plaque as a biofilm with references

2. PLAQUE AS A BIOFILM

3. INTRODUCTION
• The accumulation and metabolism of bacteria on hard oral surfaces is
considered the primary cause of dental caries, gingivitis, periodontitis
and peri-implant infections.
• In the context of the oral cavity, the bacterial deposits have been
termed dental plaque or bacterial plaque.
• In 1 mm3 of dental plaque weighing approximately 1mg, approx 1011
bacteria are present.
Socransky SS, Gibbons RJ, Dale AC et al. The microbiota of the gingival crevice area of man. I. Total microscopic and
viable counts of specific microorganisms. Arch Oral Biol 1953;8:275

4. • Dental plaque was the first biofilm to be studied in terms of
both its microbial composition and its sensitivity to
antimicrobial agents.
• In the 17th century, Antonie van Leeuwenhoek pioneered the
approach of studying biofilms by direct microscopic
observation when he reported on the diversity and high
numbers of animalcules present in scrapings taken from
around human teeth.
• Research over several decades has provided a solid foundation
for current studies of oral biofilms.
• Numerous cultural studies have reported the diversity of the
resident oral microflora, both at the genus and species level in
health and disease
HISTORY

5. AS A
PLAQUE
BIOFILM

6. PLAQUE
Bowen W.H. in the year 1976 defined dental plaque clinically as a structured,
resilient, yellow-grayish substance that adheres tenaciously to the intraoral
hard surfaces, including removable and fixed restorations.
According to WHO (1978)
Plaque is a specific but highly variable structural entity resulting from
colonization and growth of microorganisms on surfaces of teeth and
consisting of numerous microbial species and stains embedded in a
extracellular matrix.

7. PLAQUE
According to the Glossary of Periodontal Terms, 4th Edition
An organized mass, consisting mainly of microorganisms, that adheres to teeth, prostheses, and
oral surfaces and is found in the gingival crevice and periodontal pockets. Other components
include an organic, polysaccharide-protein matrix consisting of bacterial by-products such as
enzymes, food debris, desquamated cells, and inorganic components such as calcium and
phosphate.
According to Carranza, 11th Edition
Dental plaque is defined clinically as a structured, resilient yellow-grayish substance that adheres
tenaciously to the intraoral hard surfaces, including removable and fixed restorations.

8. BIOFILM
• The term biofilm describes the relatively undefinable microbial
community associated with a tooth surface or any other hard, non-
shedding material.
Wilderer & Charaklis 1989
• 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. Dental Biofilms: Difficult Therapeutic Targets
Periodontology 2000 2001;28:12–55.

9. • Biofilms are not simply organism-containing slime layers on
surfaces; instead, biofilms represent biological systems with a
high level of organization where bacteria form structured,
coordinated, functional communities.
• In essence, biofilms represent an interdependent community-
based existence.
• Mixed species biofilms may be regarded as primitive precursors
to the more complex organizations observed for eukaryotic
species.
O’Toole, G. A., H. Kaplan, and R. Kolter. 2000. Biofilm formation as microbial development. Annu.
Rev. Microbiol. 54:49–79

10. Biofilms may be found virtually anywhere...
DOCKS BOATS PIPES
STREAMS CATHETERS MOUTH

11. Based on its position on the tooth surface Dental
plaque is broadly classified as
Supragingival Plaque
Found at or above the gingival margin.
Demonstrates a stratified organization of
a multilayered accumulation of bacteria
Gram-positive cocci and short rods
predominate at the tooth surface,
Gram-negative rods and filaments, as
well as spirochetes, predominate in
the outer surface of the mature
plaque mass.
Subgingival Plaque
Found below the gingival margin,
between the tooth and the gingival pocket
epithelium.
The composition of the subgingival plaque
depends on the pocket depth.
The subgingival microbiota differs in
composition primarily because of the local
availability of blood products and an
anaerobic environment.
The apical part is more dominated by
spirochetes, cocci and rods, whereas in the
coronal part more filaments are observed.

12. PLAQUE HYPOTHESIS
Specific Plaque
Hypothesis Non Specific
Plaque Hypothesis

13. NON-SPECIFIC PLAQUE HYPOTHESIS
• A direct relationship was assumed to exist between the total number of
accumulated bacteria and the amplitude of the pathogenic effect.
• Individuals with extensive periodontal disease were either suspected of having a
weak resistance to bacterial plaque or were blamed for inadequate home care.
• Such a view of dental plaque as a biomass is referred to as the NON-SPECIFIC
PLAQUE HYPOTHESIS (Theilade 1986).
• The "non-specific plaque hypothesis" purports that many of the heterogeneous
mixture of organisms in plaque could play a role in disease, and that disease is a
result of the overall interaction of the plaque microflora with the host.
Marsh PD. Microbial ecology of dental plaque and its significance in health and disease.
Adv Dent Res 1994;8:263-71

