MICROBIAL BIOFILM IN

ENDODONTIC INFECTIONS

DEFINITION

A biofilm is a complex aggregation of microorganisms growing on a solid

substrate. Biofilms are characterized by structural heterogeneity, genetic diversity,
complex community interactions, and an extracellular matrix of polymeric
substances.
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
substances.
(Ingle 6th edition)

The term biofilm was introduced to designate the thin layered condensations of
microbes (e.g. bacteria, fungi,protozoa) that may occur on various surface
structures in nature. Free-floating bacteria existing in an aqueous environment, socalled planktonic microorganisms, are prerequisite for biofilm formation. Such
films may thus become established on any organic or inorganic surface substrate
where planktonic microorganisms prevail in a water-based solution.
(GUNNEL SVENSATER & GUNNAR BERGENHOLTZ)

Biofilms are usually found on solid substrates submerged in or

exposed to some aqueous solution. Biofilms consist of many species of bacteria
and archaea living within a matrix of excreted polymeric compounds. This matrix
protects the cells within it and facilitates communication among them through
chemical and physical signals. Some biofilms have been found to contain water

channels that help distribute nutrients and signalling molecules. This matrix is
strong enough that in some cases, biofilms can become fossilized.
A microbial biofilm is considered a community that meets the following four basic
criteria:
The microorganism living in the community;
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)

OVERVIEW OF BACTERIAL BIOFILMS

Root canal is an extraordinary microenvironment for several microbial species to

attach on dentin surface and form dense bacterial biofilms,which are prevalent on
most wet surfaces and can cause environmental problems, representing a common
cause of persistent infections.The stages of structural organization of biofilm,the
composition and activities of the colonizing microorganisms in various
environments may be different, although the establishment of a micro-community
on a surface seems to follow essentially the same series of developmental stages,

including deposition of a conditioning film, adhesion and colonization of

planktonic microorganisms in a polymeric matrix, co-adhesion of other organisms,
and detachment of biofilm microorganisms into surroundings. The success of
infected root canal treatment is dependent on inactivation of microorganisms
present in biofilm and planktonic ambiance. (J Appl Oral Sci. 2009;17(2):87-91)

Sundqvist and Fidgor (2003) reported that root canal infection is not
a random event. Species that establish a persistent endodontic infection are
selected by the phenotypic traits that they share and that are suited to the modified
environment. Some of these shared characteristics include the capacity to penetrate
and invade dentin, a growth pattern of chains or cohesive filaments, resistance to
antimicrobials used in endodontic treatment, as well as an ability to grow in monoinfections, to survive periods of starvation and to evade the host response.

In the past,bacteriological studies were conducted on free-floating

bacterial cells(planktonic state),ignoring the importance of sessile bacterial
cells(biofilm state).Biofilm can be formed wherever there is a flow of
fluid,microorganisms and a solid surface.It is one of the basic survival strategies
employed by bacteria in all natural and industrial ecosystems in response to
starvation.

The sessile bacterial cells in a biofilm state differ greatly from

their planktonic counterparts.Inside a biofilm,the bacterial cells exhibit altered

phenotypic properties and are protected from antimicrobials,environmental

stresses, bacteriophages and phagocytic amoebae.

Biofilms are responsible for most of the chronic infections and almost all
recalcitrant infections in human beings,as bacteria in biofilm are resistant to both
antibiotic therapy and host defence mechanism.

ULTRASTRUCTURE OF BIOFILM

A fully developed biofilm is described as a heterogenous arrangement of microbial

cells on a solid surface.The basic structural unit of a biofilm is the microcolonies or
cell clusters formed by the surface adherent bacterial cells.Microcolonies are
discrete units of densely packed bacterial cell(single or multispecies)
aggregates.There is a spatial distribution of bacterial cells(microcolony) of
different physiological and metabolic states within a biofilm.A glycocalyx matrix
made up of extracellular polysaccharide surrounds the microcolonies and anchors
the bacterial cell to the substrate.

85% by volume of the biofilm structure is made up of matrix

material while 15% is made up of cells.A fresh biofilm matrix is made of
biopolymers such as polysaccharides,proteins,nucleic acids and salts.The structure
and composition of a matured biofilm is known to modify according to the

environmental conditions such as growth conditions,nutritional availability,nature

of fluid movements,physicochemical properties of the substrate.

