Periodontal Diseases - Microbiology PDF

PERIODONTAL DISEASES MICROBIOLOGY Pinelopi Pani DDS, CAGS, MS OUTLINE • What is normal flora? • How does bacteria cause disease? • What are the bacterial complexes in Gingival and Periodontal diseases? • What are the most common microbial species in Periodontitis? The human body contains...

PERIODONTAL DISEASES MICROBIOLOGY Pinelopi Pani DDS, CAGS, MS OUTLINE • What is normal flora? • How does bacteria cause disease? • What are the bacterial complexes in Gingival and Periodontal diseases? • What are the most common microbial species in Periodontitis? The human body contains 10 times more bacteria than human cells . The bacterial population comprises 2 kg of the total body weight in a normal, healthy human being. Louis Pasteur • Pasteur hypothesized that the normal bacterial flora was essential to life. • He theorized that foreign microorganisms were responsible for infecting animals and humans and causing disease. • He discovered the processes of fermentation, pasteurization and vaccines. He proposed preventing the entry of microorganisms into the human body, leading Joseph Lister to develop antiseptic methods in surgery. Resident / Normal Microflora • Majority are found on exposed surfaces of the body Skin Mouth Mucous membranes • Each surface is distinctive, having characteristic biological and physical properties • Composition varies significantly within the mouth Respiratory track Digestive track WHEN DOES THE NORMAL FLORA START TO FORM IN THE ORAL CAVITY? The mouth presents a succession of different ecological situations with age, and this corresponds with changes in the composition of the normal flora. Tooth Eruption: Nondesquamating surface: Streptococcus mutans, Streptococcus parasanguis Gingival crevice area: Anaerobic species, yeasts Intra-uterine/Birth: Sterile 4-12 hrs after birth: Lactobacilli/streptococci (feeding) Puberty: Increase # of teeth/ changes in diet = complex oral flora (anaerobes: bacteroides, spirochetes) Adult age: dental plaque/ Presence of periodontal diseases influence the number and types of bacteria present Old Age: With loss of teeth, microorganisms decrease (Lactobacilli, Strept. mutans and yeasts) except with complete dentures Adult Oral Flora • AEROBIC Strept. mutans, Salivarius, sanguis and mitior, together with Staph. epidermidis Neisseria spp., diphtheroids. • ANAEROBIC Lactobacilli, bacteroides, actinomyces, veillonella and spirochetes. • Candida, as a member of the oral flora is present in 20– 80% of adults. • Protozoa may be detected in the oral cavity: Entamoeba gingivalis and Trichomonas temax. Bacterial flora around dental implants is similar to that found around the natural teeth. The Mouth as a Microbial Habitat • 700 different species • The oral environment supports microbial growth •“Incubator environment” for bacteria, viruses, fungi, protozoa • Metabolic environment determined by • Temperature • Moisture • Nutrients • pH • Oxygen levels • Metabolic inhibitors The Mouth as a Microbial Habitat • Most oral bacteria are harmless commensals under normal circumstances • Provide benefits to the host • The importance of commensal microbiota is clearly illustrated by the development of yeast infections when the normal microbiota is reduced. The Mouth as a Microbial Habitat • Under specific conditions can cause DISEASE • Increased mass • Increased pathogenicity • Suppression of commensal or beneficial bacteria • Reduced host response The Mouth as a Microbial Habitat • The normal flora occupy available colonization sites which makes it more difficult for other microorganisms (nonindigenous species) to become established. • The oral flora are the usual cause of various oral diseases in humans, including abscesses, dental caries, gingivitis, and periodontal disease. The oral flora contribute to host nutrition through the synthesis of vitamins, and they contribute to immunity by inducing low levels of circulating and secretory antibodies that may cross react with pathogens. • If oral bacteria can gain entrance into deeper tissues, they may cause abscesses of alveolar bone, lung, brain, or the extremities. • The oral bacteria exert microbial antagonism against nonindigenous species by production of inhibitory substances such as fatty acids, peroxides and bacteriocins. • Periodontal Health vs Disease HEALTH A state of balance where multiple bacteria coexist with the host and no damage occurs to the bacteria