14. SPECIFIC PLAQUE HYPOTHESIS
• The propensity of inflamed sites to undergo permanent tissue destruction was recognized
to be more specific in nature, because not all gingivitis lesions seemed invariably to
progress to periodontitis.
• Such a view of periodontitis being caused by specific pathogens is referred to as the
specific plaque hypothesis (Loesche 1979).
• The "specific plaque hypothesis“ proposes that, out of the diverse collection of
microorganisms that constitute the resident plaque microflora, only a very limited
number are actively involved in causing disease.
Problems - occasions when either disease is diagnosed in the apparent absence of the
putative pathogens, or when pathogens are present at sites with no evidence of disease.
Marsh PD. Microbial ecology of dental plaque and its significance in health and disease.
Adv Dent Res 1994;8:263-71

15. ECOLOGICAL PLAQUE HYPOTHESIS
• A modified hypothesis was proposed by Marsh (1991) in an attempt to unify
clinical and laboratory observations.
• In this hypothesis, it was proposed that a change in a key environmental
factor (or factors) will trigger a shift in the balance of the resident plaque
microflora, and this might predispose a site to disease.
Marsh PD. Microbial ecology of dental plaque and its significance in health and disease.
Adv Dent Res 1994;8:263-71

16. How does this solve our dilemma??
• Under the conditions that prevail in health, these organisms would be
only weakly competitive and may also be suppressed by inter-microbial
antagonism, so that they would comprise only a small percentage of the
plaque microflora and would not be significant clinically.
• Microbial specificity in disease would be due to the fact that only certain
species are competitive under the new (changed) environmental
conditions.

17. PLAQUE FORMATION

18. STAGES OF PLAQUE FORMATION
Formation of a
conditioning
film
Bacterial
Adhesion
Multiplication
Maturation

19. Formation of a conditioning film
Immersion of solid substratum into fluid media.
Macromolecules adsorb to the surface
Form a conditioning film – Acquired Pellicle
What does it do?
Conditioning film alters the charge and free energy of the surface
thus increasing the efficiency of bacterial adhesion.
The conditioning film alters the properties of the surface, and
bacteria interact directly with the constituent molecules.
Salivary pellicle can be detected on clean enamel surfaces within 1
minute.
By 2 hours, the pellicle is essentially in equilibrium.
Marsh PD, Moter A, Devine DA.
Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011;
55:16-35.
Electron micrographic illustration of a 4hr dental
pellicle. Brecx et al. (1981).

20. Bacterial adhesion
• According to Mergenhagen and Rosan (1985) the ability to adhere depends on a series
of interactions between:
• Surface to be colonized
• Microbe
• Ambient fluid milieu.
• Some bacteria have specific attachment structures which enable it to attach
rapidly such as:
• Extracellular Polymeric substance
• Fimbriae
• Other bacteria require prolonged
exposure to bind firmly.

21. Reversible adhesion
• Reversible adhesion involves weak, long-
range, physico-chemical interactions
between the charge on the microbial cell
surface and that produced by the
conditioning film.
• Irreversible adhesion involves
interactions between specific molecules
on the microbial cell surface (adhesins)
and complementary molecules
(receptors) present in the acquired
pellicle.
Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011; 55:16-35.

22. Multiplication
Active
Cellular
Growth of
Bacteria
Synthesis of
new outer
membrane
components
Increased
bacterial
mass

23. Multiplication
• Leads to an increase in biomass and
synthesis of exopolymers to form a
biofilm matrix.
• The matrix makes a significant
contribution to the structural integrity
and general tolerance of biofilms to
environmental factors (e.g. desiccation)
and antimicrobial agents.
• The matrix can be biologically active
and retain water, nutrients and
enzymes within the biofilm.
Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011;
55:16-35.

24. CO-ADHESION
• The primary colonizing bacteria adhered to the tooth surface provide new receptors
for attachment by other bacteria, in a process known as “co-adhesion.”
• Together with growth of adherent microorganisms, co-adhesion leads to the
development of microcolonies and eventually to a mature biofilm.
Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control.
Periodontol 2000 2011; 55:16-35.

25. CO-AGGREGATION
• Most human oral bacteria adhere to other oral bacteria. This cell-to-cell adherence
is known as coaggregation.
• Coaggregation is defined as the specific cell-to-cell recognition that occurs between
genetically distinct cell types.
• Gibbons and Nygaard (1970) discovered coaggregation among plaque bacteria and
called it interbacterial aggregation.
• The term coaggregation was coined to describe a clumping phenomenon that
occurred when sucrose-grown streptococci were paired with actinomyces and was
used to distinguish this intergeneric type of clumping from the dextran-mediated
intraspecies aggregation of actinomyces.
Kolenbrander PE, Palmer RJ Jr, Rickard AH, Jakubovics NS, Chalmers NI, Diaz PI.
Bacterial interactions and successions during plaque development. Periodontol 2000 2006;42:47-79.
Gibbons RJ, Nygaard M. Interbacterial aggregation of plaque bacteria. Arch Oral Biol 1970;15: 1397–400.
Bourgeau G, McBride BC. Dextran-mediated interbacterial aggregation between dextran-synthesizing streptococci and Actinomyces
viscosus. Infect Immun 1976: 13: 1228– 1234.

