An elaborate presentation on oral biofilm. Helpful for dental graduate and periodontology post graduate students.

1. ORAL BIOFILM
- DR AISHWARYA PANDEY
- DEPARTMENT OF PERIODONTOLOGY
- BANARAS HINDU UNIVERSITY

2. INTRODUCTION
On the basis of physical and morphologic criteria, the oral cavity
can be divided into six major ecosystems (also called niches):
1. The intraoral and supragingival hard surfaces (teeth,
implants, restorations, and prostheses)
2. Subgingival regions adjacent to a hard surface, including
the periodontal/peri-implant pocket
3. The buccal palatal epithelium and the epithelium of the
floor of the mouth
4. The dorsum of the tongue
5. The tonsils
6. The saliva

3. • The mouth is similar to other habitats within the body in
having a characteristic microbial community that provides
benefits for the host.
• The mouth is warm and moist, and is able to support the
growth of a wide range of microorganisms, including
viruses, mycoplasma, bacteria, fungi, and protozoa.
• These microorganisms colonize mucosal and dental surfaces
in the mouth to form three‐dimensional, structurally‐
organized multispecies communities that are termed
biofilms.

4. • Biofilms are composed of microbial cells encased within
a matrix of extracellular polymeric substances, such as
polysaccharides, proteins, and nucleic acids.
• The biofilms that form on teeth are referred to as
dental plaque.

5. Perturbation in a key environmental factor can disrupt the natural
stability (microbial homeostasis) of the resident microbiota at a site
and result in a rearrangement of the composition and activity of the
resident microbial community; such a change might predispose the
site to disease.

6. Role of temperature
• The mouth is maintained at a temperature of around 35–37°C,
which is suitable for the growth of a broad range of microbes.
• Temperature increase at subgingival sites during
inflammation
Alter bacterial gene expression
Alter the competitiveness of bacteria within the microbial
community,
Increased growth and protease activity of some putative
periodontal pathogens.

7. Role of Redox Potential
• Although the mouth is overtly aerobic, the majority of oral
bacteria are facultatively or obligately anaerobic.
• The distribution of these anaerobes in the mouth is generally
related to the redox potential (Eh), the measure of the degree of
oxidation–reduction at a site.
• The gingival crevice has the lowest Eh in the healthy mouth,
and harbors the largest numbers of obligate anaerobes.
• Bacterial metabolism in mature oral biofilms results in sharp
gradients of oxygen and Eh, thereby generating a mosaic of
microenvironments suitable for the growth of bacteria with a
range of oxygen tolerances.

8. Role of pH
• In the mouth, pH is a major determinant of bacterial distribution and
metabolism.
• The buffering activity of saliva plays a major role in maintaining the
intraoral pH at around neutrality, which again is suitable for the
growth of members of the resident oral microbiota.
• Changes in environmental pH frequently occur, and when they do
they drive major shifts in the proportions of bacteria within oral
biofilms.
• Even a small change in pH can alter the growth rate and pattern of
gene expression in subgingival bacteria, and increase the
competitiveness of some of the putative Gram‐negative anaerobic
pathogens at the expense of species associated with periodontal
health.

9. Definitions
• Dental plaque is defined as a structured, resilient, yellow-
grayish substance that adheres tenaciously to the intraoral
hard surfaces, including removable and fixed restorations.
• Materia alba refers to soft accumulations of bacteria, food
matter, and tissue cells that lack the organized structure of
dental plaque and that are easily displaced with a water spray.
• Calculus is a hard deposit that forms
via the mineralization of dental plaque &
that is generally covered by a layer of
unmineralized plaque.

10. Dental Plaque Biofilm
• The architecture of a dental plaque biofilm has many features in
common with other biofilms.
• It is heterogeneous in structure, with clear evidence of open fluid-
filled channels running through the plaque mass.
• Nutrients make contact with the sessile (attached) microcolonies by
diffusion from the water channels to the microcolony.
• The bacteria exist and proliferate within the intercellular matrix
through which the channels run.
• The matrix confers a specialized environment that distinguishes the
bacteria that exist within the biofilm from those that are free
floating; this is the so-called planktonic state in solutions such as
saliva or crevicular fluid.