Typically a viable,fully hydrated biofilm appears as tower or mushroom

shaped structures adherent to a substrate.The overall shape of a biofilm is
determined by the shear forces generated by the flushing of fluid media.Biofilms
formed in high-shear environments have shown that the microcolonies are
deformed by these forces to produce tadpole-shaped oscillation in the bulk fluid.

Scanning electron microscopy of (a) Root dentinal surface covered by E. faecalis biofilm
(X5,000). (b) Magnification area of previous picture (X10,000). (c,d) Aggregated bacterial cells
on and in dentinal tubules (X10,000)

Advanced microscopy of living biofilms,have revealed that single-species biofilms

growing in the laboratories to complex multispecies biofilms growing in the
natural ecosystems have similar basic community structure,with some subtle
variations.

The water channels which are regarded as a primitive circulatory

system in a biofilm,intersect the structure of biofilm to establish connections
between the microcolonies.Presence of water channels facilitates efficient
exchange of materials between bacterial cells and bulk fluid,which in turn helps to
coordinate functions in a biofilm community.The structural feature of a biofilm
that has the highest impact in chronic bacterial infection is the tendency of

microcolonies to detach from the biofilm community.During the process of

detachment,the biofilm transfer particulate constituents(cells,polymers and
precipitates) from the biofilm to the fluid bathing the biofilm.There are two main
types of detachment process:erosion(the continual detachment of single cells and
small portions of the biofilm) and sloughing(the rapid,massive loss of biofilm).

Detachment plays an important role in shaping the morphological

characteristics and structure of mature biofilm.It is also considered as an active
dispersive mechanism(seeding dispersal).These detached cells,which have
acquired the resistance traits from their parent biofilm community,can be source for
persistent infection.

CHARACTERISTICS OF BIOFILM
Bacteria in a biofIlm state show distinct capacity to survive tough growth
and environmental conditions.
This unique capacity of bacteria in a biofilm state is due to the following
features:
a) BiofIlm structure protects the residing bacteria from environmental threats
b) Structure of biofilm permits trapping of nutrients and metabolic
cooperativity between resident cells of same species and/or different species
c) Biofilm structures display organized internal compartmentalization, which
allows bacterial species with different growth requirements to survive in
each compartment

d) Bacterial cells in a biofIlm community may communicate and exchange

genetic materials to acquire new traits.

PROTECTION OF BIOFILM BACTERIA

Bacteria residing in a biofilm community experience certain degree of protection
and homeostasis. Many bacteria are capable of producing polysaccharides, either
as cell surface structures (eg., capsule) or as extracellular excretions (eg., EPS).

Extracellular polysaccharide covers biofIlm communities and creates a

micro niche favorable for the long-term survival and functioning of the bacterial
communities.

Extracellular polysaccharides protects the biofilm bacteria from a variety of

environmental stresses, such as UV radiation, pH shifts, osmotic shock, and
desiccation. Extracellular polysaccharide can sequester metals, cations, and toxins.

Metallic cations such as magnesium and calcium minimize electrostatic

repulsion between negatively charged biopolymers, increasing the cohesiveness of
the Extracellular polysaccharide matrix.

Diffusion is the predominant transport process with in cell aggregates. The

diffusion distance in a planktonic cell is on the order of magnitude of the

dimension of an individual cell, while the diffusion distance in a biofilm is on the

order of the dimension of the multicellular aggregate.

A biofilm that is 10 cells thick will exhibit a diffusion time 100 times longer than
that of a single cell.

METABOLIC COOPERATIVITY IN A BIOFILM

An important characteristic of biofilms growing in a nutrient-deprived
ecosystem is its ability to concentrate trace elements and nutrients by physical
trapping or by electrostatic interaction.

The water channel connects the outer fluid medium with the. interior of
the biofilm, ensuring nutrient availability to microbial communities deep inside the
biofilm structure.

The complex architecture of a biofilm provides the opportunity for

metabolic co-operation, and niches are formed within these spatially wellorganized systems.

