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plaque as a biofilm power point in periodontics
- 1. Plaque as abiofilm
- 2. CONTENTS • Introduction • Definition •History • Classification of dental plaque • Composition of dental plaque • Macroscopic and microscopic structure of plaque • Properties of biofilm • Formation of plaque • Factors affecting supra gingival plaque formation • Characterstics of biofilm bacteria • Conclusion
- 3. Introduction • The termbiofilm describes the relatively indefinable microbial community associated with a tooth surface or any other hard, non-shedding material. • (Wilderer & Charaklis 1989) • Biofilm consists of one or more communities of micro-organisms embedded in glycocalyx , that are attached to solid surfaces. (Costerton et al 1994)
- 4. INTRODUCTION • Hard, non-sheddingsurfaces (teeth) - accumulation of bacteria -is considered the primary cause of dental caries, gingivitis, periodontitis and peri-implant infections. • Teeth were considered as “port d entree” for periodontal pathogens • In biofilms, communities of microorganisms are spatially organised into three dimension structure are enclosed in matrix of extracellular material derived from cells themselves and from the environment
- 5. DEFINITION Dental plaque: “structured,resilient ,yellow-grayish substance that adheres tenaciously to the intraoral hard surfaces, including removable and fixed restorations.” Bowen W.H. (1976) • Bacterial aggregations on the teeth or other solid oral structures Lindhe, 2003
- 6. DEFENITION DENTAL PLAQUE “is aspecific but highly variable structural entity, resulting from sequential colonization of microorganisms on tooth surfaces, restorations & other parts of oral cavity, composed of salivary components like mucin, desquamated epithelial cells, debris & microorganisms, all embedded in extracellular gelatinous matrix.” WHO-1961
- 7. HISTORY J LeonWilliams (1897) – described dental plaque GV Black (1899) – coined term “gelatinous dental plaque” Waerhaug (1950) described the importance of bacterial plaque in the etiology of periodontal disease Loe et al (1965), landmark study on plaque , saying that plaque is main etiological agent in periodontal diseaes.
- 8. Classification of dentalplaque Listgarten (1976) Classified Dental Plaque According to its Location as • Marginal plaque • Supra gingival plaque • Subgingival plaque – Tooth associated – Tissue associated
- 9. Tooth attached UnattachedTissue Attached Gram positive – rods and cocci Gram negative rods, filaments, spirochetes Gram variable Does not extend to JE Extend to JE Extend to JE Calculus formation, root caries Gingivitis Gingivitis, periodontitis May penetrate cementum May penetrate epithelium and connective tissue
- 10. 2) On basisof pathogenicity: Cariogenic: Generally acidogenic and gram-positive. Periopathogenic: Mostly basophilic and gram-negative.
- 11. GRANTS CLASSIFICATION- ACCORDING TOLOCATION A. Coronal plaque- Coronal to the gingival margin B. Gingival plaque- forms on the external surface of the oral epithelium and attached gingiva C. Sub gingival plaque- located between the periodontal attachment and the gingival margin, within the sulcus or pocket. D. Fissure plaque- develops in pits and fissures E. Peri-implant plaque.
- 12. Classification of softdeposits • Materia Alba : refers to soft accumulations of bacteria and tissue cells that lack the organized structure of dental plaque. • Calculus : is hard deposits that form by mineralization of dental plaque and is generally covered by a layer of un mineralised plaque Schwartz et al 1969
- 13. Classification of softdeposits
- 14. COMPOSITION OF DENTALPLAQUE BIOFILM • Plaque consists of about 80% of water and 20% solids (micro-organisms and inter-cellular matrix). • 1) Micro-organisms: • Bacteria: Bacteria are the main constituents of plaque. • More than 325 bacterial species have been found in plaque. • 1 gm of plaque contains about 2 x 1011 bacteria. Socransky SS,1953), (Schroeder, De Boever-1970 • Mycoplasma species. • Protozoa. • Yeast. • Viruses.
