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Plaque as a Biofilm

plaque as a biofilm with references

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Plaque as a Biofilm

  1. 1. PLAQUE AS A BIOFILM
  2. 2. INTRODUCTION • The accumulation and metabolism of bacteria on hard oral surfaces is considered the primary cause of dental caries, gingivitis, periodontitis and peri-implant infections. • In the context of the oral cavity, the bacterial deposits have been termed dental plaque or bacterial plaque. • In 1 mm3 of dental plaque weighing approximately 1mg, approx 1011 bacteria are present. Socransky SS, Gibbons RJ, Dale AC et al. The microbiota of the gingival crevice area of man. I. Total microscopic and viable counts of specific microorganisms. Arch Oral Biol 1953;8:275
  3. 3. • Dental plaque was the first biofilm to be studied in terms of both its microbial composition and its sensitivity to antimicrobial agents. • In the 17th century, Antonie van Leeuwenhoek pioneered the approach of studying biofilms by direct microscopic observation when he reported on the diversity and high numbers of animalcules present in scrapings taken from around human teeth. • Research over several decades has provided a solid foundation for current studies of oral biofilms. • Numerous cultural studies have reported the diversity of the resident oral microflora, both at the genus and species level in health and disease HISTORY
  4. 4. AS A PLAQUE BIOFILM
  5. 5. PLAQUE Bowen W.H. in the year 1976 defined dental plaque clinically as a structured, resilient, yellow-grayish substance that adheres tenaciously to the intraoral hard surfaces, including removable and fixed restorations. According to WHO (1978) Plaque is a specific but highly variable structural entity resulting from colonization and growth of microorganisms on surfaces of teeth and consisting of numerous microbial species and stains embedded in a extracellular matrix.
  6. 6. PLAQUE According to the Glossary of Periodontal Terms, 4th Edition An organized mass, consisting mainly of microorganisms, that adheres to teeth, prostheses, and oral surfaces and is found in the gingival crevice and periodontal pockets. Other components include an organic, polysaccharide-protein matrix consisting of bacterial by-products such as enzymes, food debris, desquamated cells, and inorganic components such as calcium and phosphate. According to Carranza, 11th Edition Dental plaque is defined clinically as a structured, resilient yellow-grayish substance that adheres tenaciously to the intraoral hard surfaces, including removable and fixed restorations.
  7. 7. BIOFILM • The term biofilm describes the relatively undefinable microbial community associated with a tooth surface or any other hard, non- shedding material. Wilderer & Charaklis 1989 • Biofilms consist of one or more communities of microorganisms, embedded in a glycocalyx, that are attached to a solid surface. Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000 2001;28:12–55.
  8. 8. • Biofilms are not simply organism-containing slime layers on surfaces; instead, biofilms represent biological systems with a high level of organization where bacteria form structured, coordinated, functional communities. • In essence, biofilms represent an interdependent community- based existence. • Mixed species biofilms may be regarded as primitive precursors to the more complex organizations observed for eukaryotic species. O’Toole, G. A., H. Kaplan, and R. Kolter. 2000. Biofilm formation as microbial development. Annu. Rev. Microbiol. 54:49–79
  9. 9. Biofilms may be found virtually anywhere... DOCKS BOATS PIPES STREAMS CATHETERS MOUTH
  10. 10. Based on its position on the tooth surface Dental plaque is broadly classified as Supragingival Plaque Found at or above the gingival margin. Demonstrates a stratified organization of a multilayered accumulation of bacteria Gram-positive cocci and short rods predominate at the tooth surface, Gram-negative rods and filaments, as well as spirochetes, predominate in the outer surface of the mature plaque mass. Subgingival Plaque Found below the gingival margin, between the tooth and the gingival pocket epithelium. The composition of the subgingival plaque depends on the pocket depth. The subgingival microbiota differs in composition primarily because of the local availability of blood products and an anaerobic environment. The apical part is more dominated by spirochetes, cocci and rods, whereas in the coronal part more filaments are observed.
