GOOD

MORNING
07/25/2021 1
BIOFIL
M
PRESENTED BY:
DR. YASHIKA KALYANI
PG 1ST YEAR
07/25/2021 2
CONTENTS
• Introduction
• Composition of biofilm
• Structure of biofilm
• Formation of dental plaque biofilm
• Factors affecting supragingival plaque formation
• Characteristics of biofilm
• Microbiologic specificity of periodontal diseases
• Criteria for identification of periodontal pathogens
• Conclusion
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• References
INTRODUCTION
• 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)
• Dental plaque is composed primarily of microorganisms encased
within an extracellular matrix.
• One gram of plaque (wet weight) contains approximately 1011 bacteria.
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COMPOSITION
• It has been estimated that more than 500 distinct microbial
phylotypes can be present as natural inhabitants of dental plaque.

• Nonbacterial microorganisms that are found in plaque include

archaea, yeasts, protozoa, and viruses.

• The intercellular matrix consists of organic and inorganic materials

derived from saliva, gingival crevicular fluid, and bacterial products.

• Organic constituents of the matrix include polysaccharides, proteins,

glycoproteins, lipid material, and DNA. 07/25/2021 5
• Glycoproteins from the saliva are important components of the
pellicle that initially coats a clean tooth surface, but they also
become incorporated into the developing plaque biofilm.

• Polysaccharides produced by bacteria also contribute to the organic

portion of the matrix. Play a major role in maintaining the integrity
of the biofilm.

• The inorganic components of plaque are predominantly calcium

and phosphorus, with trace amounts of other minerals such as
sodium, potassium, and fluoride.

• As the mineral content increases, the plaque mass becomes

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calcified to form calculus.

STRUCTURE

• 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).

• 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)
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• Structureof 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

• The biofilm matrix is penetrated by fluid channels that conduct

the flow of nutrients, waste products, enzymes, metabolites, and
oxygen.
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• 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.
• Exopolysaccharides (EPS), which are produced by the bacteria in
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 materials. 07/25/2021 9
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EXOPOLYSACCHARIDES – the backbone of the
biofilm

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FORMATION OF THE DENTAL PLAQUE
BIOFILM

• The process of plaque formation can be divided into following

several phases:
(1) the formation of the pellicle on the tooth surface,
(2) the initial adhesion/attachment of bacteria, and
(3) colonization/plaque maturation.

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Formation of the pellicle

• All surfaces in the oral cavity, are coated with a layer of organic
material known as the acquired pellicle.
• Consists of molecules that can function as adhesion sites (receptors)
for bacteria.
• Can be detected on clean enamel surfaces within 1 minute after their
introduction into the mouths of volunteers.
• By 2 hours, the pellicle is essentially in equilibrium between adsorption
and detachment.
• Bacteria that adhere to tooth surfaces do not contact the enamel
directly but interact with the acquired enamel pellicle. 07/25/2021 15
Initial adhesion/attachment of bacteria

• The initial steps of transport and interaction with the surface are
essentially nonspecific.
• Small proportion of oral bacteria possess adhesins that interact
with receptors in the host pellicle.
• These organisms are the most abundant bacteria in biofilms on
tooth enamel shortly after cleaning.
• First 4–8 hours, the genus Streptococcus tends to dominate.
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• Other bacteria that are commonly present at this time include
Haemophilus spp. and Neisseria spp. (obligate aerobes), as well as
Actinomyces spp. and Veillonella spp. (facultative anaerobes)
• Considered the “primary colonizers” of tooth surfaces.
• Provide new binding sites for adhesion by other oral bacteria.
• Modify the local microenvironment in ways that can influence the
ability of other bacteria to survive in the dental plaque biofilm.
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Colonisation and plaque maturation
• The primary colonizing bacteria provide new receptors for
attachment by other bacteria (coadhesion).
• Coadhesion leads to the development of microcolonies and
eventually to a mature biofilm.
• When many genetically distinct bacteria adhere to each other and
form clumps, it is referred to as coaggregation (direct interaction-
cell–cell interaction).
• Different species—or even different strains of a single species—
have distinct sets of coaggregation partners.
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• Secondary colonizers do not initially colonize clean tooth surfaces
but rather adhere to bacteria that are already in the plaque mass.
• The transition from early supragingival dental plaque to mature
plaque sub gingivally, involves a shift in the microbial population
from primarily gram-positive organisms to high numbers of gram-
negative bacteria.
• Color-coded “complexes” of periodontal microorganisms: based on
the frequency with which different clusters of microorganisms
were recovered, and the complexes were color-coded for easy
conceptualization .
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• Early colonizers are either independent of defined complexes or
members of the yellow or purple complexes.
• Secondary colonizers fall into the green, orange, and red
complexes.
• The green and orange complexes include species recognized as
pathogens in periodontal and nonperiodontal infections.
• Red complex is associated with bleeding on probing.
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FACTORS AFFECTING SUPRAGINGIVAL
PLAQUE FORMATION
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
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formation
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 bacteria.
• Levan – Used as carbohydrate nutrient by plaque bacteria in
absence of exogenous sources.

