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Aa comitans sem15.pptx
- 1. Dr Shilpa SM 3rdyear PG 1
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- 3. Genus - Aggregatibacter Family – Pasteurellaceae Firstisolated – 1902 – Lignieres and Spitz Role in periodontal infections – Jorgen Slots Actinobacillus Actinomycetem Comitans – Topley and Wilson. 3
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- 5. Gram-negative coccobacillus approximately0 .4 x 1µm in size (Zambon 1985) Capnophilic, requiring an atmosphere containing 5-10% CO2 for good growth (slots 1982) It is microaerophilic and a facultative anaerobe and can grow under anaerobic conditions (Pulverer 1970) 5
- 6. Non sporulating Nonmotile Non-haemolytic Oxidase Catalase positive Fermentative ability Unable to grow on MacConkey’s Agar (Slots 1982) 6
- 7. Internal star-shaped orcrossed cigar morphology form embedding in the agar that gives A. actinomycetemcomitans its name. 7
- 8. FIMBRIAE Small filamentouscell surface appendages associated with bacterial colonization of host tissues.(Ofek 1980) Peritrichous arrays (Scannapieco 1987) may be more than 2 µm in length and 5nm in diameter and often occur in bundles (Preus 1988,Rosan 1988) 8
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- 10. VESICLES Lipopolysaccharide innature, originate from and are continuous with the outer membrane and released into the external environment in large numbers (Holt et al 1980) Highly leukotoxic A. actinomycetemcomitans strains--abundance of extracellular membranous vesicles Minimally or non leukotoxic strains-- few or no vesicles. (Lai et al 1981). 10
- 11. EXTRACELLULAR AMORPHOUS MATERIAL Amorphous material that frequently embeds adjacent cells in a matrix( Holt 1980) Protein, most likely a glycoprotein, and has been shown to exhibit both bone- resorbing activity (Wilson 1985) and adhesive properties (Meyer 1993). The material is easily removed by washing cells with phosphate-buffered saline. 11
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- 13. 24 groups byPulverer and Ko based on tube agglutination assays. These 24 groups were further divided into 6 agglutinating antigens. Non oral A.actinomycetemcomitans was divided into 3 serogroups by King and Tatum based on a heat stable component Taichman et al. divided A.actinomycetemcomitans into 4 serogroups based on surface antigens and proteinaceous leukotoxin Zambon divided A.actinomycetemcomitans into three serotypes − a, b and c 13
- 14. 8 biotypesbased on fermentation reactions with galactose,mannitol and xylose. 10 biotypes based on fermentation of dextrin,maltose,mannitol and xylose. Serotype a does not ferment xylose while serotype b ferments xylose. Serotype c has both xylose positive and xylose negative strains . (Saarela 1992) 14
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- 16. A.Actinomycetemcomitans may constitute Exogenousspecies ……….. Transmission studies and vigorous host response to periodontal infection by this organism and the ability of appropriate therapies to eradicate the organism completely from the oral cavity (which is not the case for indigenous pathogens) also appear to confirm this fact. 16
- 17. Periodontopathogenic potential …. Immunomodulatory bacterial products.... (Page RC 1997) A.actinomycetemcomitans contains proteinaceous products that have the ability to selectively stimulate T suppressor cells and also potentially suppress immunoglobulin production. 17
- 18. LOCALISED JP Large numbersof A. actinomycetemcomitans are routinely isolated from localized juvenile periodontitis lesions The eradication of A. actinomycetemcomitans from diseased sites is usually correlated with recovery from clinical symptoms of disease ORAL INFECTIONS 18
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- 25. Tissue culture medium (Sreenivasan et al,1993) Chemically definedmedia (Socransky et al ,1985) “A” medium – TSBV with spiramycin, fusidic acid and carbenicillin. TSBV – trypticase soy agar with serum, bacitracin and vancomycin. MGB-- (a trypticase soy broth with malachite green and bacitracin) Mandell et al 1981 Slots et al 1982 Holm 1987 25