  11. 11. PLAQUE HYPOTHESIS Specific Plaque Hypothesis Non Specific Plaque Hypothesis
  12. 12. NON-SPECIFIC PLAQUE HYPOTHESIS • A direct relationship was assumed to exist between the total number of accumulated bacteria and the amplitude of the pathogenic effect. • Individuals with extensive periodontal disease were either suspected of having a weak resistance to bacterial plaque or were blamed for inadequate home care. • Such a view of dental plaque as a biomass is referred to as the NON-SPECIFIC PLAQUE HYPOTHESIS (Theilade 1986). • The "non-specific plaque hypothesis" purports that many of the heterogeneous mixture of organisms in plaque could play a role in disease, and that disease is a result of the overall interaction of the plaque microflora with the host. Marsh PD. Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 1994;8:263-71
  13. 13. SPECIFIC PLAQUE HYPOTHESIS • The propensity of inflamed sites to undergo permanent tissue destruction was recognized to be more specific in nature, because not all gingivitis lesions seemed invariably to progress to periodontitis. • Such a view of periodontitis being caused by specific pathogens is referred to as the specific plaque hypothesis (Loesche 1979). • The "specific plaque hypothesis“ proposes that, out of the diverse collection of microorganisms that constitute the resident plaque microflora, only a very limited number are actively involved in causing disease. Problems - occasions when either disease is diagnosed in the apparent absence of the putative pathogens, or when pathogens are present at sites with no evidence of disease. Marsh PD. Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 1994;8:263-71
  14. 14. ECOLOGICAL PLAQUE HYPOTHESIS • A modified hypothesis was proposed by Marsh (1991) in an attempt to unify clinical and laboratory observations. • In this hypothesis, it was proposed that a change in a key environmental factor (or factors) will trigger a shift in the balance of the resident plaque microflora, and this might predispose a site to disease. Marsh PD. Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 1994;8:263-71
  15. 15. How does this solve our dilemma?? • Under the conditions that prevail in health, these organisms would be only weakly competitive and may also be suppressed by inter-microbial antagonism, so that they would comprise only a small percentage of the plaque microflora and would not be significant clinically. • Microbial specificity in disease would be due to the fact that only certain species are competitive under the new (changed) environmental conditions.
  16. 16. PLAQUE FORMATION
  17. 17. STAGES OF PLAQUE FORMATION Formation of a conditioning film Bacterial Adhesion Multiplication Maturation
  18. 18. Formation of a conditioning film Immersion of solid substratum into fluid media. Macromolecules adsorb to the surface Form a conditioning film – Acquired Pellicle What does it do? Conditioning film alters the charge and free energy of the surface thus increasing the efficiency of bacterial adhesion. The conditioning film alters the properties of the surface, and bacteria interact directly with the constituent molecules. Salivary pellicle can be detected on clean enamel surfaces within 1 minute. By 2 hours, the pellicle is essentially in equilibrium. Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011; 55:16-35. Electron micrographic illustration of a 4hr dental pellicle. Brecx et al. (1981).
  19. 19. Bacterial adhesion • According to Mergenhagen and Rosan (1985) the ability to adhere depends on a series of interactions between: • Surface to be colonized • Microbe • Ambient fluid milieu. • Some bacteria have specific attachment structures which enable it to attach rapidly such as: • Extracellular Polymeric substance • Fimbriae • Other bacteria require prolonged exposure to bind firmly.
  20. 20. Reversible adhesion • Reversible adhesion involves weak, long- range, physico-chemical interactions between the charge on the microbial cell surface and that produced by the conditioning film. • Irreversible adhesion involves interactions between specific molecules on the microbial cell surface (adhesins) and complementary molecules (receptors) present in the acquired pellicle. Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011; 55:16-35.
  21. 21. Multiplication Active Cellular Growth of Bacteria Synthesis of new outer membrane components Increased bacterial mass
  22. 22. Multiplication • Leads to an increase in biomass and synthesis of exopolymers to form a biofilm matrix. • The matrix makes a significant contribution to the structural integrity and general tolerance of biofilms to environmental factors (e.g. desiccation) and antimicrobial agents. • The matrix can be biologically active and retain water, nutrients and enzymes within the biofilm. Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011; 55:16-35.
  23. 23. CO-ADHESION • The primary colonizing bacteria adhered to the tooth surface provide new receptors for attachment by other bacteria, in a process known as “co-adhesion.” • Together with growth of adherent microorganisms, co-adhesion leads to the development of microcolonies and eventually to a mature biofilm. Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011; 55:16-35.
  24. 24. CO-AGGREGATION • Most human oral bacteria adhere to other oral bacteria. This cell-to-cell adherence is known as coaggregation. • Coaggregation is defined as the specific cell-to-cell recognition that occurs between genetically distinct cell types. • Gibbons and Nygaard (1970) discovered coaggregation among plaque bacteria and called it interbacterial aggregation. • The term coaggregation was coined to describe a clumping phenomenon that occurred when sucrose-grown streptococci were paired with actinomyces and was used to distinguish this intergeneric type of clumping from the dextran-mediated intraspecies aggregation of actinomyces. Kolenbrander PE, Palmer RJ Jr, Rickard AH, Jakubovics NS, Chalmers NI, Diaz PI. Bacterial interactions and successions during plaque development. Periodontol 2000 2006;42:47-79. Gibbons RJ, Nygaard M. Interbacterial aggregation of plaque bacteria. Arch Oral Biol 1970;15: 1397–400. Bourgeau G, McBride BC. Dextran-mediated interbacterial aggregation between dextran-synthesizing streptococci and Actinomyces viscosus. Infect Immun 1976: 13: 1228– 1234.
  25. 25. CO-AGGREGATION • Different species, or even different strains of a single species, have distinct sets of coaggregation partners e.g. • Streptococcus spp. And Actinomyces spp., two of the initial colonizing genera on enamel surfaces. • Fusobacteria coaggregate with all other human oral bacteria. • Veillonella spp, Capnocytophaga spp. bind to streptococci and/or actinomyces. • Each coaggregation is mediated by one or more complementary sets of adhesin–receptor pairs.
