1.1 DR. SWAPNIL BORKAR I-YEAR POST GRADUATE STUDENT DEPARTMENT OF PERIODONTICS & ORAL IMPLANTOLOGY S.D.K.S DENTAL COLLEGE NAGPUR

2. Need to understand microbial etiology Postulate of Robert Koch and Limitation Socranskys Ceriterion Microbial complex Virulence : History Functions Definitions Virulence factor : Type outer membrane Environmental Stress Biological activity Outer Membrane Proteins Outer membrane vesicles Lipopolysaccharide And Associated Macromolecules Peptidoglycan as aVF PhysiologicalVirulence of commensal opportunistic pathogens VF and Host response VF of P. gingivalis 2 3. Microbial etiology of periodontal diseases is needed to be well understood because of two major reasons Knowledge of the etiological agents of periodontal diseases would help in the selection of appropriate treatment . It would provide a useful therapeutic approach to control and prevent periodontal diseases. (manufacturing of vaccines against various pathogens prior to the onset of diseases.) 3 4. Microbiology of periodontal diseases is difficult to understand and study due to difficulty in sample collection, cultivation and identification of isolates. Periodontal infections are mixed infections and microbiota is very complex, making hard to distinguish between secondary invaders and true pathogens. Periodontal diseases appears to be episodic and thus, there is difficulty in differentiating between active and inactive site for sampling. 4 5. Robert Koch,1843-1910, Germany In 1890 Robert Koch postulated guidelines to establish a standard for evidence of causation in infectious disease (based on early work by Henle): The microorganisms should be constantly associated with the lesion of the disease. It should be possible to isolate the bacterium in pure culture from the lesions. Inoculation of such pure culture into suitable animal model should reproduce the lesion of disease. It should be possible to reisolate the microbes in pure culture from the lesions produced in the experimental animals. 5 6. Limitations of Kochs postulates : Inability to culture all organisms (large size spirochetes in periodontitis can not be grown in pure culture.) Lack of good model system :host range in restricted to humans or to animal species in which human diseases cannot be reproduced (p. gingivalis does not usually coloninize in animals.) The actual state of periodontal disease progression can be difficult to determine. 6 7. The same clinical sign and symptoms my be produce by several organisms which may give rise to variety of clinical disease features. Thus the inherent problem with kochs postulates in showing causality is the primarily concern with the infecting agent and lack of consideration for the environment or the influence of the host in disease development. 7 8. Socranskys criterion : Sigmund Socransky, a researcher at the forsyth dental center in Boston, proposed criteria by which periodontal microorganisms my be judge to be potential pathogens. Association with the lesion : association is the 1st requirement for a microbe to cause a periodontal lesion. Pathogen should be associated with disease, as evident by inc. in the no. organisms at diseased sites. Elimination of suspected organism : organism should be eliminated or decreased in sites that demonstrate clinical resolution of disease with treatment. 8 9. Host response to organisms: microbe should be able to demonstrate a host response, in the form of an alteration in the host cellular or humoral immune response. Animal studies : pathogen should be capable of causing disease in experimental animal models. Virulence factor : potential pathogens should be capable of producing virulence factor which cause destruction of the periodontal tissues. 9 10. Microbial complex It has been estimated that nearly 700 bacterial taxa, phylotypes and species, which show some structural organization in the biofilms (Patridge et al.1985), can colonize the oral cavity of humans, although it remains unclear how this multitude of bacteria compete, coexist, and or synergize to initiate chronic and aggressive periodontal disease process 10 11. 11 12. 12 Microorganism associated with periodontal health 13. Virulence : History : The term virulence is generally defined as the relative ability of a organism to cause disease or to interfere with a metabolic or physiologic function of its host. The word is derived from the Latin,virulentus, or full of poison. Vir is a latin word meaning strength. In this context it means "very destructive". Humans are the most virulent organism ever produced on this planet. Thus virulence refers to the ability of a microbe to express pathogenicity (eg; virulent), which is contrasted with nonpathogenic or avirulent organisms. 