11. CLASSIFICATION
11

12. SUPRA- GINGIVAL PLAQUE
• Supragingival plaque is found at or above the gingival
margin.
• Supragingival plaque in direct contact with the gingival
margin is referred to as marginal plaque
12

13. SUBGINGIVAL PLAQUE
• Sub gingival plaque
is found below the gingival
margin, between the tooth
and gingival sulcular tissue.
13

15. Diagram depicting the
plaque–bacteria
association between the
tooth surface and the
periodontal tissues.

16. Plaque biofilm on dental
implants
• It has been reported previously that implants revealing signs of
infection contain subgingival microbiota similar to those of the
natural teeth with periodontitis
• Implant biofilm can lead to infection at two levels: the mucosal
level (peri-implant mucositis) that causes an inflammatory
lesion residing in the mucosa and bone level (peri-implantitis)
which is explained as inflammatory lesion affecting the
supporting tissues.
• Treatment of such infections consists of an anti-infective
protocol that can be achieved through mechanical debridement
of the implant surface and subgingival chlorhexidine or
povidone iodine irrigation.

17. COMPOSITION

18. INTERCELLULAR MATRIX

19. ORGANIC MATERIAL
• Polysaccharides ( 30% ) produced by bacteria.
• Protein (30%) –Albumin (originating from GCF)
• Glycoproteins from saliva.
• Lipid (15%) consists of debris from disrupted bacteria,
host cells & food debris.
• DNA of the microbial component.

20. INORGANIC MATERIAL
•Predominantly – calcium, and phosphorous, with traces of
sodium, potassium & fluorides.
•Source of inorganic material in supragingival plaque is primarily
saliva
• Source of inorganic material in sub gingival plaque is GCF
• As the mineral content increases, the plaque mass becomes calcified
to form calculus.

21. MICRO-ORGANISMS
• Dental plaque is composed primarily of microorganisms.
• One gram of plaque (wet weight) contains approximately
1011 bacteria.
• Any individual may harbor hundreds of different species.
• Next to bacteria, nonbacterial organisms can also be found
in the dental plaque biofilm, including archaea, yeasts,
protozoa, and viruses.

22. Formation of dental biofilms
• Dental biofilms form via an ordered sequence of
events, resulting in a structurally‐ and functionally‐
organized, species‐rich microbial biofilm (Socransky &
Haffajee 2002; Marsh et al. 2011)

23. 1. Adsorption of a conditioning film (pellicle formation)
2. Reversible adhesion between the microbial cell surface and the
conditioning film
3. More permanent attachment involving interactions between specific
molecules on the microbial cell surface (adhesins) and complementary
molecules (receptors) present in the conditioning film
4. Co‐adhesion, in which secondary colonizers adhere to receptors on
already attached bacteria leading to an increase in microbial diversity
5. Multiplication of the attached cells, leading to an increase in biomass
and synthesis of exopolymers to form the biofilm matrix (plaque
maturation)
6. Detachment of attached cells to promote colonization elsewhere
The distinct stages in dental biofilm
formation include:

24. Formation of the Pellicle
• All surfaces in the oral cavity, including the hard and soft tissues,
are coated with a layer of organic material known as the
acquired pellicle.
• The pellicle on tooth surfaces consists of more than 180 peptides,
proteins, and glycoproteins, including keratins, mucins, proline-
rich proteins, phosphoproteins (e.g., statherin), histidine-rich
proteins, and other molecules that can function as adhesion sites
(receptors) for bacteria.
• It is composed of two layers: a thin basal layer that is very
difficult to remove, and a thicker globular layer, up to 1 µm or
more, that is easier to detach.

25. • Consequently, bacteria that adhere to tooth surfaces do not
contact the enamel directly but interact with the acquired
enamel pellicle.
• However, the pellicle is not merely a passive adhesion
matrix.
• Many proteins retain enzymatic activity when they are
incorporated into the pellicle, and some of these, such as
peroxidases, lysozyme, and α-amylase, may affect the
physiology and metabolism of adhering bacterial cells.

