BIOFILM IN ENDODONTICS;

CURRENT STATUS AND

FUTURE DIRECTION
PRESENTER : DR JUNIOR KASSIM
DEPARTMENT OF RESTORATIVE DENTISTRY
11-03-2022
 OUTLINE
• 1. INTRODUCTION
• 2. DEFINITION OF BIOFILM
• 3. HISTORY OF BIOFILM
• 4. CHARACTERISTICS OF A BIOFILM
• 5. CURRENT STATUS
• 6. FUTURE DIRECTION
• 7. CONCLUSION
• 8. REFERENCES
 INTRODUCTION
• Microbial communities are commonly referred to as
biofilms (Costerton et al. 1995). These communities are
found associated with humans, generally on the skin or
mucous membranes, but can also be found in natural
(e.g., rivers and streams or soil) and artificial
environments (e.g., on the surfaces of the places where
• we live and work). In general, biofilms connote the
lifestyles of aggregated, sessile, or attached microbes in
any environment and contrast free-floating, planktonic
 DEFINITION OF BIOFILM

• A biofilm is a highly organized structure consisting of

bacterial cells enclosed in a self-produced extracellular
polymeric matrix attached on a surface.
• (Flemming et al, 2016).
• Biofilms may also be considered as a layer of
condensation of microbiota or a microbial-derived
community consisting of cells that are irreversibly
attached to a substratum or interface or to each other,
and embedded in a matrix of extra-cellular
polysaccharides in addition to extracellular DNA (eDNA)
and extracellular proteins (Marsh, 2004).
 HISTORY OF BIOFILM
• In 1894:
• Miller published his findings on the bacteriological
investigation of pulps. He observed many different
microorganisms in the infected pulp space and realized
that some were uncultivable when compared with the full
range observed by microscopy, and that the flora was
different in the coronal, middle, and apical parts of the
canal system.(Fabricius et al, 1982)
• In 1965:
• Kakehashi and others convincingly demonstrated (in a
rat model) the essential role of microbes in apical
periodontitis (Kakehashi et al, 1965). This led to further
studies In the mid-1970s, using advanced anaerobic
technology,
• In 1976:
• Sundqvist extended the aetiological role of microbes to
• apical periodontitis associated with human teeth
containing necrotic pulps (Sundqvist 1976).
• In 1987:
• Using the technique of correlative light and transmission
electron microscopy, Nair presented the ultrastructural
visualization of intracanal microbial flora
• and described it as embedded in extracellular matrix
• of bacterial origin’ and existing as sessile intricate
• communities promoting a symbiotic relationship what is
now known as endodontic biofilms.
 CHARACTERISTICS OF A
BIOFILM
• Bacteria in the state of a biofilm are able to survive tough
growth and environmental conditions, due to the
following characteristics
• 1. Protection of biofilm bacteria from environmental
• threats
• 2. Enhanced tolerance to antimicrobials
• 3. Quorum sensing
 Protection of Biofilm Bacteria from
Environmental Threats
• Bacteria are capable of producing cell surface structures
(e.g., capsule) or extracellular secretions
• (e.g., extracellular polysaccharide). The extracellular
polysaccharide (EPS) can offer protection to all
• resident bacteria from various environmental stresses
such as pH shifts, osmotic shock, UV radiation
• and desiccation. It also alleviates the effect of any
harmful substances that have to diffuse through the EPS
matrix before reaching the microorganisms
 Enhanced Tolerance to Antimicrobials

• Enhanced tolerance to Long term use of antimicrobials

drugs leads to the development of resistance among
microorganisms due to altered gene expression and
transfer of resistance genes, rendering the antimicrobial
agent ineffective (Davey et al, 2000). EPS matrix in
bacterial cells acts as a barrier, trapping extracellular
enzymes such as β-lactamase and thus inactivates β-
lactam antibiotics. When there is depletion of nutrients,
• bacteria are forced into a dormant state and thereby
protected from being killed readily
 Quorum Sensing