26. CO-AGGREGATION
• Different species, or even different strains of a single
species, have distinct sets of coaggregation partners
e.g.
• Streptococcus spp. And Actinomyces spp., two of the initial
colonizing genera on enamel surfaces.
• Fusobacteria coaggregate with all other human oral bacteria.
• Veillonella spp, Capnocytophaga spp. bind to streptococci
and/or actinomyces.
• Each coaggregation is mediated by one or more
complementary sets of adhesin–receptor pairs.

27. CO-AGGREGATION BRIDGES
• The basic coaggregation principle exhibited by
sequential coaggregation is the principle of
bridging.
• Each newly accreted cell becomes itself a new
surface and therefore may act as a co-
aggregation bridge to the next potentially
accreting cell type that passes by.
• A coaggregation bridge is formed when the
common partner bears two or more types of
coaggregation mediators.
• These mediators can be various types of
receptor polysaccharides, or various types of
adhesins, or a mixture of the two.

30. Maturation
• The close proximity of cells to one another facilitates the development of numerous synergistic and
antagonistic interactions between neighbouring species and food chains.
• The metabolism of the microorganisms produces gradients within the plaque; for example, in
nutrients and fermentation products.
• These gradients result in a mosaic of microenvironments.
• These processes lead to the establishment of a mature biofilm with a relatively stable composition
Marsh PD, Moter A, Devine DA.
Dental plaque biofilms: communities, conflic
t and control. Periodontol 2000 2011; 55:16-
35.

31. Detachment
• Bacteria are able to sense changes to their environment.
• If conditions deteriorate, some species (e.g. Aggregatibacter
actinomycetemcomitans) respond by upregulating enzymes that cleave their
adhesins, enabling the cell to detach and colonize elsewhere.
Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011;
55:16-35.

32. STRUCTURE
OF DENTAL
PLAQUE

33. SUPRAGINGIVAL PLAQUE
The first cellular material adhering to the pellicle on the tooth surface consists of coccoid
bacteria with numbers of epithelial cells and polymorphonuclear leukocytes
During the first few hours, bacteria that resist detachment from the pellicle start to
proliferate and form small colonies of morphologically similar organisms.
Since other types of organisms also proliferate in an adjacent region, the pellicle
becomes populated by a mixture of different microorganisms
Clumps of organisms of different species become attached to the tooth surface or to the already
attached microorganism, contributing to the complexity of the plaque composition after a few days
Another feature of older plaque is the presence of dead and lysed bacteria which may provide
additional nutrients to the still viable bacteria (Theilade & Theilade 1970).
The material present between the bacteria in dental plaque is called the intermicrobial matrix and
accounts for approximately 25% of the plaque volume.

34. SUBGINGIVAL PLAQUE
Between subgingival
plaque and the tooth an
electron-dense organic
material is interposed,
termed as CUTICLE.
This cuticle contains the
remains of the epithelial
attachment originally
connecting the junctional
epithelium to the tooth,
with the addition of
material deposited
fromthe gingival exudate
(Frank & Cimasoni 1970)
A densely packed
accumulation of
microorganisms is seen
adjacent to the cuticule
The bacteria comprise
Gram-positive and
Gram-negative cocci,
rods, and fliamentous
organisms.
Spirochetes and various
flagellated bacteria may
also be encountered,
especially at the apical
extension of the plaque
When a periodontal
pocket has formed
filamentous
microorganisms
dominate, but cocci and
rods also occur.
In the deeper parts of
the periodontal pocket,
the filamentous
organisms become
fewer in number, and in
the apical portion they
seem to be virtually
absent.
A characteristic feature
of subgingival plaque is
the presence of
leukocytes interposed
between the surfaces of
the bacterial deposit
and the gingival sulcular
epithelium

35. ULTRASTRUCTURE OF PLAQUE
• Listgarten described the ultrastructural characteristics of
mature plaque present on extracted teeth that were
associated with healthy periodontal tissues and various
degrees of periodontal disease.
• ‘Corn cob’ formations were occasionally seen as a feature of
plaque present on teeth associated with gingivitis.
• ‘Bristle-brush’ formations, composed of a central axis of a
filamentous bacterium with perpendicularly associated
short filaments, were commonly seen in the subgingival
plaque of teeth associated with periodontitis.
• It is evident that the close proximity of different bacterial
cell types allows the formation of microenvironments in
which cell–cell interactions easily occur.
Listgarten MA. Structure of the microbial flora associated with periodontal health and disease in man. A light and electron microscopic
study. J Periodontol 1976: 47: 1–18.

36. NATURE OF DENTAL PLAQUE –
THE BIOFILM WAY OF LIFE

37. • Biofilms consist of one or more communities of microorganisms, embedded
in a glycocalyx, that are attached to a solid surface.
• The biofilm allows microorganisms to stick to and multiply on surfaces.
• Thus, attached bacteria (sessile) growing in a biofilm display a wide range of
characteristics that provide a number of advantages over single cell
(planktonic) bacteria.
• The interactions among bacterial species living in biofilms take place at
several levels including physical contact, metabolic exchange, small signal
molecule mediated communication, and exchange of genetic information.

