Biofilm

Biofilm in Endodontics – Dr. Nithin Mathew

In Endodontics
Dr. Nithin Mathew

• Introduction & Definition
• Ultra-Structure
• Characteristics
• Development of Biofilm
• Protection of Biofilm Bacteria from
Environmental Threats
• Nutrient Trapping And Metabolic
Cooperativity In A Biofilm
• Exchange of Genetic Material
• Quorum Sensing
CONTENTS
3
• Resistance of Microbes in the Biofilm to
Antimicrobials
• Resistance Of Microbes
• Benefits Of Biofilm To Microbes
• Oral Biofilm
• Endodontic Biofilm
• Conclusion
• References

• Biofilm is a mode of microbial growth where dynamic communities of interacting sessile cells
are irreversibly attached to a solid substratum, as well as each other, and are embedded in a
self made matrix of extracellular polymeric substance (EPS). - Ingle
• Biofilm is defined as a community of microcolonies of microorganisms in an aqueous solution
that is surrounded by a matrix of glycocalyx, which also attaches the bacterial cells to a solid
substratum. - Grossman
DEFINITION
4

• Biofilms are likely to represent a natural scenario for bacterial communication.
(Davey and O’Toole, 2000)
• A microbial biofilm is considered a community that meets the following four basic criteria:
1. Must possess the abilities to self-organize (Autopoiesis)
2. Resist environmental perturbations (Homeostasis)
3. Must be more effective in association than in isolation (Synergy)
4. Respond to environmental changes as a unit rather than single individuals
(Communality).
5

• Biofilms are characterized by
• Surface attachment
• Extracellular matrix of polymeric substances
• Structural heterogenicity
• Genetic diversity
• Complex community interactions
6

• Biofilms grow virtually everywhere, in almost any environment where there is a combination
of :
• Moisture
• Nutrient supply
• Surface
• Sites include :
• Natural materials above and below ground
• Metals & plastics
• Medical implant materials
• Plant and body tissue
7
Staphylococcus aureus biofilm
on the surface of a catheter

• Composed primarily of microbial cells and glycocalyx like matrix (Extracellular polymeric
substance)
• Fully developed biofilm is described as heterogeneous arrangement of microbial cells on a solid
surface.
• Basic structural unit of a biofilm is the microcolonies or cell cluster formed by the surface
adherent bacterial cells.
• 85% : - Matrix (polysaccharides, proteins, nucleic acid & salts)
• 15% : - Bacterial cells
ULTRA STRUCTURE OF BIOFILM
8

• Viable, fully hydrated biofilm appears as ‘tower’ or ‘mushroom’ shaped
structure adherent to the substrate .
• Water channels : regarded as circulatory system in a biofilm : establish
connection between microcolonies.
• Facilitates efficient exchange of materials between bacterial
cells and bulk fluid :
• Coordinate functions in a biofilm community.
9

10

Matrix
• A glycocalyx matrix made up of extracellular polymeric substance (EPS)
surrounds the microcolonies and anchors the bacterial cell to the substrate.
• Functions:
• Maintains the integrity of biofilms
• Prevents dessication
• Resists antimicrobial agents
• Create a nutritionally rich environment
• Acts as a buffer and retains extracellular enzymes
11

• Slow bacterial growth enhance EPS production because EPS is highly hydrated,
it prevents desiccation in some natural biofilms.
• By maintaining a highly hydrated layer surrounding the biofilm, the EPS will
prevent lethal desiccation in some natural biofilms and may thus protect
against diurnal variations in humidity.
(J Microbiology 2001)
• EPS : mechanical stability of the biofilms, enabling them to withstand
considerable shear forces.
(Mayer et al. 1999)
12

• Studies : different organisms produce differing amounts of EPS and that the
amount of EPS increases with age of the biofilm.
• EPS production is known to be affected by
• Nutrient status of the growth medium
• Excess available carbon and limitation of nitrogen, potassium, or
phosphate promote EPS synthesis
• EPS may associate with metal ions, divalent cations, other macromolecules
(such as proteins, DNA, lipids)
13

• Bacterial cells within matrix produce Beta-lactamase against
antibiotics
• Also produce catalases and superoxide dismutase against oxidising ions
of phagocytes
• Elastases and cellulases produced by bacteria are concentrated in the
matrix and produce tissue damage.
14