• Quorum sensing is a bacterial cell-to-cell communication

system (Majumdar et al, 2017). Using chemical signaling
molecules, bacteria are able to communicate with one
another. For example, molecules such as the
competence-stimulating peptide (CSP) are important to
co-ordinate gene expression. Quorum sensing allows
bacteria to monitor the environment for other bacteria
and allow alteration of one’s behavior in a population-
wide scale.
 CURRENT UNDERSTANDING OF
ENDODONTIC BIOFILM
• The current status of endodontic biofilm will be discuss
under the following:
• 1. Current understanding of biofilm
• 2. Current criteria for defining a biofilm
• 3. Endodontic biofilm
• 4. Classification of endodontic biofilm
• 5. Current methods of studing biofilm
• 6. Endodontic biofilm management
 1. Current understanding of
biofilm
• A fully developed biofilm is described as a
heterogeneous arrangement of microbial cells on a solid
surface. The basic structural unit, microcolonies or cell
clusters, is formed by the surface-adherent bacterial
cells.(Wingeder et al, 1999).
• Biofilm is composed of matrix material consisting of
proteins, polysaccharides, nucleic acids, and salt, which
makes up 85% by volume, while 15% is made up of
cells.(Whitchurch et al, 2002)
• Biofilm are formed whenever there is free flow of fluid,
microorganism and a solid surface. it is one of the basic
survival strategies employed by bacteria.
• Biofilm formation involves four stages
• STAGE 1. Adsorption of macromolecule in the planktonic
phase of surface( transport of the microbe to the
substrate surface)
• STAGE 2. Adhesion and co-adhesion of microbes and
attachment strengthened by polymer production and
unfolding of cell surface structure(none specific
microbial- substrate adherence)
• STAGE 3: Multiplication and metabolism of attached
microorganisms
• STAGE 4: Detachment of biofilm microb
 2. Current criteria for defining a
biofilm
• Caldwell et al. In 1997, highlighted four characteristics of
biofilm as follows:
• (A). Autopoiesis – Must possess the ability to self-organize
• (B). Homeostasis – Should resist environmental
perturbations
• (C). Synergy – Must be more effective in association than in
isolation
• (D). Communality – Should respond to environmental
changes as a unit rather than as single individuals.
• The typical example of a biofilm is dental plaque.(Caldwell et
al. 1997)
 3. Endodontic Biofilm
• More than 1000 different bacterial species have been
identified in the oral cavity by culture and independent
molecular microbiology, but with advanced massively
parallel DNA pyrosequencing techniques, the number
may be higher (Paster et al, 2000). Although other
organisms exist, baterial predorminate endodontic
biofilm.
• From current available evidence, Enterococcus faecalis
is the only species that has been widely studied for its
capacity to form biofilms.(Duggan et al 2007)
• Microorganism involved in endodontic biofilm formation
• 1. Enterococcus faecalis
• 2. coagulase- negative staphylococcus
• 3. streptococci
• 4. Actinomyces species
• 5. P. Propionicun
• others are fungi, F. nucleatum, P. gingivalis
Role of Enterococcus faecalis in
biofilm formation:
• E. faecalis is a gram-positive, facultative anaerobic coccus
that is strongly associated with endodontic infections. Being
an opportunistic pathogen, it causes nosocomial infections
and is frequently isolated from the failed root canals
undergoing retreatment.
• (Sundqvist et al 1998)
• They can grow in extremely alkaline pH, salt concentrated
environment, in a temperature range of 10–45°C, and
survive a temperature of 60°C for 30 min. E. faecalis is able
to suppress the action of lymphocytes, potentially
contributing to endodontic failure.
• (Sedgley et al 2005)
• E. faecalis has the ability to form biofilm that can resist
calcium hydroxide dressing by maintaining pH
homeostasis, but at a pH of 11.5 or greater, E. faecalis is
unable to survive.
 The development of E. faecalis
biofilm on the root canal dentin
• It involves three stages as follows:
• Stage 1: Microcolonies are formed as E. faecalis cells
adhere on the root canal dentin surface
• Stage 2: Bacterial-mediated dissolution of the mineral
fraction from the dentin substrate leads to localized
increase in the calcium and phosphate ions causing
mineralization (or calcification) of the E. faecalis biofilm
• Stage 3: Due to this interaction of bacteria and their
metabolic products on dentin, E. faecalis biofilm is
mineralized.(Distel et al, 2002.)
 4. Classification of endodontic
biofilm
• Endodontic bacterial biofilms are classified as:
• 1. Intracanal biofilms
• 2. Extraradicular biofilms
• 3. Periapical biofilms
• 4. Foreign body–centered biofilm
• Intracanal Biofilm
• Intracanal biofilms are microbial biofilms formed on the root
canal dentin of the infected tooth. Identification of biofilm in
root canals was earlier reported by Nair 1987 under
transmission electron microscopy.
• (Ramachandran et al, 1987).
• Major bulk of the organisms existed as loose collections of
filaments, spirochetes, cocci, and rods. Apart from these,
bacterial condensations were seen as a palisade structure
similar to dental plaque seen on tooth surface.[49] The
extracellular matrix material of bacterial origin was also
found.
Intra radicular biofilm
magnified view of intracanal
biofilm
• Extraradicular Biofilm
• Extraradicular biofilms formed on the external root
surface adjacent to the root apex of endodontically
infected teeth or teeth with persistent periapical lesion
• they are usually caused by Actinomyces speciess and
propionibacterium
• P. gingivalis, F. Nucleatum and T. Forsythensis can also
be seen.
extra radicular biofilm
• Periapical Biofilms
• These are isolated biofilm around the root apex which
can be seen in the absence of root canal infection
• organism involved in periapical biofilm include
• P. propionicum, actinomycosis. the aggregation of
Actinomyces cells is influenced by PH, ionic strength and
cell concentration which facilitate biofilm formation.
• Foreign body–centered biofilm
• Foreign body–centered biofilm is found when bacteria
adhere to an artificial biomaterial surface and form
biofilm structures.[83] It is also known as biomaterial-
centered infection.
• It is a major complication associated with prosthesis and
also in implant-supported prosthesis. Biomaterial-
centered infection reveals opportunistic invasion by
nosocomial organisms. (Takemura et al 2004)
 5. Current methods of studing
biofilm
• (I). Cuture
• (II). Microscopy
• (III). Immunological method
• (IV). Molecular biology
• (V). Colorimetric techniques
• (VI). Fourier transform infrared (FTIR) spectroscopy
• (VII). Solid-state nuclear magnetic resonance (NMR)
• (VIII). confocal laser scanning microscopy (CLSM)
• (IX). flow cytometry
• (X).fluorescence in situ hybridization (FISH)
• (XI).Green fluorescent protein (GFP) tagging
 Microscopic Techniques