38. PROPERTIES OF BIOFILMS

39. STRUCTURE OF A BIOFILM
• Earlier electron microscopy was the method of choice to examine microbial biofilms.
Disadvantage: Sample preparation for electron microscopy resulted in dehydrated
samples leading to biofilm collapse.
• Nowadays, Confocal Scanning Laser Microscopes (CSLM) are used for the
visualization of fully hydrated samples, which revealed the elaborate three-
dimensional structure of biofilms.
Davey ME, OToole GA. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 2000: 64: 847–
867.

40. 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).
• Individual microcolonies can consist of a single species but more frequently are
composed of several different species.
• Structure of the Biofilm depends on environmental parameters under which
they are formed. These include:
• Surface and interface properties,
• Nutrient availability,
• Composition of the microbial community,
• Hydrodynamics
Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000
2001;28:12–55.

41. • For example, under high shear stresses, such as on the surface of teeth during chewing, the
biofilm is typically stratified and compacted.
• At low shear force, the colonies are shaped liked towers or mushrooms, while at high shear
force, the colonies are elongated.
• Biofilms grown under laminar flow were found to be patchy and consisted of rough round cell
aggregates separated by interstitial voids.
• Biofilms grown in the turbulent flow cells were also patchy, but elongated “streamers” that
oscillated in the bulk fluid were observed.
Davey ME, O Toole GA. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 2000: 64: 847–
867.

42. • As a surface becomes colonized with individual cells, the bacteria form
microcolonies, which then secrete a sticky extracellular polymeric
substance.
• Upon secretion of the extracellular polymeric substance, the biofilm
matures by becoming larger and taking on a distinctive architecture.
Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47.

43. • Earlier studies of thick biofilms (0.5 mm) that develop in sewage treatment plants indicated the
presence of voids or water channels between the microcolonies present in these biofilms.
• The water channels permit the passage of nutrients and other agents throughout the biofilm acting as
a primitive ‘‘circulatory’’ system.
• Nutrients make contact with the sessile (attached) microcolonies by diffusion from the water channel
to the microcolony rather than from the matrix.
• The interstitial voids or channels are, in essence, the lifeline of the system, since they provide a means
of circulating nutrients as well as exchanging metabolic products with the bulk fluid layer .
Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000
2001;28:12–55.

44. EXOPOLYSACCHARIDES
– the backbone of the biofilm
• The bulk of the biofilm consists of the
matrix or glycocalyx and is composed
predominantly of water and aqueous
solutes.
• The “dry” material is a mixture of
exopolysaccharides, proteins, salts,
and cell material.
• Exopolysaccharides (EPS), which are
produced by the bacteria in the
biofilm, are the major components of
the biofilm making up 50–95% of the
dry weight.

45. They play a major role in…..
maintaining the integrity of the biofilm
preventing desiccation and attack by harmful agents.
Binds essential nutrients to create a local nutritionally rich environment.
Acts as a buffer
Assists in retention of extracellular enzymes enhancing substrate utilization
by bacterial cells.

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

47. MICROBIAL INTERACTIONS
• In biofilms, microorganisms are in close physical proximity to one another and interact as
a consequence.
• Metabolic interactions can occur at many levels and include nutritional co-operation,
environmental modification through oxygen detoxification, and small-molecule signal-
mediated gene regulation.
• Many conventional metabolic interactions (synergistic and antagonistic) have been
described among oral bacteria, and the development of food chains or food webs is
common, in which the metabolic product of one organism becomes the primary nutrient
for a second.

48. • Bacteria collaborate in order to catabolize complex host molecules (proteins,
glycoproteins)
• Obligately anaerobic bacteria such as P. gingivalis can survive in aerobic
environments if they partner with and co-aggregate to oxygen-consuming
species such as Neisseria.
• Antagonistic interactions involve the production of inhibitory compounds
(bacteriocins, acids, H2O2, etc.) to inhibit neighbouring cells, and can provide the
producer cells with a competitive advantage.

49. QUORUM SENSING
• Some of the functions of biofilms are dependent on the ability of the
bacteria and microcolonies within the biofilm to communicate with
one another.
• Quorum sensing in bacteria “involves the regulation of expression of
specific genes through the accumulation of signaling compounds that
mediate intercellular communication” (Prosser 1999).

50. QUORUM SENSING
• In quorum sensing bacteria secrete
a signaling molecule that
accumulates in the local
environment and triggers a
response such as a change in the
expression of specific genes once
they reach a critical threshold
concentration.
• The threshold concentration is
reached only at a high-cell density,
and therefore bacteria sense that
the population has reached a
critical mass, or quorum.

