biofilms

1. Biofilm in Endodontics

2. Contents INTRODUCTION ULTRA STRUCTURE OF BIOFILM STAGES OF BIOFILM FORMATION CHARACTERISTICS OF BIOFILM QUORUM SENSING ORAL DISEASES IN CONSEQUENCE OF BIOFILM RESISTANCE OF MICROBES BENEFITS OF BIOFILM TO MIRCOBES ENDODOTNICS BIOFILM CONCLUSION REFERENCES

3. Introduction MICROBIAL BIOFILM: - A structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface.

4. Dental caries and periodontal disease are the most common intraoral diseases and among the most prevalent disorders known to man. Both are associated with bacteria contained in dental biofilms. (Biofilm Formation, Identification and Removal) 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 6th )

5. Ultra Structure of Biofilm • A fully developed biofilm is described as heterogeneous arrangement of microbial cells on a solid surface. • The 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.

6. A gylcocalyx matrix made up of extracellular polymeric substance (EPS) surrounds the microcolonies and anchors the bacterial cell to the substrate. • Typically, a viable, fully hydrated biofilm appears as ‘tower’ or ‘mushroom’ shaped structure adherent to the substrate . • The water channels which are regarded as circulatory system in a biofilm, intersect the structure of biofilm to establish connection between microcolonies.

8. Stages of Biofilm Formation • Bacteria can form biofilm on any surface that is bathed in a nutrition containing fluid. • Biofilm development involves selection and adaptation by bacteria already present in the intraoral environment. • Acid producing Strep. mutans and lactobacilli are selected in a low pH environment, leading to acid production by the bacteria in the subsequent biofilm and the initiation of the caries process. • Similarly, anaerobic microorganisms are selected when there is an increase in gingival crevicular fluid, nutrients, and pH, all of which contribute to the establishment of periodontopathogens and periodontal disease.

9. Three major component involved in biofilm formation involves: - • Bacterial cells • A solid surface • A fluid medium

10. Step 1: - Adsorption of inorganic and organic molecules to the solid surface, forming a thin layer termed as conditioning layer. • Step 2: - 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. • Step 3: - Bacterial growth and expansion.

11. Formation of conditioning layer Planktonic bacterial cell attatchment Detachment ( seeding dispersal ) Bacterial growth and Biofilm Expansion

13. Detachment: - • The rate of detachment of microorganisms from dental biofilms is not clear. • The number of microorganisms in the planktonic phase (saliva) ≈10-100 million per milliliter • These microorganisms originate from dental and soft tissue biofilms so, the detachment of microorganisms should be seen as a continuous process during development. • The fact that microorganisms detach regularly has implications for their spreading and colonization to other sites/ seeding dispersal.

14. Characteristics of Biofilm • Bacteria in the biofilm state show unique capacity to survive tough growth and environmental conditions. • This capacity of bacteria in a biofilm is due to the following features: - • 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.

15. Characteristic of a Mature Biofilm • A mature biofilm is a metabolically active community of microorganism where individuals share duties and benefits. • Bacterial cells exhibit a considerable variation in its genetic and biochemical constitution compared to its planktonic counterpart. • Microbial interactions: - • Co-aggregation • Co-adhesion

16. Protection of Biofilm Bacteria From Environmental Threats • 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.

17. Nutrient Trapping and Metabolic Cooperativity in a Biofilm • 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. • Juxtapositioning of various microorganism provides cross feeding and metabolic cooperativity between different species. E.g. Production of growth factors. Production of different arrays of lytic enzymes.

19. Internal Compartmentalization • Environmental niches that supports the physiological requirement of different bacterial species are available 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.

21. Exchange of Genetic Material • Bacterial biofilm provides a setting for the residing bacterial cells to communicate with each other. • 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. • Exchange of genetic material between bacterial species residing in a biofilm will result in the evolution of microbial communities with different traits.

22. The Phenotype of Biofilm Bacteria IsDistinct from that of PlanktonicBacteria • Certain bacterial species have been found to display new, and more virulent types when growing in biofilms, and most importantly, bacteria within biofilms have an inherently increased resistance to anti-microbial agents compared with the same bacteria grown under planktonic conditions. • It is becoming increasingly clear that the adhesion of microorganisms to a surface triggers an altered expression of a large number of genes and their phenotypes are thus changed. • The character of such surface-induced changes is dependent both on the microorganisms and the nature of the surface involved.

