DENTAL PLAQUE - PART 2 (BIOFILM)

DENTAL PLAQUE - II
GUIDED BY:
DR. RUPINDER KAUR
DR. DIVYA JAGGI
PRESENTED BY:
DR.MALVIKA THAKUR
PG II YEAR

Page  2
CONTENTS
1. Introduction
2. Properties of biofilm
3. Factors affecting biofilm development and behavior
4. Microbial specificity of periodontal disease
5. Biofims and Host in conflict
6. Possible strategies to control oral biofilm
7. Detection of dental plaque
8. Conclusion
9. References

Page  3
Matrix embedded microbial populations, adherent to each other
and/or to surfaces or interfaces (Costerton et al. 1999)
Biofilms consist of one or more communities of microorganisms,
embedded in a glycocalyx, that are attached to a solid surface.
(Sigmund S. Socransky & Anne D. Haffajee. 2001)
INTRODUCTION
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Page  4
A microbial biofilm is considered a community that meets the
following four basic criteria:
AUTOPOIESIS HOMEOSTASIS
SYNERGY COMMUNALITY
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Page  5
Schematic representation of the types of interaction that
occur in a microbial community, such as dental plaque
11/12/2016
BENEFITS OF MICROBIAL COMMUNIY LIFESTYLE
3/45

Page  6
PROPERTIES OF BIOFILM
ATTACHMENT
OF BACTERIA
PHYSIOLOGICAL
HETEROGENICITY
QUORUM
SENSING
ANTIBIOTIC
RESISTANCE
GENE TRANSFER
STRUCTURE OF
BIOFILM
MICROBIAL
INTERACTIONS
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Page  7
1. STRUCTURE OF A BIOFILM
 Biofilms are composed of microcolonies of bacterial cells (15–20%
by volume) that are non-randomly distributed in a matrix or
glycocalyx (75–80% volume).
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 The bacterial vitality varies
throughout the biofilm,
with the most viable
bacteria present in the
central part of plaque , and
lining the voids and
channels (Auschill et al
2001)

Page  8
 The biofilm matrix is
penetrated by fluid
channels that conduct the
flow of nutrients, waste
products, enzymes,
metabolites, and oxygen.
6/45
 Structure of the Biofilm depends on environmental parameters
under which they are formed. These include:
• Surface and interface properties
• Nutrient availability
• Composition of the microbial community
• Hydrodynamics

Page  9
The bacteria in a biofilm use a communication system termed
quorum sensing that involves sending out chemical signals .
These chemical signals trigger the bacteria to produce potentially
harmful proteins and enzymes, virulence factors that help the
intraoral biofilm bypass host defense systems
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Page  10
EXOPOLYSACCHARIDES – the backbone of the biofilm
 The bulk of the biofilm consists of the matrix or glycocalyx and
is composed predominantly of water and aqueous solutes
 The “dry” material is a mixture of exopolysaccharides, proteins,
salts, and cell material.
 Exopolysaccharides (EPS), which are produced by the bacteria in
the biofilm, are the major components of the biofilm making up
50–95% of the dry weight.
Maintaining the integrity of the biofilm
Preventing desiccation and attack by harmful agents.
Binds essential nutrients to create a local nutritionally rich
environment.
Acts as a buffer
Assists in retention of extracellular enzymes enhancing substrate
utilization by bacterial cells
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Page  11
2. ATTACHMENT OF BACTERIA
 The key characteristic of a biofilm - the microcolonies within the
biofilm attach to a solid surface.
 Many bacterial species possess surface structures such as fimbriae
and fibrils that aid in their attachment to different surfaces.
 Fimbriae have been detected on a number of oral species including
P. gingivalis, A. actinomycetemcomitans and some strains of
streptococci.
 Oral species that possess fibrils include S. salivarius, the S. mitis
group, Pr. intermedia, Pr. nigrescens, and Streptococcus mutans.
Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic
Targets Periodontology 2000 2001;28:12–55.
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Page  12
VARIABLES IMPORTANT IN CELL ATTACHMENT
AND BIOFILM FORMATION
PROPERTIES OF
THE
SUBSTRATUM
PROPERTIES OF
THE BULK
FLUID
PROPERTIES OF
THE CELL
 Texture or
roughness
 Hydrophobicity
 Conditioning
 Flow velocity
 pH
 Temperature
 Cations Presence
 Cell surface
hydrophobicity
 Fimbriae
 Flagella
 Extracellular
polymeric
substances of
antimicrobial
agents

Page  13
3. PHYSIOLOGICAL HETEROGENEITY
 Cells of the same microbial species can exhibit extremely
different physiologic states in a biofilm even though separated
by as little as 10 μm.
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Clinical Periodontology and Implant Dentistry by Jan Lindhe, 5th Edition.
 Studies to date indicate that
sessile cells growing in
mixed biofilms can exist in
an almost infinite range of
chemical and physical
microhabitats within
microbial communities.