51. QUORUM SENSING
• Two types of signalling molecules have been detected from dental plaque
bacteria:
• Peptides released by gram-positive organisms during growth
• A “universal” signal molecule autoinducer.
• Responses are induced only when a threshold concentration of the peptide
is attained, and thus the peptides act as cell density, or quorum, sensors.
• The streptococcal peptides are known as competence stimulating peptides.
Carranza’s Clinical Periodontology, 11th Edition

52. QUORUM SENSING
• In 2001, Schauder et al. proposed that a small molecule called autoinducer-
2 was a universal signal, mediating messages among the species in mixed
species communities.
• This is distinct from the regulation of gene expression mediated by
autoinducer- 1, a family of acyl homoserine lactones, which regulate gene
expression in genetically identical cells.
Kolenbrander PE, Palmer RJ Jr, Rickard AH, Jakubovics NS, Chalmers NI, Diaz PI.
Bacterial interactions and successions during plaque development. Periodontol 2000 2006;42:47-79
Schauder S, Shokat K, Surette MG, Bassler BL. The LuxS family of bacterial autoinducers: biosynthesis of a novel
quorum-sensing signal molecule. Mol Microbiol 2001: 41:463–476.

53. QUORUM SENSING
• The possible role of quorum sensing in influencing the properties of biofilms
was first suggested by Cooper et al.
• Quorum sensing also has the potential to influence community structure by
encouraging the growth of beneficial species (to the biofilm) and
discouraging the growth of competitors.
• Quorum sensing therefore appears to play diverse roles:
• Modulating the expression of genes for antibiotic resistance,
• Encouraging the growth of beneficial species to the biofilm,
• Discouraging the growth of competitors.
Carranza’s Clinical Periodontology, 11th Edition

54. EXCHANGE OF GENETIC INFORMATION
• Signaling is not the only way of
transferring information in biofilms.
• The high density of bacterial cells
growing in biofilms facilitates exchange
of genetic information between cells of
the same species and across species or
even genera.
• Conjugation, transformation, plasmid
transfer, and transposon transfer have
all been shown to occur in naturally
occurring mixed species biofilms.
Clinical Periodontology and Implant Dentistry by Jan Lindhe, 5th Edition.

55. • Cells also communicate and interact with one another in biofilms via horizontal gene
transfer.
• The transfer of conjugative transposons encoding tetracycline resistance between
streptococci has been demonstrated in model biofilms
• Gene transfer between Treponema denticola and S. gordonii has also been
demonstrated in the laboratory.
• The presence of “pathogenicity islands” in periodontal pathogens such as P.
gingivalis is also indirect evidence for horizontal gene transfer having occurred in
plaque biofilms at some distant time in the past, and may explain the evolution of
more virulent strains.
Chen T, Hosogi Y, Nishikawa K, Abbey K, Fleischmann RD, Walling J, Duncan MJ. Comparative whole-genome analysis
of virulent and avirulent strains of Porphyromonas gingivalis. J Bacteriol 2004: 186: 5473–5479.
Wang BY, Chi B, Kuramitsu HK. Genetic exchange between Treponema denticola and Streptococcus gordonii in biofilms. Oral
Microbiol Immunol 2002: 17: 108– 112.
Roberts AP, Cheah G, Ready D, Pratten J, Wilson M, Mullany P. Transfer of TN916-like elements in microcosm dental
plaques. Antimicrob Agents Chemother 2001: 45: 2943–2946

56. ATTACHMENT OF BACTERIA
• The key characteristic of a biofilm is that the microcolonies within the biofilm attach to a solid
surface.
• Many bacterial species possess surface structures such as fimbriae and fibrils that aid in their
attachment to different surfaces.
• Fimbriae have been detected on a number of oral species including P. gingivalis, A.
actinomycetemcomitans and some strains of streptococci.
• Oral species that possess fibrils include S. salivarius, the S. mitis group, Pr. intermedia, Pr.
nigrescens, and Streptococcus mutans.
Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000 2001;28:12–55.

57. Both physical and chemical factors affect the
attachment of biofilms to a surface…..
PHYSICAL PROPERTIES
• Roughness of the surface
• Increases surface area and hence
increases colonization.
• Roughness also provides
protection from shear forces and
increases the difficulty of
cleaning.
CHEMICAL COMPOSITION
• Since it may contain beneficial or
detrimental components.
• For example, metals such as brass
(an alloy of copper and zinc) have
antimicrobial properties due to
dezincification and the
antimicrobial properties of copper.
• Polyvinyl chloride on the other
hand, contains carbon, hydrogen
and chloride, which encourage
bacterial growth.

58. INCREASED ANTIBIOTIC RESISTANCE
• Organisms growing in biofilms are more resistant
to antibiotics than the same species growing in a
planktonic (unattached) state.
• Estimates of 1000–1500 times greater resistance
for biofilm-grown cells than planktonic grown cells
have been suggested.
• The mechanisms of this increased resistance differ
from species to species, from antibiotic to
antibiotic, and for biofilm growing in different
habitats.
Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000
2001;28:12–55.

59. MECHANISMS OF ANTIBIOTIC RESISTANCE
Recently, the notion of a subpopulation
of cells within a biofilm that are ‘‘super-
resistant’’ was proposed. Such cells could
explain remarkably elevated levels of
resistance to certain antibiotics that have
been suggested in the literature
Slower rate of growth of bacterial
species in a biofilm, which makes
them less susceptible to many but not
all antibiotics.
The exopolymer of the biofilm matrix,
although not a significant physical
barrier to the diffusion of antibiotics,
does have certain properties that can
retard antibiotic penetration.
Hydrodynamics and the turnover rate
of the microcolonies has an impact on
antibiotic effectiveness.
Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000 2001;28:12–55.