Bacterial microcolonies in a biofilm structure are exposed to distinct

environmental signals. For example, cells located near the center of a microcolony
are more likely to experience low oxygen tensions compared to cells located near
the surface. Moreover, due to the juxta positioning of different microorganisms,

cross feeding and metabolic co-operativity between different species of

microorganisms are seen in a biofilm.

Studies have reported the production of essential growth factors such as

hemin by W. recta to support the growth of fastidious organisms such as
P.gingivalis in a biofilm. In addition, each bacterial species residing in a biofIlm
possess different array of lytic enzymes, and a biofilm as a unit is equipped with a
wide spectrum of enzymes that can degrade complex organic materials. For
instance, bacterial species possessing proteolytic enzymes make nutrients available
to all other bacteria in a protein-rich environment.

ORGANIZED

INTERNAL COMPARTMENTALIZATION

IN

BIOFILM
A mature biofilm structure displays gradients in the distribution of nutrients,
pH, oxygen, metabolic products, and signaling molecules within the biofilm.
Cell-cell communication in a biofilm. .Some bacteria can produce chemical
signals (green) and other bacteria from the same species or from different species
or strain can respond to them (red).
This would create different microniche that can accommodate diverse
bacterial species within a biofilm. The gradients in nutrients, chemicals, and gases,
observed in a biofilm structure, are influenced by the type of nutrients and the
physiological requirements of the residing microorganisms.

In a multispecies biofilm involving aerobic and anaerobic bacteria, oxygen is

consumed by the aerobic and facultative anaerobic species, making the
environment rich in carbon dioxide and other gases.
When the aerobic bacteria residing on the surface of the biofilm consumes
all available oxygen, the interior of the biofilm can be absolutely anaerobic that it
can even support the growth of obligatory anaerobes.Despite the fact that oral
cavity is abundant in oxygen, anaerobic microbes are found to dominate oral
biofilms because of the possible redox gradient formed within the biofilm
structure.

Bacterial cells residing in a biofilm communicate, exchange genetic materials,

and acquire new traits
Bacterial biofilm provides a setting for the residing bacterial cells to
communicate with each other. Some of these signals, produced by cells, may be
interpreted not just by members of the same species, but by other microbial species
too. Communications between bacterial cells residing in a biofilm is attained
through signaling molecules, by a process called Quorum sensing. Quorum
sensing is mediated by low molecular weight molecules, which in sufficient
concentration can alter the metabolic activity of neighboring cells, and coordinate
the functions of resident bacterial cells within a biofilm. Exchange of genetic
materials between bacterial species residing in a biofilm will result in the evolution
of microbial communities with different traits.

Planktonic bacteria; The concentration of chemical signals secreted by the

planktonic cells is low.

Close proximity of microbial cells in a biofilm facilitates genetic exchange

between bacteria of genetically distant genera. Even the possibility of gene transfer
between a commensal organism (Bacillus subtilis) and oral biofilm bacteria
(Streptococcus species) has been demonstrated.

The horizontal gene transfer is of importance in human diseases caused by

bacterial biofilm as it can result in the generation of antibiotic-resistant
bacterial population.

Biofilm bacteria; Biofilm cells are held together in dense populations, so the
secreted chemical signals higher concentrations. Signal molecules then re-cross the
cell membranes and trigger changes in genetic activity.

Gene transfer between bacteria residing in a biofilm is thought to be

mediated by bacterial conjugation. The presence of diverse bacterial species in a
biofilm presents a pool of genetic codes for nutrient breakdown, antibiotic
resistance, and xenobiotic metabolism.

Cell-cell communication can result in the coordinated behavior of microbial

population residing in a biofilm.

DEVELOPMENT OF BIOFILM

The three major components involved in biofilm formation are bacterial

cells, a solid surface, and a fluid medium.

Diagram

showing

different

factors

influencing

interaction

Stages in the development of biofilm

initial

bacteria-substrate

Stage 1; the first step involved in the development of biofilm is the adsorption of
inorganic and organic molecules to the solid surface creating what is termed a
conditioning layer

Stage 2: During dental plaque formation, the tooth surface is conditioned by the
saliva pellicle. Once the conditioning layer is formed, the next step in biofilm
formation is the adhesion of microbial cells to this layer.