or the host tissues. Periodontal Health vs Disease DISEASE A disruption of this balance that causes alterations to the host and bacterial biofilm which ultimately results in destruction of teeth and the connective tissues of the periodontium. Bacterial adherence 6 Major Niches • Supragingival surfaces • Subgingival regions • Soft tissue epithelium • Tongue • Tonsils • Saliva Disease Producing Microorganisms Attach to the tooth surfaces and colonize Cause carious lesions of the enamel and root surfaces, in pits and fissures, and on smooth surfaces. Cause inflammatory changes in the periodontium that can lead to the destruction of the tissues that support the teeth. Supragingival vs Subgingival Environments • Different metabolic environments!!! • Different nutrient sources • Different pH • Different oxygen levels ENVIRONMENT DICTATES ECOLOGY DIFFERENT ENVIRONMENTS Bacteria embedded in a matrix of polymer Dental Plaque Biofilm Complex microbial community (bacterial and salivary origin) Structurally and functionally organized HOW DO BACTERIA CAUSE DISEASE? Acquisition Adhesion or Retention Avoidance of Elimination Multiplication Prosperity and Longterm Survival Elaboration of Virulence factors Initial Survival Acquisition, Adherence and Retention • The ability of an organism to attach to a surface. • Early colonizers are Gram-positive cocci bacteria which produce EPS (extracellular polysaccharide) that will glue the cells to the surface and eventually form the biofilm matrix. • Biofilms are responsible for the interaction of microbes with each other as well as with interfaces. • Initial adhesion is dictated by physicochemical variables. • Initial adhesion between bacteria and nonliving surfaces is usually mediated by nonspecific (e.g. hydrophobic) interactions, whereas adhesion to living surfaces is usually accomplished through specific molecular docking mechanisms. • Later on, during the biofilm growth, cellto-cell adhesion can be mediated by specific adhesins or cell surface modifications such as pili or fimbriae. • Co-aggregation of genetically distinct bacteria via specific molecules has been shown common in the development of oral mixed-species biofilms. Initial Survival • Bacteria requires not only a stable surface, it also needs a source of nutrients. • Gingival crevicular fluid (GCF) is a rich source of nutrients such as essential energy sources like glucose and iron. But it also contains host-derived elements which can decrease bacterial survival such as antibodies and complement. Prosperity and long term survival • The development of a “biofilm”. • A mature biofilm with its complex architecture provides niches with distinct physicochemical conditions, differing e.g. in oxygen availability, in concentration of diffusible substrates and metabolic side products, in pH, and in the cell density. • Cells in different regions of a biofilm can exhibit different patterns of gene expression “Quorum Sensing”. • The natural biofilm is a complex, highly differentiated, multicultural community much like our own city. • A mixed species biofilm is a dynamic community harboring bacteria that stay and leave with purpose, compete and cooperate, share their genetic material, and fill distinct niches within the biofilm. Avoidance of Elimination A key survival strategy of all pathogens!!! Survival Strategies for Microbes • Some organisms are able to sequester themselves within the host tissues, such as epithelial cells, WBCs, or the dentinal tubules, thus providing a pool for recolonization or periodontal pockets once biomechanical therapy has been provided to the patient. • The use of antibiotics is counter-productive in chronic periodontitis, because by the time the active agent has penetrated the biofilm, its concentration is too low to be effective and resistant strains of bacteria can emerge. Multiplication Potential periodontal pathogens need to reach a certain critical mass within the subgingival biofilm in order to survive. Further multiplication is required for disease to occur. Elaboration of Virulence Factors Latin virulentus, which means "full of poison.