  26. 26. CO-AGGREGATION BRIDGES • The basic coaggregation principle exhibited by sequential coaggregation is the principle of bridging. • Each newly accreted cell becomes itself a new surface and therefore may act as a co- aggregation bridge to the next potentially accreting cell type that passes by. • A coaggregation bridge is formed when the common partner bears two or more types of coaggregation mediators. • These mediators can be various types of receptor polysaccharides, or various types of adhesins, or a mixture of the two.
  27. 27. Maturation • The close proximity of cells to one another facilitates the development of numerous synergistic and antagonistic interactions between neighbouring species and food chains. • The metabolism of the microorganisms produces gradients within the plaque; for example, in nutrients and fermentation products. • These gradients result in a mosaic of microenvironments. • These processes lead to the establishment of a mature biofilm with a relatively stable composition Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflic t and control. Periodontol 2000 2011; 55:16- 35.
  28. 28. Detachment • Bacteria are able to sense changes to their environment. • If conditions deteriorate, some species (e.g. Aggregatibacter actinomycetemcomitans) respond by upregulating enzymes that cleave their adhesins, enabling the cell to detach and colonize elsewhere. Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011; 55:16-35.
  29. 29. STRUCTURE OF DENTAL PLAQUE
  30. 30. SUPRAGINGIVAL PLAQUE The first cellular material adhering to the pellicle on the tooth surface consists of coccoid bacteria with numbers of epithelial cells and polymorphonuclear leukocytes During the first few hours, bacteria that resist detachment from the pellicle start to proliferate and form small colonies of morphologically similar organisms. Since other types of organisms also proliferate in an adjacent region, the pellicle becomes populated by a mixture of different microorganisms Clumps of organisms of different species become attached to the tooth surface or to the already attached microorganism, contributing to the complexity of the plaque composition after a few days Another feature of older plaque is the presence of dead and lysed bacteria which may provide additional nutrients to the still viable bacteria (Theilade & Theilade 1970). The material present between the bacteria in dental plaque is called the intermicrobial matrix and accounts for approximately 25% of the plaque volume.
  31. 31. SUBGINGIVAL PLAQUE Between subgingival plaque and the tooth an electron-dense organic material is interposed, termed as CUTICLE. This cuticle contains the remains of the epithelial attachment originally connecting the junctional epithelium to the tooth, with the addition of material deposited fromthe gingival exudate (Frank & Cimasoni 1970) A densely packed accumulation of microorganisms is seen adjacent to the cuticule The bacteria comprise Gram-positive and Gram-negative cocci, rods, and fliamentous organisms. Spirochetes and various flagellated bacteria may also be encountered, especially at the apical extension of the plaque When a periodontal pocket has formed filamentous microorganisms dominate, but cocci and rods also occur. In the deeper parts of the periodontal pocket, the filamentous organisms become fewer in number, and in the apical portion they seem to be virtually absent. A characteristic feature of subgingival plaque is the presence of leukocytes interposed between the surfaces of the bacterial deposit and the gingival sulcular epithelium
  32. 32. ULTRASTRUCTURE OF PLAQUE • Listgarten described the ultrastructural characteristics of mature plaque present on extracted teeth that were associated with healthy periodontal tissues and various degrees of periodontal disease. • ‘Corn cob’ formations were occasionally seen as a feature of plaque present on teeth associated with gingivitis. • ‘Bristle-brush’ formations, composed of a central axis of a filamentous bacterium with perpendicularly associated short filaments, were commonly seen in the subgingival plaque of teeth associated with periodontitis. • It is evident that the close proximity of different bacterial cell types allows the formation of microenvironments in which cell–cell interactions easily occur. Listgarten MA. Structure of the microbial flora associated with periodontal health and disease in man. A light and electron microscopic study. J Periodontol 1976: 47: 1–18.
  33. 33. NATURE OF DENTAL PLAQUE – THE BIOFILM WAY OF LIFE
  34. 34. • Biofilms consist of one or more communities of microorganisms, embedded in a glycocalyx, that are attached to a solid surface. • The biofilm allows microorganisms to stick to and multiply on surfaces. • Thus, attached bacteria (sessile) growing in a biofilm display a wide range of characteristics that provide a number of advantages over single cell (planktonic) bacteria. • The interactions among bacterial species living in biofilms take place at several levels including physical contact, metabolic exchange, small signal molecule mediated communication, and exchange of genetic information.
  35. 35. PROPERTIES OF BIOFILMS
  36. 36. STRUCTURE OF A BIOFILM • Earlier electron microscopy was the method of choice to examine microbial biofilms. Disadvantage: Sample preparation for electron microscopy resulted in dehydrated samples leading to biofilm collapse. • Nowadays, Confocal Scanning Laser Microscopes (CSLM) are used for the visualization of fully hydrated samples, which revealed the elaborate three- dimensional structure of biofilms. Davey ME, OToole GA. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 2000: 64: 847– 867.