- 15. COMPOSITION OF DENTALPLAQUE BIOFILM 2) Inter-cellular matrix: • It forms about 20 to 30% of the plaque mass • Constituents of inter-cellular matrix are: • Organic materials: polysaccharides (mainly dextran) , glycoproteins, proteins and lipids. • Inorganic materials: calcium and phosphorus are the main inorganic constituents of plaque. Other minerals like sodium, potassium and fluoride are also found in traces.
- 16. The main sourcesfrom where inter-cellular substances of plaque are derived, are: • Saliva: Glycoproteins and inorganic constituents of supra- gingival plaque are derived from saliva. • Gingival crevicular fluid: Albumin and inorganic components of sub-gingival plaque are derived from gingival crevicular fluid. • Bacterial products: Polysaccharides. • Diet and breakdown products of cell wall: Lipids. • External sources: Fluoride is mainly derived from fluoridated toothpastes and mouth washes.
- 17. Differences between supraand sub-gingival plaque Supra-gingival plaque Sub-gingival plaque Present above the gingival margin Present below the gingival margin Always tooth-associated May be tooth-associated or tissue-associated Primary source of nutrition for bacteria is saliva and ingested food Primary source of nutrition for bacteria is gingival crevicular fluid Mainly responsible for gingivitis (marginal plaque) or calculus formation and dental caries Responsible for calculus formation and root caries(tooth-associated plaque) and destruction of soft tissue (tissue-associated plaque)
- 18. Macroscopic Structure ofplaque biofilm • The inter-cellular matrix forms a hydrated gel Acts as a barrier in biofilm.. bacterial products are retained and concentrated bacteria gets protection from antimicrobial agents. bacteria in it can proliferate and colonize.
- 19. Microscopic Structure ofplaque: • Light microscopy, transmission electron microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy • The inner layer of plaque (attached to tooth surface)- mainly consists of densely packed coccoid and rod shaped bacteria • The outer layer -in addition has filamentous bacteria • On microscopy - “corn-cob appearance.” • Between rod shaped bacterial cells forming the inner core of the structure and the coccal cells that attach along the surface of these rod shaped cells. • The term corn-cob -was coined by Jones (1971).
- 20. Properties of Biofilm •Micro colonies of bacterial cells • Metabolic cooperativity • Water channels are present which form primitive circulatory system • Resistant to host defense • Resistant to antibiotics and antimicrobials
- 21. quorum sensing. • Bacteriain biofilm communicate with each other by a process called quorum sensing. • Enables bacteria to monitor each other's presence and to modulate their gene expression in response to the number of bacteria . • portions of the biofilm can become detached in order to maintain a cell density compatible with continued survival
- 22. MICROBES IN ORALCAVITY AND THEIR BIOFILM MODE OF LIVING • Streptococcus salaivarus and Streptococcus mitis- first colonizers of the oral cavity • Supragingival plaque demonstrates a stratified organisation of a multilayered accumulation of bacterial morphotypes • Gram positive cocci and short rods predominate the tooth surface of plaque • Gram negative rods and filaments and spirochaets predominate the outer surface of the mature plaque •
- 23. MICROBES IN ORALCAVITY AND THEIR BIOFILM MODE OF LIVING Subgingival microbiota differs in composition from supragingival plaque primarily because of the local availability of blood products and low reduction -oxidation(redox) potential which characterstics the anerobic environment.