“ What are virulence factors? • The virulence factors are typically proteins or other molecules of bacterial origin, produced to cause disease in the host and allow the pathogen to thrive • Virulence factors can be enzymes, metabolic waste products or toxins. How do virulence factors cause disease? • Production of toxic cell constituents (endotoxins, surface components, capsular components, enzymes, etc.) • Disruption of host cell membranes or stimulate endocytosis into host cells • Inhibition of the host's immune system defenses Common Virulence Factors Adhesion and colonization Fimbriae, Capsule, Microbial antagonisms/synergy Evasion of host response Tissue Destruction Leukoaggressins, Chemotaxis Inhibitors, Phagocytosis Inhibitors, Proteases against: Immunoglobulins, Complement components, Modulation of bacterial antigens Surface Fc receptors, Fibroblast Inhibitors, Leukotoxins, T suppressor cell stimulation, Intracellular invasion of host cells, Plasma Proteinase Casacade Collagenase, Hyaluronidase, Lipopolysaccharide (LPS), Sulfur Compounds, Endotoxin mediated bone resorption, Acid Phosphatases, Epithelial cell toxin, Fibrolysin, Siderophores, Induction of destructive hostderived cytokines, Proteinase inhibitors, Catalases, Mannan, Metabolic byproducts SUMMARY The ability of bacteria to cause disease is based on: • The number and type of infecting bacteria •The route of entry into the body • The effects of host defense mechanisms • Extrinsic characteristics of bacteria • Intrinsic characteristics of the bacteria “virulence factors” WHAT HAPPENS DURING PERIODONTAL DISEASE? • 40 subgingival microorganisms • Complexes colonize and proliferate at an early stage • Orange complex is the bridge of early and late colonizers • Red complex bacteria are associated with BOP and PD Socransky 1998, JCP The composition of the different color-coded complexes is based on the frequency with which the organisms are encountered together: • The blue & purple complex consists of grampositive cocci and rods • The yellow complex is comprised of grampositive facultative cocci • The green complex contains gram-positive and negative rods and cocci, but non-motile species • The orange complex demonstrates a higher concentration of gram-negative rods and cocci, with some motile species present • The red complex is exclusively gram-negative, motile anaerobes Socransky 1998, JCP MICROBIAL COMPOSITION Bacteria that are associated with periodontal health include primary or early colonizers • Streptococcus sanguis • Streptococcus mitis • Veillonella spp. • Gemella spp • Capnocytophaga PERIODONTAL HEALTH Primarily organisms from Blue, Yellow, Green and Orange Complexes: • Streptococcus sanguis • Streptococcus oralis • Capnocytophaga ochracea • Actinomyces naeslundii • Eikenella corrodens • Fusobacterium Nucleatum GINGIVITIS • Aggregatibacter actinomycetemcomitans • Porphyromonas gingivalis • Tannerella forsythia • Treponema denticola • Prevotella intermedia • Fusobacterium nucleatum • Eikenella corrodens • Campylobacter rectus (Wolinella recta) • Peptostreptococcus micros • Streptococcus intermedius PERIODONTITIS WHAT ROLE DOES PLAQUE PLAY IN THE ETIOLOGY OF GINGIVAL/ PERIODONTAL DISEASES? THE NON-SPECIFIC PLAQUE HYPOTHESIS ONLY THE QUANTITY OF BACTERIA MASS IS IMPORTANT, AND THE ABUNDANT AMOUNT OF PLAQUE CAN CAUSE DISEASE. QUANTITY > QUALITY THE NON-SPECIFIC PLAQUE HYPOTHESIS • Periodontal disease were believed to occur due to accumulation of plaque over the period of time. • Hypothesis states that the periodontal breakdown results from “elaboration of noxious products by entire plaque flora”- Miller 1890 • Updated Hypothesis- “Any microbial colonization of sufficient quantity in the gingival crevice causes at least gingivitis ”- Theilade 1986 • This was supported by the fact the non-pathogenic plaque was never observed. THE NON-SPECIFIC PLAQUE HYPOTHESIS • WHY DO SOME PATIENTS HAVE PLAQUE AND CALCULUS ACCUMULATION FOR SEVERAL YEARS, BUT THEY DEVELOP ONLY GINGIVITIS WITHOUT ANY ATTACHMENT LOSS? • WHY DO SOME PATIENTS SHOW SEVERE ATTACHMENT LOSS WITH RELATIVELY GOOD ORAL HYGIENE? • WHY DO THE SEVERITY AND SPEED OF PROGRESSION DIFFER FROM TOOTH TO TOOTH IN THE SAME INDIVIDUAL OR EVEN SHOW DIFFERENT PATTERN AROUND DIFFERENT SURFACE AT THE SAME TOOTH? THE SPECIFIC PLAQUE HYPOTHESIS SPECIFIC MICRO-ORGANISMS WERE NECESSARY FOR THE DEVELOPMENT OF PERIODONTAL DISEASE - Loesche 1976 QUALITY > QUANTITY THE SPECIFIC PLAQUE HYPOTHESIS • Hypothesis states that “only certain plaque is pathogenic and its pathogenicity depends on the presence of amount of specific bacteria”- Slots 1977 • Some suspected periodontal pathogens can be isolated from clinically “healthy” mouths • Not all genotypes of the same periodontal pathogen have the same capacity to induce disease • This hypothesis was widely accepted due to recognition of A. actinomycetemcomitams as a pathogen in Localized Aggressive Periodontitis THE NON-SPECIFIC PLAQUE HYPOTHESIS • Why can some suspected periodontal pathogens be isolated from clinically “healthy” mouths? • Not all genotypes of the same periodontal pathogen have the same capacity to induce disease. THE ECOLOGICAL PLAQUE HYPOTHESIS A CHANGE IN KEY ENVIRONMENTAL FACTORS WILL CAUSE A SHIFT IN THE BALANCE OF RESIDENTIAL BACTERIAL SPECIES, WHICH COULD THEN PREDISPOSE A SITE TO DISEASE. - Marsch 1994 • Some researchers consider the pathogenic flora to occur due to environmental disturbances that results in predisposition to disease. • The selection of pathogenic bacteria is related to changes in the environment. • Any species with a relevant traits can cause the disease process. KEYSTONE PLAQUE HYPOTHESIS Disease occurs with: • Specific pathogenic bacteria • Ecological changes • Host specific changes Hajishengallis et al. 2012 • Certain low-abundance microbial pathogens can orchestrate inflammatory disease by remodeling a normally benign microbiota into a dysbiotic one. • A shift in the ecology of the biome results in an imbalance and exorbitantly high numbers of certain keystone pathogens. TOP 3 BACTERIA CAUSING PERIODONTAL DISEASE RECOGNIZED BY THE AAP • Porphyromonas gingivalis • Tannerella forsythia (Bacteroides forsythus) • Aggregatibacter actinomycetemcomitans • Also, overwhelming evidence for Spirochetes, Prevotella intermedia, Fusobacterium nucleatum PORPHYROMONAS GINGIVALIS (P.g.) Black pigmented Gram-negative Anaerobic Major site of colonization is the gingival sulcus Evidence for its role in the pathogenesis of chronic periodontitis. • Considered as one of the putative periodontal pathogens and usually found in pockets and colonizes deeper in the tissues • Frequently isolated from subgingival plaque • Major site of colonization is the gingival sulcus • Cell surface adhesion molecules on the surface of Porphyromonas interact with other bacteria, epithelial cells, and extracellular matrix proteins • P. gingivalis is thought to spread through tissue, destroy tissue, and evade host defenses Microbial and Biochemical Properties: •Non motile, Gram negative, rod shaped •Anaerobic and forms black colonies of agar samples PORPHYROMONAS GINGIVALIS (P.g.) • P. gingivalis cytotoxic metabolic end products, which include butyrate, propionate, have low molecular weights which allows them to easily penetrate periodontal tissue and disrupt the host cell activity. • Elevated in periodontal lesions, rare in health • Elimination or suppression resulted in successful therapy PORPHYROMONAS GINGIVALIS (P.g.) Virulence Factors: • Bacterial capsule has the major virulence effect. The antiphagocytic activity against the host cells allows the microbe in penetration in the host tissue while it multiplies there • Lipopolysaccharides (LPS), Ig and complement proteases, capsule, other antiphagocytic products: Involved in evading (modulating) host responses • Fimbriae, exopolysaccharides, hemagglutins, vesicles, outer membrane: Involved in colonization and attachment • Proteinases, hemolysins: Involved in multiplying • Collagenase, trypsin like protease, gelatinase, protein antigens, Proteinases “gingipains”, fibrinolytic, keratinolytic, and other hydrolytic activities: Involved in damaging host tissue and spreading PORPHYROMONAS GINGIVALIS (P.g.) TANNERELLA FORSYTHIA (T.f.) Formerly Bacteroides forsythus Spindle-shaped Non-pigmented Gram-negative Difficult to culture Non-motile Highly pleomorphic rod Anaerobic Associated with “refractory” periodontitis • First isolated at “The Forsyth Institute” and formerly named Bacteriodes Forsythus • Commonly seen in endodontic infection, early stage and progressing periodontitis and periimplantitis Microbial and Biochemical Properties: • Non motile, Gram negative, spindle shaped • Obligate anaerobes and non pigmented TANNERELLA FORSYTHIA (T.f.) Virulence Factors: • Trypsin like and Proteolytic enzymes • Leucine rich surface protein (BspA): triggers bone resorption pro-inflammatory enzymes • Apoptosis inducing activity • Hemagglutinin TANNERELLA FORSYTHIA (T.f.) Aggrecatibacter actinomycetemcomitans (A.a.) Formerly Actinobacillus actinomycetemcomitans “Star”- shaped internal structure Gramp-negative rod Facultative anaerobe Short, straight or curved Growth enhanced by the presence of CO2 Ferments most sugars Possibly