biofilm (2).pptx

Bio film –Current
Concepts
Dr S.Muthukumar

Overview
1.History
2.Definition
3.Biofilm as an ecosystem
4.Structure and components
5.Implications in periodontal disease
6.Development
7.Dental plaque as a bio film
8.Propeties
9.Study methods
10.Molecular genetics
11.Biofilm and dental implants
12.Therapeutic strategies

HISTORY
 Dates back to 1684 when
identified the bacteria
(animicules) from dental plaque (scruf)
 Mid 1800 – Robert koch developed
nutrient medium for growth and isolation
of microbes.
 Concentrated on planktonic growth

• Henrici(1933) & Heukelekian and
A.Heller(1940) reported the growth of
microbes on surface
• 1940 - Claude ZoBell described
fundamental characteristics of attached
microbial community
• Harremoes (1977) used the term biofilm

Bacteria
Free-
Floating Attached to
a surface

 Recent research technology – Study
Bacteria in their Natural environment.
 Revealed that most bacteria live in complex
communities called Biofilms

DEFINITION
“ 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 )

 Once a bacterium attaches to a surface, it activates a whole
different set of genes that gives the bacterium different
characteristics from those that it had as a free-floating
organism
 It has been estimated that more than 99% of all bacteria on
the earth live as attached bacteria
 Biofilms can be found on medical and dental implants living
in intravenous and urinary catheters, contact lenses, and
prosthetic devices,such as heart valves, biliary stents,

 Ecosystem – complex of organisms in a specified
environment and the nonmicrobial surroundings with
which the organisms are associated.
Habitat - it is the site at which a population or community
grows, reproduces or survives.
Niche - functional role of an organism in a habitat.
Ecosystem

Hierarchy
.
Single organism
Population
Community
Ecosystem

 Habitat affect the community and the
community affect its habitat.
 Microbial ecology is concerned with the
interrelationships between microorganisms
and their environment (ecosystem).

 Microbes exist as
biofilm in the oral
cavity and various
factors control their
establishment,
composition and their
re-establishment once
they get disturbed

Influence of habitat on
microbial composition
 Around 700 species are identified
 Composition of oral micro flora varies
significantly at distinct surfaces within the
mouth.

Three similar clusters are identified (Mager et al
2003)
1st - dorsum and lateral aspect of tongue and saliva
2nd - other soft tissues of oral cavity.
3rd - supra and sub gingival plaque.

Direct Relationship
Composition of the
Micro flora.
Oral
Environment

 This relationship is dynamic, and a change in a
key environmental factor can alter the
competitiveness of individual species, a possible
consequence of which is the selection of a
previously minor component of the microbial
community
 This situation essentially describes the
relationship between dental plaque and the host in

Biofilm – Analogy to City
 Planktonic (nomad) vs Biofilm (city)
 Initial colonization followed by lateral spread, vertical direction growth
 Shared resources and activities only possible through biofilm
 Protection from other species, host, and harsh environment
 Need communication – quorum sensing, exchange of genetic information

The Nature of Biofilms
• Protection from Competing microrganisms
• Environmental factors, host defense
• Toxic substances, such as lethal chemicals, antibiotics
• Facilitate processing and uptake of nutrients, cross-
feeding, removal of harmful metabolic products
• Development of an appropriate physico-chemical
environment

THE STRUCTURE OF BIOFILM
COMMUNITIES

Composition
of Bioflims
Micro
colonies
(15-20%
glycocaly
x (75-
80%
Exo
polysacc
harides
(EPS)
50-95%
“circulato
ry”
system
Shaped
matrix
Passage of
nutrients and
other agents
Backbone of the biofilm
Structure of Biofilms

 Bulk consist of matrix – mixture of water (80-90%) and
solutes
 Dry material - exopolysaccharides, proteins, salts and
cell material (bacteria).
 Bacterial colonies (70%) – more than 500 species have
been identified.
 Non bacterial organisms like mycoplasma, yeasts,
viruses also detected.

Exopolysaccharides
 Exopolysaccharides (back bone)produced by the bacteria,
are the major components, making up 50–95% of the
dryweight .
 EPS includes insoluble glucans, fructans and
heteropolymers.
 can produce several different polysaccharides depending on
the physiological state and the presence of specific

 Glucans are synthesized by glucosyltransferase (GTF).
GTFs can be secreted or adsorbed onto other bacteria,
acquired pellicle.
 Fructans are produced by fructosyltransferases (FTF) which
are short lived and act as nutrient storage compounds for
use by other bacteria.

 Some exopolysaccharides are neutral, such as the mutan whereas
others are highly charged polyanionic macromolecules.
 Different ionic charge and concentrations will alter the confirmation
and cause rapid changes in the three-dimensional gel network of
polysaccharides.
 Exopolysaccharides can exist in both ordered or disordered forms.
At high temperatures at very low ionic concentrations, the
disordered form predominates.