  37. 37. STRUCTURE • Biofilms are composed of microcolonies of bacterial cells (15–20% by volume) that are non-randomly distributed in a matrix or glycocalyx (75–80% volume). • Individual microcolonies can consist of a single species but more frequently are composed of several different species. • Structure of the Biofilm depends on environmental parameters under which they are formed. These include: • Surface and interface properties, • Nutrient availability, • Composition of the microbial community, • Hydrodynamics Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000 2001;28:12–55.
  38. 38. • For example, under high shear stresses, such as on the surface of teeth during chewing, the biofilm is typically stratified and compacted. • At low shear force, the colonies are shaped liked towers or mushrooms, while at high shear force, the colonies are elongated. • Biofilms grown under laminar flow were found to be patchy and consisted of rough round cell aggregates separated by interstitial voids. • Biofilms grown in the turbulent flow cells were also patchy, but elongated “streamers” that oscillated in the bulk fluid were observed. Davey ME, O Toole GA. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 2000: 64: 847– 867.
  39. 39. • As a surface becomes colonized with individual cells, the bacteria form microcolonies, which then secrete a sticky extracellular polymeric substance. • Upon secretion of the extracellular polymeric substance, the biofilm matures by becoming larger and taking on a distinctive architecture. Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47.
  40. 40. • Earlier studies of thick biofilms (0.5 mm) that develop in sewage treatment plants indicated the presence of voids or water channels between the microcolonies present in these biofilms. • The water channels permit the passage of nutrients and other agents throughout the biofilm acting as a primitive ‘‘circulatory’’ system. • Nutrients make contact with the sessile (attached) microcolonies by diffusion from the water channel to the microcolony rather than from the matrix. • The interstitial voids or channels are, in essence, the lifeline of the system, since they provide a means of circulating nutrients as well as exchanging metabolic products with the bulk fluid layer . Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000 2001;28:12–55.
  41. 41. EXOPOLYSACCHARIDES – the backbone of the biofilm • The bulk of the biofilm consists of the matrix or glycocalyx and is composed predominantly of water and aqueous solutes. • The “dry” material is a mixture of exopolysaccharides, proteins, salts, and cell material. • Exopolysaccharides (EPS), which are produced by the bacteria in the biofilm, are the major components of the biofilm making up 50–95% of the dry weight.
  42. 42. They play a major role in….. maintaining the integrity of the biofilm preventing desiccation and attack by harmful agents. Binds essential nutrients to create a local nutritionally rich environment. Acts as a buffer Assists in retention of extracellular enzymes enhancing substrate utilization by bacterial cells.
  43. 43. PHYSIOLOGICAL HETEROGENEITY WITHIN BIOFILMS • Cells of the same microbial species can exhibit extremely different physiologic states in a biofilm even though separated by as little as 10 μm. • Studies to date indicate that sessile cells growing in mixed biofilms can exist in an almost infinite range of chemical and physical microhabitats within microbial communities. Clinical Periodontology and Implant Dentistry by Jan Lindhe, 5th Edition.
  44. 44. MICROBIAL INTERACTIONS • In biofilms, microorganisms are in close physical proximity to one another and interact as a consequence. • Metabolic interactions can occur at many levels and include nutritional co-operation, environmental modification through oxygen detoxification, and small-molecule signal- mediated gene regulation. • Many conventional metabolic interactions (synergistic and antagonistic) have been described among oral bacteria, and the development of food chains or food webs is common, in which the metabolic product of one organism becomes the primary nutrient for a second.
  45. 45. • Bacteria collaborate in order to catabolize complex host molecules (proteins, glycoproteins) • Obligately anaerobic bacteria such as P. gingivalis can survive in aerobic environments if they partner with and co-aggregate to oxygen-consuming species such as Neisseria. • Antagonistic interactions involve the production of inhibitory compounds (bacteriocins, acids, H2O2, etc.) to inhibit neighbouring cells, and can provide the producer cells with a competitive advantage.
  46. 46. QUORUM SENSING • Some of the functions of biofilms are dependent on the ability of the bacteria and microcolonies within the biofilm to communicate with one another. • Quorum sensing in bacteria “involves the regulation of expression of specific genes through the accumulation of signaling compounds that mediate intercellular communication” (Prosser 1999).
  47. 47. QUORUM SENSING • In quorum sensing bacteria secrete a signaling molecule that accumulates in the local environment and triggers a response such as a change in the expression of specific genes once they reach a critical threshold concentration. • The threshold concentration is reached only at a high-cell density, and therefore bacteria sense that the population has reached a critical mass, or quorum.