- 24. TECHNIQUES TO IDENTIFYBACTERIA WITHIN DENTAL PLAQUE PLAQUE IDENTIFIED • Direct vision • By instrumentation: • Disclosing agents: • Now techniques -specific visulization of individual bacteria within mixed populations Specific labelling is achieved using nucleic acid probes (fluorescence in situ hybridisation )- FISH or Specific antibodies (immunofluroscence) •
- 25. ACCUMULATION OF DENTALPLAQUE BIOFILM The process of plaque formation can be divided into several phases: 1. Formation of pellicle on the tooth surface 2. Intial adhesion and attachment of bacteria 3. Colonisation/plaque maturation
- 26. 1.Formation of pellicleon the tooth surface • Acquired pellicle - a homogenous, membranous, acellular film that covers the tooth surface and frequently form the interface between the tooth ,the dental plaque and calculus. Schluger • Derived from components of saliva and crevicular fluid as well as bacterial and host tissue cell products and food debris. FUNCTIONS OF DENTAL PELLICLE • Protective barrier • Lubrication • Preventing tissue desiccation • Substrate to which bacteria attaches
- 27. FORMATION OF PELLICLE •All hard and soft tissues oral cavity are coated with a layer of organic material known as the acquired pellicle . Pellicle consists of peptides Proteins Glycoproteins including keratins,mucins,proline rich proteins phospho proteins (eg statherin),histidine rich proteins • Pellicle function as adhesion sites (receptors)for bacteria.
- 28. FORMATION OF PELLICLE •Pellicle forms by selective adsorption of the environmental macromolecules. Scannapieo FA et al . • Mechanism involved are: Electrostatic forces Van der waals Hydrophobic forces
- 29. 2.INTIAL ADHESION ANDATTACHMENT OF BACTERIA • Is the specific interaction between microbial cell surface ‘adhesion’ molecules and receptors in the salivary pellicle. • Only a small proportion of bacteria possess- adhesins -the most abudant bacteria in biofilms. The first organism to colonise the mouth are the streptococcus species – (obligate aerobes)- Hemophillus species,Nisseria species -Can survive only in the presence of oxygen facultative anerobes-Actinomyces species and Vellionella species.-Organisms that can grow in the presence or absence of oxygen- • These species are conidered as the primary colonisers on the tooth surface.
- 30. INTIAL ADHESION ANDATTACHMENT OF BACTERIA • The metabolic activity of primary colonisers modifies the local microenvironment - by removing oxygen ,it provides condition of low oxygen tension that permit the survival and growth of obligate anerobes. • The Intial steps in colonising of teeth by bacteria- • Phase 1: Transport to surface • Phase2:Initial adhesion • Phase 3:Strong attachment •
- 31. The Intial stepsin colonising of teeth by bacteria- • The first step in transport of bacterium to tooth surface occurs by Browinian motion Sedimentation of microorganisms Through liquid flow Active bacterial movement
- 32. The Intial stepsin colonising of teeth by bacteria- • • The second stage results in an intial reversible adhesion of bacterium,this results when bacterial cell comes in close proximity to the surface.(ie approx 50nm) • The behaviour of bacterial cells can be described by the Derjaguin-Lanadau-Verweey-Owerbeek (DLVO) theory of colloid stability. According to this theory the total interaction energy (Total Gibbs Energy) GTOT is the sum of attractive forces(GA) and the electrostatic repulsion(GR)
- 33. The Intial stepsin colonising of teeth by bacteria • THIRD STAGE-STRONG ATTACHMENT • After initial adhesion a firm anchorage between bacterium and surface is established • The binding is also mediated by adhesions (proteins) and complementary receptors (proteins,glycoproteins or polysaccharide) in the acquired pellicle.
- 34. 3.COLONISATION AND PLAQUEMATURATION • The primary colonizing bacteria adhered to the tooth surface provide new receptors for attachement by other bacteria - coadhesion leads to development of microcolonies and eventually to a mature biofilm • Cell –cell adhesion between genitically distinct oral bacteria also occurs-results in clumps or coaggregates . • Eg fuso bacterium coaggregate with all other oral bacteria • while Vellionella spp, Capnocytophaga spp and Prevotella bind to streptococci and actinomyces.