• Bacteria in the biofilm : unique capacity to survive tough growth and environmental conditions.
• Biofilm structure protects the residing bacteria from environmental threats.
• Structure of biofilm permits trapping of nutrients and metabolic cooperativity between
resident cells of the same species and or different species.
• Biofilm structure displays organized internal compartmentalization.
• Bacterial cells in a biofilm community may communicate and exchange genetic material to
acquire new traits.
CHARACTERISTICS OF BIOFILM
15

• Bacteria can form biofilm on any surface that is bathed in a nutrition containing fluid.
• 3 major components involved in biofilm formation
• Bacterial cells
• A solid surface
• A fluid medium
• Influenced by the physicochemical properties of the components involved in the biofilm.
DEVELOPMENT OF BIOFILM
16

• Adsorption of inorganic and organic molecules to the solid surface, forming a thin layer termed
as conditioning layer.
• During dental plaque formation, the tooth surface is conditioned by the saliva pellicle.
STAGE 1
17

• Adhesion of microbial cells to this layer.
• Phase 1 - Transport of microbes to surface.
• Phase 2 - Initial non specific microbial-substrate adherence phase.
• Phase 3 - Specific microbial-substrate adherence phase.
• Pioneer organisms : streptococci (major population)
STAGE 2
18

• Several factors that affect bacterial attachment to a solid substrate.
• ph
• Temperature
• Surface energy of the substrate
• Flow rate of the fluid passing over the surface
• Nutrient availability
• Length of time the bacteria is in contact with the surface
• Bacterial growth stage
• Bacterial cell surface charge
• Surface hydrophobicity.
STAGE 2 : Phase 1 : Transport of microbes to surface
19

• Physicochemical properties such as surface energy and charge density determine the nature of
initial bacteria-substrate interaction
• Microbial adherence to a substrate is also mediated by bacterial surface structures such as
fimbriae, pili, flagella, and EPS (glycocalyx).
• Bacterial surface structures form bridges between the bacteria and the conditioning film.
STAGE 2 : Phase 1 : Transport of microbes to surface
20

• Bridges formed are a combination of
• Electrostatic attraction
• Covalent and hydrogen bonding
• Dipole interaction
• Hydrophobic interaction
• Bacteria with surface structures:
• P. gingivalis, S. mitis, Streptococcus salivarius, P. intermedia, P. nigrescens, S.
mutans and A. naeslundii
STAGE 2 : Phase 2 : Initial non specific microbial-substrate adherence phase
21

• Initial bonds : not strong
• With time these bonds gains in strength, making the bacteria-substrate attachment
irreversible.
• Finally,a specific bacterial adhesion with a substrate is produced via polysaccharide adhesin or
ligand formation.
• Adhesin or ligand on the bacterial cell surface will bind to receptors on the substrate.
• Specific bacterial adhesion is less affected by many environmental factors such as electrolyte,
pH, or temperature.
STAGE 2 : Phase 3 : Specific microbial-substrate adherence phase
22

• Bacterial growth and expansion.
• Monolayer of microbes attracts secondary colonizers forming microcolony, and the collection
of microcolonies gives rise to the final structure of biofilm.
• The lateral and vertical growth of indwellers gives rise to microcolonies similar to towers.
STAGE 3
23

• A mature biofilm will be a metabolically active community of microorganisms where
individuals share duties and benefits.
• Some microorganisms help in adhering to the solid support, while some others
create bridges between different species
• The bacterial cells in a matured biofilm will exhibit considerable variation in its genetic and
biochemical constitutions compared to its planktonic counterparts.
STAGE 3
24

• Two types of microbial interactions occur at the cellular
level
• Co-adhesion
• The process of recognition between a suspended
cell and a cell already attached to substratum.
• Co-Aggregation
• Genetically distinct cells in suspension recognize
each other and clump together.
25

26

• During detachment, the biofilm transfer particulate constituents from the biofilm to the fluid
bathing the biofilm.
• Detachment plays an important role in shaping the morphological characteristics and structure
of mature biofilm.
• Also considered as an active dispersive mechanism
• Aka Seeding Dispersal
STAGE 4 : Detachment / Dispersion
27