  48. 48. QUORUM SENSING • Two types of signalling molecules have been detected from dental plaque bacteria: • Peptides released by gram-positive organisms during growth • A “universal” signal molecule autoinducer. • Responses are induced only when a threshold concentration of the peptide is attained, and thus the peptides act as cell density, or quorum, sensors. • The streptococcal peptides are known as competence stimulating peptides. Carranza’s Clinical Periodontology, 11th Edition
  49. 49. QUORUM SENSING • In 2001, Schauder et al. proposed that a small molecule called autoinducer- 2 was a universal signal, mediating messages among the species in mixed species communities. • This is distinct from the regulation of gene expression mediated by autoinducer- 1, a family of acyl homoserine lactones, which regulate gene expression in genetically identical cells. Kolenbrander PE, Palmer RJ Jr, Rickard AH, Jakubovics NS, Chalmers NI, Diaz PI. Bacterial interactions and successions during plaque development. Periodontol 2000 2006;42:47-79 Schauder S, Shokat K, Surette MG, Bassler BL. The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule. Mol Microbiol 2001: 41:463–476.
  50. 50. QUORUM SENSING • The possible role of quorum sensing in influencing the properties of biofilms was first suggested by Cooper et al. • Quorum sensing also has the potential to influence community structure by encouraging the growth of beneficial species (to the biofilm) and discouraging the growth of competitors. • Quorum sensing therefore appears to play diverse roles: • Modulating the expression of genes for antibiotic resistance, • Encouraging the growth of beneficial species to the biofilm, • Discouraging the growth of competitors. Carranza’s Clinical Periodontology, 11th Edition
  51. 51. EXCHANGE OF GENETIC INFORMATION • Signaling is not the only way of transferring information in biofilms. • The high density of bacterial cells growing in biofilms facilitates exchange of genetic information between cells of the same species and across species or even genera. • Conjugation, transformation, plasmid transfer, and transposon transfer have all been shown to occur in naturally occurring mixed species biofilms. Clinical Periodontology and Implant Dentistry by Jan Lindhe, 5th Edition.
  52. 52. • Cells also communicate and interact with one another in biofilms via horizontal gene transfer. • The transfer of conjugative transposons encoding tetracycline resistance between streptococci has been demonstrated in model biofilms • Gene transfer between Treponema denticola and S. gordonii has also been demonstrated in the laboratory. • The presence of “pathogenicity islands” in periodontal pathogens such as P. gingivalis is also indirect evidence for horizontal gene transfer having occurred in plaque biofilms at some distant time in the past, and may explain the evolution of more virulent strains. Chen T, Hosogi Y, Nishikawa K, Abbey K, Fleischmann RD, Walling J, Duncan MJ. Comparative whole-genome analysis of virulent and avirulent strains of Porphyromonas gingivalis. J Bacteriol 2004: 186: 5473–5479. Wang BY, Chi B, Kuramitsu HK. Genetic exchange between Treponema denticola and Streptococcus gordonii in biofilms. Oral Microbiol Immunol 2002: 17: 108– 112. Roberts AP, Cheah G, Ready D, Pratten J, Wilson M, Mullany P. Transfer of TN916-like elements in microcosm dental plaques. Antimicrob Agents Chemother 2001: 45: 2943–2946
  53. 53. ATTACHMENT OF BACTERIA • The key characteristic of a biofilm is that the microcolonies within the biofilm attach to a solid surface. • Many bacterial species possess surface structures such as fimbriae and fibrils that aid in their attachment to different surfaces. • Fimbriae have been detected on a number of oral species including P. gingivalis, A. actinomycetemcomitans and some strains of streptococci. • Oral species that possess fibrils include S. salivarius, the S. mitis group, Pr. intermedia, Pr. nigrescens, and Streptococcus mutans. Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000 2001;28:12–55.
  54. 54. Both physical and chemical factors affect the attachment of biofilms to a surface….. PHYSICAL PROPERTIES • Roughness of the surface • Increases surface area and hence increases colonization. • Roughness also provides protection from shear forces and increases the difficulty of cleaning. CHEMICAL COMPOSITION • Since it may contain beneficial or detrimental components. • For example, metals such as brass (an alloy of copper and zinc) have antimicrobial properties due to dezincification and the antimicrobial properties of copper. • Polyvinyl chloride on the other hand, contains carbon, hydrogen and chloride, which encourage bacterial growth.
  55. 55. INCREASED ANTIBIOTIC RESISTANCE • Organisms growing in biofilms are more resistant to antibiotics than the same species growing in a planktonic (unattached) state. • Estimates of 1000–1500 times greater resistance for biofilm-grown cells than planktonic grown cells have been suggested. • The mechanisms of this increased resistance differ from species to species, from antibiotic to antibiotic, and for biofilm growing in different habitats. Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000 2001;28:12–55.