- 26. YEAST EXTRACT: -enhances thegrowth (Sreenivasan 1993,Slots 1982,Van Steenbergen 1986,Mashimo 1985 -1.2% yeast extract CYSTEINE -enhances the growth (Sreenivasan 1993) HORMONES Steroid hormones, including estrogen, progesterone and testosterone-- Enhances the growth. (Kornman1980,1982 ) 26
- 27. IRON -expresses iron- binding proteinsand has hemin-binding activity.(Graber 1998) pH good growth between pH 7.0-8.0, with optimal growth at pH 7.5 (Sreenivasan 1993) SALT CONC. demonstrates optimal growth between 85.1 mEq/l and 170 mEq/l (Sreenivasan 1993) 27
- 28. Microorganism Unique ecological niche Subverthosts normal defenses Replicate in new environnment Express pathogenic traits VIRULENCE FACTORS 28
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- 61. Culture: TSBV agaris the medium of choice for culturing A.actinomycetemcomitans Immunodiagnostic methods include indirect immunofluorescence, flow cytometry, Evalusite ™ test, bacterial concentration fluorescence immunoassay Nucleic acid probes: Digixogenin labeled whole genomic DNA, Radiolabelled cloned DNA, Radiolabelled oligonucleotide • Polymerase chain reaction 61
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- 66. Non surgical therapyhas the least effect on A.actinomycetemcomitans counts in heavily infected periodontal lesions. This is because Periodontal therapy often fails to effectively control subgingival A.actinomycetemcomitans. Modified Widman flap surgery is shown to have about 50 % effect. Superior performance of resective periodontal surgery may be due to the excision of A.actinomycetemcomitans – infected gingival tissue and reduction of pocket depth. 66
- 67. Systemic amoxicillin-metronidazole combinationshows striking clinical results in treatment of localized aggressive periodontitis, adult periodontitis and refractory periodontitis, even in the absence of other periodontal therapy. The prescribed regimen is 250 mg amoxicillin and 250 mg metronidazole- thrice daily for 8 days. 67
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Editor's Notes
- #4 Aggregatibacter actinomycetemcomitans is a major putative periodontopathic bacteria. It is closely associated with periodontitis in young individuals and in cases of refractory adult periodontitis it belongs to the FAMILY Pasteurellaceae and GENUS Aggregatibacter. Aggregatibacter actnomycetemcomitans was first described by Klinger. It was first named Bacterium actinomycetemcomitans .Aggregatibacter actinomycetemcomitans is more closely related to Haemophilus than to the genus Actinobacillus. Hence A.actinomycetemcomitans is not a true Actinobacillus. That is the reason why it was renamed as Aggregatibacter
- #6 Microaerophilic-requires oxygen but in lower levels ie..2% Capnophilic- requires co2 in more levels A facultative anaerobe is an organism that makes ATP by aerobic respiration if oxygen is present, but is capable of switching to fermentation if oxygen is absent.
- #7 The fermentative ability of A. actinomycetemcomitans strains to utilize galactose, dextrin, maltose, mannitol and xylose permits the biotyping of this organism into several biotypes and serves to distinguish this organism from other members of the oral flora.
- #8 The colonies were rough surfaced. The colonial variation is associated with fimbriation.
- #9 A significant feature of A. actinomycetemcomitans is its surface ultrastructure which includes fimbriae, vesicles, and extracellular amorphous material
- #10 Freshly isolated strains are fimbriated, but in uitro subculture results in organisms that lack fimbriae. Fimbriated associated proteins of aa such as --54 kDa,304 a, aa 310 was found to be associated with adhesion(ishihara 1997,Inouye 1990)
- #11 A prominent feature of the surface of A. actinomycetemcomitans is vesicles (blebs) vesicles per se exhibit leukotoxic activity A. actinomycetemcomitans vesicles also exhibit adhesive properties; this observation prompted the hypothesis that vesicles function as delivery vehicles for A. actinomycetemcomitans toxic materials.(Meyer 1983)
- #12 Associated with the surface of certain aaa.. Furthermore, A. actinomycetemcomitans strains, which normally exhibit low levels of adhesion, exhibit increased levels of adhesion when suspended in extracellular amorphous material, a phenomenon termed conveyed adhesion.