60. ADVANTAGES OF BIOFILM
They are the preferred method of growth for many and perhaps most species
of bacteria .
ESTABLISHMENTOFSYNTROPHIC
RELATIONSHIPS
FACILITATES
ACQUISITIONOFNEW
GENETICTRAITS
PROTECTSAGAINST
Host defense
mechanisms,
Potentially
toxic
substances in
the
environment,
such as lethal
chemicals or
antibiotics.
Processing and
uptake of nutrients,
Cross-feeding (one
species providing
nutrients for
another),
Removal of
potentially harmful
metabolic products
(often by utilization
by other bacteria)
Development of an
appropriate
physicochemical
environment (such
as a properly
reduced oxidation
reduction potential)
An effective
means of
exchanging
nutrients and
metabolites thus
enhancing
nutrient
availability.
opportunity for
metabolic
cooperation in
niches formed
within these
systems
facilitates inter-
species substrate
exchange
Most bacteria in
natural settings
reside within
biofilms, it
follows that
conjugation is a
likely mechanism
by which bacteria
in biofilms
transfer genes
within or
between
populations

61. ORAL BIOFILMS IN HEALTH AND DISEASE

62. BIOFILMS ASSOCIATED WITH ORAL HEALTH
• In the case of the oral cavity, attempts to characterize the normal microbial flora have met
with challenges:
Over 700 species have been detected in the oral
cavity, over half of which have never been
cultivated.
There is substantial diversity in the content of the
microflora between individuals and between
different oral sites within the same individual.
Dietary changes combined with poor hygiene can
cause a shift in the composition of the oral
microflora.
The oral microbiome changes as humans age.
Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47.

63. • Such variations make it difficult to identify a typical oral microbiome for a healthy
individual.
• While agreement on exactly which species could be used as markers of oral health is
yet to be achieved, an overall picture of what types of bacteria are commonly found
in healthy individuals is starting to emerge.
• A positive association has been observed between oral health and the presence of
• Veillonella
• Capnocytophaga ochracea
• Streptococcus salivarius
• Streptococcus mitis,
• Gemella haemolysans
• Granulicatella adiacens
Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47.

64. BIOFILMS IN ORAL DISEASE
• Just as entire microbial communities can be associated with health,
current research also indicates that entire microbial communities can be
associated with disease.
• The transition to gingivitis is evident by inflammatory changes and is
accompanied first by the appearance of gram-negative rods and
filaments, then by spirochetal and motile microorganisms.

65. • The initial microbiota of experimental gingivitis consists of gram-positive rods,
gram-positive cocci, and gram-negative cocci.
• In chronic periodontitis, the bacteria most often detected at high levels include
P. gingivalis, T. forsythia, P. intermedia, P. nigrescens, C. rectus, Eikenella
corrodens, F. nucleatum, A. actinomycetemcomitans (often serotype b), P. micra,
E. nodatum, Leptotrichia buccalis, Treponema (T. denticola), Selenomonas spp. (S.
noxia), and Enteric spp.

66. MICROBIAL COMPLEXES
• The association of bacteria within mixed biofilms is not random,
rather there are specific associations among bacterial species.
• Socransky et al. (1998) demonstrated the presence of specific
microbial groups within dental plaque.
• Six closely associated groups of bacterial species were recognized.

67. Socransky SS, Haffajee AD. Periodontal Microbial Ecology Periodontology 2000 2005;38:135–87

68. • Initial colonization appears to involve members of the yellow, green, and purple
complexes along with Actinomyces species.
• These groups of species are early colonizers of the tooth surface, and their growth usually
precedes the multiplication of the predominantly gram negative orange and red
complexes
• As the disease progresses, members of the orange and then red complexes become more
dominant.
• Certain complexes are observed together more frequently than others in subgingival
plaque.
• For example, it is extremely unlikely to find red complex species in the absence of
members of the orange complex.
• In contrast, members of the Actinomyces, yellow, green and purple complexes are often
observed without members of the red complex or even the red and orange complexes.

69. • The lack of a beneficial organism in a biofilm may be just as
important as the presence of a pathogen in the contribution
to disease.
• Because of these revelations, a hypothesis has been
developed linking certain diseases to a shift in membership
of the local microbiota.
Socransky SS, Haffajee AD. 2005

70. THE MICROBIAL SHIFT HYPOTHESIS
• Also known as dysbiosis.
• It refers to the concept that some diseases
are due to a decrease in the number of
beneficial symbionts and ⁄ or an increase
in the number of pathogens.
• Dysbiosis in the oral cavity can lead to
periodontitis.
• The long-standing paradigm is that, as
periodontitis develops, the oral microbiota
shifts from one consisting primarily of
gram-positive aerobes to one consisting
primarily of gram-negative anaerobes.
Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47.