• Light microscopy is the fundamental technique used for

the examination of biofilms, either directly on in vitro
samples or in vivo histopathological sections. It is a
relatively inexpensive, rapid, and readily available
method. In a microscopic method, the bacterial biofilm is
• stained with a suitable dye that is fluorescent (e.g.,
propidium iodide) or non-fluorescent in nature (e.g.,
safranin). Most high-resolution light microscopy
• will enable counting of bacterial cells on a substrate
surface
 Colorimetric Techniques
• The colorimetric assay is a semiquantitative method that
applies the principles of dye uptake by bacterial cells to
determine the biofilm biomass. In this assay, after the
bacterial biofilm is stained with a dye (e.g., crystal violet),
it is disrupted using a known quantity of alcohol or a
surfactant (sodium dodecyl sulfate), and the intensity of
the eluted dye is measured using a spectrophotometer.
This is an easy assay that allows the rapid quantification
of biofilm bacteria.
Light microscopic image of a monospecies biofilm of E.
faecalis grown on a polycarbonate
membrane( after staining with crystal violet)
 Confocal Scanning Laser
Microscopy (CSLM)
• Confocal Scanning Laser Microscopy (CSLM) has
become the preferred technique to study the architecture
of biofilms because it provides a powerful microscopy
tool to analyze microbial communities in situ. It is an
optical imaging technique for increasing optical
resolution and contrast of a micrograph by means of
using a spatial pinhole to block out-of-focus light in
image formation. Usually CSLM is applied with
fluorescent probe techniques that take advantage of the
optical geometry construction of the CSLM.
Confocal Scanning Laser Microscopy
(CSLM)
 Fluorescence in situ hybridization (FISH).

• Fluorescence in situ hybridization (FISH) is a laboratory

technique for detecting and locating a specific DNA
sequence on a chromosome. The technique relies on
exposing chromosomes to a small DNA sequence called
a probe that has a fluorescent molecule attached to it.
The probe sequence binds to its corresponding
sequence on the chromosome.The microbial
compositions of biofilm communities, such as those
growing in the oral cavity or root canal of teeth, are
generally diverse.
• Thus, it is imperative that in situ
determination of the different species
present and their distribution in a three-
dimensional space are accomplished for
subsequent analysis and interpretations.
Identification of bacteria Biofilm by fluorescence in situ
hybridization (FISH).