• Because of flow effects, biofilm cells may be dispersed either by
• Shedding of daughter cells from actively growing cells
• Detachment as a result of nutrient levels
• Quorum sensing
• Shearing of biofilm aggregates
28

• Brading et al have emphasized the importance of physical forces in detachment, stating that the
three main processes for detachment are
• Erosion or shearing (continuous removal of small portions of the biofilm)
• Sloughing (rapid and massive removal), and
• Abrasion (detachment due to collision of particles from the bulk fluid with the biofilm)
( JADA 1996 )
29

30

31

• Environmental niches that supports the physiological requirement of different bacterial
species in a biofilm.
• A mature biofilm structure displays gradients in the distribution of nutrition, pH, oxygen,
metabolic products and signaling molecules within the biofilm.
• This would create different microniches that can accommodate diverse bacterial species within
a biofilm.
Internal Compartmentalization
32

• Bacteria residing in a biofilm experiences certain degree of protection and homeostasis.
• Bacteria are capable of producing polysaccharides, either as cell surface structures (eg.
Capsules) or as extracellular excretions (eg. EPS).
• EPS creates microniche.
• Protects from environmental stresses.
• Sequestration of metallic cations and toxins.
• Physically prevents diffusion of certain compounds by acting as ion exchanger.
Protection of Biofilm Bacteria from Environmental Threats
33

• An important characteristics of biofilm growing in a nutrient deprived ecosystem is its ability
to concentrate trace elements and nutrients by physical trapping or by electrostatic interaction.
• Highly permeable and interconnected water channels provide an excellent means of material
exchange.
• The complex architecture of biofilm provides opportunity for metabolic cooperation and niches
are formed within these spatially well-organized systems.
Nutrient Trapping And Metabolic Cooperativity In A Biofilm
34

• Juxta positioning of various microorganism provides cross feeding and metabolic cooperativity
between different species.
• Production of growth factors
• Production of different arrays of lytic enzymes
Nutrient Trapping And Metabolic Cooperativity In A Biofilm
35

• Bacterial biofilm provides a setting for the residing bacterial cells to communicate with each
other.
• The ability to communicate through quorum-sensing has been shown in some oral streptococci
and some periodontal pathogens. (Loo et al. 2000)
EXCHANGE OF GENETIC MATERIAL
36
• For most oral biofilm micro-organisms,
• Presence and function of signal transduction pathways
• Quorum-sensing communication

• Some of these signals produced by cells may also be interpreted by cells of different species by
a process called quorum sensing.
• Quorum sensing is mediated by low molecular weight molecules which in sufficient
concentration can alter metabolic activity of neighboring cells and coordinates in the function
of resident bacterial cells within a biofilm.
37

• Quorum sensing
• Involves the regulation of expression of specific genes through the accumulation of
signaling compounds that mediate intercellular communication
• Dependent on cell density and mediated through signaling compounds.
38
• The signals are thought to allow cross-talk between
species, causing them to increase their production of
exopolysaccharide and the factors that increase their
virulence.

• Quorum sensing gives biofilms their distinct properties.
• Exchange of genetic material between bacterial species
• Evolution of microbial communities with different traits
39

• Quorum sensing is involved in the regulation of
• Genetic competence
• Mating
• Bacteriocin production
• Sporulation
• Stress responses
• Virulence expression
• Biofilm formation
• Bioluminescence
40

AUTOINDUCER-2
• Autoinducer-2 is an universal signal that may foster the growth of certain species in a mixed
species community
• LuxS genes encode for AI2 and have been detected in several genera of Gm+ and Gm- bacteria

AUTOINDUCER-2
A clean tooth surface, with the associated acquired pellicle, is colonized by commensal species
The commensal cells produce picomolar amounts of autoinducer-2, and this fosters mutualistic
interdigitated growth leading to rapid expansion

AUTOINDUCER-2
As commensal cell numbers and diversity increase, the likelihood of pathogens integrating into the
biofilm increases; integration is through Coaggregation
The pathogens produce higher concentrations of autoinducer-2 than the commensals, and this high
concentration retards mutualistic growth between the commensal species which are unable to compete
with the invading, rapidly multiplying pathogens

AUTOINDUCER-2
• Commensal oral bacteria respond to low levels of AI2 whereas periodonto-pathogenic bacteria
responds to higher levels.