13 14. The absence of a susceptible host renders a potentially virulent microbe, avirulent. As early as 1924, Kolmer attempted to define virulence as characteristic comprising at least two intrinsic microbial factors, toxicity, and aggressiveness, or the ability to invade a susceptible host. Poulin & Combs defined the concept of virulence in terms of the type of molecules being produced by the microbe. As such, they defined virulence in terms of virulence factors, that is, components of a microbe which when present harm the host, but when absent (i.e. mutation) impair this ability. This mutation does not affect the ability of the microbe to grow and reproduce. 14 15. Thus virulence is not a separate property of the microbe, but is a complex interaction between the microbe and its host; this interaction being dependent upon many extrinsic factors of the environment. Virulence factors can have a multitude of functions: The ability to induce microbe-host interactions (attachment) The ability to invade the host The ability to grow in the confines of a host cell The ability to evade/interfere with host defenses 15 16. Virulence is defined as: Property of invasive power [H Zinsser. 1914] Strength of the pathogenic activity [WW Ford 1927] Relative capacity to enter and multiply in a given host [T. Smith. 1934] Pathogenicity: the capacity of a microbe to produce disease [Watson-DW Brandly 1949 ] Synonym for pathogenicity: the capacity of a microbe to cause disease [Youmans, GP., Paterson, PY Sommers, HM. 1975] 16 17. Degree of pathogenicity [Wood-WB and Davis-BD. 1980] Relative capacity to overcome available defenses [Sparling PF. 1983] Percent of death per infection [Ewald, PW. 1993] Disease severity as assessed by reduction in host fitness postinfection [Read AF. 1994] Measure of the capacity to infect or damage a host [Lipsitch M Moxon ER. 1997] Relative capacity to cause damage in a host [Casadevall A Pirofski LA1999] 17 18. Virulence factor is defined as: Microbial products that permit a pathogen to cause disease [H. Smith. 1977] A component of a pathogen that when deleted specifically impairs virulence but not viability [Youmans, GP., Paterson, PY, Sommers, HM. 1975] A component of a pathogen that damages the host; can include components essential for viability including modulins [Casadevall A Pirofski LA1999, Henderson B; Poole S;Wilson M 1996] 18 19. VIRULENCE FACTORS Important to the role of these prokaryotes in attacking the host is the ability of several of them to directly attack host tissues by proteolytic digestion, as well as their ability to elaborate large amounts and types of virulence factors- LPS outer membrane proteins vesicles Toxins enzymes, 19 20. Which act both directly and indirectly through the activation of a variety of macromolecules that themselves are destructive to the host. The elaboration of several of these virulence factors appears to be closely regulated by the expression of host factors (i.e., hemin) that appear in several in vivo animal models of pathogenesis to control the virulence of the specific microbial species. 20 21. 21 22. For the most part the establishment of a bacterial infection whether in the respiratory, alimentary, urogenital tract, or in the oral cavity (associated with hard and soft tissue surfaces) requires at least five integrated events: (1) an initial colonization of the tissue surface; (2) penetration of this surface either directly or indirectly; (3) emergence and multiplication of the invading bacterium in this environment; (4) the eventual damage of the host's tissues; and (5) the survival of the invading bacterium in the ecological niche by evasion of the host's defense mechanisms. 22 23. 23 24. 24 25. 25 26. 26 27. Colonization The ability to adhere to host cells and resist physical removal or the establishment of pathogen at the appropriate portal of entry. Pathogens usually colonize host tissues that are in contact with the external environment. 27 28. Virulence factor that promote Bacterial Colonization Using pili (fimbriae) to adhere to host cells Using adhesins to adhere to host cells Using biofilms to adhere to host cells 28 29. Invasiveness The ability of a pathogen to invade tissue Invasiveness encompasses 1. Mechanisms for colonization (adherence and initial multiplication) 2. Production of extracellular substances (invasins) that promote the immediate invasion of tissues 3. Ability to bypass or overcome host defense mechanisms which facilitate the actual invasive process. 29 30. 30 31. 31 32. 32 33. 33 34. 34 35. 35 36. 36 37. 