Amongst the pioneer organisms, the oralis group of streptococci is the major
population to form a bacterial monolayer on the salivary pellicle coated tooth
surface.

Factors that affect bacterial attachment to a solid substrate. These factors

include 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, and
surface hydrophobicity.

Physicochemical properties such as surface energy and charge density

determine the nature of initial bacteria-substrate interaction (Phase 1: transport of
microbe to substrate surface).

In addition, the microbial adherence to a substrate is also mediated by

bacterial surface structures such as fimbriae, pili, flagella, and EPS (glycocalyx).

Molecular-specific interactions between bacterial surface structures and

substrate become active in this phase (Phase 2: initial non-specific microbialsubstrate adherence phase).

Initially, the bonds between the bacteria and the substrate may not be strong.
However, 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 (Phase 3: specific
microbial substrate adherence phase). In this phase, 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 3; the bacterial growth and biofilm expansion. During this stage, the
monolayer of microbes attracts secondary colonizers forming microcolony, and the
collection of micro colonies gives rise to the final structure of biofilm.

The lateral and vertical growth of indwellers gives rise to microcolonies

similar to towers. A mature biofilm will be a metabolically active community of
microorganisms where individuals share duties and benefits.

The bacterial cells in a matured biofilm will exhibit considerable variation in

its genetic and biochemical constitutions compared to its planktonic counterparts.
Two types of microbial interactions occur at the cellular level during the formation
of biofllm. One is the process of recognition between a suspended cell and a cell
already attached to substratum. This type of interaction is termed co-adhesion.
In the second type of interaction, genetically distinct cells in suspension
recognize each other and clump together. This type of interaction is called
coaggregation.

RESISTANCE

OF

MICROBES

IN

BIOFILM

TO

ANTIMICROBIALS

The nature of biofilm structure and physiological characteristics of the

resident microorganisms offer an inherent resistance to antimicrobial agents, such
as antibiotics, disinfectants, or germicides.

The resistance to antimicrobial agents has been found to amplify more than
thousand times for microbes in biofilm, when compared to planktonic cells.

An identical biofilm exposed to vancomycin and rifampin for 72 hours at

concentrations exceeding the minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC) for the microorganism.

ENDODONTIC BIOFILMS

Endodontic microbiota is established to be less diverse compared to the oral

microbiota. This transition in the microbial population is more conspicuous with
the progressionof infection. Progression of infection alters the nutritional and
environmental status within the root canal.

The root canal enviroment apparently becomes more anaerobic and the
nutrition level will be depleted.

These changes will offer a tough ecological niche for the surviving
microorganisms. Furthermore, clinical investigations have shown that the complete
disinfection of root canal is very difficult to achieve. Microbes are found to persist
in the anatomical complexities such as isthmuses and deltas and in the apical
portion of root canal system. Often, bacterial activities may not be confined to
intracanal spaces, but also access regions beyond the apical foramen.

These anatomical and geometrical complexities in the root canal systems

shelter the bacteria from cleaning and shaping procedures.

Additionally, biofilm mode of bacterial growth offers other advantages such as

a) resistance to antimicrobial agents
b) increase in the local concentration of nutrients
c) opportunity for genetic material exchange
d) ability to communicate between bacterial

populations of same and/or

different species
e) produce growth factors across species boundaries.

Endodontic bacterial biofilms can be categorized as

1. Intra canal biofilms
2. Extra radicular biofilms
3. Periapical biofilms
4. Biomaterial centered infections.

INTRACANAL MICROBIAL BIOFILMS

lntracanal microbial biofilms are microbial biofilms formed on the root canal
dentine of an endodontically infected tooth.

A detailed description on the intracanal bacterial biofilm was documented by

Nair in 1987.It was suggested that the intracanal micro biota in an endodontically
infected teeth existed as both loose collection and biofilm structures, made up of
cocci, rods, and filamentous bacteria.