23. It is evident that oral microorganisms have the capacity to respond and adapt to changing environmental conditions. • As biofilms form on a surface, the nature of the biofilm gives rise to diverse physical– chemical gradients with examples including the concentration of nutrients, metabolic end products, oxygen, growth factors and biocides. • Individual microorganisms are able to sense and process the chemical information from the environment and thereby adjust their phenotypic properties.

24. Quorum Sensing • Quorum sensing, a bacterial cell-to-cell communication mechanism for controlling cellular functions, is of particular interest because of the presence of dense aggregates of bacteria in biofilms. • The signalling is mediated by diffusible molecules which, when present in sufficient concentrations, serve to modify gene expression in neighbouring microorganisms. • Quorum sensing signalling is known to be involved in the regulation of several microbial properties,including virulence and the ability to form biofilms, incorporate extracellular DNA and cope with environmental stress.

25. S.gordonii, Streptococcus mitis, Porphyromonasgingivalis, Fusobacteriumnucleatum and Prevotellaintermedia possess the ability to communicate through quorum sensing. • The known peptide-signal molecules produced by oral streptococci are primarily used for intra-species communication.

26. Oral Diseases as Consequences ofEcological Changes in Biofilms • While seemingly a contradictory quality, dental biofilms are essential for maintenance of both oral health and oral disease conditions. • Currently the development of oral diseases are considered to be a consequence of ecologically driven imbalances in dental microbial biofilms. • In the case of caries for example, a low pH environment caused by microbial fermentation of carbohydrates selects populations of acid-tolerant and acid-producing strains that in turn increase acid formation and may result in demineralization of the tooth structure.

27. In the case of marginal periodontitis, accumulation of dental plaque enhancesinflammation and increases the flow of gingival crevicular fluid. This environmental change may favor growth of various proteolytic bacteria, which compete other members of the micro-community to become pathogenic by virtue of a numerical dominance. As far as endodontic infections are concerned, acute exacerbations of endodontic lesions may be explained by a shift in the flow of nutrients to the root canal space, giving rise to ecological changes, which promote growth of proteolytic bacteria.

28. The role of Candida albicans in dental biofilm is of particular interest given the increase in opportunistic C.albicans infections in immuno-compromised patients. The growth and survival of C. albicans in dental biofilms has been shown to be influenced by the concentration of aerobic and anaerobic bacteria present.

29. Resistance of Microbes in the Biofilm to Antimicrobials • The nature of biofilm structure and physiological characteristics of resident microorganisms offer an inherent resistance to antimicrobial agents. • Mechanism responsible for resistance: • Resistance associated with extracellular polymeric matrix. • Resistance associated with growth rate and nutrient availability. • Resistance associated with adoption of resistance phenotype.

31. Resistance associated with EPS : – - diffusion barrier - direct neutralization of antimicrobials - inactivation by modified enzymes produced by bacteria. Resistance associated with growth rate and nutrient availability • Susceptibility towards antimicrobial is directly proportional to growth rate. • Resistance thickness of biofilm Resistance associated with adoption of resistant phenotype • Up regulation of EPS • Nutrient limitation • Sub lethal dose • Increase responsiveness of stress response genes • Shock proteins • Activation of multi drug efflux pump

32. Benefits of Biofilm to Microbes • 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.

33. EndodonticBiofilm Endodontic microbiota is established to be less diverse as compare to oral microbiota. Progression of infection alters the nutritional and environmental status within the canal. Complete disinfection is difficult to achieve because of anatomical complexities. Often bacterial activities may not confined to intracanal space but also get access beyond the apical foramina.

34. Bacteria Associated with in vivo Dentin Caries and Root Canal Infection that can Invade Root Dentinal Tubules in vitro BacteriumReference • Streptococcus sanguinisAkpata and Blechman, 1982 Ørstavik and Haapasalo, 1990 Perez et al., 1993 • Streptococcus gordonii Love, 1996b Love et al., 1996 Love et al., 1997 • EnterococcusfaecalisAkpata and Blechman, 1982 Haapasalo and Ørstavik, 1987 Ørstavik and Haapasalo, 1990 • Streptococcus sobrinusNagaoka et al., 1995 • Lactobacilluscasei Nagaoka et al., 1995 • Actinomycesviscosus (naeslundii) Nagaoka et al., 1995 • Streptococcusmutans Love et al., 1997 Crit. Rev. Oral Biol. Med. 2002; 13; 171

35. Categories of Endodontic Biofilms Intracanal biofilms Extra radicular biofilms Periapical biofilms Biomaterial centered infections

36. Intracanal Microbial Biofilm Theses are microbial biofilm formed on the root canal dentin of an endodontically infected tooth. First described by the Nair in 1987. Intracanalmicrobiota - Loose collection - Biofilm structure Monolayer and/or mutilayer bacterial biofilm are found to adhere to the dentinal wall. Extracellular matrix of bacterial origin is also found to interspersed with the cell aggregate.