• The nature of biofilm structure and physiological characteristics of resident microorganisms
offer an inherent resistance to antimicrobial agents, such as antibiotics, disinfectants, or
germicides.
• Mechanism responsible for resistance:
1. Resistance associated with extracellular polymeric matrix.
2. Resistance associated with growth rate and nutrient availability.
3. Resistance associated with adoption of resistance phenotype.
Resistance of Microbes in the Biofilm to Antimicrobials
45

• Resistance associated with EPS :
• Diffusion barrier
• Direct neutralization of antimicrobials
• Inactivation by modified enzymes produced by bacteria.
46

• Resistance associated with growth rate and nutrient availability :
• Susceptibility towards antimicrobial is directly proportional to growth rate.
• Resistance increases as thickness of biofilm increases
47

• Resistance associated with adoption of resistant phenotype :
• Up regulation of EPS production
• Nutrient limitation
• Sub lethal dose
•Diminished bacterial growth rate
•Increase responsiveness of stress response genes
• Activation of multi drug efflux pump
Act as inducers/transcriptional activators of more tolerant phenotypes : multi-drug resistance.

• Helps the bacteria to survive in unfavorable environmental and nutritional conditions.
• Resistance to antimicrobial agents.
• Increase in local concentration of nutrients.
• Opportunity of genetic material exchange.
• Ability to communicate between bacterial population of same and or different species.
• Produce growth factors across species boundaries.
Benefits Of Biofilm To Microbes
49

• Oral bacteria have the capacity to form biofilms on distinct surfaces ranging from hard to soft
tissues.
• Oral biofilms are formed in three basic steps :
• Pellicle formation
• Bacterial colonization
• Biofilm maturation
• Composition :
• 80% : Water
• 20% : Inorganic & organic (80% bacteria)
ORAL BIOFILMS
50

• Organic : Carbohydrates, proteins, and lipids.
• Carbohydrates : produced by bacteria : glucans, fructans, or levans.
• Contribute to the adherence of microorganisms to each other and are the stored
form of energy in biofilm bacteria.
• Proteins (supragingival biofilm) : derived from saliva
• Proteins (subgingival biofilm) : derived from gingival sulcular fluid.
• Lipid : Endotoxins (LPS) from Gram-negative bacteria.
• Inorganic : Calcium, phosphorus, magnesium and fluoride.
51

• Saliva contains proline-rich proteins : aggregate together to form micelle like globules called
salivary micelle-like globules (SMGs).
• SMGs : adsorbed to the clean tooth surface to form acquired enamel pellicle, which acts as a
‘‘foundation’’ for the future multilayered biofilm.
• Presence of calcium facilitates the formation of larger globules by bridging.
52

• Acquired Pellicle attracts Gram-positive cocci (S. mutans and S. sanguis : pioneer organisms)
• Filamentous bacterium (F. nucleatum and slender rods) adheres to primary colonizers.
53
• Gradually, the filamentous form grows into the
cocci layer and replaces many of the cocci.
• Vibrios and spirochetes appear as the biofilm
thickens.

• Coaggregation of F. nucleatum with coccoid bacteria gives rise to “CORNCOB” structure, which
is unique in plaque biofilms.
54
• Presence of these bacteria makes it possible for other non-aggregating
bacteria to coexist in the biofilm, by acting as coaggregating bridges.
• The existence of anaerobic bacteria in an aerobic environment is made
possible by the coexistence of aerobic and anaerobic bacteria.