37 38. The identification of pathogen (s) of an infectious disease, including periodontal diseases, leads inevitably to the question how do these organisms cause the disease? By far the greatest number of studies that have sought virulence factors of known or presumed periodontal pathogens have examined factors produced by P. gingivalis. Holt and Ebersol 2000: have provided a global overview of the microbial factors that are thought to cause virulence in bacterial infections and have described specific examples of such factors that are produced by periodontal species, in particular, the Red Complex species P. gingivalis,T. forsythia, andT. denticola The classic progression of the development of periodontitis with its associated formation of an inflammatory lesion is characterized by a highly reproducible microbiological progression of a Gram- positive microbiota to a highly pathogenic Gram-negative one. 38 39. Among these "putative periodontopathic species" are members of the genera Porphyromonas Bacteroides Fusobacterium Wolinella Actinobacillus Capnocytophaga, and Eikenella. While members of the genera Actinomyces and Streptococcus may not be directly involved in the microbial progression, these species do appear to be essential to the construction of the network of microbial species that comprise the subgingival plaque matrix. The Gram-negative eubacteria that are now recognized as putative periodontopathogens have developed a plethora of mechanisms by which they can invade the host, and evade destruction or neutralization by the host's defenses and survive in this complex environment of diverse microbial species that compete and synergize for survival. 39 40. These mechanisms include the development of surface slimes and capsules that function to interfere with host defenses (phagocytosis), as well as initiate the formation of abscesses. Essential in many cases to the emergence and survival of a bacterium in a host environment is its adherence to tissue surfaces. The periodontal pocket is unique in that it contains four types of colonizing surfaces: mineralized tissues (cementum and dentin), keratinized epithelium (oral sulcular epithelium), nonkeratinized epithelium (junctional epithelium), as well as the surfaces of the eubacteria themselves. Without a doubt, all of the species that invade or "pass through the oral cavity have access to the periodontal pocket; however, only those eubacteria that are able to successfully colonize these different surfaces will survive in this environment. 40 41. Adhesion to these surfaces usually (but not always) occurs by the interaction of thin surface appendages to specific host receptors, fimbriae and capsules. The invading bacterium has also developed complex macromolecules in its surface layer that play an active role in the infective process. These macromolecules include lipopolysaccharides, outer membrane-associated proteins, enzymatic molecules, toxins, and bacteriocins. The outer membrane macromolecules are not only detrimental to host cell integrity and viability, but may also have a direct effect on the character of the resident microbiota. 41 42. OUTER MEMBRANE Both the Gram-positive cell wall and Gram negative cell wall (cell envelope) are complex structures. The Gram-negative cell envelope consists of a trilaminar outer membrane and an underlying peptidoglycan layer. The thin peptidoglycan layer that maintains cell shape is linked to the outer membrane by a lipoprotein of low molecular weight through its diaminopimelic acid in the peptide of the peptidoglycan. 42 43. The linkage of the lipoprotein is through an interaction between the N-terminal lipid of the lipoprotein and the hydrophobic (i.e., lipophilic) domain of the outer membrane. Several unique and physiologically essential proteins are major components of the outer membrane. These include the porins, integral/matrix proteins, and the lipopolysaccharide. 43 44. Environmental Stress In addition to the possible regulation of bacterial virulence through the surfaced layer, eubacteria also regulate virulence by controlling the synthesis of specific outer membrane proteins (OMPS). They accomplish this by the action of specific signals from their immediate growth environment. Numerous investigators have shown that iron regulates the expression of unique OMPS that are essential for a variety of cell functions, including transport and virulence. McKee et al have shown that the virulence of P. gingivalis strain W50 as expressed in a mouse abscess model was regulated by the level of hemin in the growth medium 44 45. Excess hemin (i.e., concentrations above 2.5 g/ml) in the growth medium resulted in increased P. gingivalis virulence, while low hemin concentrations (i.e., up to 0.5 g/ml) produced cells with decreased virulence. Bramanti and Holt (unpublished) showed that the pretreatment of mice with the iron chelating agent Desferal protected them from lethal doses of bacteria grown in either limited or excess hemin. These data provide strong evidence that support the role of iron and iron availability in the pathogenesis of P. gingivalis. 