Microbiology aspect in endodontics

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Microbiology aspect in endodontics

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Microbiology aspect in endodontics

  1. 1. Microbiology aspectinendodontic Thalerngsak Samaksamarn Department of restorative dentistry Faculty of dentistry. KKU
  2. 2. Terminology Colonization : the establishment of bacteria or orther microorganism in a living host. Infection : damage the host and produce clinical signs and symptom Pathogenicity : The capacity of organisms to produce disease within a particular host Virulence : the degree of pathogenicity in a host under defind circumstance
  3. 3. State of disease Spread of infection No.&Virulence of bacteria Associated anatomical structure Abscess formation PA Pathology Host defense Status of host defensive system
  4. 4. Entries of bacteriato the pulp system
  5. 5. •Caries•Periodontal disease•Trauma and restoration leakage•Anachoreasis
  6. 6. Dental caries Facultative gram positive bacteria Propionibacterium, Eubacteruim, Arachnia, Lactobacilli, Bifidobacterium , Actinomyces Some of gram negative bacterial such as Bacteroides Hoshino et al 1985
  7. 7. DENTAL CARIES the most common pathway to the root canal system for microbes. When the tooth is intact, enamel and dentin protect it against invasion of the pulp space. As caries approaches the pulp, reparative dentin is laid down to avert exposure, but this rarely can prevent microbial entry without caries excavation
  8. 8. Dentinal tubules 1 to 4 μm in diameter, bacteria are less than 1 μm in diameter. the protective cementum layer is missing or if it has been lost through trauma, the dentinal tubules may be exposed and may serve as a pathway for microbial invasion of the pulp space. Bacterial movement is restricted by :outflow of dentinal fluid, : odontoblastic processes, : mineralized crystals : macromolecules, including immunoglobulins in the tubules.
  9. 9. •Accessory canal•Furcation canal•Lateral canal•Apical foramen•Exposed dentinal tubule Anerobic bacteria  Gram negative rod, spirochete
  10. 10. Inflammation and local necrosis have beendemonstrated in pulp adjacent to entry pathway. Immune system via circulation Whole pulp tissue change due to periodontal disease
  11. 11. Necrotic pulpPeriodontal lesion involve apical foramen Guldenor et al 1985
  12. 12. Pulp exposure due to trauma give access tooral bacteria, this will cause bacterial invasionto the pulp  inflammation Pulp necrosis Kakehashi et al 1965
  13. 13. Healthy exposed vital pulp, the penetration oftissue by bacteria is relative slow ≤2 mm./wk Cvek et al 1978
  14. 14. Laboratory experiments indicate that bacteriacan enter through even minor crack in enamelor dentine. Love et al 1996Dentinal tubules exposed by tooth fractureduring cavity preparation or marginal leakageare the potential pathway. Bender & Seltzer 1959 Smulson & Sieraski 1989
  15. 15. Anachoresis “Blood borne bacteria is preferentially localized in areas of inflammation.” Burke & Knigton 1960 Gier & Mitchell 1968 Intravenous bacterial injection into bloodstream could be demonstrated pulp that were inflamed due to deep cavity preparation and chemical irritation.
  16. 16. Polymicrobia l Microorganism Endodontics failurePulpal and periapicaldisease In 1890 W.D. Miller, the father of oral microbiology, was the first investigator to associate the presence of bacteria with pulpal disease. A classic study published in 1965 by Kakehashi et al
  17. 17. Polymicrobial the number of microorganisms detected in endodontic infections increased to a range of three to 12 organisms per infected root canal associated with an apical lesion The number of colony forming units (CFU) is usually 102 to 108 A positive correlation exists between the number of bacteria in an infected root canal and the size of periradicular radiolucencies
  18. 18.  Strict anaerobes: function at low oxidation-reduction potential and grow only in the absence of oxygen, but they vary in their sensitivity to oxygen. Obligate anaerobic bacteria lack the enzymes superoxide dismutase and catalase. Some species of bacteria are microaerophilic; they can grow in the presence of oxygen, but they derive most of their energy from anaerobic energy pathways. Facultative anaerobic bacteria :can grow in the presence or absence of oxygen. Obligate aerobic bacteria : have both superoxide dismutase and catalase and require oxygen for growth
  19. 19.  Intact teeth with necrotic pulp : strict anaerobes more than 90% of the bacteria ( Sundqvist 1989) The apical 5 mm of carious exposed teeth : 67% of the bacteria were strict anaerobes ( Baumgaetner 1999) Gram-negative bacteria, especially species of Porphyromonas and Prevotella that are dark (black) pigmented, have been associated with endodontic infections.
  20. 20. Type of microorganisms found inendodontic Infections ตาราง 2 Pisano and
  21. 21. Type of microorganisms found inendodontic Infections Pisano and