 The exopolysaccharides can be degraded and utilized
by bacteria within the biofilm
 One distinguishing feature of oral biofilms is that many
of the microorganisms can both synthesize and
degrade the exopolysaccharides

 Maintain integrity of the biofilm
 Adhesive
 Confers protection
 Prevents dessication
 bind essential nutrients such as cations to
create a local nutritionally rich environment
favoring specific microorganisms.
 act as a buffer and assist in retaining
extracellular enzymes (and their substrates),
enhancing substrate utilization by bacterial
cells.
Functions of EPS

Components
 Surface
 Bulk fluid
 Microbial community

1.Surface
Influenced by the nature of the surface, genetic
background (which might alter the surface receptors),
possible introduction of artificial surfaces, hygiene
practices etc
- Non Shedding Surface
- Shedding Surface

2.Bulk fluid
Passes over the biofilm, providing nutrients to the colonizing
organisms, removal of waste products, and transport of cells
to new colonizing sites.
Stationary sub layer
Layer of fluid in motion

3.Microbial community
 The vast majority of the micro-organisms in dental plaque are bacteria.
 Many of these ‘unculturable’ organisms are found at sub-gingival sites, especially
in disease, and represent novel groups of bacteria (often unnamed at present, e.g.
the TM7 group) the properties of which we know little or nothing about.
 These sensitive molecular techniques have expanded our concept of the diversity
of the microflora of the mouth. Over 700 distinct types (taxa) have now been
distinguished, although not all of these are ever found in a single mouth, let alone
in an individual sample of dental plaque.
 An individual plaque sample from a healthy site would more typically contain
around 30 species of bacteria.

 Most periodontal sites either all or none of the species
belonging to the same complex
 Red complex seldom detected in the absence of orange
complex; higher the orange complex higher the red
complex.
 Yellow and green cluster show similar preference for
each other; weaker relation with orange and red
complex.
 Purple complex, loose relation with all the other
complexes.

Distribution of different complexes in subgingival plaque sample
Kigure et al (1995)

Role of dental plaque in oral
health
 Dental plaque is part of the natural resident microflora of the
body.
 The resident microflora also reduces the risk of infection by
barrier to colonisation by exogenous (and often pathogenic)
phenomenon termed ‘colonisation resistance’).
 Mechanisms contributing to this colonisation resistance include
effective competition for nutrients and attachment sites, the
of inhibitory factors, and creation of unfavourable growth
for invading species by the normal microflora.

Early colonizers of human
mouth

Healthy Gingivae
Constitute early colonizers can withstand high oxygen
concentrations and removal mechanisms
Enables adhesion of subsequent bacterial species
8.2
3.2
1.4
Streptococcus
Actinomyces and
Corynebacterium
Veillonella and Neisseria

Role of dental plaque in
disease
 In the absence of effective oral hygiene, plaque can accumulate
to levels that are no longer compatible with health, thereby
predisposing sites to dental caries or periodontal diseases.
 There is a shift in the balance of the microflora away from those
species that are found at healthy sites .
 Unlike most classical medical infections, the microflora from sites
with dental disease is diverse, and no single species is diagnostic
or predictive.

Gingivitis
 Anaerobes predominate due to decrease in oxygen levels created by
increased biofilm thickness
8.2
7.8
1.4
Strptococcus spp,
Actinomyces spp
Fusobacterium spp,
Prevotela spp
Campylobacter,
Capnocytophaga

Periodontitis
 Viruses have also been detected
 Increase in the amount of pathogenic organisms
8.2
6.5
0.8
Red complex
Fusobacterium,
Campylobacter,Aggregatibac
ter,E.corrodens
Streptococcus, Actinimyces
spp

Health
Gram positive
Coccus
Non motile
Aerobes
Saccharolytic
Gingivitis Periodontitis
Gram negative
Rods and spirochetes
Motile
Anaerobes
Proteolytic

In Periodontitis………….
 Mean count of supra and sub gingival
plaque increases in periodontitis.
 Increased number of
Taxa in both the sites
Actinomyces, green complex in supragingival
films
orange and red complex in subginigival films

In Dental Health vs
Disease plaque

Microbial succession
Hypothesized relationship between the addition of species during microbial
succession leading to the development of gingival inflammation. In turn, the
increased inflammation would result in increased growth of colonizing species.

 Specific plaque hypothesis– of the diverse organisms in the microflora,
only a very limited number are actively involved in the disease (Loesche
1976) .
 Non specific plaque hypothesis– many organisms play a role, and the
disease is a result of overall interaction of plaque and the host (Theilade
1986) .
 Ecologic plaque hypothesis– change in the key environmental factor(s)
will trigger a shift in the balance of resident flora, and this might predispose
to the disease (Marsh 1991)
Implications in Periodontal
Disease

Formation of Dental Plaque
Biofilms
 The application of novel imaging and molecular
techniques has increased our understanding of
how dental plaque functions and develops as a
biofilm.
 Distinct phases of biofilm development are
recognised

Tooth surface
Acquired Pellicle
Stage 1: Pellicle formation Stage 2: Initial Adherence
Tooth surface
Acquired Pellicle
Stage 3: Aggregation
Tooth surface
Acquired Pellicle
Stage 4: Maturation
Tooth surface
Acquired Pellicle
Stage 5: Dispersal
Tooth surface
Acquired Pellicle

Development of dental
plaque Biofilm

Step I. Formation of
pellicle
 Selective adsorption of host molecules forming a
conditioning film (<1 ᶙm thick) by van der Walls,
electrostatic and hydrophobic forces.
 Contains proteins, glycoproteins like statherin, proline
rich proteins etc.
 Usually <1ᶙm thick, takes 90-120 min for the adsorption.
Intended to protect the tissues from desiccation, act as
receptors favoring bacterial attachment.