Page  14
The residents in the microbial community display extensive
interactions while forming biofilm structures, carrying out
physiological functions, and inducing microbial pathogenesis.
These interactions, include
1. Competition between bacteria for nutrients
2. Synergistic interactions which may stimulate the growth or survival of
one or more residents
3. Production of an antagonist by one resident which inhibits the growth of
another
4. Neutralization of a virulence factor produced by one organism by
another resident
5. Interference in the growth-dependent signaling mechanisms of one
organism by another
4. MICROBIAL INTERACTIONS
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Page  15
NUTRIENTS AS THE BASIS FOR BACTERIAL
INTERSPECIES INTERACTION WITHIN BIOFILM

Page  16
GENERAL METABOLIC PRODUCTS WHICH
INFLUENCE BIOFILM RESIDENT INTERACTIONS
 Antagonistic effect - S. sanguinis group are producers of H 2O2 , a
nonspecific antimicrobial agent - an antagonistic effect on other
coresidents, such as S. mutans.
Synergistic effect - lactic acid produced by S. mutans can be readily
metabolized by members of the Veillonella family.
• Co-operative metabolic interactions -
F. nucleatum & P. intermedia
grow at pH range of 5.0 to
7.0. P. gingivalis susceptible
to - pH levels < 6.5.
Using glutamic
and aspartic
acids (GCF &
saliva)
Generate
Ammonia +
organic acids.
This contributes
to a more
neutral pH
prevents ↓ in pH even in
presence of lactic acid
bacteria & fermentable
carbohydrates
Acid-sensitive species - P.
gingivalis are protected
against acid attack

Page  17
BACTERIOCINS
Proteinaceous bactericidal substances produced by bacteria to
inhibit the growth of closely related bacterial species or strains
(Hojo et al 2009)
Regulated by genetic and environmental factors
Enable bacteria to select their neighbours, promote the
establishment of a community with specific bacterial species.
Inhibition of growth of P.gingivalis, T.forsythia, S.salivarius,
S.sanguinis by bacteriocin produced by L.paracaesi
Nigrescin, produced by P.nigrescens display bactericidal effect
against P.gingivalis, P.intermedia, T.forsythia, Actinomyces spp.
Bacteriocin production also reported by P.intermedia, A.a,
C.ochracea, F.nucleatum, E.corrodens, H.influenzae
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Page  18
Bacterial co-aggregation influences localization within
biofilms
Gibbons and coworkers(1970), demonstrated that strains of P.
gingivalis aggregated with several oral streptococci, this might
be an important mechanism - P. gingivalis could become
incorporated into dental plaque that is composed primarily of
the initial streptococcal colonizers.
T. denticola does not appear to form biofilms on most inert
surfaces, in contrast to P. gingivalis, which does so readily.
However, in the presence of P. gingivalis , T. denticola is
incorporated into the biofilm.
T. forsythia is a weak colonizer of inert surfaces but will
become incorporated into biofilms in the presence of F.
nucleatum.
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Page  19
5. QUORUM SENSING
 It is defined as the cell density dependent regulation of gene
expression in response to soluble signals called autoinducers (Bassler
1999)
 It has been defined by Miller (2001) as “the regulation of gene
expression in response to fluctuations in cell population density”.
 Quorum sensing can occur within a single bacterial species as well
as b/w diverse species.
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Page  20
 Quorum sensing has been described in both G+ve & G-ve bacteria.
 Cell-cell communication may occur b/w and within bacterial
species (Miller, 2001)
 Quorum sensing-controlled behaviors are those that only occur
when bacteria are at high cell population densities.
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Page  21
 Three types of molecules :
1. Acyl-homoserine lactones (AHLs) - signaling molecules
used by many G-ve bacteria, it synthesized by Lux-I family
protiens.
2. Autoinducer peptides (AIPs) - signaling molecules used by
G +ve bacteria
3. Autoinducer-2 (AI-2) - used by both G-ve & G+ve
bacteria, chemically it is furanosyl borate diester. Synthsized
by protein Lux-S.
Schauder, S. and B. L. Bassler (2001). "The languages of
bacteria."
QUORUM SENSING MOLECULES
19/45