- #16 A.actinomycetemcomitans population is genetically heterogenous. S
- #17 The distinction between exogenous and indigenous periodontal pathogens is important because the nature of periodontal infection significantly influences the clinical approach to disease management. ……..A.actinomycetemcomitans may constitute exogenous species because of the rare occurrence in periodontally healthy individuals.
- #18 may be due to their ability to manipulate the immune response of the host. T lymphocytes are believed to be the regulators of immune response. immunomodulatory bacterial products are the lipopolysaccharides of gram negative bacteria, which can activate B cells, monocytes, macrophages and polymorphonuclear neutrophils . Super antigens are T cell modulating components of bacteria and viruses. These super antigens, though potent T cell stimulators are ultimately immunosuppressive [14]. This feature is also thought to be present in A.actinomycetemcomitans
- #19 actinornyceterncornitans is an important pathogen in severe and recurrent forms of periodontitis. The prevalence of A. actinornyceterncomitans….. Among the five currently recognized serotypes of A. actinornycetemcornitans (52), serotype b strains often predominate in periodontal lesions of localized JP.
- #21 The evidence supporting the role of this microbe as a pathogen in periodontal disease based on Sockransky’s criteria has been presented in this table. However, conflicting views are seen in this regard. Some reports find no apparent association between A.actinomycetemcomitans and progressive adult periodontitis while some reports do
- #22 Prior to 1962, A. actinomycetemcomitans was considered an agent incapable of causing infection except in association with A. israelii King & Tatum ,Kaplan 1989--- recognized the ability of A. actinomycetemcomitans to cause infections by itself and documented 32 cases of infection (80). extraoral infections by A. actinomycetemcomitans have been reported from the brain, meninges, septicemia, urinary tract infections, vertebral osteomyelitis and abscesses of the abdomen, brain, face, hand and thyroid gland. Few complications---CHF,Embolism
- #23 model of a pathway from dental plaque and periodontal disease to infective endocarditis and atherosclerosis and coronary heart disease
- #26 Understanding of the physiological and metabolic requirements of A. actinomycetemcomitans adds an additional dimension in our understanding of the pathogenic process and the biology of this bacterium that adapts it for pathogenesis. Appropriate culture medium:-Microorganisms require a suitable culture medium that can support their nutritional needs. By understanding the growth requirements of a given organism, it is possible to establish the necessary conditions in vitro to support their optimal growth. A selective medium that utilises…as inhibitory agents was one of the earliest media developed……followed by the dev of ….both were useful but other gram negative orgns—sucha s Capnocytophaga and Neisseria were growing..so another medium was developed To know the precise nutritional req of a microorganism—chemically defined media req Since aaa growth is fastidious and it supports the growth of streptococcus and p.gingivalis Advt---commercialy avialble and supports the growth of aa
- #27 Yeast extract has been consistently used to supplement media for the growth of A. actinomycetemcomitans Periodontal disease is correlated with pregnancy, adolescence and birth control drugs, conditions in which physiological levels of steroid hormones and iron undergo dramatic changes.
- #28 ----However, the addition of iron salts and protein-bound iron to trypticase soy broth did not influence the growth or protein profiles of A. actinomycetemcomituns, as compared with cultures grown in trypticase soy broth alone (Sreenivasan 1993) Perhaps, the concentration of iron in trypticase soy broth is sufficient for A. actinomycetemcomitans --- downregulates expression of a 70-kDa membrane protein in iron-limited conditions . --The NaCl concentration is an environmental parameter that affects the growth of many bacteria. The concentration of sodium in the gingival fluid ranges from 90 mEq/l in health to 136 mEq/l following inflammation. These results suggest that A. actinomycetemcomitans may thrive under conditions of gingival inflammation and suggests an adaptation favoring pathogenesis.
- #29 Virulence is the ability of an organism to cause infection. Virulence factors are attributes of a microorganism that enable it to colonize a particular niche in its host, overcome the host defenses and initiate a disease processIn order to produce periodontal disease A.actinomycetemcomitans infect periodontal sites by attaching to epithelial cells, existing microbes (or) the tooth surface, by competing with the resident flora in an effective manner and also by overcoming the cellular and humoral host defense mechanisms
- #30 A. actinomycetemcomitans has been shown to possess a myriad of virulence factors that enhance its survival in the oral cavity and enable it to circumvent the host’s protective strategies. Many of these virulence factors may be involved in the pathogenesis of periodontitis.