45 46. Biological Activity The majority of the studies have been concentrated on P. gingivalis. Unfortunately, many of these studies have involved the use of cell extracts, and for the most part interpretation of the results remains unclear since these extracts contain a variety of bacterial components in addition to the outer membrane. In one of the few studies of the biological activity of the oral Eikenella spp., E. corrodens, Progulske et al1984. isolated both the LPS and outer membrane and found both exhibited potent bone resorptive activity 46 47. Outer Membrane Proteins Porphyromonas and Bacteroides Kennell and Holt and Williams and Holt have made an exhaustive examination of the distribution of both the omps and major outer membrane proteins (momps) in a large number of the oral P. gingivalis strains. What was evident in their results was that the distribution of both outer membrane proteins in these P. gingivalis strains was very complex when compared with, for example, E. coli. In addition, there was significant strain specificity. . 47 48. Fusobacterium Several studies of the cell envelope of F. nucleatum have revealed that in addition to the usual complement of omps, F. nucleatum also contained at least two momps in the 40 to 46 kDa region Actinobacillus One of the few studies of the protein composition of the cell envelope from A. actinomycetemcomitans was that of DiRienzo and Spieler. The cell envelope polypeptides were remarkably conserved across the strains, with three momps (Env-1, -2, and -3) being observed. 48 49. Eikenella Progulske and Holt 1984 andWagley et al have examined the distribution of omps of E. corrodens and observed that the momps occurred at approximately 33.5 kDa. Wolinella Borinski and Holt 1990. have studied the surface of clinical isolates and ATCC strains of W. recta. The membrane consisted of three momps with relative molecular migrations of 43, 47, and 51 kDa calculated by linear regression analysis. 49 50. 50 51. Treponema One of the few studies of the chemistry and biology of the outer sheath of Treponema denticola was that of Masuda and Kawata. The outer sheath of the oral treponemes consisted of tripartate vesicles with a polygonal fine structure in a hexagonal array 51 photomicrograph of negatively stained Treponema denticola, strain GM-1.The coiled cell is surrounded by a thin, loose-fitting outer sheath (OS) that encloses a series of endoflagella (EF) that emerge from each end of the cell.The cytoplasmic region is enclosed in the protoplasmic cylinder (PS). Note in the inset that the outer sheath is constructed of a regular hexagonal array of subunits, the components of which probably comprise the major polypeptide of the sheath, the 64 kDa protein. Bar =100 nm; inset bar = 10 nm. 52. Outer MembraneVesicles Structure and Composition The putative periodontopathic bacteria all release outer membrane vesicles into their surroundings. These vesicles are a direct outgrowth or evagination of the outer membrane. The vesicle has the capacity to entrap the contents of the periplasmic space (location of the cells numerous bacterial proteolytic and hydrolytic enzymes), and along with its LPS and other outer membrane proteins is a formidable virulence factor. 52 53. 53 54. Biologic Activity The fact that it is capable of concentrating many of the hydrolytic, phosphorylytic, and proteolytic enzymes from the periplasmic space in structures that range in size between 20 to 500 nm in diameter makes it a significant potential participant in the progressive events of periodontal disease. The vesicle could interact with other bacteria or penetrate into host tissues, resulting in the release of small peptides that are used by many different oral bacteria for growth. This, along with its LPS, permits it to find its way into deeper tissues of the periodontium and other host tissues where it can elaborate its complement of destructive enzymes and outer membrane-associated LPS 54 55. 55 56. LIPOPOLYSACCHARIDE AND ASSOCIATED MACROMOLECULES Introduction and structure The lipopolysaccharide (LPS, endotoxin) has been one of the most studied of the prokaryotic outer membrane macromolecules. The cell envelope consists of two basic layers: the outer membrane and a thin peptidoglycan layer. The outer membrane contains diffusion pores, matrix proteins, lipopolysaccharide, and lipoproteins. The lipoprotein chemically connects the outer membrane with the peptidoglycan and functions to stabilize it. 56 57. 57 58. Interactions of LPS Bone Resorption Cytokine Stimulation Lymphocyte Stimulation Fibroblast and Epithelial Cell Interactions 58 59. The LPS appears to be a significant mediator of inflammatory host responses, especially the stimulation of a variety of cytokines that are closely linked to the inflammatory process. Meikle et al. 1986 have proposed that the destruction of the periodontium is the result of the interaction of bacterial products (LPS, outer membrane, etc.) and inflammatory cells. The outcome of this interaction is the production of IL-1, a cytokine responsible for the production of a variety of metalloproteases, lymphocyte differentiation and proliferation, eicosanoid production, and bone resorption. Clearly, the outcome of these interactions is tissue damage and bone destruction 59 60. Peptidoglycan as aVirulence Factor Barnard and Holt1984. have provided the only observations that the isolated peptidoglycan from several of the putative periodontal pathogens functions as a virulence factor. All of the peptidoglycans produced a dose-dependent PGE2 response. 