Page  24
 This communication controls various functions reflecting the
needs of a specific bacterial species to inhabit a particular niche
such as the production of virulence factors, or by the transmission
and acquisition of the generic information needed to produce
virulence factors from other species in the biofilm development
(Passador et al., 1993; Reading et al., 2006).
 Several strains of P. intermedia, T. forsythia, F. nucleatum and P.
gingivalis were found to produce quorum sensing signal
molecules (Frias et al., 2001; Sharma et al., 2005).
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Page  26
26
Strategies for quorum sensing inhibition
3 strategies can be applied
Targeting AHL signal
dissemination
Targeting the signal
receptor
Targeting signal
generation
Signal precursor
Signal
Signal receptor
Signal precursor Signal precursor
Signal Signal
Signal receptor Signal receptor
X
X
X
Anti- activator
proteins
• AHL
degradation
enzymes
• RNA dependant
regulation
Interference
of signal
receptor
• Furanones
• L-canavanine
• Human
hormones
Bacterial
components
• Transgenic
plants
• Synthetic
analogues
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Page  27
6. ANTIBIOTIC RESISTANCE
 Bacteria growing in a biofilm are
highly resistant to antibiotics, up
to 1,000-1,500 times more
resistant than the same bacteria
not growing in a biofilm.
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 MIC of chlorhexidine and amine fluoride was 300 and 75
times greater respectively, when S.sobrinus was grown in
biofilm compared to planktonic cells
 Biofilms of P.gingivalis tolerated 160 times the MIC of
metronidazole than planktonic cells

Page  29
.
The biofilm matrix may restrict the penetration of a
charged antimicrobial agent (diffusion-reaction theory)
Agent may also bind to, and inhibit, the organisms at
the surface of the biofilm, leaving cells in the depths
of the biofilm relatively unaffected.
The novel phenotype expressed in a biofilm may result
in the drug target being modified or not expressed, or the
organism may use alternative metabolic strategies
27/45
Bacteria replicate only slowly in an established biofilm
and, as a consequence, are inherently less susceptible
than faster dividing cells.
In addition, samples of gingival crevicular fluid
(GCF) can contain sufficient ß lactamase to
inactivate the concentrations of antibiotic delivered
to the site
A susceptible pathogen can be rendered resistant if
neighbouring, non-pathogenic cells produce a
neutralising or drug-degrading enzyme.

Page  30
7. EXCHANGE OF GENETIC INFORMATION
Conjugation,
transformation and
transduction have all
been shown to occur in
naturally occurring mixed
species biofilms. Clinical
Periodontology and Implant
Dentistry by Jan Lindhe, 5th
Edition.
28/45

Page  31
Cells also communicate and interact with one another in biofilms
via horizontal gene transfer.
Gene transfer between Treponema denticola and S. gordonii has
also been demonstrated in the laboratory. Wang BY, Chi B,
Kuramitsu HK. Genetic exchange between Treponema denticola
and Streptococcus gordonii in biofilms. Oral Microbiol Immunol
2002: 17: 108– 112.
 The presence of “pathogenicity islands” in periodontal pathogens
such as P. gingivalis is also indirect evidence for horizontal gene
transfer having occurred in plaque biofilms at some distant time in
the past, and may explain the evolution of more virulent strains.
Chen T, Hosogi Y, Nishikawa K, Abbey K, Fleischmann RD,
Walling J, Duncan MJ. Comparative whole-genome analysis of
virulent and avirulent strains of Porphyromonas gingivalis. J
Bacteriol 2004: 186: 5473–5479.
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Page  32
Detachment
Can be Movement of Individual cells or Biofilm en masse
Brading et al have emphasized the importance of physical
forces in detachment, stating that the three main processes for
detachment are (JADA 1996)
 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)
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Page  33
Individual Cell Transfer
 Erosion - detachment of single cells in a
continuous predictable fashion
 Sloughing - sporadic detachment of
large groups of cells or
 Intermediate process whereby large
pieces of biofilm are shed from the
biofilm in a predictable manner, resulting
in detached clusters consisting of about
104 cells.
31/45
 This possibility has been
demonstrated in vitro studies of
mixed biofilm that showed
movement of intact biofilm
structures across solid surfaces
while remaining attached to them.
 Advantage - formation of the
biofilm is not reliant on planktonic
cells, which are known to be more
susceptible to antimicrobial agents
Stoodley 1991
En masse transfer

