complementary receptors on the
interacting surface.
Associated with filamentous appendages or fimbria, or with
HMW proteins extending from the microbial surface

Early colonizers of the tooth surfaces are connected by
microbial surface fimbriae (Nyvad and Fejerskov, 1987)

Fimbriae possess hydrophobic domains consisting of nonpolar amino acids which confer adhesion.

3.Intermicrobial Co-aggregation
Kollenbrander 1990- Intrageneric co aggregation by
streptococci could contribute to early plaque formation.

Fusobacteria and streptococci are capable of intrageneric
coaggregation.

Individual variables influencing plaque formation
Heavy (fast) and light (slow) plaque formers.
Simonsson et al (1987) –
Clinical wettability of the tooth surfaces
 Saliva induced aggregation of oral bacteria
 Relative salivary flow conditions around teeth

Diet
 Chewing fibrous food
 Smoking
 Presence of copper amalgam
 Tongue & palate brushing
 Colloid stability of bacteria in the saliva
 Chemical composition of the pellicle
 Retention depth of the gingival area

Growth dynamics of dental Plaque
 During the first 2 to 8 hours, adherent pioneering streptococci
saturate the salivary pellicle binding sites and thus cover 3 to
30% of the enamel surface (Liljemark 1996)
 After 1 day, the term biofilm is fully deserved because
organization takes place within it.
 As bacterial densities approach 2 to 6 million bacteria/mm2
on the enamel surface, a marked increase in growth rate can be
observed to 32 million bacteria/mm2
Denovo supra gingival plaque formation clinical
aspects
Clinically, early undisturbed plaque formation on teeth follows an
exponential growth curve when measured planimetrically
(Quirynen 1989)
First 24 h – Negligible 
Next 3 days ,increase is much faster, but from then on there is a
tendency to slow down.
After 96 h - 30% of total tooth crown area covered by plaque.
After the fourth day- Shift towards anaerobic and gram negative
flora, including an influx of fusobacteria,filaments,spiral forms &
spirochetes .
The slow start of the plaque growth curve can be partly explained
by a colony of bacteria needing to reach a certain size before it is
clinically detected.
During the night, plaque growth is reduced by 50%
(Quirynen 1989 )
Variation within the dentition
- Faster In the lower jaw
- In molar areas.
- On the buccal tooth surfaces
- In the interdental region
Impact of gingival inflammation
- More plaque facing inflamed gingival margins than on healthy
gingiva (Ramberg 1994,Quirynen 1993)
- GCF during inflammation  Nutrient supply (Saxton 1973)
- Inflammatory odema of gingival margin constitutes an anatomic
shelter for growing plaque (Quirynen et al 1991)
-  amounts of plasma protein in the pellicle
Denovo sub gingival plaque formation
Difficult to study/sterilize/
Intd. of oral implants
Complex subgingival microbiota introduction 1 week after
abutment insertion.
Followed by slow increase in no. of pathogenic species
(Quirynen 2005)
Subgingival flora largely dependent on the remaining
presence of teeth and degree of periodontitis in the remaining
natural dentition.
Microbial interactions in Dental Plaque
Microbial metabolism within plaque will produce localized
gradients in factors affecting the growth of the species, ranging
from depletion of essential nutrients with the simultaneous
accumulation of toxic or inhibitory by products, to the
consumption of oxygen enabling the growth of obligate
anaerobes.
Synergistic Interactions
1.Complete degradation of host molecules.
2.Nutritional Interdependence
3.Coaggregation
Antagonistic Interactions
1.Bacteriocins
2.Organic acids, Hydrogen peroxide and enzymes
3.Low pH.
Synergistic Interactions
Nutrients in Plaque
•
•
•
•
A direct effect - specific nutrient provides an important
energy, nitrogen, or carbon source for one or more of the
micro-organisms in the environment;
An effect where the products of metabolism of an organism
provide a significant nutrient for other organisms
An indirect effect, where the products of bacterial metabolism
of a nutrient alter the environment in a way which influences
the resident microflora
An effect whereby a substrate is provided for the production of
specific polymers by biofilm cells.
Synergistic Interactions
o The early colonizers (Streptococci,Actinomyces species) use
oxygen and lower the redox potential of the environment which
then favors the growth of anaerobic species (Diaz2000)
o Gram positive species uses sugars as an energy source and saliva
as a carbon source.
o Anaerobic and asaccharolytic bacteria in mature plaque use
amino acids and small peptides as energy sources (Loesche 1968)
o Host also functions as important source of nutrients.
Steroid hormone - P.intermedia in sub gingival plaque. (Kornman
(Carlson 1983).
(Bramanti 1991)
1982)
Principle of bacterial transmission /translocation
/cross infection
Jeopardizes the
outcome of periodontal therapy
demonstrated translocation of A.Acommitans
by periodontal probe in patients with Lap
Inoculation of non infected pockets with A.Acommitans by a
single course of probing with a probe previously inserted in an
infected pocket of the same patient.

Christersson et al 1985
reported comparable composition of
subgingival microbiota (P.gingivalis, P.intermedia, B.forsythus)
around teeth and implants from partially edentulous patients with
different types of periodontal infections.