60 61. Herpes virus in PDL disease Since the mid 1990s, herpes viruses have emerged as putative pathogens in various types of periodontal disease JPR 2000 In particular, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) seem to pay important roles in the etiopathogensis of severe types of periodontitis. progressive periodontitis in adults localized and generalized aggressive (juvenile) periodontitis HIV-associated periodontitis acute necrotitising ulcerative gingivitis periodontal abscesses and some rare types of advanced periodontitis associated with medical disorders. 61 62. HCMV infects periodontal monocytes/macrophages and T-lymphocytes, and EBV infects periodontal B-lymphocytes. Herpes-virus-associated periodontal sites tend to harbor elevated levels of periodontopathic bacterisa, including P. gingivalis,T. forsythia,T. denticola, Prevotella intermedia,A.A. comitans. 62 PATHOGENESIS OF HERPESVIRUS ASSOCIATED PERIODONTAL DISEASE 63. Physiological virulence of commensal opportunistic pathogens Porphyromonas gingivalis P. gingivalis appears to play a significant role in the progression of chronic periodontitis. In fact, Darveau et al1997, classified this small, gram-negative, black-pigmented anaerobe as a bona fide periodontal pathogen. Specifically, P. gingivalis has been shown to be capable of adhering to a variety of host tissues and cells, and to invade these cells and multiply. 63 64. To accomplish this, P. gingivalis employs several bacterial components: fimbriae, proteases, hemaglutinins, and lipopoysaccharide. P. gingivalis is capable of coaggregating with Actinomyces naeslundii, Streptococcus gordonii, and S. mitis. Various other oral streptococci can also coaggregate with P. gingivalis 64 65. Namikoshi et al 2003, have shown that a 40 kDa outer membrane protein from P. ginigvalis is critical for coaggregation with s. gordinii. Coaggregation of P. gingivalis with both S. oralis and S. gordonii is inhibited by chlorhexidine and hydrogen peroxide. P. gingivalis effect changes in host cell structures/functions, and bacterial macromolecular responses appear to contribute substantially to the success and breadth of P. gingivalis as periodontal pathogen across human population. 65 66. Treponema denticola T. denticola are rapidly motile, obligatory anaerobic Gram- negative bacteria and have been estimated to account for approximately 50% of the total bacteria present in a periodontal lesion. During periods of oral health the number and distribution of these types of bacteria are low or nearly undetectable. However, during gingivitis and the progression to periodontitis there is large increase in the number, proportion of the total population, and diversity of species. 66 67. Armitage et al was one of the first to establish a positive relationship between the percentage of spirochetes in periodontal pockets and clinical measures of inflammation, increased dental plaque, increasing gingival exudate, bleeding on probing, periodontal pocket depth, and the loss of connective tissue attachment. T. denticola interacts with other oral bacterial species, notably P. gingivalis and F. nucleatum. This coaggregation likely plays a role in the progression of PDL disease (i.e. biofilm formation) Kigure et al 1995 67 68. T. denticola possesses several peptidases associated with its outer sheath. A prolyl-phehnylalanine specific protease appears to be important in T.denticola virulence Uitto et al 1986: called this protease a chemotrypsin-like protease. This spirochete displays numerous properties that enhance its ability to interact with host tissue and contribute to changing the inflammatory environment of infected periodontal sites. 68 69. Tannerella forsythia They are the third member of the Red Complex as detailed by Socransky et al 1998: Tanner et al described it as a gram negative anaerobic fusiform isolated from the human oral cavity. It was frequently isolated along with P. gingivalis from cases of active chronic periodontitis Grossi et al 2001 and has been associated with severe periodontal disease. T. forsythia colonization has been associated with Dialister Pneumosintes, a new periodontal putative pathogen, JPR 2002 particularly in older individuals with destructive periodontal disease. 69 70. Actinobacter actinomycetemcomitans A. actinomycetemcomitans armamentarium of virulence factors ensures its survival in the oral cavity and enables it to promote disease. Factors that promote A. actinomycetemcomitans colonization and persistence in the oral cavity include adhesins, bacteriocins, invasins and antibiotic resistance. It can interact with and adhere to all components of the oral cavity (the tooth surface, other oral bacteria, epithelial cells or the extracellular matrix). The adherence is mediated by a number of distinct adhesins that are elements of the cell surface (outer membrane proteins, vesicles, fimbriae or amorphous material). 