Step II. Initial adhesion
and attachment of bacteria
 First step involves transportation of bacteria. Few are
mobile and the majority of the organisms are transported
by the bulk fluid.
 Long range, between bacteria and pellicle coated
enamel. The strength of this interaction is weak and
span around 10-20 nm.
Physical phase

 Next step involves the closer movement of bacteria so
that specific short range interaction occur.
 This is to happen if water is removed between the two
surfaces, brought about by bacterial cell components.
This initial attachment of bacteria to surfaces is the initial part
of
adhesion, which makes the molecular or cellular phase of adhesion
possible.

 Adhesion between a substrate and bacteria
can attractive or repulsive depending on the
net forces acting, can be explained by
DLVO
or
Extended DLVO theory

 The DLVO theory has been used to describe the net interaction
(VTOT ) between a cell and a surface as a balance between two
additive factors: VA resulting from van der Waals interactions
(generally attractive) and repulsive interactions (VR) from the
overlap between the electrical double layer of the cell and the
substratum.
G TOT= GA+GB
 Cannot explain all molecular interactions

Three stages
1. Secondary minimum (reversible attraction)
2. Positive maximum (energy barrier)
3. Primary minimum (irreversible
attraction)

 Extended DLVO theory has been suggested in which
the hydrophobic/hydrophilic interactions are included.
So, the total adhesion energy can be expressed as:
ΔG adh= ΔGvdW+ ΔGdE+
ΔGAB

 GEL is calculated from Zeta potentials of interacting
surfaces.
 Calculation of G∆LW and G∆AB relies on contact angle
measurements.
 With various liquids on the interacting surfaces application of
a thermodynamic approach becomes essential.
 It considers free energy states of microorganisms in
suspension and in an adhering state.

 ∆Gadh is negative (nature tends to minimize free
energy), adhesion is thermodynamically favoured and
will proceed spontaneously.

Using contact angle measurements the interfacial free
energies of adhesion
ƔGadh = Ɣsb-Ɣsi-Ɣbi
Sb bacterial interfacial free energy
Si surface liquid interfacial free energy
Bi bacterial liquid interfacial free energy

Cellular phase
 Initial colonizers include S.sanguinis, S.oralis, S.mitis (within minutes)
followed by Actinomyces and Neisseria spp (about 2 hours). Obligate
anaerobes are very rare.
 These bacteria multiply forming micro colonies and get embedded in the
matrix.
 This step involves firm anchorage between the bacteria and components of
acquired pellicle.

 Involves specific adhesins on the bacteria with salivary
receptors on the pellicle.
 Can be:
 Direct interaction between bacteria and pellicle proteins
 Bacterial proteins interacting with pellicle
 Involvement of cryptitopes

Involvement of
cryptitopes
 Molecules undergo conformational change
when they adsorb to the tooth surface,
exposing their new receptors, called as
cryptitopes.
 Binding of A.naeslundii with acidic PRP, only
when the latter adsorbed to the surface.

Direct interaction between
bacteria and pellicle
proteins
Pili, fimbriae present on the bacterial cell can act
as adhesins and bind to components of pellicle.
Type I and II fimbriae of A.viscosus binding to
proline rich proteins on the pellicle.
Binding of PRP-1 to S.gordonii and A.viscosus.

Bacterial proteins
interacting with pellicle
 Lectin like bacterial proteins interacts with
oligosaccharide or pellicle associated
glycoproteins facilitating adhesion.
 Binding of S.oralis by galactose binding lectin
trisaccharide structure containing sialic acid,
galactose and N-acetyl galactosamine.
 Binding of Actinomyces via β – galactoside.

Surface Proteins
 S. gordonii: Amylase-binding protein,
AbpA
S. gordonii

Factors limiting
colonization
 Available physical space
 Preemptive colonization – prior colonization by one
species excludes another (colonization resistance).
 Environmental resistance – restriction in number of
individual species or biomass imposed by physical,
chemical or biological factors of the ecosystem.
Combination of all these factors determine the
members in each habitat

Step III. Plaque colonization
and maturation
 Multitude of interactions occur between bacteria as plaque matures.
 Both intra and inter generic interactions seen between early and
secondary colonizers.
 Cell to cell interactions seen between various organisms.
 Coaggregation leads to unusual combination of bacteria like corn-
cobs' (Gram-positive filaments covered by Gram positive cocci),
'rosettes (coccal bacteria covered by small Gram-positive curved
rods), or 'bristle brushes' (large filaments surrounded by Gram-
negative rods or short filaments)

Test-tube brush found in subgingival plaque
Tannerella sp. (yellow) in a test-tube brush

Test-tube brush with Lactobacillus
sp. (red rods) as central structures.
F. nucleatum (green) and
Bacteroides cluster filaments
radiating from the central structures.
Transversal view of
Streptococcus sp. (green)
aggregation around a central
cell (not stained) in
supragingival plaque

Co-adhesion
 Some bacteria are unable to bind directly to the conditioning film,
but are able to interact with molecules on bacteria that are
already attached (co-adhesion), also by adhesin-receptor
interactions.
 One bacterium, Fusobacterium nucleatum, can co-adhere with
almost all other bacteria found in dental plaque, and is
considered to be a key bridging organism between early and
later colonisers.

 Co-aggregation is the
interaction between planktonic
micro-organisms of a different
strain or species
 Co-adhesion is the interaction
between a sessile, already
adhering organism and
planktonic micro-oganisms of
a different strain or species

CO-AGGREGATION
PROPERTIES
1. Specificity
It is highly specific and not random. Mediated by receptors and
adhesins
Receptors - usually polysaccharide
Adhesins – lectin
Two types
Lactose inhibitable
Lactose non inhibitable

2. Functional similarity of adhesins
Even though structurally distinct but
functionally similar adhesins on each species
can bind to the same receptors on common
partner.