Monolayer and/or multilayered bacterial biofilms were found to adhere to

the dentinal wall of the root canal. The extracellular matrix material of bacterial
origin was also found interspersed with the cell aggregates in the biofilm

Studies have established the 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 under nutrient-rich environment (aerobic and anaerobic) produced

typical biofilrn structures with characteristic surface aggregates of bacterial cells
and water channels. Viable bacterial cells were present on the surface of the
biofilm. Under nutrient-deprived environment (aerobic and anaerobic), irregular
growth of adherent cell clumps were observed.

Nutrient- deprived condition after 1 week,

Nutrient- deprived condition after 4 weeks,

Nutrient- rich condition after 1 week

Nutrient-rich condition after 4 weeks.

Laser scanning confocal microscopy displayed many dead bacterial cells and
pockets of viable bacterial cells in this biofilm structure.

In vitro experiments have revealed distinct stages in the development of E.

faecalis biofIlm on root canal dentine.

In stage 1, E. faecalis cells adhered and formed microcolonies on the root

canal dentine surface. In stage 2, they induced bacterial-mediated dissolution of the
mineral fraction from the dentine substrate. This localized increase in the calcium
and phosphate ions will promote mineralization (or calcification) of the E.faecalis
biofilm in stage 3.

EXTRARADICULAR MICROBIAL BIOFILMS

Extraradicular microbial biofilms also termed root surface biofilms are
microbial biofilms formed on the root (cementum) surface adjacent to the root
apex of endodontically infected teeth.

The extraradicular biofilm structures were dominated by cocci and short

rods, with cocci attached to the tooth substrate. Filamentous and fibrillar forms
were also observed in the biofilm.

A smooth, structureless biofilm structure consisting of extracellular matrix

material with embedded bacterial cells was noticed to coat the apex of the root tip
adjacent to the apical foramen. There was no obvious difference in the biofilm
structures formed on the apical root surface of teeth with and without sinus tracts.

Bacterial biofilms in the areas of the root surfaces between fibers and cells
and in crypts and holes. The biofilm contained varying degrees of extracellular
matrix materials (glycocalyx).

The root surface biofilms were mostly multispecies in nature associated with
periapical inflammation and delayed periapical healing in orthograde treatment .

PERIAPICAL MICROBIAL BIOFILMS

Periapical microbial biofilms are isolated biofilms found in the periapical
region of an endodontically infected teeth. Periapical biofilms may or may not be
dependent on the root canal. The micro biota in the majority of teeth associated
with apical periodontitis is restricted to the root canal, as most of the microbial
species that infect the root canal are opportunistic pathogens that do not have the

ability to survive host

defense mechanism in the periapical tissues.

Members of the genus Actinomyces and the species P. propionicum have

been demonstrated in asymptomatic periapical lesions refractory to endodontic
treatment. These microorganisms have the ability to overcome host defense
mechanisms, thrive in the inflamed periapical tissue, and subsequently induce a
periapical infection.

Clinical investigation detected Actinomyces in 72 of 129 (55.8%) clinical

samples. Of those, 41 of 51 (80.4%) were from infected root canals, 22 of 48
(45.8%) were from abscesses, and 9 of 30 (30%) were associated with cellulites.

BIOMATERIAL CENTERED INFECTION

Biomaterial centred infection is caused when bacteria adheres to an artificial
biomaterial surface and forms biofilm structure.Biomaterial centred infection is
one of the major complications associated with prosthesis and implant related
infections.In endodontics,biomaterial centred biofilms would form on root canal
obturating materials.These biofilms can be intraradicular or extraradicular
depending upon whether the obturating material is within the root canal space or
has it extruded beyond the root apex.Studies have shown that gram-positive
facultative anaerobs have the ability to colonize and form extracellular matrices on
gutta-percha points,while serum plays a crucial role in biofilm formation.

Biofilm formations on the root canal walls of an extracted tooth with attached periapical tissue
lesion.

CONCLUSION
The application of the biofilm concept to endodontic microbiology will play a
crucial role in helping us to understand, not only the pathogenic potential of the
root canal microbiota,but also the basis for new approaches to infection control.
How bacteria adapt their properties under different disease conditions as well as
how biofilms are organized in root canals are important issues to be addressed on
the road to a clearer understanding of how the root canal bacteria resist endodontic
treatment measures.