Page  34
Factors affecting biofilm
development and behavior
1. ROLE OF SALIVA
 Saliva contains – mixture of glycoproteins – mucin.
 Bacteria – enzymes (glycosidases) – split off carb. – utilized as nutrients.
 Remaining protein – contributes to plaque matrix
 Neuraminidase – separates sialic acid from salivary glycoprotein.
 Loss of sialic acid - ↓ salivary viscosity
- Formation of precipitate – factor in plaque
formation
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Page  35
2. ROLE OF INGESTED NUTRIENTS
 Most readily utilized nutrients – diffuse easily into plaque – sucrose,
glucose, fructose, maltose & less amt. of lactose.
 Dextran – greater quantity, adhesive properties , relative insolubility &
resistance to destruction by bactera.
 Levan – Used as carbohydrate nutrient by plaque bacteria in absence of
exogenous sources.
3. DIET AND PLAQUE FORMATION
 Consistency affects the rate of plaque formation : Forms rapidly on soft
diets, hard chewy food retard it.
 Dietary supplements of sucrose ↑ plaque formation and affect its bacterial
composition. i.e ECM
 Plaque formation occurs on high protein fat diets and carbohydrate - free
diets but in smaller amounts.
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Page  36
 IMPORTANCE OF FOOD CHAINS
• Stimulation of growth of other bacteria (eg) stimulation of
growth of T.denticola by butyric acid produced by P.gingivalis
• Increasing the virulence of organisms (eg) more virulent strains
of P.gingivalis in the presence of S.gordonii.
• Removal of toxic metabolites (eg) protection from hydrogen
peroxide by A.neaslundii
• Utilization of metabolic products for maintaining structural
integrity (eg) succinic acid produced by T.denticola integrated
onto the cell wall of P.gingivalis
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Page  37
MICROBIAL SPECIFICITY
Non
Specific
Plaque
Hypothesis
Early
1930’s
Specific
Plaque
Hypothesis
Loesche
1976
Modern
Version of
Specific
Plaque
Hypothesis
Socransky
1979
Unified
Theory
Thelaide
1986
Ecological
Plaque
Hypothesis
PD Marsh
& Martin
1999
Keystone
Pathogenic
Hpothesis
Hajishenga
llis et al
2012
4/42
35/45

Page  38
THEORY DRAWBACK
Non-Specific Plaque
Hypothesis(NSPH),
Individuals with longstanding plaque and gingivitis do not
develop periodontitis, while others, with minimal plaque, had
lower resistance to disease.
The Specific Plaque
Hypothesis(SPH),
Many of the organisms observed in periodontal health were also
observed at diseased sites (Slots, 1977)
The Ecological Plaque
hypothesis (EPH)
Does not address the role of genetic factors of the host that
contribute to the composition of dental plaque and to
susceptibility to disease
The Keystone Pathogen
Hypothesis(KPH).
P.Gingivalis is one of the easily culturable micro organisms in
plaque. Over 700 bacterial speicesare found in dental plaque. So
it can be that any one of the uncultured micro-organisms can
also create conditions ideal to the growth of
periodontopathogens.
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Page  39
MICROBIAL SHIFT/DYSBIOSIS
Concept that some
diseases are due to a
decrease in the number of
beneficial symbionts and ⁄
or an increase in the
number of pathogens.
This model proposes that
periodontitis is initiated by
a dysbiotic microbial
community (rather than by
select periodontal
pathogens) within which
different microbial members
or specific gene
combinations have distinct
roles that synergize to
shape a microbiota that
causes disease.
37/45

Page  40
MICROBIAL SHIFT LEADING TO PERIODONTITIS
GRAM +VE AEROBES GRAM -VE ANAEROBES
 Gradually changes the symbiotic host–microbe relationship to a
pathogenic one.
Prevotella intermedia
Fusobacterium
nucleatum
P. Gingivalis
Tannerella forsythia
Treponema denticola
38/45