26. Initial Adhesion/Attachment of
Bacteria
The initial steps in colonization of teeth by bacteria
occur in three phases.
Phase 1 is transport to the surface
Phase 2 is initial reversible adhesion
Phase 3 is strong attachment.

27. Phase 1: Transport to the Surface
• The first stage involves the initial transport of the bacterium
to the tooth surface.
• Random contacts may occur via :
• Brownian motion
• Sedimentation of microorganisms
• Liquid flow
• Active bacterial movement (chemotactic activity).

28. Phase 2: Initial Adhesion
• The second stage results in an initial reversible adhesion of the bacterium.
• This is initiated when the bacterial cell comes into close proximity to the surface
(separation distance, ≈50 nm).
• Long- and short-range forces, including van der Waals attractive forces and
electrostatic repulsive forces, operate at this distance.
• The behavior of bacterial cells can be reasonably described by the Derjaguin–
Landau–Verwey–Overbeek (DLVO) theory of colloid stability.
• According to this theory, the total interaction energy (also called the total Gibbs
energy [GTOT]), is the sum of the attractive forces (GA) and the electrostatic
repulsion (GR).
• At the physiologic ionic strength of saliva, the van der Waals forces result in a net
attraction of bacterial cells at distances of tens of nanometers from the surface.
• At distances of approximately 10 nm from the surface, bacterial cells are reversibly
bound.

29. Phase 3: Strong Attachment
• After initial adhesion, a firm anchorage between the bacterium
and the surface is established.
• The binding between the bacteria and the pellicle is mediated
by specific adhesins on the bacterial cell surface (usually
proteins) and complementary receptors (proteins,
glycoproteins, or polysaccharides) in the acquired pellicle.
• Many proteins in the acquired pellicle can act as receptors for
streptococci, including α-amylase, acid proline-rich proteins,
statherin, and salivary agglutinin glycoprotein gp340.

30. Colonization and Plaque
Maturation
• Once attached, the pioneer colonizers start to multiply.
• The metabolism of these bacteria that attach early modifies the local
environment, for example by making it more anaerobic following their
consumption of oxygen and the production of reduced end products of
metabolism.
• As the biofilm develops, adhesins on the cell surface of more
fastidious secondary colonizers, such as obligate anaerobes, bind to
receptors on bacteria by a process termed co‐adhesion or
co‐aggregation which is the cell-to cell recognition of genetically
distinct partner cell types.
• A key organism in plaque biofilm development is Fusobacterium
nucleatum.
• This species can co‐adhere to most oral bacteria, and acts as an important
bridging organism between early and late colonizing species.

31. Characteristic cell associations can be seen in mature dental plaque,
such as “corn‐cob” (in which coccal‐shaped cells attach along the
tip of filamentous organisms) and “test‐tube brush” (rod‐ shaped
bacteria sticking out perpendicularly from bacterial filaments)
formations.
CORNCOB STRUCTURE TEST TUBE BRUSH

34. Schematic representation of the
different stages in the formation of
dental biofilms
1. Pellicle
forms on a
clean tooth
surface
2i Bacteria are
transported
passively to the
tooth surfaces
2ii where they
may be held
reversibly by
weak, long‐range
forces of
attraction.

35. 4 Secondary colonizers
attach to the already
attached primary
colonizers by molecular
interactions
3 Attachment becomes more
permanent through specific
stereochemical molecular interactions
between adhesins on the bacterium
and complementary receptors in the

36. 5 Growth results in biofilm maturation,
facilitating a wide range of
intermicrobial interactions (synergistic
6 On occasions, cells
can detach to colonize
elsewhere.

37. Diagrammatic representation of a mature plaque

38. Properties of plaque biofilms
• Physiological heterogeneity
• Greater phenotypic resistance
• Interbacterial communication
• Resistance to antimicrobial agents

39. Physiological heterogeneity
• A wide variety of markedly heterogeneous microniches or
microenvironments that are close together can exist within a
biofilm under variable environmental conditions.
• In this complex heterogeneous community structure,
microorganisms live together, cooperate, interact and
communicate through a system of signals that direct the
phenotype and regulate gene expression.
• The clinical importance of this intricate biofilm ecosystem lies in
the ability of these microbial communities to change in response
to treatment intervention.