  56. 56. MECHANISMS OF ANTIBIOTIC RESISTANCE Recently, the notion of a subpopulation of cells within a biofilm that are ‘‘super- resistant’’ was proposed. Such cells could explain remarkably elevated levels of resistance to certain antibiotics that have been suggested in the literature Slower rate of growth of bacterial species in a biofilm, which makes them less susceptible to many but not all antibiotics. The exopolymer of the biofilm matrix, although not a significant physical barrier to the diffusion of antibiotics, does have certain properties that can retard antibiotic penetration. Hydrodynamics and the turnover rate of the microcolonies has an impact on antibiotic effectiveness. Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000 2001;28:12–55.
  57. 57. ADVANTAGES OF BIOFILM They are the preferred method of growth for many and perhaps most species of bacteria . ESTABLISHMENTOFSYNTROPHIC RELATIONSHIPS FACILITATES ACQUISITIONOFNEW GENETICTRAITS PROTECTSAGAINST Host defense mechanisms, Potentially toxic substances in the environment, such as lethal chemicals or antibiotics. Processing and uptake of nutrients, Cross-feeding (one species providing nutrients for another), Removal of potentially harmful metabolic products (often by utilization by other bacteria) Development of an appropriate physicochemical environment (such as a properly reduced oxidation reduction potential) An effective means of exchanging nutrients and metabolites thus enhancing nutrient availability. opportunity for metabolic cooperation in niches formed within these systems facilitates inter- species substrate exchange Most bacteria in natural settings reside within biofilms, it follows that conjugation is a likely mechanism by which bacteria in biofilms transfer genes within or between populations
  58. 58. ORAL BIOFILMS IN HEALTH AND DISEASE
  59. 59. BIOFILMS ASSOCIATED WITH ORAL HEALTH • In the case of the oral cavity, attempts to characterize the normal microbial flora have met with challenges: Over 700 species have been detected in the oral cavity, over half of which have never been cultivated. There is substantial diversity in the content of the microflora between individuals and between different oral sites within the same individual. Dietary changes combined with poor hygiene can cause a shift in the composition of the oral microflora. The oral microbiome changes as humans age. Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47.
  60. 60. • Such variations make it difficult to identify a typical oral microbiome for a healthy individual. • While agreement on exactly which species could be used as markers of oral health is yet to be achieved, an overall picture of what types of bacteria are commonly found in healthy individuals is starting to emerge. • A positive association has been observed between oral health and the presence of • Veillonella • Capnocytophaga ochracea • Streptococcus salivarius • Streptococcus mitis, • Gemella haemolysans • Granulicatella adiacens Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47.
  61. 61. BIOFILMS IN ORAL DISEASE • Just as entire microbial communities can be associated with health, current research also indicates that entire microbial communities can be associated with disease. • The transition to gingivitis is evident by inflammatory changes and is accompanied first by the appearance of gram-negative rods and filaments, then by spirochetal and motile microorganisms.
  62. 62. • The initial microbiota of experimental gingivitis consists of gram-positive rods, gram-positive cocci, and gram-negative cocci. • In chronic periodontitis, the bacteria most often detected at high levels include P. gingivalis, T. forsythia, P. intermedia, P. nigrescens, C. rectus, Eikenella corrodens, F. nucleatum, A. actinomycetemcomitans (often serotype b), P. micra, E. nodatum, Leptotrichia buccalis, Treponema (T. denticola), Selenomonas spp. (S. noxia), and Enteric spp.
  63. 63. MICROBIAL COMPLEXES • The association of bacteria within mixed biofilms is not random, rather there are specific associations among bacterial species. • Socransky et al. (1998) demonstrated the presence of specific microbial groups within dental plaque. • Six closely associated groups of bacterial species were recognized.
  64. 64. Socransky SS, Haffajee AD. Periodontal Microbial Ecology Periodontology 2000 2005;38:135–87
  65. 65. • Initial colonization appears to involve members of the yellow, green, and purple complexes along with Actinomyces species. • These groups of species are early colonizers of the tooth surface, and their growth usually precedes the multiplication of the predominantly gram negative orange and red complexes • As the disease progresses, members of the orange and then red complexes become more dominant. • Certain complexes are observed together more frequently than others in subgingival plaque. • For example, it is extremely unlikely to find red complex species in the absence of members of the orange complex. • In contrast, members of the Actinomyces, yellow, green and purple complexes are often observed without members of the red complex or even the red and orange complexes.
  66. 66. • The lack of a beneficial organism in a biofilm may be just as important as the presence of a pathogen in the contribution to disease. • Because of these revelations, a hypothesis has been developed linking certain diseases to a shift in membership of the local microbiota. Socransky SS, Haffajee AD. 2005
  67. 67. THE MICROBIAL SHIFT HYPOTHESIS • Also known as dysbiosis. • It refers to the concept that some diseases are due to a decrease in the number of beneficial symbionts and ⁄ or an increase in the number of pathogens. • Dysbiosis in the oral cavity can lead to periodontitis. • The long-standing paradigm is that, as periodontitis develops, the oral microbiota shifts from one consisting primarily of gram-positive aerobes to one consisting primarily of gram-negative anaerobes. Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47.