Lactobacillus &
Streptococcus
Enterococcus
(STR405) (Green)
(LAC722) (Red)
 Scanning electron microscopy (SEM)

• Scanning electron microscopy provide a vast amount of

information on the structure of microbial ecosystems.it is
a combination of traditional and morden microscopic
technique. The use of SEM analysis has increased due
to its rapidity and sensitivity to detect structural changes
in microbial ecosystems. As seen in the figure below,it
clearly demonstrates the heterogeneous architecture of
the canal biofilm. The biofilm is adherent adjacent to the
dentinal tubule lumen and is characterized by cocci,
filaments, and both yeast and hyphal cell-forming
networks of extracellular matrix strands
Scanning electron microscopy (SEM) image of a resilient
root canal microbial community
in the apex of an infected root cana
 6.Endodontic Biofilm
Management
• Generally, the strategies designed to liminate bacterial
biofilms are directed at
• 1. Inactivating resident bacteria within the biofilm
structure
• 2. disrupting biofilm structure
• 3. killing resident bacteria. These objectives can be met
by employing a variety of antimicrobial agents and/or
treatment strategies. these include:
• (a). Root canal irrigant
• (b). Instrumentation
• (c). Antimicrobia photodynamic therapy
• (d). use of Antibacteria nano paticles
• (e). Laser assisted root canal disinfection
• (f) Ozone
• ROOT CANAL IRRIGANT
• This include
• . Proteolytic Irrigant
• Antiseptic
• Demineralizing agent.
• Combination of Irrigating Solution
• Natural Agents (Phytotherapeutic or Ethnopharmacological
Approaches
• Nanoparticles Based Disinfection
• Miscellaneous Interventions e.g Enzymatic irrigation was
introduced by Niazi and coworkers
• INSTRUMENTATION
• Sonics and Ultrasonics
• Ultrasonic agitation can cause dis-agglomeration of the
bacterial biofilm, thus re-suspending the bacteria in planktonic
form which are then, more susceptible to antimicrobial irrigants.
• Also, any cavitation that may be produced, would cause
temporary weakening of the cell membrane, thereby increasing
the bacterial cell permeability to antimicrobial irrigants (Chen et
al, 2014).There is wide variability in the endodontic literature
with regards to effectiveness of sonic and ultrasonics in
removal of smear layer as well as antibacterial activity. I
• ANTIMICROBIA PHOTODYNAMIC
THERAPY
• Photodynamic therapy (PDT) is based on the concept
that a nontoxic dye, termed a photosensitizer (PS), can
be preferentially localized in a tissue and subsequently
be activated by light of an appropriate wavelength to
generate cytotoxic singlet oxygen and other reactive
oxygen species to create a desired therapeutic effect.
• The photooxidative effect caused by the photosensitizer
on bacteria has
• damaging effect on the bacteria DNA (Menezes et al.
1990), cell membrane
• (Wakayama et al. 1980), protease activity, and
lipopolysaccharide (LPS) (K€omerik
• et al. 2000). Thus eliminating the biofilm
• ANTIBACTERIA NANO PARTICLES
• Nanoparticles are microscopic particles with one or more
dimensions in the range of 1–100 nm. They possess unique
physico-chemical properties that are very
• different from their bulk or powder counterparts.
Nanoparticles that are antimicrobial, for example, possess a
broad spectrum of antimicrobial action and possess a much
lower potential of inducing microbial resistance than
conventional antibiotics. Magnesium oxide and calcium
oxide slurries have demonstrated bactericidal action against
both gram-positive and gram-negative bacteria (Sawai
2003)
• LASER ASSISTED ROOT CANAL
DISINFECTION
• A laser (Light Amplification by Stimulated Emission of
Radiation) is a device that emits light through a process of
optical amplification. The photons in a laser beam
• are emitted as a coherent, unidirectional, monochromatic
light beam that can be collimated into an intensely focused
ray of energy. Lasers have been primarily used in root
canal treatment to enhance elimination of microorganisms
from the root canal system(Miserendino et al, 1995)
• Infrared lasers such as CO2, Nd:YAG, diode, and erbium
lasers have been the lasers of choice for disinfection.
• The bactericidal effect of laser depends upon the