3. Co-aggregation bridging
This is formed when the common partner bears two
or more types of co-aggregation mediators.
It can be
various polysaccharide receptors
Or
Various adhesins
Or
Mixture of two

4. Co-Aggregation competition
Competition occurs when multiple cell
types recognize the same co-aggregation
mediator on the common co-aggregation
partner.

Early Colonizers
 Includes Streptococcus spp, Actinomyces spp,
Veillonella spp, Hemophilus spp and Propionibacterium
spp.
 Streptococcus , only genus that have extensive intra
and inter generic co aggregation.
 They bind to the pellicle and provide site for attachment

F. nucleatum and Late
colonizers
F. nucleatum categorized as bridging organism :
1.more numerous in healthy sites and they are
found in increased number in diseased sites
2.coaggregates well with all early and late colonizers
3. provide ideal anaerobic condition for the growth of
P.gingivalis
Kolenbrander 2002

Interactions among dental
plaque bacteria

Factors limiting colonisation of
Biofilm
1. Factors related to attachment surface.
2. Factors related to Biofilm community
3. Factors related to bulk fluid
4. Environmental factors

Factors related to attachment surface
Physical factors
Surface roughness - se surface area
- protection from shear force
- se difficulty in cleaning
• Chemical composition of the surface.
- brass reduces attachment
- polyvinyl chloride encourages biofilm growth.
Type of tissue and the genetic background of the host
which might alter the receptors

Factors related to Biofilm
community
Role of Exopolysaccharides
a. Maintain biofilm structure – networked cross linked linear
macromolecules
b. Chemical composition and tertiary structure determine the
adhesive character and hydrophilic or hydrophobic nature.
c. Protect the microorganism – from desiccation, harmful agents
d. Create a nutritional environment
- by binding cations
- by retaining extracellular enzymes
e. Act as a buffer

Role of Micro organisms
 Pre emptive colonization
 Through Co-aggregation
 Through metabolic interactions

Factors related to bulk fluid
Saliva - for supragingival
GCF - for subgingival
Bulk fluid provides nutrients, remove waste
products and act as a vehicle for transport of
bacterial cells.

Bulk fluid influence
through….
1. Cohesiveness of fluid
2. Composition
- nutrient content
- antibacterial agents
3. Hydrodynamics
- Shear force
High shear forces (turbulent flow) - thinner
and denser - elongated with streamers capable of
oscillation or patches of ripples.
Low shear forces (laminar flow)- thicker
with voids - like tower or mushroom

Environmental Factors
1. Addition of nutrients
2. Osmolarity
3. pH
4. Iron availability
5. Oxygen tension
6. Temperature
7. Physical barrier - availability of
space
8. Chemical & biologic barrier

Open architecture in
supragingival biofilms
 Polymer containing channels or pores
linking the biofilm and tooth surface
 Bacterial vitality varies throughout the film;
most viable in the center and lining the
voids
 Heterogeneity in pH over relatively short
distance facilitating survival of fastidious

 Sub gingival plaque not viewed directly by confocal
microscopy due to limited access.
 Light microscopy revealed a complex organization of
attached microorganisms in which there exist distinct tooth
associated and epithelial associated biofilms.
Socransky & Haffajee (2002)

Supra and sub gingival
biofilm
 One surface (tooth)
 Saliva: bulk fluid
 Microbes: increased
number of
Actinomyces and
S.sanguis
 3 surfaces (tooth,
tissue and zone
intermediate)
 GCF :bulk fluid
 Microbes : increased
number of orange
complex organisms
Socransky et al 1998

Biofilm Properties
 Metabolic communication
 Bacterial competitive interactions
 Barrier function
 Genetic exchange
 Cell-cell communication
 Detachment

Importance of food chains
 Provide metabolites which serve as energy
source for other members. (eg) lactate utilization
by Veillonella produced by streptococci
 Formation of symbiotic relationship
 Making the environment favorable (eg)
generation of ammonia by F.nucleatum
elevating pH
favorable for the growth of P.gingivalis

 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

Bacterial competitive
interactions
 Both synergistic and antagonistic interactions seen
 Antagonism can be mediated by metabolites or through
bacteriocins
 Can influence localization of residents in the film

Bacterial competitive
interactions
Bacterial synergism
Bacterial antagonism

Metabolic products
 Streptococci produce hydrogen per oxide which
are toxic to many bacteria
 S.oligofermentus lactic acid
hydrogen peroxide toxic to S.mutans
 Short chain fatty acids like lactic acid lowers pH,
having a disadvantageous effect n less aciduric
bacteria.

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.
 Usually narrow spectrum with few exceptions like antibiotics

 Streptococcus produces mutacins (mutacin I – V) active
against S.sanguinis
 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

Clinical significance
 Bacteria determine their neighbours
 Prevention of pathogenic biofilm formation bacteriocins
produced by S.pyogenes and S.salivarius are
structurally similar, antagonize each other when
colonise at the same time, inhibit the growth of other
via antagonizing growth dependent signaling, prevent
the biofilm formation of the former by the latter.
 Maintain ecological balance

 Gene regulation in response to cell density,
which influence various function.
 Most commonly quorum sensing molecules
include CSP and AI – 2

Definition
 It is defined as the cell density dependent regulation of
gene expression in response to soluble signals called
autoinducers (Bassler 1999)
 Quorum sensing can occur within a single
bacterial species as well as between diverse species,
and can regulate a host of different processes,
essentially serving as a simple communication
network.