  68. 68. CLINICAL CONSIDERATIONS OF BIOFILMS – Extension to the Dental Environment
  69. 69. MICROBIAL CONTAMINATION OF DENTAL UNIT WATERLINES • The ability of bacteria to colonize surfaces and to form biofilm in water supply tubes, including DUWL, is a common phenomenon. • Microorganisms from contaminated DUWL are transmitted with aerosol and splatter. • This increases the risk for cross-infection in clinical practice, especially in view of the ever-increasing number of immunocompromised persons. Szymańska J, Sitkowska J, Dutkiewicz J. Microbial contamination of dental unit waterlines. Ann Agric Environ Med 2008;15:173-9
  70. 70. Szymańska J, Sitkowska J, Dutkiewicz J. Microbial contamination of dental unit waterlines. Ann Agric Environ Med 2008;15:173-9
  71. 71. • A wide range of chemical disinfectants have been used in DUWLs: • Hydrogen peroxide, • Hydrogen peroxide with silver ions, • Chlorine dioxide, • Peracetic acid, • Sodium hypochlorite • Chlorhexidine gluconate • Liaqat and Sabri (2009) found that, overall, combination of chlorhexidine with povidone iodine was very effective in eliminating/reducing the biofilm bacteria at 1000 μg/mL as compared to other combinations. Dallolio L, Scuderi A , Rini MS,Valente S,Farruggia P, Sabattini et al Int. J. Environ. Res. Public Health 2014, 11, 2064-76. Liaqat I, Sabri AN. In vitro efficacy of biocides against dental unit water line (DUWL) biofilm bacteria. Asian J Exp Sci. 2009;1:67–75
  72. 72. FIGHTING ORAL BIOFILMS: ADJUNCTIVE TREATMENTS FOR PERIODONTITIS
  73. 73. PHYSICAL REMOVAL OF MICROORGANISMS – Mechanical Debridement • Fortunately, biofilms in the oral cavity, unlike many other biofilms, are readily accessible allowing their physical removal. • Indeed, the most common form of periodontal therapy is the removal of supra and subgingival plaque by procedures such as self performed oral hygiene, scaling and root planing or periodontal surgery. Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000 2001;28:12–55.
  74. 74. CALCULUS REMOVAL AND PREVENTION OF ITS FORMATION • Calculus in itself appears to be non-pathogenic, but, due to its rough surface, harbors oral biofilms associated with oral diseases. • Thus, prevention and removal of supra- and subgingival calculus are critical in the management of periodontal diseases. • Complete removal of subgingival calculus in patients with periodontitis remains an elusive goal. • Hand instruments, sonic scalers, ultrasonic scalers and Er:YAG lasers have been used for removal of supra- and subgingival calculus. Flemmig TF, Beikler T. Control of oral biofilms. Periodontol 2000 2011;55:9-15.
  75. 75. • Scaling and root planing is the primary therapy of choice for most clinicians, and it is widely considered the gold standard for treating periodontitis. • Because of the underlying microbial basis of periodontitis, it is becoming more conventional to use antimicrobial therapy as an adjunct to scaling and root planning. • Additionally, because the host inflammatory response also plays a major role in disease progression, treatments aimed at suppressing inflammation can be used.
  76. 76. ANTIBIOTICS • Often used as an adjunct to scaling and root planing, and they can be applied locally or administered systemically. • Meta-analysis confirmed that minocycline and tetracycline were the most effective local adjunctive therapies when measured in terms of probing depth reduction and clinical attachment level gain. • Antibiotics yield only modest results clinically. • It must be kept in mind that periodontitis is a biofilm- associated disease, and biofilms are notoriously difficult to treat with antibiotics. Bonito AJ, Lux L, Lohr KN. Impact of local adjuncts to scaling and root planing in periodontal disease therapy:a systematic review. J Periodontol 2005: 76: 1227–36.
  77. 77. ANTISEPTICS • Antiseptics such as chlorhexidine, bleach, povidone-iodine can be used as an alternative to antibiotics. • An in vitro biofilm study showed that P. gingivalis was completely eradicated after 30 minutes of exposure to chlorhexidine, povidone- iodine or Listerine. Bercy P, Lasserre J. 2007
  78. 78. HOST MODULATION THERAPY • Aims to modulate the host by suppressing the inflammatory response. • Matrix metalloproteinases, mediate tissue destruction by degrading plasma membrane proteins and extracellular matrix proteins such as collagen significantly contribute to the tissue destruction and alveolar bone loss. • Members of the tetracycline family of antibiotics possess the ability to inhibit matrix metalloproteinases. • A meta-analysis subsequently determined that adjunctive administration of sub-antimicrobial doses of doxycycline conferred a statistically significant benefit over scaling and root planing alone. Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47. Reddy MS, Geurs NC, Gunsolley JC. Periodontal host modulation with antiproteinase, anti-inflammatory, and bone- sparing agents. A systematic review. Ann Periodontol 2003: 8: 12–37.