• Calcified dental biofilm is termed as Calculus.
• Formed by the precipitation of calcium phosphates within the organic plaque matrix.
• Factors that regulate the deposition of minerals on dental biofilms are
• Physicochemical factors :
• Plaque pH
• Local saturation of Ca, P and fluoride ions
• Biological factors such as presence of crystallization nucleators/inhibitors.
• The localized supersaturation of calcium and phosphate ions provides the driving force for
mineralization.
55

• The commensal bacterial biofilms inhibits colonization by exogenous pathogenic
microorganisms by a phenomenon termed Colonization Resistance.
• Dental biofilm (plaque) formed on the tooth surface is harmless under normal conditions.
• A shift in microenvironment due to repeated use of ‘‘habit forming’’ substances, diet, and
host immune response can lead to biofilm-mediated infections or diseases in the oral cavity.
• According to the ‘‘ecological plaque hypothesis,’’ any environmental change that favours
increasing colonization by potential pathogenic bacteria would cause disease.
56

• A decline in the host defense mechanisms caused by disease or immuno-suppressive
medicaments may also render generally ‘‘harmless commensals’’ to become ‘‘opportunistic
pathogens.’’
• Ex. diseases caused by biofilm community:
• Dental caries
• Gingivitis
• Periodontitis
• Peri-implantitis
• Apical periodontitis
57

• Less diverse compared to the oral microbiota
• Progression of infection alters the nutritional and environmental status within the root canal.
• Root canal environment : more anaerobic and depleted nutritional levels.
• Tough ecological niche for the surviving microorganisms.
• Microbes : anatomical complexities (isthmuses and deltas and in the apical portion of RCS)
• Shelter the adhering bacteria from cleaning and shaping procedures.
• Bacterial activities not confined to intracanal spaces, but also beyond the apical foramen.
ENDODONTIC BIOFILM
58

• In addition, the physical conditions available to support the growth of bacteria,
• pH
• Ionic concentration
• Nutrient availability
• Oxygen supply ( JOE 2002 )
59

• Biofilm mode of bacterial growth : Advantages
1. Resistance to antimicrobial agents
2. Increase in the local concentration of nutrients
3. Opportunity for genetic material exchange
4. Ability to communicate between bacterial populations of same and/or different species
5. Produce growth factors across species boundaries
60

• Biofilms offer their member cells several benefits, the foremost of which is protection from
killing by antimicrobial agents
• 3 Mechanisms that confer antimicrobial tolerance to cells living in a biofilm (JOE 2002)
• FIRST : barrier properties of the EPS matrix.
• Extracellular enzymes such as ß lactamase may become trapped and concentrated in the
matrix, thereby inactivating ß lactam antibiotics
61

• SECOND mechanism involves the physiological state of biofilm microorganisms.
• Bacterial cells residing within a biofilm grow more slowly than planktonic cells, as a result,
biofilm cells take up antimicrobial agents more slowly.
62
• THIRD suggested mechanism : microorganisms within the biofilm experience
metabolic heterogeneity.

• Interaction between E.faecalis biofilm and root canal dentin substrate
(Kishen et al, J BioMed Research 2006)
• By examining the shift in chemical composition of biofilm structure with time.
• By studying the topography and ultrastructure of the biofilm and dentin substrate.
• Study Showed :
• E.faecalis formed biofilm on root canal dentin
• Bacteria induced dissolution of the mineral fraction from the dentin substrate
• A reprecipitated apatite layer was formed in the biofilm structure
63

• Endodontic bacterial biofilms can be categorized as
1. Intracanal biofilms
2. Extra radicular biofilms
3. Periapical biofilms
4. Biomaterial centered infections.
64

• Microbial biofilms formed on the root canal dentine of an endodontically infected tooth.
INTRACANAL BIOFILMS
65
• First documented by Nair et al in 1987
• Biofilm : Cocci, rods, and filamentous bacteria.
• Monolayer and/or multi-layered bacterial biofilms were
found to adhere to the dentinal wall of the root canal.

• Ability of E. faecalis to resist starvation and develop biofilms under different environmental
and nutrient conditions (aerobic, anaerobic, nutrient-rich, and nutrient-deprived conditions).
• E. faecalis biofilms were modified according to the prevailing environmental and nutrient
conditions.
• Nutrient-rich environment (aerobic and anaerobic)
• Surface aggregates of bacterial cells and water channels.
• Viable bacterial cells were present on the surface of the biofilm.
• Nutrient-deprived environment (aerobic and anaerobic)
• Irregular growth of adherent cell clumps were observed.
66

67
Nutrient-rich
condition after 1 week
Nutrient-rich
Nutrient-deprived
Nutrient-deprived

• Stage I : Adherence and micro colonies
• Stage II : Dissolution of dentin and
• Stage III : Mineralization of biofilm
• Carbonated apatite structure
• Obvious signs of dentine surface degradation under nutrient-
deprived environment
• Interaction of bacteria and their metabolic products on
dentine.
68