37. Bacterial microcolonies are formed by co-aggregation of single/ several morphological type of bacteria. Studies have established the ability of E.faecalis to resist starvation and develop biofilms under difficult environmental and nutritional conditions. Physiochemical characteristics of E.faecalisbiofilm modifies according to the prevailing environment and nutrient conditions.

38. E.faecalis under nutritional rich environment produce typical biofilm structures with characteristic surface aggregates of bacteria cells and water channels. • Under nutrition deprived environment, irregular growth of adherent cell clumps were observed.

39. Extraradicular Microbial Biofilms • Also termed as root surface biofilms, formed on the root surface (cementum) adjacent to root apex of endodontically infected teeth. Are associated with: - • Teeth with asymptomatic periapical periodontitis • Chronic periapical abscess associated with sinus tract. Characteristics: - • Root surface biofilm are mostly multispecies in nature. • Smooth and structureless.

40. Bacterial biofilm is found in the areas of the root surfaces between fibres & cells and crypts & holes. The extraradicularbiofilms are dominated by cocci and short rods, with cocci being attached to the tooth substrate. A smooth, structurelessbiofilm structure consisting of extracellular matrix material with embedded bacterial cells to coat the apex of root tip adjacent to the apical foramina.

41. Periapical Microbial Biofilms • Periapical microbial biofilm are isolated biofilm found in the periapical region of an endodontically infected teeth. • Periapical biofilm may or may not be dependent on the root canal. • Microorganism involved are: • Actinomyces • P.propionicum

42. These microorganism have ability to overcome the host defence mechanism, thrive in the inflamed periapical tissue and subseqently induce a peripapical infection. • Actinomyces species in tissue grow in microscopic or macroscopic aggregates, which may reach in diameter of 3-4 mm.

43. They are commonly referred to as sulfur granules, because of the yellow granular appearance. Microscopically granules gives the appearance of rays projecting out from the centre mass of filaments called as ray fungus.

44. Biomaterial Centered Infection Biomaterial centered infection (BCI) occurs when bacteria adheres to an artificial biomaterial surface and forms biofilms. Presence of biomaterial in close proximity to the host immune system can increase the susceptibility to BCI. BCI occurs when pathogenic bacterial population reaches a critical size and overcomes the host defense mechanism. Because biofilms are extremely resistant to host defense mechanism and antibiotic treatment, BCI is rarely resolved.

45. BCI usually involves opportunistic invasion by nosocomial organism, e.g. staphylococcus, enterococci, streptococci, P.aeruginosa, fungus. • 3 phases of bacterial adhesion to biomaterial surface: • Phase1: transport of bacteria to biomaterial surface. • Phase2: initial, non specific adhesion phase. • Phase3: specific adhesion phase. • Factors affecting: • pH & concentration of electrolytes. • Bacterial strains that do not produce extracellular polimeric substance (EPS) are less adherent and less pathogenic.

46. Future Developments • Novel areas for future research include: • The development of inhibitors and antiplaque agents that are more effective against surface-associated micro-organisms. • Interference with communication networks that coordinate or regulate microbial activities within biofilms. • Preventing colonization of selected organisms. • Affecting biofilm architecture, for example, by the use of enzymes that can degrade the exopolymers that comprise the plaque matrix. Caries Res 2004;38:204–211

47. The neutralization of parameters that select for the species that are implicated in disease. • The identification of pathogenic clones could also improve diagnosis and might predict sites that are more susceptible to disease. Caries Res 2004;38:204–211

48. Conclusion Dental biofilm is complex, with a well-organized structure. For oral and systemic health, the development and maturation of dental biofilm should be impeded by regular and meticulous removal. The application of the biofilm concept to endodontic microbiology plays 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.

49. References • Endodontics; Ingle, 6th edition: 268-286; 2008. • Dental Plaque Biofilms; Jill S. Nield-Gehrig • Biofilm Formation, Identification and Removal; Fiona M. Collins. • Dental Plaque as a Microbial Biofilm; P.D. Marsh:Caries Res 2004;38:204–211. • Invasion of dentinal tubules by oral bacteria; R.M. Love and H.F. Jenkinson: Crit. Rev. Oral Biol. Med. 2002; 13; 171