Page  41
Biofilm and host in conflict
Oral microflora has a harmonious and +vely beneficial
relationship with the host - microbial homeostasis.
Plaque
Accumulation
Inflammatory
response by
Host
GCF flow ↑
Introduction of complex
host molecules
(transferrin, Hb) into
GCF
These get
catabolized
Used as a nutrient
source by the
proteolytic G –ve
Bac
This proteolytic
metabolism
leads to
↑ local ph
↓ in the redox
potential,
Promotes
upregulation of
virulence factors
(e.g. Gingipain
activity by P.
Gingivalis)
Favours growth at the
expense of beneficial
species(i.e. ↑ the
competitiveness of
the potential
pathogens)
If sustained
Re-arrangement in
community structure &
selective ↑ in the
proportions of the
anaerobic & proteolytic
components
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Page  42
Gingivitis
Reduced
plaque
Increased
plaque
Reduced
inflammation
Increased
inflammation
Low GCF
flow
High GCF,
bleeding,
raised pH &
temperature,
low Eh
Gram +ve
bacteria
Gram -ve
anaerobes
Gingival health
Gingival health
Periodontitis
Stress
Environmental
change
Ecologica
l
shift
A schematic representation of the ecological plaque hypothesis in relation to
periodontal disease
Plaque biofilm accumulation can produce an inflammatory host response; this causes changes in the local
environmental conditions and introduces novel host proteins and glycoproteins that favour the growth of
proteolytic and anaerobic G –ve bacteria. In order to prevent or control disease, the underlying factors
responsible for driving the selection of the putative pathogens must be addressed, otherwise disease will
recur.

Page  43
POSSIBLE STRATEGIES TO
CONTROL ORAL BIOFILM
1. Inhibiting Adherence with Antagonists
2. Passive Immunization
3. Replacement Therapy
4. Regulating the Levels of Nonpathogenic Bacteria to Influence
Virulence
5. Probiotic Approaches
6. Interference with Signaling Mechanisms
7. Targeted Antimicrobial Therapy via a Novel STAMP
Technology
41/45

Page  44
CONTROL
OF
NUTRIENTS
• Addition of base – generating nutrients (arginine)
• Reduction of GCF flow through anti-
inflammatory agents
• Inhibition of key microbial enzymes
CONTROL
OF BIOFILM
pH
• Sugar substitutes
• Antimicrobial agents
• Fluorides
• Stimulate base production
CONTROL
OF REDOX
POTENTIAL
• Redox agents
• eg: methylene blue
• Oxygenating agents
42/45

Page  45
METHODS OF DETECTION OF
DENTAL PLAQUE
VISUAL
PERIODONT
AL PROBE
OR
EXPLORER
DISCLOSING
AGENTS
43/45

Page  46
1. DIRECT VISION : -
 Thin plaque – may be translucent & therefore not visible
 Stained plaque – may be acquired e.g- tobacco stained
 Thick plaque – tooth may appear dull & dirty
2. USE OF EXPLORER : -
 Tactile Examination – when calcification has started it appears slightly
rough, otherwise it may feel slippery due to coating of soft , slimy
plaque
 Removal Of Plaque – when no plaque is visible , an explorer can be
passed over the tooth surface & when plaque is present it will adhere to
explorer tip.this technique is used when evaluating plaque index.
 This can be done by running the explorer or probe along the gingival 3
rd of the tooth
44/45

Page  47
3. Disclosing Agents:
1) Two tone
2) Erythrosine
3) Bismark
4) Benders
5) Basic Fuschin
6) Disclosing Tablets – Dental Mart, oral B
144/45

Page  48
CONCLUSION
Dental plaque biofilm cannot be eliminated permanently.
 However, the pathogenic nature of the dental plaque biofilm
can be reduced by reducing the bioburden (total microbial load
and different pathogenic isolates within that dental plaque
biofilm) and maintaining a normal flora with appropriate oral
hygiene methods that include daily brushing,flossing and
rinsing with antimicrobial mouthrinses.
This can result in the prevention or management of the
associated sequelae, including the development of periodontal
diseases and possibly the impact of periodontal diseases on
specific systemic disorders.
45/45

Page  49
REFERENCES
1. Glickman; Clinical periodontology 4th edition: Saunders
2. Carranza, Newman ;Clinical Periodontology, 8th edition: Saunders
3. Newman, Takei, Klokkevold, Carranza; Clinical Periodontology; 10th Edition:
Elseveir
4. Jan Lindhe, Niklaus P. Lang, Thorkildkarring; Clinical Periodontology And Implant
Dentistry: 5th Edition: Blackwell
5. Socransky SS, Haffajee AD. Dental biofilms: difficult therapeutic targets. Peridntol
2000 2002 : 28; 12-55.
6. Marsh PD, Moter A, Devine DA. Dental plaque biofilms: commuinties, conflict and
control. Periodontol 2000 2011; 55: 16-35
7. Max A. Listgarten , The structure of dental plaque, Periodontology 2000, Vol. 5,
1994, 52-65
8. Interspecies Interactions within Oral Microbial Communities
9. Howard K. et al Microbiology And Molecular Biology Reviews, Dec. 2007, P. 653–
670

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