Why the name quorum??
 Accumulation of a stimulatory
concentration of an extra-cellular
autoinducer can only occur when a
minimum number of cells i,e critical cell
density called a “quorum,” is present.

Quorum sensing is
dependent on cell density
Low level of signalling
molecules
Increased level of
molecules
Activation of gene
expression
Less
cell
density

HOW BACTERIA TALK TO
EACH OTHER:
Bacteria
Inducer Receptor
AI
Transcription
of genes

 Quorum sensing-controlled behaviors are those that only
occur when bacteria are at high cell population densities.
 These behaviors are ones that are unproductive when
undertaken by an individual bacterium but become
effective by the simultaneous action of a group of cells.

ROLE OF QS
There is an increase in
 Virulence and pathogenicity
 Secondary metabolite production
 Motility
 Conjugation
 Biofilm formation
 Growth inhibition

Key players in quorum
sensing
Autoinducers
• AHL
• Autoinducer 2
• Cyclic
dipeptides
• Bradyoxetin
• Other types
Autoinducer
synthases
• AHL synthases
• AI2 synthases
• Synthases of
other types of
autoinducers
Quorum sensing
Regulators
• Lux R type
• Lux P/Q type

QUORUM SENSING
SYSTEMS
1. QS used for intra species communication
-a. QS used by gm –ve bacteria
-b. QS used by gm +ve bacteria
2. QS used for inter species communication

It is divided into three
major classes
(1) LuxI/LuxR–type
in Gram-negative bacteria, which use AHL
(2) Oligopeptide-two-component-type quorum sensing
in Gram-positive bacteria, which use small peptides
(3) luxS-encoded autoinducer 2 (AI-2) quorum sensing
in both Gram-negative and Gram-positive bacteria.

QUORUM SENSING IN
GRAM-NEGATIVE
ORGANISMS

Quorum-sensing systems in
Gram-negative micro-organisms.
S
cheie A A , Petersen F C CROBM 2004;15:4-12

QUORUM SENSING IN GRAM-POSITIVE
ORGANISMS
 Two types of quorum sensing systems for gram positive
bacteria.
 First type : Three components
 Signaling peptide – Autoinducing peptide
 Two component signal transduction systems (that detects
and responds to signals) , histidine kinase and a cytoplasmic
response regulator protein
 AIP is not permeable through the membrane and therefore an
oligopeptide transporter , largely an ABC transporter is
required to secrete AIP in to the extracellular environment.

Quorum-sensing systems in
Gram-positive micro-organisms.
Scheie A A , Petersen F C CROBM 2004;15:4-12

 The second type of Quorum sensing system
 Small double trytophan signalling peptide Pheromone
XIP
 Oligopeptide transporter system
 Transcription regulator ComR, proximal regulator sigX
Master regulator ComX

Types of Quorum sensing
molecules
Autoinducer 1
1st detected in Vibrio fisheri - by
Chemically – N – Acyl Homoserine Lactone(AHL)
Proteins involved are designated as
Lux I & Lux R
Lux I - Catalyses the synthesis of AHL
Lux R - transcriptional regulator
Autoinducer 1 is not common in oral biofilm.
It usually regulates gene expression in genetically
identical cells.

Autoinducer 2
First observed by Schauder et al 2001
• Collection of molecules formed from
spontaneous rearrangement of 4,5
dihydroxy-2-3 pentanedione (DPP)
• Produced by both gran +ve & -ve organism
• Gene responsible for it production - lux S -
protein - LuxS

 In the absence of two component response circuit (receptor
protein)
auto inducer does not function in cell-cell communication but
functions in basic metabolism - catalyses methyl cycle.
 Autoinducer 2 mediate gene expression in mixed communities.
 It is also density dependent
 Commensal bacteria respond to low levels and pathogenic
bacteria
respond to high levels of autoinducer 2

Other functions of autoinducer 2
1. Regulate iron uptake in Aa
2. Regulate hemin (iron source) acquisition in
Pg.
3. Regulate enzymes involved in stress related
function.
4. Control the formation of multi species
biofilm.
5. Induces expression of leukotoxon in Aa and
modulate protease activities in Pg.

Competence stimulating
peptide(CSP)
• Competence-stimulating peptide (CSP) is a small
soluble peptide having from 14-23 amino acid residues
and is potentially produced by many species of
streptococci.
• Implicated in bacteriocin production, virulence and
biofilm formation.

 Staphylococcus aureus is one of the most common commensal
Gram-positive organism
 The QS system that this bacterium utilizes is one of the most
studied systems in Gram-positive organisms.
 The accessory gene regulator (Agr) system regulates toxin and
protease secretion in staphylococci.
 At low cell density, the bacteria express proteins required for
attachment and colonization, and as the cell density becomes
higher, this expression profile switches to express proteins
involved in toxin and protease secretion (Novick, 2003).

QS used for inter species
communication

Intra- and
Interspecies Cell-Cell
Communication

 AHLs and peptide autoinducers are highly
specific and are used for intraspecies cell-cell
communication.
 AI-2 and its synthase LuxS, on the other
hand, exist in over 40 species of gram-
negative and gram-positive bacteria, and AI-2
is proposed to act as a more universal
interspecies chemical language.