• Recent investigation : Ability of E. faecalis to coaggregate with F. nucleatum.
• Coexist in a microbial community : endodontic infection.
(Siren et al. Int Endod J, 1997)
• Investigations have also demonstrated biting force-induced retrograde fluid movement into the
apical portion of the root canal (apical retrograde fluid movement)
(Kishen et al. Int Endod J, 2005)
• Cyclic influx of ion-rich tissue fluid into the apical portion of the root canal can promote
persistence of bacteria as biofilms and their mineralization.
69

• Biofilms formed on the root (cementum) surface adjacent to the root apex of endodontically
infected teeth.
• Reported in
• Asymptomatic periapical periodontitis
• Chronic apical abscesses associated with sinus tracts.
• Cocci and short rods, with cocci attached to the tooth substrate.
• Filamentous and fibrillar forms were also observed in the biofilm.
• Calcified biofilms was also reported by Riccuci et al.
EXTRA-RADICULAR BIOFILM
70

• No obvious difference in the biofilm structures formed on the apical root surface of teeth with
and without sinus tracts.
71

• Isolated biofilms found in the periapical region of an endodontically infected teeth.
PERIAPICAL MICROBIAL BIOFILMS
72
• May or may not be dependent on the root canal.
• Actinomyces and P. propionicum : Asymptomatic periapical
lesions
• Ability to overcome host defense mechanisms, thrive
in the inflamed periapical tissue, and subsequently
induce a periapical infection.

• Actinomyces species : Grow in microscopic or macroscopic
aggregates.
• Commonly referred to as ‘‘sulfur granules,’’ because of
the yellow granular appearance.
• Microscopically : appearance of rays projecting out from
a central mass of filaments. (Ray fungus)
PERIAPICAL MICROBIAL BIOFILMS
73

• Caused when bacteria adheres to an artificial biomaterial surface and forms biofilm
structures.
• Presence of biomaterials in close proximity to the host immune system can increase the
susceptibility to BCI.
• BCI is one of the major complications associated with prosthesis and/or implant-related
infections.
BIOMATERIAL-CENTERED INFECTION (BCI)
74

• BC biofilms : formed on root canal obturating materials.
• Intraradicular
• Extraradicular
depending upon whether the obturating material is within
the root canal space or has it extruded beyond the root apex.
• Filaments, long rods, and spirochete-shaped bacteria were predominant
in the biofilm formed on guttapercha.
BCI IN ENDODONTICS
75

• A study investigated the initial biofilm-forming ability of root canal isolates on gutta-percha points
in vitro.
• E. faecalis and S. sanguinis biofilms were significantly thicker than others.
• P. gingivalis, and P. intermedia did not form biofilms on gutta-percha.
BCI IN ENDODONTICS
• Suggests that Gram-positive facultative anaerobes have the
ability to colonize and form extracellular matrices on gutta-
percha points
• Serum plays a crucial role in biofilm formation.
(Takemura et al. Eur J Oral Sci 2004)
Filamentous or spirochete shaped
bacteria on surface of GuttaPercha

• Extraradicular microbial biofilms formed on tissue or biomaterial surface were related to
refractory periapical disease
(Moine et al. Shock 2004)
77

• The stages of biofilm formation follows basically the same model in various micro-organisms,
the biofilm architecture and molecular mechanisms involved in biofilm formation appear to
differ.
• Information on the genetic regulation of oral biofilm formation is still lacking.
• A better understanding of these processes is necessary to the development of novel strategies
for oral disease prevention and control based on interference of two-component signal
transduction systems or quorum-sensing.
CONCLUSION
78

• Ingle’s Endodontics : 6th Edition
• Endodontic Practice : Grossman : 13th Edition
• Pathways of the Pulp : Cohen : 11th Edition
• Principles and Practice of Endodontics : Walton & Torabinejad : 3rd Edition
• Endodontic Microbiology : Fauad : 2nd Edition
• Essential Endodontology : Orstavik & Pitt Ford : 2nd Edition
REFERENCES
79

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