Papaioannou et al (1996)
 Vehicles-
Salivary flow, oral hygiene aids, dental instruments
1.Salivary flow
Continuous outflow of crevicular fluid from the pocket makes the
spontaneous entrance of saliva impossible.
2.Oral hygiene aids
Daily used tooth brushes harbor a complex microbiota including
periodontopathogens (Muller et al 1989).Contaminated brushes -health
risk, especially for immunosuppressed patients (Glass and Lare 1986)
3. Dental instruments
Barnett et al 1982 examined instruments after probing a single deep
pocket (> 6mm) All specimen contained pathogenic flora
gm – cocci,filaments and rods, flagellated filaments, spirochetes
Clinical relevance of intra oral translocation- full
mouth disinfection as treatment (Quirynen et al 1995)
To eradicate or at least suppress all periodontopathogens within a 24 hr period.
Combination of :
 Full mouth SRP 2 visits in 24 hrs- To reduce the number of
subgingival pathogenic organisms (Mousques et al 1980)

Subgingival irrigation (repeated thrice within 10 min) of all
pockets with 1 % chlorhexidine gel for 1 min to suppress the
bacteria in this niche

Mouth rinsing with 0.2% chlorhexidine solution for 2 min to
reduce the bacteria in the saliva (Rindomm et al 1976)

Recolonisation of the pockets was retarded by optimal oral
hygiene supported by mouth rinsing with 0.2 % chlorhexidine for
first 2 months.
Result:
 Additional pocket reduction (>=1.5mm)
 Gain in attachment (>= 1 mm) up to 8 months after therapy
(Vanderkhove et al 1996)
 Larger reduction in spirochetes, motile rods, several
pathogenic organisms (A.A.comitans, P.gingivalis,
P.intermedia, B.forsythus, E.corrodens, F.nucleatum, P.micros,
C.rectus Eubacterium species)
 Beneficial bacteria increased significantly more in the group
of patients with full mouth disinfection (Bollen et al 1996)
Microbial Homeostasis In Dental Plaque
The ability to maintain community stability in a variable
environment has been termed microbial homeostasis.
It results from a balance of dynamic microbial interactions,
including synergism and antagonism.
Factors involved in the maintenance of
microbial homeostasis
Plaque and Disease
Pathogenic Potential Of plaque
1.Total microscopic count - 10 8 microorganisms/mg of plaque.
Viable counts of gingival sulcus by plaque is 1.6 * 10 7 to 4.1 * 10 7
organisms/mg
2. Enzymes produced by the microbiota
Act on the intercellular substance of Junctional epithelium.
Facilitate penetration of microbial products into the gingival
connective tissue
3. Metabolites - Plaque microorganisms metabolize carbohydrates,
amino acids and proteins, and various metabolites accumulate in
plaque
4. Cell wall components- Endotoxin and mucopeptide complex
of gram positive bacteria.
5. Chemotactic factors- These are present in plaque and may
attract neutrophills migrating from the connective tissue into the
gingival sulcus, white blood cells release lysosomal enzymes
that destroy tissue.
6. Bacterial antigens
7. Viruses ,Protozoa, Candida.
Plaque Microflora And Disease
Accumulation of Plaque

Increase in Plaque mass

Less penetration of saliva into plaque

Less enamel protection

Beakdown of Microbial homeostasis

Shifts in composition of microflora
Streptococci-dominated microflora (Slots, 1977) to one with higher
levels of Actinomyces spp. and an increase in capnophilic and
obligately anaerobic bacteria such as Capnocytophaga,
Fusobacterium, and Prevotella species (Savitt and Socransky, 1984; Moore
et aL, 1987).
Depending on the type of disease, bacteria belonging to the genera
Actinobacillus, Campy lobacter, Selenomonas, Treponema, and
Wolinella may be isolated.
Microbial specificity of periodontal
diseases
Nonspecific plaque hypotheses
In the mid 1900s periodontal diseases were believed to result
from an accumulation of plaque over time, eventually in
conjunction with a diminished host response and increased host
susceptibility with age (Lovdal 1958, Russel 1967, Schie 1959)
Periodontal disease results from the elaboration of noxious
products by the entire plaque flora (Loesche 1976)
Large amounts of plaque would produce large amounts of noxious
products which would essential overwhelm the hosts defenses.
Control of periodontal disease depends on the control of the
amount of plaque accumulation
The current standard treatment of periodontitis by debridement
and oral hygiene measures still focuses on the removal of plaque
and its products and is founded in the non specific plaque
hypotheses
Contraindications Of Theory
1.Individuals with considerable amounts of plaque and calculus as
well as gingivitis never developed destructive periodontitis.
2. Individuals with who did present with periodontitis demonstrated
considerable site specificity in the pattern of disease. Some sites
were unaffected, whereas advanced disease was found in adjacent
sites.
Specific plaque hypotheses
Only certain plaque is pathogenic, and its pathogenicity depends
on the presence of or increase in specific microorganisms (Loesche
1976)
Plaque harboring specific bacterial pathogens results in a
periodontal disease because these organisms produce substances
that mediate destruction of host tissues.
Acceptance of the specific plaque hypothesesA.A comitans as a pathogen in localized aggressive periodontitis
(Newman 1977)
Modified hypothesis ( "ecological plaque hypothesis")
(Marsh, 1991)
A change in a key environmental factor (or factors) will trigger a
shift in the balance of the resident plaque microflora, and this
might predispose a site to disease
Inflammatory response
results in an
environmental change,
subgingivally, which
produces a shift in the
balance of the resident
microflora. Such
a shift predisposes a site
to disease
Denture plaque
Conclusion
Dental Plaque functions as a true microbial community ; the
interactions of the component species results in a metabolic
efficiency and diversity that is greater than the sum of its
constituent species. The nature of a biofilm helps explain why
periodontal diseases have been so difficult to prevent and treat. An
improved understanding of biofilm will lead to new strategies for
management of these widespread diseases.
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13. JCP 2002 32,suppl 6 7-15 Dental Plaque Biological Significance Of A Biofilm And Community Life Stly(marsh PD Et Al)
14.Dental Biofilms:difficult Therapeutic Targets Socransky Periodontology 2000 Vol 28 2002,12-55
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Paul E. Kolenbrander
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