 Many gram-negative bacteria use AHL autoinducers and also produce AI-2.
 Likewise, many gram-positive bacteria have oligopeptide signaling systems
as well as AI-2.
 Making and responding to combinations of these and potentially other types
of chemical signals could permit bacteria to take a census of their own
population numbers and also the population density of other species in the
vicinity.
 A distinct response to each signal, or a response that is based on a
combinatorial sampling of a variety of signals, could enable bacteria to
continu-ously modulate behavior depending on the species present in a
consortium.

Quorum sensing in oral
bacteria
Several of the oral pathogens clearly produce
AI-2 and may possess the AI-2 quorum-sensing
circuit.
(P. E. Kolenbrander and E. P.
Greenberg)

Proposed role of
Quorum Sensing in
periodontal diseases

ROLE IN Biofilm Formation
 Biofilm formation involves four sequential steps:
 Surface attachment,
 Microcolony formation,
 Gaining depth as the colony matures and
 Formation of the architecture of the biofilm
(Stanley
2004).

Importance of strepto in plaque dev has lead to the
forefront of biofilm research
Ganesh kumar et al used a microtitre plate system to
determine
the influence of various factors on biofilm formation by the
oral
isolates of s.gordonii.
He found that the gene comD encoded a sensor kinase
that is
required for the development of competence for genetic
transformation and the regulation of QS system which
depends
on CSP.

Genetic dissection of biofim development has
demonstrated
that QS is required for biofilm development in both gram
positive and Negative bacteria
• Potential of AI2 to
communicate cell density to a
mixed community of bacteria
Streptococcus
mutans
• Biofilm growth is stimulated
by sialic acid – constituent of
the novel sialic acid
transporter system involved in
Qs
Tannerella
forsythia

Are signal molecules
freely diffusible in a
biofilm?
 Biofilm cells are usually encased in an extracellular
matrix, consisting of a mixture of secreted proteins,
polysaccharides, nucleic acids and dead cells.
 Acyl-HSLs are assumed to diffuse freely through this
matrix, although, depending upon the relative
hydrophobicity of matrix components it could serve as
a sink, sequestering signal molecules.

Do all cells in a biofilm produce
signal molecules at the same
rate?
 The availability and the composition of the
substrate pool will depend upon the metabolic
state of the cell.
 Cells buried in the interior of a biofilm show
decreased metabolic activity.
 Therefore, one might predict that the levels of
acyl-HSL synthesis would differ in the interior of
the biofilm compared with the metabolically
active exterior.

Antibiotic resistance -
characteristic feature of
biofilm
 BIC 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
Scaling and root planing
cornerstone of
periodontal therapy

 .
• The biofilm matrix may restrict the penetration of a
charged antimicrobial agent (diffusion-reaction theory)
• The 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

• 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.

Role of QS in antibiotic
resistance
Three hypotheses for mechanisms of antibiotic resistance in
biofilms

Multi—Layered Dilutions of Antibiotics

GENETIC EXCHANGE
 Interaction that could have major consequence for the
physiology of biofilms.
 Can occur through conjugation, transduction and
transformation collectively called as horizontal gene
transfer.
 Mechanisms by which antibiotic resistance genes can
be transferred.

 Transposons – elements capable of excision from the
chromosome of the donor genome, transfer to recipient cell
and get integrates its genome.
 Integron – gene cassette system – mechanism that allows
bacteria to accumulate diverse genes at a common locus,
useful in acquiring antibiotic resistance, are site specific
recombinase of Intl family .
 Genomic islands – regions of genome acquired horizontally.
 Combination of these.
Mobile genetic elements

 Plasmid - It is an extra chromosomal genetic
element consisting of DNA situated in the
cytoplasm in free state and reproducing
independently.
 They are grouped into incompatibility groups
(inc groups) based on their inability to co-exist
in the same cell.

 Bacteriophage -
viruses that
parasitize bacteria
and consist of
nucleic acid core
and a protein
coat.

Genetic exchange
Conjugation
Bacterium
Bacterium
Transduction
Bacterium
Bacterium
Transformation
DNA outside the cell is fragmented and
combined with bacterial DNA
plasmid
bacteriophage

Detachment
Can be Movement of
Individual cells
or
Biofilm en masse

Individual Cell Transfer
 The detachment of cells from biofilm is essential to allow colonization of
new habitats by bacteria.
 Cells detach in different fashions.
Erosion - detachment of single cells in a continuous predictable fashion
the
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.

 The detachment rate was shown to be about six clusters
per mm2 of surface per hour.
 Can be within the oral cavity and from subject to subject
(bacterial translocation)
 Microbes show centers of spread called bacterial
reservoir.
 Both horizontal (spouse to spouse for P.gingivalis) and
vertical transmission (parent to child for A.a) has been
demonstrated.

En masse transfer
 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 in that formation of the biofilm is not reliant on
planktonic cells, which are known to be more susceptible
to antimicrobial agents .
Stoodley
1991

Methods to grow biofilm
1. Static systems
- good method to inspect the biofilm in early
development
- cannot be used to study different stages of
biofilm development
2. Flow cell systems (nutrients constantly flowing
system)
- Can be used to examine biofilm development
under different growth conditions
Methods to measure biofilm environment
- miniature micro electrode – to measure pH and
solutes - micro sensor - to measure the conc: of
oxygen and other gases

DETECTION OF BACTERIAL
SPECIES IN BIOFILM

Chronology of various diagnostic
techniques

Molecular Genetics of
Biofilm
Genes required for biofilm development are
mainly
1. Surface adhesion for cell to cell & cell to surface
interactions
2. For quorum sensing
3. environmental sensing two component systems
4. General stress response.