related to systemic infections Evidence of antibody titers in localized aggressive periodontitis patients • A.a. was first demonstrated in dental plaque in 1975 • It was recognized as the cause of juvenile periodontitis by Newman et al 1975 and Slots in 1976 • Aggregatibacter actinomycetemcomitans was reclassified from Actinobacillus actinomycetemcomitans in 2006 Aggrecatibacter Actinomycetemcomitans (A.a.) Microbial and Biochemical Properties: • Gram negative, “Star” shaped internal structure • Non motile, facultative anaerobe • 0.4x1 micron in size • Capnophilic • Evidence of antibody titers in localized aggressive periodontitis patients Aggrecatibacter Actinomycetemcomitans (A.a.) Serotypes: • a, b, c, d, e, f- six serotypes • “a” strain, frequently in oral cavity, variable leukotoxin expression • “a” and “b” are most commonly associated with oral infections while “c” is seen in extra-oral infections • “b” strain, most frequently in localized aggressive periodontitis, strong leukotoxin expression while “a” is commonly seen in chronic periodontitis • “c” strain, low leukotoxin expression • Intrafamilial transmission seen- family members can be affected by the same serotype Aggrecatibacter Actinomycetemcomitans (A.a.) Suspected Virulence Factors: • • • • • • • • Leukotoxin (RTX): Induces apoptosis of WBC Cytolethal distending toxin (CDT) Chaperonin 60 LPS: apoptosis, bone resorption. etc. OMP, vesicles Fimbriae Collagenase Immunosupprssive Factor Aggrecatibacter Actinomycetemcomitans (A.a.) Factors promoting colonization and persistence: •Adhesins •Invasins •Bacteriocins •Antibiotic resistance Factors that destroy host tissues: •Cytotoxins •Heat Shock Proteins (HSP) •Collagenase •Bone resorption agents Factors that interfere with host defense: •Leukotoxins: Promote WBCs apoptosis •Lipopolysaccharides (LPS)- found in outer membrane of Gram-negative bacteria •Chemotactic Inhibitors •Immunosuppressive proteins •Fc binding proteins •Cytolethal distending toxins (CDT) Aggrecatibacter Actinomycetemcomitans (A.a.) SPIROCHETES Treponema Denticola (T.d.) Gram-negative Increased numbers in deep periodontal pockets Anaerobic Difficult to distinguish individual species (15 subgingival species) Spiral Highly motile Main virulence factor: dentilisin Associated with Necrotizing Periodontal Disease PREVOTELLA INTERMEDIA (P.i.) Strains of “P. intermedia” separated into two species, P. intermedia and P. nigrescens Hemagglutination activity: induce an increased release of MMP-8 and MMP-9 in pockets Gram-negative Adherence Anaerobic Round-ended Non-motile Induces alveolar bone loss Successful periodontal therapy leads to decreased levels of these organisms Strains are divided into two species: P. Intermedia and P. Nigrescins • Found in chronic periodontitis and induces hemagglutination activity • Induces alveolar bone loss • Appropriate treatment can lead to decrease in P. Intermedia • Microbial and Biochemical Properties: • Gram negative, round ended, non motile • Anaerobic, less virulent and less pathogenic than P. gingivalis PREVOTELLA INTERMEDIA (P.i.) FUSOBACTERIUM NUCLEATUM (F.n.) Gram-negative Anaerobic Spindle-shaped rod Apoptosis activity Bridging species Induces invasion of epithelium cells The most common isolates found in subgingival plaque samples (7-10%) Recognized as part of the subgingival microbiota for over 100 years Prevalent in subjects with periodontitis and periodontal abscesses TAKE-HOME MESSAGE • Bacterial Plaque is the main etiology of Periodontal Disease! • An immune-inflammatory response develops in the periodontal tissues in the chronic presence of dental plaque bacteria and results in the destruction of the periodontal structures. • General oral cleanliness and health depends on the daily removal of bacterial deposits. • The accumulation of microorganisms (bacteria) on the teeth and soft tissues contribute to: • • Disease • Unpleasant esthetics • Halitosis TAKE-HOME MESSAGE • The bacteria resident in the biofilm known as dental plaque represent the most significant environmental risk factor for both dental caries and periodontal inflammatory disease. • The association of microbial species with the onset of periodontal disease has greatly strengthened the belief that the periodontal diseases are opportunistic infections. • Laboratory Studies on germ-free animals (Gnotobiotics) -In the absence of bacteria there is: Optimal dental/ periodontal health THANK YOU [email protected]