40. Greater phenotypic resistance
• Bacteria growing as a sessile community in biofilms exhibit a
phenotype that differs from that shown by bacteria that grow in
suspension, or planktonically, expressing genes that are never
expressed in planktonic form and that give them resistance to
antibiotics, to environmental stress and to host defense
(antibodies and phagocytic cells).
• This resistance is maintained even when they are released from
the biofilm.

41. Interbacterial communication
• Bacteria that live together in a biofilm are able to
communicate with each other either by using chemical signals
or by transferring genetic material through conjugation
mechanisms.
• Quorum sensing is an important phenomenon that occurs via
chemical signals.
• This is a phenomenon by which bacteria perceive bacterial
population density existing in their close surroundings through
specific bio-sensory mechanisms. When this density reaches a
critical level, a specific, genetically set bacterial response is
triggered.

42. Quorum sensing may give biofilms their distinct properties:
Alteration of physiological properties of bacteria in the
community through quorum sensing.
Has the potential to influence community structure, by
encouraging the growth of beneficial species (to the biofilm)
and discouraging the growth of competitors.
Expression of genes for antibiotic resistance at high cell
densities may provide protection.
42

43. Resistance to antimicrobial
agents
• One of the characteristics of biofilms is their great antimicrobial
resistance. This quality may be due to several circumstances:
• slower rate of growth of bacterial species in a biofilm, which makes them less
susceptible to many but not all antibiotics
• lower -non-effective- concentration at which antimicrobials reach the deepest
areas of the biofilm
• strongly charged or chemically highly reactive agents can fail to reach the
deeper zones of the biofilm because the biofilm acts as an ion-exchange resin
that removes such molecules from solution
• the quiescent state of bacteria in the deep regions of the biofilm, due to the lower
supply of nutrients, which makes them less susceptible to antimicrobial action.
• antibiotic resistance may be spread through a biofilm via the intercellular
exchange of DNA.

44. 1. Synergistic/Agonistic interactions:
• Streptococcus and Actinomyces produce lactate and formate as
metabolic byproducts which are used in the metabolism of
Veillonella and Campylobacter respectively.
• Veillonella produces menadione which is used by P. gingivalis
and P. intermedia.
• Campylobacter produces protoheme which is used by P.
gingivalis.
• P. gingivalis produces isobutyrate which is utilized by
Treponema.
• Treponema and Capnocytophaga produce succinate which is
used by P. gingivalis.
Metabolic interactions among bacterial
species in plaque biofilm

45. 2. Antagonistic interactions:
• S. sanguis produces H2O2 which kills Aggregatibacter
actinomycetemcomitans.
• Aggregatibacter actinomycetemcomitans produces bacteriocin
which kills S. sanguis.

46. Factors That Affect Plaque
Biofilm Formation
 Topography
 Surface microroughness
 Variation within dentition
 Impact of Gingival Inflammation and Saliva
 Impact of Patient’s Age

47. Topography
• Early plaque formation on teeth follows a typical topographic
pattern, with initial growth along the gingival margin and from the
interdental spaces (i.e., the areas protected from shear forces).
• Later, a further extension in the coronal direction can be observed.
This pattern may change severely when the tooth surface contains
irregularities that offer a favorable growth path.
• Plaque formation may also originate from grooves, cracks,
perikymata, or pits.
• Scanning electron microscopy studies clearly revealed that the
early colonization of the enamel surface starts from surface
irregularities in which bacteria shelter from shear forces.

48. Surface Microroughness
• Rough intraoral surfaces (e.g., crown margins, implant abutments,
denture bases) accumulate and retain more plaque and calculus in
terms of thickness, area, and colony-forming units.
• Smoothing an intraoral surface decreases the rate of plaque
formation.
• Below a certain surface roughness (Ra < 0.2 µm), however, further
smoothing does not result in an additional reduction in plaque
formation.
• There seems to be a threshold level for surface roughness (Ra ≈ 0.2
µm) above which bacterial adhesion will be facilitated.