Genes & proteins expressed in Streptococcus
gordonii - a primary colonizer
AbpA, AbpB - amylase binding protein
Hsa - sialic acid binding proteins
bind to salivary mucins & platelets.
SSaB - adhesin
LuxS - autoinducer 2
brpA - biofilm regulatory protein A
Com D - competence for genetic transformation
HK/RR 11 - two component regulatory system – role
in biofilm development.
mvt T - DNA replication/ repair
PBP2B, PBP5, glmM bacc A- peptidoglycan biosynthesis

Genes & proteins expressed in Aa
flp - rcp-tad - gene cluster for long thick fimbrils
moaA, moeA - synthesis of molybdenum
cofactor (Moco)
OMP34 - heat modifiable surface protein
pgaa ABCD - for synthesis of PGA - a linear
polymer of N-acetyl – D- glucosamine in β
linkage(component of extracellular matrix)
Crp (cyclic AMP receptor protein) - global regulatory
protein of sugar metabolism.

BIOFILM FORMATION ON
DENTAL IMPLANTS

 Rougher surfaces (crowns, implant abutments,and denture bases)
accumulate and retain more plaque (applying parameters such as
thickness, area, and colony-forming units), after several days of
undisturbed plaque
 Formation, rough surfaces harbour a more mature plaque
characterized by an increased proportion of rods, motile organisms,
and spirochetes,and
 As a consequence of the former, tooth surfaces with rough surfaces
are more frequently surrounded by an inflamed periodontium,
characterized by a higher bleeding index, an increased crevicular
fluid production, and/or an inflammatory infiltrate.

 The effect of substratum SFE on supra and subgingival plaque
maturation around implants was investigated by comparing 3-
month-old plaque from abutments with either a high (titanium) or
a low (teflon coating) SFE (Quirynen et al. 1993).
 Low-SFE substrata harboured a significantly less mature plaque
supra – as well as subgingivally,characterized by a higher
proportion of cocci and a lower proportion of motile organisms
and spirochetes

 The reduced biofilm formation on surfaces with a low SFE
could partially be explained by a low binding strength between
bacteria and substratum,probably because of a cohesive
failure within the conditioning layer (Christersson et al. 1989;
Busscher et al. 1995)

 Seven clinical RCT studies on the impact of different
potential implant surfaces on invivo biofilm formation. Even
though the surfaces had similar roughness characteristics,
these RCT studies clearly highlighted significant
differences both in the amount as well as in the
composition of the flora on different implant/abutment
surfaces. This can be explained by the antibacterial
properties of some materials. Titanium for example has a
bacteriostatic effect on oral bacteria (Bundy et al. 1980;
Leonhardt &Dahlen 1995).
 The three parameters surface roughness, SFE and
composition of biomatrials influence the formation of
biofilm around the implants.

Therapeutic Stratergies
 Biofilm – mainly commensal with limited number of
pathogens
 Selective inhibition and modulation of microbial composition
to be followed
 Many under experiments

Oral biofilm formation (a) and prospects for future
intervention (b)

1. Surface modification
2. Quorum- quenching
a. Signal transduction interference
b. Structural mimics
3. Replacement therapy
4. Regulating the levels of non-pathogenic bacteria
5. Immunization
6. Targeted Antimicrobial Therapy
7. Probiotic Approaches
8. New Approaches under development
a. Photodynamic Therapy
b. Prebiotics

APPROACHES TO REDUCING
ADHERENCE

Surface modification
Altering the tooth surface or the salivary pellicle to impede bacterial
colonization.
1. Change the surface characteristics by manipulating the protein film on
the enamel, thereby reducing bacterial adhesion.
2. Functional groups like phosphate and phosphonate may be used to
anchor water-soluble,protein repelling substances to the mineral
surface(olsson, 1998).
3. In vitro studies have shown that the combination of an alkylphosphate
and a non-ionic sufactant alters the surface characteristics of the tooth,
making it less attractive for micro-organisms.

Inhibiting Adherence with Antagonist
 Aim is to prevent the incorporation of potentially pathogenic
organisms into biofilm.
 Reducing the colonization by S.mutans can limit formation of
biofilm to some extent.
 A dodecapeptide analogue of active binding site of SpaP
protein of S.mutans available which inhibits attachment of
S.mutans synthesized.

History
 Zhang and colleagues(2004) showed that for ‘autoinducer
inactivation’, a gene from Bacillus sp. encodes a factor that can
inactivate AHLs, and hence paralyses the quorum-sensing
system.
 The authors have now further characterized AiiA by using it to
digest several AHLs. In each case they found the molecular mass
of the AHL to be increased by 18 after the enzymatic digestion,
indicating the addition of a water molecule. This is consistent with
hydrolysis of the ester bond of the homoserine lactone ring, This
approach then became widely applicable for other QS systems.