71. CLINICAL CONSIDERATIONS OF BIOFILMS – Extension to the
Dental Environment

72. MICROBIAL CONTAMINATION OF DENTAL
UNIT WATERLINES
• The ability of bacteria to colonize surfaces and to form biofilm in water supply
tubes, including DUWL, is a common phenomenon.
• Microorganisms from contaminated DUWL are transmitted with aerosol and
splatter.
• This increases the risk for cross-infection in clinical practice, especially in view of
the ever-increasing number of immunocompromised persons.
Szymańska J, Sitkowska J, Dutkiewicz J. Microbial contamination of dental unit waterlines.
Ann Agric Environ Med 2008;15:173-9

73. Szymańska J, Sitkowska J, Dutkiewicz J. Microbial contamination of dental unit waterlines.
Ann Agric Environ Med 2008;15:173-9

74. • A wide range of chemical disinfectants have been used in DUWLs:
• Hydrogen peroxide,
• Hydrogen peroxide with silver ions,
• Chlorine dioxide,
• Peracetic acid,
• Sodium hypochlorite
• Chlorhexidine gluconate
• Liaqat and Sabri (2009) found that, overall, combination of chlorhexidine
with povidone iodine was very effective in eliminating/reducing the biofilm
bacteria at 1000 μg/mL as compared to other combinations.
Dallolio L, Scuderi A , Rini MS,Valente S,Farruggia P, Sabattini et al
Int. J. Environ. Res. Public Health 2014, 11, 2064-76.
Liaqat I, Sabri AN. In vitro efficacy of biocides against dental unit water line (DUWL) biofilm bacteria.
Asian J Exp Sci. 2009;1:67–75

75. FIGHTING ORAL BIOFILMS: ADJUNCTIVE
TREATMENTS FOR PERIODONTITIS

76. PHYSICAL REMOVAL OF MICROORGANISMS
– Mechanical Debridement
• Fortunately, biofilms in the oral cavity, unlike many other biofilms,
are readily accessible allowing their physical removal.
• Indeed, the most common form of periodontal therapy is the
removal of supra and subgingival plaque by procedures such as self
performed oral hygiene, scaling and root planing or periodontal
surgery.
Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets
Periodontology 2000 2001;28:12–55.

77. CALCULUS REMOVAL AND PREVENTION
OF ITS FORMATION
• Calculus in itself appears to be non-pathogenic, but, due to its rough surface,
harbors oral biofilms associated with oral diseases.
• Thus, prevention and removal of supra- and subgingival calculus are critical in the
management of periodontal diseases.
• Complete removal of subgingival calculus in patients with periodontitis remains an
elusive goal.
• Hand instruments, sonic scalers, ultrasonic scalers and Er:YAG lasers have been used
for removal of supra- and subgingival calculus.
Flemmig TF, Beikler T. Control of oral biofilms. Periodontol 2000 2011;55:9-15.

78. • Scaling and root planing is the primary therapy of choice for most clinicians, and it is
widely considered the gold standard for treating periodontitis.
• Because of the underlying microbial basis of periodontitis, it is becoming more
conventional to use antimicrobial therapy as an adjunct to scaling and root planning.
• Additionally, because the host inflammatory response also plays a major role in
disease progression, treatments aimed at suppressing inflammation can be used.

79. ANTIBIOTICS
• Often used as an adjunct to scaling and root planing,
and they can be applied locally or administered
systemically.
• Meta-analysis confirmed that minocycline and
tetracycline were the most effective local adjunctive
therapies when measured in terms of probing depth
reduction and clinical attachment level gain.
• Antibiotics yield only modest results clinically.
• It must be kept in mind that periodontitis is a biofilm-
associated disease, and biofilms are notoriously
difficult to treat with antibiotics.
Bonito AJ, Lux L, Lohr KN. Impact of local adjuncts to scaling and root planing in
periodontal disease therapy:a systematic review.
J Periodontol 2005: 76: 1227–36.

80. ANTISEPTICS
• Antiseptics such as chlorhexidine,
bleach, povidone-iodine can be used
as an alternative to antibiotics.
• An in vitro biofilm study showed that
P. gingivalis was completely
eradicated after 30 minutes of
exposure to chlorhexidine, povidone-
iodine or Listerine.
Bercy P, Lasserre J. 2007

81. HOST MODULATION THERAPY
• Aims to modulate the host by suppressing the inflammatory response.
• Matrix metalloproteinases, mediate tissue destruction by
degrading plasma membrane proteins and extracellular matrix proteins such
as collagen
significantly contribute to the tissue destruction and alveolar bone loss.
• Members of the tetracycline family of antibiotics possess the ability to
inhibit matrix metalloproteinases.
• A meta-analysis subsequently determined that adjunctive administration of
sub-antimicrobial doses of doxycycline conferred a statistically significant
benefit over scaling and root planing alone.
Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47.
Reddy MS, Geurs NC, Gunsolley JC. Periodontal host modulation with antiproteinase, anti-inflammatory, and bone-
sparing agents. A systematic review. Ann Periodontol 2003: 8: 12–37.