49. Variation Within the Dentition
• Within a dental arch, large differences in plaque growth rate can
be detected.
• In general, early plaque formation occurs faster:
 in the lower jaw (as compared with the upper jaw)
 in molar areas
 on the buccal tooth surfaces (as compared with palatal sites,
especially in the upper jaw)
 in the interdental regions (as compared with the buccal or
lingual surfaces).

50. Impact of Gingival Inflammation
and Saliva
• In vivo plaque formation is more rapid on tooth surfaces facing
inflamed gingival margins. than on those adjacent to healthy
gingivae.
• It is suggested that the increase in crevicular fluid production
enhances plaque formation.
• Probably some substances from this exudate (e.g., minerals,
proteins, carbohydrates) favor both the initial adhesion and the
growth of the early colonizing bacteria.
• Xerostomia decreases the oral pH and increases the
development of plaque. In absence of regular salivary flow,
plaque accumulation increases.

51. Impact of Patient’s Age
• Recent reports clearly indicate that a subject’s age does not
influence de novo plaque formation.
• In a study by Fransson and colleagues, no differences could be
detected in de novo plaque formation between a group of young
(20 to 25 years old) patients and a group of older (65 to 80 years
old) subjects who abolished mechanical tooth cleaning measures
for 21 days, neither in amount nor in composition.
• However, the developed plaque in the older patient group resulted
in more severe gingival inflammation, which seems to indicate an
increased susceptibility to gingivitis with aging.

52. NON-SPECIFIC
PLAQUE
HYPOTHESIS
SPECIFIC
PLAQUE
HYPOTHESIS
52
ECOLOGICAL
PLAQUE
HYPOTHESIS
Microbiologic Specificity of
Periodontal Diseases
KEYSTONE
PATHOGEN
HYPOTHESIS

53. Nonspecific Plaque Hypothesis
• Proposed by WALTER LOESCHE(1976).
• According to the nonspecific plaque hypothesis, periodontal
disease results from the “elaboration of noxious products by the
entire plaque flora.”
• When only small amounts of plaque are present, the noxious
products are neutralized by the host.
• Similarly, large amounts of plaque would cause a higher production
of noxious products, which would essentially overwhelm the host’s
defenses.

54. • Several observations contradicted these conclusions.
• 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.
• In the presence of a uniform host response, these findings were
inconsistent with the concept that all plaque was equally pathogenic.

55. Specific Plaque Hypothesis
 Specific plaque hypothesis states that only certain plaque is
pathogenic, and its pathogenicity depends on the presence
of or increase in specific microorganisms.
- NEWMAN AND SOCRANSKY (1977)
 Plaque harboring specific bacterial pathogens results in
periodontal disease.
• A. actinomycetemcomitans as a pathogen in localized
aggressive periodontitis.

56. Ecologic Plaque Hypothesis
• During the 1990s, Marsh and coworkers a developed the
“ecologic plaque hypothesis” as an attempt to unify the existing
theories regarding the role of dental plaque in oral disease.
• 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.

57.  This hypothesis postulated dynamic relationship
between environmental cause & ecological shifts
within the biofilm.
 It also introduced the concept that the disease can be
prevented not only by inhibiting the putative pathogens, but
also interfering with the environmental factors driving the
selection & enrichment of these bacteria.

58. Ecologic plaque hypothesis in relation to periodontal
diseases: gingivitis and periodontitis

59. Keystone Pathogen Hypothesis and
Polymicrobial Synergy and Dysbiosis
Model
• The keystone pathogen hypothesis indicates that certain low-
abundance microbial pathogens can orchestrate inflammatory
disease by remodeling a normally benign microbiota into a
dysbiotic one.
• In this model, interspecies communication between keystone
pathogens and other members of the community (known as
accessory pathogens) is considered one important factor that leads
to overgrowth of the more pathogenic microbiota and to a dysbiotic
microbial community.
• According to this, 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.

60. REFERENCES
• Carranza’s clinical periodontology, 12th edition
• Lindhe J, ed. Textbook of Clinical
Periodontology & implant dentistry, 6th ed.