- #31 The first challenge any microorganism faces is the ability to adhere tightly to a specific substrate. Without adhesion, the organism is quickly cleared from the surface. The oral cavity poses distinct problems for microbes, as eating, drinking, talking and salivary flow induce many shear forces that remove microorganisms not firmly attached. The bacterial surface components involved in adhesion are called adhesins.
- #33 Binding occurs very rapidly, reaching saturation levels within 1 hour after infection
- #36 The major component of the extracellular matrix is collagen (89).. A. actinomycetemcomitans binds to immobilized collagen types I, 11, and V but not to type IV collagen Actinomycetemcomitans also binds to fibronectin, but not to the plasma protein, fibrinogen (150, 201). Binding, therefore, is highly specific (The fiber-forming collagen types I, 11,111, V and XI are predominant in connective tissue, whereas type 1V, which differs significantly in structure from the fiber-forming collagens, is the major component of the basement membrane)
- #37 Moreover, the tetB determinant was capable of being transferred by conjugation to other A. actinomycetemcomitans strains and to Haemophilus influenzae These data suggest that antibiotic resistance in A. actinomycetemcomitans is on the rise and likely to be responsible for treatment failures in the future
- #38 Bacteriocins are proteins produced by bacteria that are lethal for other strains and species of bacteria. These toxic agents can confer a colonization advantage for the bacterium by lessening the ecological pressures associated with competition by other organisms for both nutrients and space. MOA:;-The mode of action is to increase the permeability of the cell membranes of target bacteria, which leads to leakage of DNA, RNA and macromolecules essential for growth.
- #41 The surface associated material of A.actinomycetemcomitans has several putative virulence factors. This material has potent osteolytic activity. It contains a protein which blocks cell cycle progression….. This may play an important role in inflammatory bone resorption by promoting osteoclast formation in periodontal disease
- #42 collagen is the most abundant constituent of the extracellular matrix. A major feature of periodontal disease is a marked reduction in gingival collagen fiber density
- #43 One of the most important cell types within the gingival connective tissue is the fibroblast. Fibroblasts are a major source of collagen and confer structural integrity to the tissue. Many oral bacteria express toxins that inhibit human fibroblast proliferation.
- #44 The Fc region of antibody is important in the binding of antibody to specific receptors on polymorphonuclear leukocytes. If other proteinscompete for binding to this region of polymorphonuclear leukocytes, binding of the antibody may be inhibited and, thereby, inhibit phagocytosis. ----molecules on the surface of A. actinomycetemcomitans ,the binding inhibits the ability of opsonizing antibodies to bind polymorphonuclear leukocytes and reduces phagocytosis by 90%. It is believed that the Fc receptors also play a role in complement activation.
- #45 One of the most studied virulence factors of A. actinomycetemcomitans is leukotoxin
- #46 The Repeats-in-Toxin (RTX) exoprotein is produced by several gram negative bacteria
- #47 It is a heat labile toxin. It is protease sensitive…Target cells include human polymorphonuclear leukocytes, monocytes and macrophages. Human platelets, fibroblasts, endothelial and epithelial cells are resistant to the effects of ltx A
- #48 Two ltx A mediated mechanisms of cell death are known to exist: Necrosis and apoptosis. The ltx A forms pores in the target cell membrane leading to water influx and osmotic lysis. This is the case when the ltx A is present in high concentrations. At low concentrations, ltx A mediates cell death via apoptosis.
- #51 . Lipopolysaccharides (endotoxins) have a high potential for causing destruction of an array of host cells and tissues. Tissue destruction is a key feature of periodontal diseases; thus, the lipopolysaccharide of A. actinomycetemcomitans has been extensively characterized (86). It causes skin necrosis (Schwartzmann reaction), bone resorption and platelet aggregation,and it activates macrophages. It is known to inhibit collagen and DNA synthesis as well as stimulation of bone resorption in a dose dependent fashion macrophages that migrate to gingival sites of A. actinomycetemcomitans infection will be stimulated to produce these cytokines, which may then be involved in gingival inflammation and alveolar bone resorption
- #52 Host defense mechanisms play a major role in controlling concentrations of bacterial communities in dental plaque.