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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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4. 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.) 07/25/2021 25
CHARACTERISTICS OF BIOFILM BACTERIA

METABOLISM OF DENTAL PLAQUE BACTERIA

• Antagonistic effect - S. sanguinis group are producers of H2O2 , 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.

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Co-operative metabolic interactions

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COMMUNICATION BETWEEN BIOFILM BACTERIA
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. 07/25/2021 29
• Quorum sensing has been described in both Gram +ve & Gram -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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Quorum sensing molecules
Three types of molecules :
• Acyl-homoserine lactones (AHLs) - signaling molecules used
by many G-ve bacteria, it is synthesized by Lux-I family
protiens.
• Autoinducer peptides (AIPs) - signaling molecules used by G
+ve bacteria
• Autoinducer-2 (AI-2) - used by both G-ve & G+ve bacteria,
chemically it is furanosyl borate diester. Synthsized by protein
Lux-S. 07/25/2021 31
• 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.
• 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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BIOFILMS AND ANTIMICROBIAL RESISTANCE
• The resistance of bacteria to antimicrobial agents is dramatically
increased in the biofilm.
• Organisms in a biofilm are 1000 to 1500 times more resistant as
compared with antibiotics in their planktonic state.
• An important mechanism of resistance appears to be the slower
rate of growth of bacterial species in a biofilm, which makes them
less susceptible to many but not all antibiotics.
• The biofilm matrix, (physical barrier) does have certain properties
that can retard antibiotic penetration.
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• Extracellular enzymes such as β-lactamases, formaldehyde lyase,
and formaldehyde dehydrogenase may become trapped and
concentrated in the extracellular matrix, and inactivate some
antibiotics.
• “Superresistant” bacteria were identified within a biofilm. These
cells have multidrug resistance pumps that can extrude
antimicrobial agents from the cell.
• Antibiotic resistance may be spread through a biofilm via the
intercellular exchange of DNA.
• Conjugation, transformation , plasmid transfer, and transposon
transfer have all been shown to occur in biofilms.
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MICROBIOLOGIC SPECIFICITY OF
PERIODONTAL DISEASES

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Nonspecific Plaque Hypothesis
• Mid-1900s, periodontal diseases were thought to result from an
accumulation of plaque over time, eventually in conjunction with a
diminished host response and increased host susceptibility with
age.
• According to the nonspecific plaque hypothesis, periodontal
disease results from the “elaboration of noxious products by the
entire plaque flora.”
• Several observations contradicted these conclusions. 07/25/2021 38
• First, some individuals with considerable amounts of plaque and
calculus, as well as gingivitis, never developed destructive
periodontitis.
• Furthermore, individuals who did present with periodontitis
demonstrated considerable site specificity with regard to the
pattern of disease.
• Some sites were unaffected, whereas advanced disease was found
in adjacent sites.
• Although the nonspecific plaque hypothesis has been discarded in
favor of other etiologic hypotheses, most of the therapeutic
interventions are still based on the basic principles of the
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nonspecific plaque hypothesis.

Specific Plaque Hypothesis
• Underlines the importance of the qualitative composition of the
resident microbiota.
• The pathogenicity of dental plaque depends on the presence of or
an increase in specific microorganisms.
• This concept encapsulates that plaque that harbors specific
bacterial pathogens may provoke periodontal disease because key
organisms produce substances that mediate the destruction of
host tissues.
• Disease association studies do not reveal whether the presence of
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specific bacteria causes or correlates with the presence of disease.

• In addition, these studies have shown that periodontal disease can
occur even in the absence of defined “pathogens,” such as red
complex bacteria, and conversely that “pathogens” may be present
in the absence of disease.