- 35. COLONISATION AND PLAQUEMATURATION • Eg of coaggregation between secondary colonizers with early colonizers include coaggregation of F.nucleatum with S.sanguins Prevotella loescheii with A. oris Capnocytophaga ochracea with A.oris Secondary colonizers such as Prevotella intermedia,P.loescheii,Capnocytophaga spp,F.nucleatum and P.gingivalis do not initially colonise tooth surface but adhere to bacteria already in plaque mass.
- 36. Plaque as biofilm: •The life cycle of a biofilm is characterized by attachment of planktonic bacteria to a surface and phenotypic differentiation to produce a mature, thick and spatially structured biofilm. Distinct stages in plaque biofilm formation start with acquired pellicle formation. Reversible adhesion involving weak long-range physicochemical interactions between the cell surface and the pellicle, which can lead to stronger adhesin- receptor mediated attachment Colonization Co-adhesion resulting in attachment of secondary colonizers to already attached cells Multiplication and biofilm formation (including the synthesis of exopolysaccharides) On occasion, detachment and re-colonization
- 37. DNA Hybridistion Method,defined colour – coded “complexes” of periodontal microorganism that tend to be found in health and disease • ACTINOM YCES • SPECIES V. Parvula A.odontol yticus S.Mitis S.Oralis S.Sanguis Streptococcus sp. S.gordonii S.intermedius PRIMARY COLONIZERS
- 38. • E.Corrodens • Capnocyptophagaspp • A.actinomycetemcomita ns P.Gingivalis T.Forsythus T.denticola P.Intermedia P.Nigrescen s P.Micros F.nucleatum E.nodatum C.rectus C.showae SECONDARY COLONIZERS
- 39. FACTORS AFFECTING SUPRAGINGIVAL PLAQUE FORMATION TOPOGRAPHY OF SUPRAGINGIVAL PLAQUE • Intial growth from the gingival margin and from the interdental space(areas are protected against shear forces SURFACE MICROROUGHNESS • Rough intra oral surfaces accumulate and retain more plaque and calculus • Smoothening an intra oral surface decreases the rate of plaque formation • INDIVIDUAL VARIABLES INFLUENCING PLAQUE FORMATION • The rate of plaque formation differs significantly between subjects. • Heavy (fast) and Light (slow) plaque formers • The saliva of light plaque formers -reduced colloidal stability of bacterial suspensions. • Heavy plaque formers -more plaque with a more complex supra gingival structure with higher proportions of gram negative rods.
- 40. FACTORS AFFECTING SUPRAGINGIVAL PLAQUE FORMATION VARIATION WITH DENTITION • Early plaque formation occurs faster in lower jaw when compared to upper jaw • More on the molars,on the buccal surfaces than palatal side and in the interdental areas when compared to buccal and lingual surfaces. • IMPACT OF GINGIVAL INFLAMMATION • Early plaque formation more on the tooth surfaces facing inflamed gingival margins .ie GCF flow may favour plaque formation,by the substances from this exudates(eg.minerals,proteins or carbohydrates) •
- 41. FACTORS AFFECTING SUPRAGINGIVAL PLAQUE FORMATION IMPACT ON PATIENTS AGE • plaque more in older patients resulted in more severe gingival inflammation. SPONTANEOUS TOOTH CLEANING • plaque remains on the occlusal sufaces of molars even after chewing fibrous foods.Absence of spontaneous tooth cleaning resulted in plaque accumulation. • DENOVO SUBGINGIVAL PLAQUE FORMATION • Subgingival irregularities sheltered microorganisms which favoured plaque accumulation. Biopsies have shown an elevated proportion of inflammatory cells in the JE and connective tissue facing rough surfaces. •
- 42. CHARACTERSTICS OF BIOFILMBACTERIA LIFE IN A “SLIME- CITY”) Metabolism of dental plaque bacteria Communication between biofilm bacteria Interaction between dental plaque bacteria Biofilm and Antimicrobial Resistance
- 43. 1.Metabolism of dentalplaque bacteria • Nutrients for dental plaque bacteria orginate from saliva or GC • The transition of gram positive to gram negative organisms observed in the structural development of plaque. • The early colonizers ( eg, Streptococcus and Actinomyces)-use oxygen and lower the redox potential of the environment which favours the growth of anerobic species. • Gram +ve bacteria use sugars as energy source. • Bacteria in mature plaque are anerobic and asaccharolytic( do not break down sugars)- use amino acids and small peptides as energy sources. •
- 44. Metabolism of dentalplaque bacteria Metabolic interactions are seen among bacteria • Lactate and formate are byproducts of metabolism of streptococci and actinomyces sps and are used for metabolism of Veilonella spp and Aa. • The growth of P gingivalis is enchanced by metabolic byproducts such as succinate from C .ochrecea and protoheme from campylobacter. Metabolic interaction also occurs between host and plaque microorganisms: • Bacterial enzymes that degrade host proteins release ammonia ,which is used by bacteria as a nitrogen source. • Hemin iron from the breakdown of host hemoglobin may be important in the metabolism of of P gingivalis. • Increased steroid hormones are seen association with P.intermedia found in subgingival plaque.