- #53 The host’s first line of defense against invading bacteria is the recruitment of phagocytes to the area. This process, known as chemotaxis, involves a number of steps, including the binding of chemotactic signals, upregulation of adhesin receptors, binding to the endothelium and movement of the phagocytic cells into the underlying tissues. The ability to disrupt chemotaxis permits the invading organism to survive this major challenge from the host. actinomycetemcomitans is capable of inhibiting neutrophils from producing antibacterial agents . It produces a heat stable protein that inhibits hydrogen peroxide production by leukocytes.
- #54 A.actinomycetemcomitans can penetrate the gingival epithelium. in vivo studies showed that A. actinomycetemcomitans occurs in very specific locations…. and that it exhibits a definite pattern of penetration. The primary receptor of A.actinomycetemcomitans invasion is transferrin receptor. invasion of KB cells by E. coli clones containing A. actinomycetemcomitans DNA was enhanced by transferrin or lactoferrin
- #56 Survival of all living forms depends upon cell replacement, and this is controlled by the processes of the cell cycle and cell division. In eukaryotic cells, the cell cycle is a complex process that is divided into the stages G1, S, G2 and mitosis (M). The S phase is the part of the cell cycle in which DNA is replicated. The G1 and G2 phases are important for generating the enzymes required for DNA replication and for checking the quality of the replicated DNA, respectively. Such an important process is likely to be targeted by bacteria----
- #57 Cytolethal distending toxin is encoded by a locus of three genes, cdtABC. The toxin itself is encoded by cdtB , while cdtA and cdtC appear to encode proteins that mediate interaction between the cytolethal distending toxin complex and the host cell surface
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- #59 Saliva is considered to be the most important transport vehicle for A.actinomycetemcomitans, as it can be cultured from salivary samples It can survive in saliva during transportation to a new host.Mucosal contact or toothbrush sharing may allow implantation of bacteria to potential growth locales.The salivary and subgingival serotypes of A.actinomycetemcomitans are the same in a patient.
- #60 Subgingival prevalence of A.actinomycetemcomitans is found to be as high as 80 %. The dynamics of subgingival A.actinomycetemcomitans is the result of a complex bacterium – host interaction.
- #61 . The dynamics of subgingival A.actinomycetemcomitans is the result of a complex bacterium – host interaction
- #62 – an antibody based sandwich enzyme linked immunosorbent assay These DNA probe methods are rapid and are efficient tools for clinical detection of periodontopathic bacteria, mainly A.actinomycetemcomitans and Porphyromonas gingivalis. For A.actinomycetemcomitans , the DNA from ATCC 43718, JP2, ATCC 29524, 310a, 146 HE are used for the probes.
- #63 data exist that A.actinomycetemcomitans is an etiologic agent of periodontal disease. Systemically administered antibiotics are recommended for elimination of this bacterium from the subgingival and adjacent intra oral areas….
- #64 This indicates that monitoring the levels of these periodontopathic bacteria and the elimination of all these three microorganisms is a prerequisite for successful treatment
- #65 Antibiotic resistance has been described amongst bacterial species colonizing the periodontal pockets.
- #66 For tetracycline resistant strains, combination of amoxicillin and metronidazole is the drug of choice. Mechanical debridement (scaling and root planning) in combination with amoxicillin – metronidazole therapy is effective in subgingival suppression of A.actinomycetemcomitans in patients with severe periodontitis
- #67 Scaling and root planing alone cannot remove A.actinomycetemcomitans from lesions of localized aggressive periodontitis .bcz of the ability of the organism to invade gingival tissue and evade the effect of mechanical debridement and periodontal healing.
- #68 Systemic metronidazole has good anti A.actinomycetemcomitans activity in localized aggressive periodontitis patients
- #69 A.a has multiple mechanisims that are responsible for the destruction of periodontal connective tissue. It is capable of attachment, colonization and most importantly by elaborating factors that interfere with hosts defence system. The ability of LtxA to cause death of all subsets with hematopoetic origin might contribute to help the bacterium to survive the host immune response and also to release compounds essential for bacterial growth. Further research is required to establish its genetic, molecular and biochemical features in detail.