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Ecologic Plaque Hypothesis
• 1990s, Marsh and coworkers.
• According to the ecologic plaque hypothesis, both the total
amount of dental plaque and the specific microbial composition of
plaque may contribute to the transition from health to disease.
• The health associated dental plaque microbiota is considered to be
relatively stable over time and in a state of dynamic equilibrium or
“microbial homeostasis.”
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• Changes in the host status, such as inflammation, tissue
degradation, and/or high gingival crevicular fluid flow, may lead to
a shift in the microbial population in plaque.
• As a result of microenvironmental changes, the number of
beneficial species may decrease, whereas the number of
potentially pathogenic species increases.
• This gradual shift in the entire microbial community, known as
dysbiosis, may result in a chronic disease state such as
periodontitis.
• Disease is associated with the overgrowth of specific members of
the dental plaque biofilm when the local microenvironment
changes, but it is not necessarily the same species in each case.
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Keystone Pathogen Hypothesis and Polymicrobial Synergy and
Dysbiosis Model
• Indicates that certain low abundance microbial pathogens can
orchestrate inflammatory disease by remodeling a normally benign
microbiota into a dysbiotic one.
• Certain pathogens may trigger the disruption of microbial
homeostasis, thereby leading to the development of periodontal
disease, even when they are present only in low numbers.
• P. gingivalis subverts the host immune system and changes the
microbial composition of dental plaque, ultimately leading to
periodontal bone loss.
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• On this basis, P. gingivalis was labeled a “keystone” pathogen; this means
that it is an organism that is central to the disease process, even when it is
at a relatively low abundance.
• Three “keystone pathogen” mechanisms are proposed for P. gingivalis.
• First, it is suggested that P. gingivalis can manipulate the Toll-like receptor 4
(TLR4) response.
• P. gingivalis can act as an agonist of TLR4, activating the immune system, or
as an antagonist of TLR4, dampening the immune response.
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• Second, P. gingivalis can inhibit the synthesis of interleukin-8 (also
in response to other bacteria).
• This phenomenon is called “local chemokine paralysis” and leads
to delayed polymorphonuclear leukocytes transmigration,
impairing the host to confront the microbial challenge.
• Third, P. gingivalis is able to interfere with the complement system,
a component of the innate immune system.

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• The keystone pathogen hypothesis has been extended to include the
concept of a disrupted homeostasis in addition to the important roles
of keystone pathogens in the polymicrobial synergy and dysbiosis
model of disease.
• Interspecies communication between keystone pathogens and other
members of the community (accessory pathogens) is considered one
important factor that leads to overgrowth of the more pathogenic
microbiota and to a dysbiotic microbial community.
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CRITERIA FOR THE IDENTIFICATION OF
PERIODONTOPATHOGENS

• 1870s, Robert Koch: Koch's postulates:

1. Be routinely isolated from diseased individuals
2. Be grown in pure culture in the laboratory
3. Produce a similar disease when inoculated into susceptible
laboratory animals
4. Be recovered from lesions in a diseased laboratory animal
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• In the case of periodontitis, three primary problems exist:
(1)the inability to culture all of the organisms that have been
associated with disease (e.g., many of the oral spirochetes),
(2) the difficulties inherent in defining and culturing sites of active
disease, and
(3) the lack of a good animal model system for the study of
periodontitis
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• Sigmund Socransky’s modifiocation
• According to his criteria, a potential pathogen must do the
following:
1. Be associated with disease, as evidenced by increase in the
number of organisms at diseased sites.
2. Be eliminated or decreased in sites that demonstrate the clinical
resolution of disease with treatment.
3. Induce a host response in the form of an alteration in the host
cellular or humoral immune response.
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4. Be capable of causing disease in experimental animal models.
5. Produce demonstrable virulence factors that are responsible for
enabling the microorganism to cause the destruction of the
periodontal tissues

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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 mouth rinses.
• 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
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disorders.
REFERENCES

• Newman, Takei, Klokkevold, Carranza; Clinical Periodontology;

13th Edition: Elseveir
• Jan Lindhe, Niklaus P. Lang, Thorkildkarring; Clinical
Periodontology And Implant Dentistry: 6th Edition: Blackwell
• Marsh PD, Moter A, Devine DA. Dental plaque biofilms:
commuinties, conflict and control. Periodontol 2000 2011; 55: 16-35
• Interspecies Interactions within Oral Microbial Communities
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THANK YOU
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