Negative regulators of QS
Anti- activator
proteins
• AHL
degradation
enzymes
• RNA dependant
regulation
Interference
of QS e
• Furanones
• L-canavanine
• Human
hormones
Bacterial
components
• Transgenic
plants
• Synthetic
analogues

Mechanism of small
Quorum quenching
inhibitors
synthetic AIPs
Interferes with the
signal or decreases
the receptor
concentration
Triclosan and
closantel
Inhibits enoyl-ACP
reductase an
intermediate in
AHLbiosynthesis
Structural mimics of
QS inhibitors Enzeym inhibitors
Inhibitor of histidine
kinase sensor

 For therapeutic purposes, it is necessary to attack the
established biofilm.
 Therefore, genes essential for viability represent the
traditional targets for anti-microbial drug design.
 Potential agents include, among others, microbial fatty
acid biosynthesis inhibitors, bacteriophages, and anti-
microbial peptides (Hancock, 1999; Payne et al., 2001;
Sulakvelidze and Morris, 2001).

 For prophylactic purposes, it seems reasonable to target
processes involved in the actual biofilm formation of
single- or mixed-bacterial communities that have the
potential to cause or favor disease, without perturbing
the balance of the normal flora.
 In this respect, two-component systems and quorum-
sensing seem to represent promising future targets.
 Interferece of signal transduction.

Signal Transduction Intereference
 Two-component signal transduction systems and histidine
kinases represent potential prophylactic targets.
 The histidine kinases and response regulators of the two-
component system exhibit both conserved and variable
domains.
 The input domain in the histidine kinase can be targeted by
signal analogues but other sources of receiving phosphate
by response regulators cannot be prevented, hence
response regulator itself will be a bettter target.

In a typical two-component signal transduction system, the
transmembrane domain of the histidine kinase recognizes a
stimulus.

Structural mimics
Halogenated furanones are similar in structure to the homoserine
lactones that are used as quorum-sensing signal molecules by gram-ve
micro-organisms. Mechanism
1. Furanones compete with homoserine lactone molecules for binding to
transcriptional activator.
2. Accelerating degradation of the transcriptional regulator that binds to the
signal.
3.De-acylation of the signal molecules and use of signal analogues.

Replacement Therapy
 Stratergy to replace potential pathogenic micro-organisms with genetically modified
organisms that are less virulent.
Requirements:
 definite pathogen to replace the replacement organism must not cause disease itself
 It must colonize persistently
 It must replace the pathogen effectively
 It must possess a high degree of genetic stability.
 Eg. Animal studies involving lactate-dehydrogenase-deficient and uerolytic S.mutans
strains have shown promising results in caries prevention.

Regulating the levels of non pathogenic
bacteria to influence virulence
 Incorporation of some organisms into biofilms is dependent
upon other antecedent biofilm residents.
 Identification of such dependencies of known pathogens and
targeting these antecedent organisms.
 Targeting strains of F.nucleatum to avoid the colonisation of
late colonizers.

Immunization
 The aim is to inhibit adhesion or reduce the virulence of putative
microbial etiologic agents.
 Micro-organisms could be cleared from the oral cavity by antibodies
prior to colonization, antibodies could block adhesins or receptors
involved in adhesion, or modify metabolically important functions or
virulence factors.
 Active or passive immunization in humans impedes select target
micro-organisms in both caries (koga et al 2002, smith 2002) and
periodontal disease (Booth et al )
 Problem: poly-microbial infections, ability of microorganisms to form
bio-film and undergo transformation leading to altered antigenecity.

Targeted antimicrobial
therapy
 Eckert et al developed a novel technology for a new
class of antimicrobials STAMPs (specifically targeted
antimicrobial peptides).
 Fusion peptide: killing moiety, non specific and targeting
moiety, specific.
 Pheromone produced by S.mutans, namely CSP as a
STAMP targeting domain to mediate S.mutans specific
delivery of antimicrobial peptide domain (Eckert 2006)

Probiotic approaches
 Developed by Hillman to prevent dental caries uses
organisms that naturally occur in plaque and considered
to be safe (GRAS).
 Implantation of S.sanguis in plaque as they are
associated with reduced periodontopathic organisms
 Implantation of lactobacillus spp.

New approaches under
development include
 Photodynamic therapy, in which a dye is activated by low power laser
light to release free radicals
 The use of probiotics (introduction of beneficial bacteria) or prebiotics
(nutrients that favour the growth of beneficial bacteria)
 Molecules that prevent the attachment of potentially pathogenic
organisms. The aim should be to reduce the levels and activity of
deleterious microbes, while retaining the benefits of this natural biofilm.

Future Directions
 Developing oral prophylactic strategies through interference with
two-component systems or quorum-sensing of biofilm micro-
organisms represents an interesting future challenge.
 Unlike strategies that target microbial viability, such approaches
may interfere with microbial adaptive pathways without killing the
micro-organisms.

Conclusion
In order to adjust a complex microbial ecosystem to
one that is compatible to health, it is essential to define
the range of species that colonize the area, recognise
their relationship with each other and the host, and
develop effective strategies to guide these ecosystems
to those compatible with long-term oral health.

 Dental biofilms: difficult therapeutic targets.
Socransky &Haffajee Periodontology 2000, Vol. 28,
2002
 Periodontal microbial ecology. Socransky &Haffajee
Periodontology 2000, Vol. 38, 2005
 Subgingival biofilm formation. Kuboniwa & Lamont.
Periodontology 2000, Vol. 52, 2010
 Microbial ecology of dental plaque and its significance in
health and disease. Marsh. Adv Dent Res 8(2):263-271,
July, 1994
 Overview of microbial biofilms. Costerton. Journal of
Industrial Microbiology (1995) 15, 137-140
References

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