  79. 79. PHOTODYNAMIC THERAPY • This technique uses long-wavelength visible light (red light) to activate photosensitizing agents (photosensitizers) that produce reactive oxygen species, such as free radicals and singlet oxygen. • These toxic oxygen derivatives then react with essential cellular components such as DNA, proteins and lipids, leading to cell death. • An in vitro study showed that antimicrobial photodynamic therapy • destroys P. gingivalis, • inactivates a virulence-associated protease • the destructive host inflammatory mediators tumor necrosis factor- and interleukin-1 β Konopka K, Goslinski T. 2007
  80. 80. • Because this treatment appears to simultaneously destroy bacterial pathogens and suppress the destructive host inflammatory response, there has been a strong interest in the therapeutic potential of antimicrobial photodynamic therapy. • With this technology, researchers have been able to successfully characterize the microflora associated with health and disease in subgingival and supragingival plaque. • More importantly, this technique has been used to determine the nature of the microbial biofilm before and after treatment of periodontitis. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. 1998 Haffajee AD, Socransky SS, Patel MR, Song X 2008
  81. 81. ASSESSING THE EFFICACY OF TREATMENT • Essentially, the parameters used to assess the efficacy of treatment fall within two broad categories: biological and clinical. • Biologically, it is important to determine whether treatment altered the content of the microflora and helped to resolve the host inflammatory response. • The clinical parameters typically measured – probing depth reduction, clinical attachment level gain, reduction of bleeding on probing, and prevention of tooth loss Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36–47.
  82. 82. CONCLUSION • Dental biofilm has all of the properties of biofilms in other habitats in nature. • It has a solid substratum, it has the mixed microcolonies growing in a glycocalyx, and it has the bulk fluid interface provided by saliva. • The interactions between bacterial species in a biofilm and between bacterial species and the nonbacterial habitat are dynamic. • They reflect a back and forth interplay between host and colonizing species • Understanding of the ecologic relationships within intraoral biofilms and between biofilm composition and the host has been slowed by the difficulty in obtaining sufficient ‘snapshots’ of the microbial composition of biofilms taken from carefully monitored clinical situations.
  83. 83. • A greater understanding of the significance of dental plaque as a mixed species biofilm will have the potential to impact significantly on clinical practice. • When assessing treatment options, an appreciation of the ecology of the oral cavity will enable the enlightened clinician to take a more holistic approach and consider the nutrition, physiology, host defenses, and general well-being of the patient, as these will affect the balance and activity of the resident oral microflora.
  84. 84. REFERENCES • Carranza’s Clinical Periodontology, 11th Edition • Clinical Periodontology and Implant Dentistry by Jan Lindhe, 5th Edition. • Sigmund S. Socransky & Anne D. Haffajee. Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000 2001;28:12–55. • Berezow AB, Darveau RP. Microbial shift and Periodontitis. Periodontology 2000 2011;55: 36– 47. • Costerton, J. W. 1995. Overview of microbial biofilms. J. Ind. Microbiol. 15:137–140. • Davey ME, O Toole GA. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 2000: 64: 847– 867. • Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011; 55:16-35. • Roberts AP, Cheah G, Ready D, Pratten J, Wilson M, Mullany P. Transfer of TN916-like elements in microcosm dental plaques. Antimicrob Agents Chemother 2001: 45: 2943–2946. • Wang BY, Chi B, Kuramitsu HK. Genetic exchange between Treponema denticola and Streptococcus gordonii in biofilms. Oral Microbiol Immunol 2002: 17: 108– 112. • Socransky SS, Haffajee AD. Periodontal Microbial Ecology Periodontology 2000 2005;38:135–87
  85. 85. REFERENCES • Chen T, Hosogi Y, Nishikawa K, Abbey K, Fleischmann RD, Walling J, Duncan MJ. Comparative whole-genome analysis f virulent and avirulent strains of Porphyromonas gingivalis. J Bacteriol 2004: 186: 5473–5479. • Kolenbrander PE, Palmer RJ Jr, Rickard AH, Jakubovics NS, Chalmers NI, Diaz PI. Bacterial interactions and successions during plaque development. Periodontol 2000 2006;42:47-79. • Marsh PD. Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 1994;8:263-71. • Gibbons RJ, Nygaard M. Interbacterial aggregation of plaque bacteria. Arch Oral Biol 1970;15: 1397–400. • Listgarten MA. Structure of the microbial flora associated with periodontal health and disease in man. A light and electron microscopic study. J Periodontol 1976: 47: 1–18. • Szymańska J, Sitkowska J, Dutkiewicz J. Microbial contamination of dental unit waterlines. Ann Agric Environ Med 2008;15:173-9. • Schauder S, Shokat K, Surette MG, Bassler BL. The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule. Mol Microbiol 2001: 41: 463–476. • Reddy MS, Geurs NC, Gunsolley JC. Periodontal host modulation with antiproteinase, anti- inflammatory, and bone-sparing agents. A systematic review. Ann Periodontol 2003: 8: 12–37.

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