82. PHOTODYNAMIC THERAPY
• This technique uses long-wavelength visible light (red light) to activate
photosensitizing agents (photosensitizers) that produce reactive oxygen species,
such as free radicals and singlet oxygen.
• These toxic oxygen derivatives then react with essential cellular components such as
DNA, proteins and lipids, leading to cell death.
• An in vitro study showed that antimicrobial photodynamic therapy
• destroys P. gingivalis,
• inactivates a virulence-associated protease
• the destructive host inflammatory mediators tumor necrosis factor- and
interleukin-1 β
Konopka K, Goslinski T. 2007

83. • Because this treatment appears to simultaneously destroy bacterial
pathogens and suppress the destructive host inflammatory response, there
has been a strong interest in the therapeutic potential of antimicrobial
photodynamic therapy.
• With this technology, researchers have been able to successfully
characterize the microflora associated with health and disease in
subgingival and supragingival plaque.
• More importantly, this technique has been used to determine the nature of
the microbial biofilm before and after treatment of periodontitis.
Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. 1998
Haffajee AD, Socransky SS, Patel MR, Song X 2008

84. ASSESSING THE EFFICACY OF TREATMENT
• Essentially, the parameters used to assess the efficacy of treatment fall within
two broad categories: biological and clinical.
• Biologically, it is important to determine whether treatment altered the content
of the microflora and helped to resolve the host inflammatory response.
• The clinical parameters typically measured – probing depth reduction, clinical
attachment level gain, reduction of bleeding on probing, and prevention of tooth
loss
Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47.

85. CONCLUSION
• Dental biofilm has all of the properties of biofilms in other habitats in nature.
• It has a solid substratum, it has the mixed microcolonies growing in a glycocalyx, and
it has the bulk fluid interface provided by saliva.
• The interactions between bacterial species in a biofilm and between bacterial
species and the nonbacterial habitat are dynamic.
• They reflect a back and forth interplay between host and colonizing species
• Understanding of the ecologic relationships within intraoral biofilms and between
biofilm composition and the host has been slowed by the difficulty in obtaining
sufficient ‘snapshots’ of the microbial composition of biofilms taken from carefully
monitored clinical situations.

86. • A greater understanding of the significance of dental plaque as a mixed
species biofilm will have the potential to impact significantly on clinical
practice.
• When assessing treatment options, an appreciation of the ecology of the
oral cavity will enable the enlightened clinician to take a more holistic
approach and consider the nutrition, physiology, host defenses, and
general well-being of the patient, as these will affect the balance and
activity of the resident oral microflora.

87. REFERENCES
• Carranza’s Clinical Periodontology, 11th Edition
• Clinical Periodontology and Implant Dentistry by Jan Lindhe, 5th Edition.
• Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets
Periodontology 2000 2001;28:12–55.
• Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–
47.
• Costerton, J. W. 1995. Overview of microbial biofilms. J. Ind. Microbiol. 15:137–140.
• Davey ME, O Toole GA. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol
Biol Rev 2000: 64: 847– 867.
• Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control.
Periodontol 2000 2011; 55:16-35.
• Roberts AP, Cheah G, Ready D, Pratten J, Wilson M, Mullany P. Transfer of TN916-like elements
in microcosm dental plaques. Antimicrob Agents Chemother 2001: 45: 2943–2946.
• Wang BY, Chi B, Kuramitsu HK. Genetic exchange between Treponema denticola and
Streptococcus gordonii in biofilms. Oral Microbiol Immunol 2002: 17: 108– 112.
• Socransky SS, Haffajee AD. Periodontal Microbial Ecology Periodontology 2000 2005;38:135–87

88. REFERENCES
• Chen T, Hosogi Y, Nishikawa K, Abbey K, Fleischmann RD, Walling J, Duncan MJ. Comparative
whole-genome analysis f virulent and avirulent strains of Porphyromonas gingivalis. J Bacteriol
2004: 186: 5473–5479.
• Kolenbrander PE, Palmer RJ Jr, Rickard AH, Jakubovics NS, Chalmers NI, Diaz PI.
Bacterial interactions and successions during plaque development. Periodontol
2000 2006;42:47-79.
• Marsh PD. Microbial ecology of dental plaque and its significance in health and disease. Adv
Dent Res 1994;8:263-71.
• Gibbons RJ, Nygaard M. Interbacterial aggregation of plaque bacteria. Arch Oral Biol 1970;15:
1397–400.
• Listgarten MA. Structure of the microbial flora associated with periodontal health and disease
in man. A light and electron microscopic study. J Periodontol 1976: 47: 1–18.
• Szymańska J, Sitkowska J, Dutkiewicz J. Microbial contamination of dental unit waterlines. Ann
Agric Environ Med 2008;15:173-9.
• Schauder S, Shokat K, Surette MG, Bassler BL. The LuxS family of bacterial autoinducers:
biosynthesis of a novel quorum-sensing signal molecule. Mol Microbiol 2001: 41: 463–476.
• Reddy MS, Geurs NC, Gunsolley JC. Periodontal host modulation with antiproteinase, anti-
inflammatory, and bone-sparing agents. A systematic review. Ann Periodontol 2003: 8: 12–37.