Microbial Biofilm in Endodontic Infections

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.