70 71. A. actinomycetemcomitans enhances its chance of colonization by producing actinobacillin, an antibiotic that is active against both streptococci and Actinomyces, primary colonizers of the tooth surface. A. actinomycetemcomitans resistance to tetracyclines, a drug often used in the treatment of periodontal disease, is on the rise Periodontal pathogens or their pathogenic products must be able to pass through the epithelial cell barrier in order to reach and cause destruction to underlying tissues (the gingiva, cementum, periodontal ligament and alveolar bone). A. actinomycetemcomitans is able to elicit its own uptake into epithelial cells and its spread to adjacent cells by usurping normal epithelial cell function. 71 72. Once the organisms are firmly established in the gingiva, the host responds to the bacterial onslaught, especially to the bacterial lipopolysaccharide, by a marked and continual inflammatory response, which results in the destruction of the periodontal tissues. A. actinomycetemcomitans has at least three individual factors that cause bone resorption (lipopolysaccharide, proteolysis-sensitive factor, and GroEL), as well as a number of activities (collagenase, fibroblast cytotoxin, etc.) that elicit detrimental effects on connective tissue and the extracellular matrix. It is of considerable interest to know that A. actinomycetemcomitans possesses so many virulence factors, but unfortunate that only a few have been extensively studied. 72 73. VIRULENCE FACTORS AND HOST RESPONSE Neutrophil-Mediated Host Response to Porphyromonas gingivalis: Alpdogan Kantarci andThomas E.Van Dyke Virulence factors of Porphyromonas gingivalis such as the gingipains, fimbrillin peptides, capsule polysaccharides, lipopolysaccharides, haemagglutinating and haemolysing activities, toxic products of metabolism, outer membrane vesicles, and other enzymes have important roles in eliciting host responses in various ways. 73 74. These factors significantly affect epithelial/endothelial cells, but their major effect is observed on the modulation of neutrophil response Periodontitis represents an important model for neutrophil- mediated host tissue injury. In this model, neutrophils, primed or stimulated by the presence or persistence of infection, express an elevated and excessive response 74 75. This, in turn, leads to tissue destruction mediated by neutrophil activity. It is essential to understand the mechanisms underlying the interactions between the neutrophils and the microbial virulence factors to be able to develop rational, novel treatment strategies. Bacterial pathogens involved in periodontal diseases exert a part of their destructive effect by triggering and inducing host cells to elevate their secretion of MMPs. 75 76. Pathogen-secreted phospholipase (PLC) is one bacterial product that may trigger this host response. Ding et al1995 Bacterial PLC may induce degranulation of PMN, MMPs and increase MMP expression in oral epithelial cells. The released proteases can be converted into active form by the proteases of plaque bacteria. Thereby, the pathogenic oral bacteria may indirectly participate in the destruction of periodontal tissues 76 77. Periodontal disease: bacterial virulence factors, host response and impact on systemic health Graves et al 2000 Teeth are coated with a biofilm that contains periodontal pathogens. Pathogens express virulence factors which enable them to invade and replicate within epithelial cells and to invade the underlying connective tissue. This stimulates production of prostaglandins and cytokines that induce tissue loss. In addition, these bacteria have the potential to modulate the course of systemic diseases such as atherosclerosis and to contribute to low birthweight and preterm labor. 77 78. Detection of important virulence factors by using the hosts immunologic response It has recently been recognized, that many pathogens express virulence genes only when they are in their human or animal host. The virulence factors that were expressed only when in the host were consistently being missed by traditional methods. Periodontitis is a disease attributable to multiple infectious agents and interconnected cellular and humoral host immune responses. 78 79. However, it has been difficult to unravel the precise role of various putative pathogens and host responses in the pathogenesis of periodontitis. Even though specific infectious agent are of key importance in the development of periodontitis, it is unlikely that a single agent or even a small group of pathogens are the sole cause or modulator of this heterogeneous disease. 79 80. 80