wavelength and energy level of the laser used, as well as
its thermal effect.
• OZONE:
• Ozone (O3) is an energized, unstable gaseous form of
oxygen, which dissociates readily back into oxygen (O2),
liberating a reactive form of oxygen, called singlet
• oxygen (O1). The singlet oxygen is highly reactive and is
capable of oxidizing cells.
• Ozone has been suggested as a means of destroying
microorganisms without promoting the development of
drug resistance (Restaino et al. 1995)
 FUTURE DIRECTION
• Despite the increasing knowledge of the microbial status
of root canal systems, much still remains unknown. The
reported success rates of root canal treatment have not
undergone significant improvement (Ng et al 2007).
From the clinical perspective, it is important to
understand the aetiopathogenesis of periradicular
periodontitis as a disease caused by microbial infection
of the root canal system.
• Even though we know that root canal biofilms are
complex, the literature unfortunately does not seem to
offer due credence to understanding the dynamics
between the components of a biofilm.
• Crosstalk between bacteria is a paradigm that has not be
sufficiently studied thus far in the context of endodontic
disease.
• The there is the need for better understanding of the
interactions between microbes in biofilms and how each
organism influences the other.
• This, coupled with targeted therapeutic strategies may
help improve the success rates of root canal treatment.
Such strategies must focus on a step-wise approach
from mono- to multispecies biofilms so as to develop a
sufficient knowledge base on their mechanisms at a
cellular level.
 REFERENCE
• 1. Loesche WJ. Chemotherapy of dental plaque infections.
Oral Sci Rev. 1976;9:65–107. [PubMed] [Google Scholar]
• 2. Flemming, H.C.; Wingender, J.; Szewzyk, U.; Steinberg, P.;
Rice, S.A.; Kjelleberg, S. Biofilms: An emergent form of
bacterial life. Nat. Rev. Microbiol. 2016, 14, 563–575. [Google
Scholar] [CrossRef] [PubMed]
• 3. Costerton, J.W.; Stewart, P.S. Battling biofilms. Sci. Am.
2001, 285, 74–81. [Google Scholar] [CrossRef] [PubMed]
• 2. Bowden GH. Microbiology of root surface caries in humans.
J Dent Res. 1990;69:1205–10. [PubMed] [Google Scholar]
• Marsh PD. Microbial community aspects of dental plaque. In:
Newman HN, Wilson M, editors. Dental Plaque Revisited.
Cardiff, UK: BioLine; 1999. pp. 237–53. [Google Scholar]
• Donlan RM. Biofilms: Microbial life on surfaces. Emerg Infect
Dis. 2002;8:881–90. [PMC free article] [PubMed] [Google
Scholar]
• Wingender J, Neu TR, Flemming HC. What are bacterial
extracellular polymeric substances? In: Wingender J, Neu
TR, Flemming HC, editors. Microbial Extracellular Polymeric
Substances: Characterization, Structure and Function.
Springer Verlag: Springer-Verlag; 1999. pp. 1–19. [Google
Scholar]
• Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS.
Extracellular DNA required for bacterial biofilm formation.
Science. 2002;295:1487. [PubMed] [Google Scholar]
• Paster BJ, Dewhirst FE. Molecular microbial diagnosis.
Periodontol 2000. 2009;51:38–44. [PMC free article]
[PubMed] [Google Scholar]
• Ramachandran Nair PN. Light and electron microscopic
studies of root canal flora and periapical lesions. J
Endod. 1987;13:29–39. [PubMed] [Google Scholar
• Duggan JM, Sedgley CM. Biofilm formation of oral and
endodontic Enterococcus faecalis. J Endod. 2007;33:815–8.
[PubMed] [Google Scholar]
• Distel JW, Hatton JF, Gillespie MJ. Biofilm formation in medicated
root canals. J Endod. 2002;28:689–93. [PubMed] [Google
Scholar]
• Majors PD, McLean JS, Pinchuk GE, Fredrickson JK, Gorby YA,
Minard KR, et al. NMR methods for in situ biofilm metabolism
studies. J Microbiol Methods. 2005;62:337–44. [PubMed] [Google
Scholar]
• Majumdar, S.; Pal, S. Cross-species communication in bacterial
world. J. Cell Commun. Signal. 2017, 11,
• 187–190. [CrossRef] [PubMed
• . Chen, J.E.; Nurbakhsh, B.; Layton, G.; Bussmann, M.;
Kishen, A. Irrigation dynamics associated with positive
• pressure, apical negative pressure and passive
ultrasonic irrigations: A computational fluid dynamics
• analysis. Aust. Endod. J. 2014, 40, 54–60. [CrossRef]
[PubMed].