- 45. 2.Communication between biofilmbacteria In a biofilm bacteria have the capacity to communicate with each other ,eg. Quarum sensing. • Bacteria secrete a signaling molecule into the local environment and triggers a response once they reach a threshold concentration, and the bacteria sense that the population has reached a critical mass or Quroum. • Two types of signaling molecules detected from dental plaque • Peptides released by gram +ve bacteria • Universal signal molecule autoinducer2 (AI-2) • Peptide signals are released by streptococci and are recognised by the cells of the same strain and are known as competence stimulating peptides. • AI- 2 is produced and detected by many bacteria,,and has demonstrated to play a role in mutulastic interaction between S oralis and Aa. •
- 46. 3 Interaction betweendental plaque bacteria • Long and short fimbriae of P.gingivalis are required for adhesion and biofilm formation • long fimbriae is down regulated in the presence of S.cristatus • & short fimbriae are down regulated by S.gordoni,S mitis or or S.sanguinins • In multispecies biofilms-oral streptococci compete with S .mutans by excreting the strongly oxidizing molecule H2O2.S.oligofermantans can convert lactic acid produced by S.mutans into H2O2,which then kills the S.mutans cells.
- 47. 4.Biofilm and AntimicrobialResistance • Resitance of bacteria to antibiotics is seen in bacteria in biofilm than in their planktonic state. • slower rate of growth of bacteria in biofilms , make them resistant and less susceptible to antibiotics. • highly reactive agents fail to reach deeper zones of biofilm,because biofilm act as an ion exchange resin removing such molecules from solutions. • Extra cellular enzymes like β lactamases,formaldehyde lyase and formaldehyde dehydrogenase may become trapped and concentrated in the extracellular matrix,thus inactivating some antibiotics • super resistant bacteria are identified within biofilm,these cells have multiresistant pumps that can extrude antimicrobial agents .
- 48. MICROBIOLOGIC SPECIFICITY OFPERIODONTAL DISEASES 1880 1900 1930 1960 1990 2000 Sp pathogens identified for many diseases Search begins for oral pathogens in plaque Non sp plaque Hypothesis Diseases linked to constitutional defects Sp plaque hypothesis Treatment aimed at Causative agent Golden age of microbiology Plaque control Biofilm
- 49. MICROBIOLOGIC SPECIFICITY OF PERIODONTALDISEASES –Nonspecific plaque hypothesis N – onspecific plaque hypothesisPP97 Proposed by Walter Loesche in 1976(correlated both age and amount of plaque in development of peridontal disease • ProposAccording to this hypothesis “,periodontal disease results from the elaboration of noxious products by entire plaque flora.” • Small amount of plaque -neutralized by host • large amount of plaque- overwhelm host defences. Short comings of nonspecific plaque hypothesis • Some individuals with large amount of plaque and calculus as well as gingivitis never developed destructive periodontitis, • In destructive periodontitis ,certain sites showed more bone loss,these finding were inconsistent with the findings that all plaque are pathogenic. • .
- 50. Specific plaque hypothesis •Specific plaque hypothesis states that only certain plaque is pathogenic, and its pathogenicity depends on the presence of or increase in specific microorganisms. NEWMAN MG , SOCRANSKY SS (1977),ADI et al ,(1978) • Concept predicts that plaque harbouring specific pathogens result in periodontal disease • Presence of Aa in localized aggressive periodontitis and “red complex” bacteria in the pathogenesis of periodontitis is in accordance to specific plaque hypothesis.
- 51. Ecologic plaque hypothesis •Marsh and coworkers 1990 ,developed ecologic plaque hypothesis. • 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 • (onset of immune disorders, changes in hormonal imbalance like pregnancy or environmental factors like smoking and diet) • States that -disease can be prevented not only by inhibiting the putative pathogens, but also interfering with the environmental factors driving the selection & enrichment of these bacteria.
- 52. Methods of toothand tissue destruction by plaque • By enzymes: • Enzymes produced by the microorganisms-Hyaluronidase and proteases -They may cause breakdown of mucopolysaccharides and proteins present in the inter-cellular substance of junctional epithelium. • Endotoxins: • Endotoxin is a lipopolysaccharide complex and is a constituent of the cell wall of all gram–ve bacteria. Endotoxin has a chemotectic effect, so it induces phagocytosis by neutrophillic granulocytes • Metabolites: • Microorganisms in the plaque leads to the accumulation of various metabolites due to their action on carbohydrates and proteins. Hydrogen sulfide and ammonia are two such metabolites produced by the plaque microorganisms.
- 53. VIRULENCE FACTORS OFPERIODONTAL PATHOGENS • Eliminating the specific microorganisms will not cure the disease, but focus on the specific molecules (virulence factors) that contribute to the disease must be eliminated. Virulence factors of periodontal microorganisms: Factors that promote colonization(adhesins) Toxins and enzymes that degrade host tissues Mechanism that protect pathogenic bacteria from host
- 54. ADHESIVE SURFACE PROTEINSOR FIBRILS • Bacteria must adhere to cells or or tissues provides surface structures for attachment like Fimbriae or pili • Eg.strains of P.gingivalis produces two types of fimbriae,known as major and minor fimbriae. • Major and minor fimbriae interact with oral streptococci such as S.gordonii • Major fimbriae also binds to host extracellular matrix proteins.
- 55. FACTORS THAT PROMOTETISSUE DESTRUCTION • Many bacterial proteins that interact with the host cells are recognized by the immune system and trigger immune responses. • Fimbriae of P.gingivalis and Aa are highly antigenic which produces tissue inflammation which contributes to tissue destruction. • Trypsin like protease( P.gingivalis, T.forcythia)- provides proteases in the form of aminoacids-produces extensive degradation of the host. • P.gingivalis produces three enzymes kown as gingipains responsible for 85% of the total host protein degradation activity. •
- 56. MECHANISM THAT PROTECTPATHOGENIC BACTERIA FROM HOST • Production of an extracellular capsule • Proteolytic degradation of host innate or acquired immunity components • Modulation of host responses by binding serum components on the bacterial cell surface • Invasion of gingival epithelial cells- polysaccaharide capsules-which protects the bacterial cells from the host immune system. • Gingipains produced by P.gingivalis are shown to degrade complements
- 57. Conclusion Dental biofilm isa complex, organized microbial community that is the primary etiologic factor for the most frequently occurring oral diseases. Although the dental biofilm cannot be eliminated, it can be controlled with comprehensive mechanical and chemotherapeutic oral hygiene practices Daily brushing, interdental cleaning, and antimicrobial mouthrinses that carry the ADA Seal of Acceptance increases the likelihood of periodontal disease prevention and reduction
- 58.