  22. 22. Failed Endodontic Treatment complete periapical healing occurred in : 94% of roots with negative culture : 68% of cases with positive culture ( Sjogren 1997 ) Enterococcus faecalis has been the predominant microbe in canals undergoing retreatment E. faecalis was found in 77% of cases, confirming that this microbe is the most prevalent species in fai led endodontic treatment
  23. 23. Sundqvist 1998
  24. 24. Bacterial virulence factors Newman, M. B., H. H.
  25. 25. Bacterial virulence factors Bacteroides มี polysaccharide capsule เป็น virulence factor ที่สำำคัญ (Samaranayake 2002) Porphyromonas endodontalis มี capsule ที่ทำำให้ต้ำนทำนต่อ phagocytosis และยังมี proteolytic capability ที่สำมำรถทำำลำย immunoglobulin G และ E และ complement factors C3 และ C5 ได้ นอกจำกนี้ยงมี toxic metabolic ั products เช่น butyrate และ propionate ที่สำมำรถพบได้ในบริเวณที่มี
  26. 26. Type of microorganisms found inendodontic Infections  Aerobes  Streptococcus sp.: S.mitis, S.salivarius  Anaerobic streptococci : Peptostreptococcus  Enterococci: E. faecalis  Bacteroides sp.: Porphyromonas, Prevotella  Provotella nigrescens  Actinomyces  Fungi: Candida albicans  Fusobacteria  Spirochetes Biofilm
  27. 27. Aerobes Beta hemolytic และ non- hemolytic streptococci ก่อโรค ได้น้อย  peptidoglycans กระตุ้น lymphokine เช่น osteoclast- activating factor, prostaglandin  lipoteichoic acid  กระตุ้น complementbone resorption S.mitis from root canal
  28. 28. Anaerobes: Enterococci เป็น facultative bacteria, frequency in Endodontic failure (Ingle and K.Bakland 2002) Common in GI tract streptococcus faecalis กำำจัดออกจำก คลองรำกฟันได้ยำก(resistant to antibiotic) ต้ำนทำนต่อ benzylpenicillin, ampicillin, clindamycin,
  29. 29. Anaerobes: Enterococci sensitive ต่อ erythromycin และ vancomycin (Dahlen, Samuelsson et al. 2000) Molander and Dahlen 2003 :Tx with calcium hydroxide+ erythromycin / tetracycline
  30. 30. Anaerobes: Bacteroides เป็น strictly anaerobes, short chain, gram negative rods and coccobacilli, common in dental plaque, non motile, no spore มี polysaccharide capsule เป็น virulence factor  serious anaerobic infection เช่น sepsis, abscess
  31. 31. Anaerobes: Bacteroides Polymicrobial infection (facultative anaerobes  reduced oxygen Bacteriodes growth) Sensitive to metronidazole และ clindamycin Resistance to penicillin (beta- lactamase)
  32. 32. Anaerobes: Bacteroides 2 genera คือ Porphyromonas และ Prevotella Porphyromonas เป็น asacharolytic bacteria, short chain, gram negative rods, non motile, no spore  P.gingivalis common in subgingival sulcus  periodontal infection  P.endodontalis (first from root canal infection) common in dental root canal, periodontal pocket, dental plaque (Samaranayake
  33. 33. Anaerobes: Bacteroides Prevotella เป็น saccharolytic bacteria, gram negative rods, non motile  Prevotella melaninogenica (Bacteroides melaninogenica)= black pigment (melanin) พบได้ใน ช่องปาก  P.intermedia พบได้ใน periodontal disease (Samaranayake
  34. 34. Anaerobes: Prevotella nigrescens ปัจจุบันแยกออกจาก Prevotella intermedius, importance in endodontic infection (Shah (1992), Bae (1997), Dougherty (1998), Baumgartner (1999)) Endotoxin: lipopolysaccharide (LPS) periapical lesion, pulp necrosis, inflammation, bone resorption, pain, edema (Schein and
  35. 35. Anaerobes: Prevotella nigrescens LPS+Peptidoglycans กระตุ้น hormone- like cytokines tisssue destruction (Henderson and Wilson (1998), Matsushita et.al. (1998)) กระตุ้น B lymphocytes และระบบ complement cascade collagenase and interleukin (macrophage cells) ผลิต pain mediators เช่น histamine, bradykinin และ prostaglandin
  36. 36. Anaerobes: Actinomyces เป็น anerobic =microaerophilic gram- positive filamentous bacteria, ไม่ก่อโรค ในช่องปาก non motile, no spore Common in nasophalynx และ gingival crevice  70-80% chronic infection, granulomatous และ endogenous infection of oral cavity (Samaranayake 2002)
  37. 37. Anaerobes: Actinomyces found in endodoctic failure case (resistant to routine antibiotic) (Baumgartner 1991, Gohean 1990, Barnard 1996, Siqueira et.al.2002) sulfer granules exudates, in biopsy branching filamentous form (acid-fast staining) Tx: surgical curettage or resurgical with long term antibiotic (Gohean, Pantera et al. 1990; Baumgartner and Falkler 1991; Barnard,
  38. 38. Fungi  Candida albicans  Debelian, Olsen et al. 1997 พบว่า candida สามารถพบได้ในผนังคลอง รากฟันในรูป blastospores และ hyphal structures ซึงสามารถแพร่ ่ ผ่าน dentinal tubule ได้
  39. 39. Fusobacteria: Fusobacteriumnucleatum  เป็น a Gram-negative, non- spore-forming, non motile, obligatory anaerobic rod, primary root canal infections. (Moraes, Siqueira et al. 2002)  All of F nucleatum associated with severe pain, swelling and flare-ups case (Chavez de Paz Villanueva 2002)
  40. 40. Spirochetes Oral spirochaete (ซึ่งมีได้ทง เชื้อตัว ั้ เล็ก กลาง และใหญ่) non culturable Common in root canal infections, pericoronitis, gingivitis และ periodontitis (10% in endodontic abscesses). (Dahle, Tronstad et al. 1993)
  41. 41. Primary endodonticinfectionPrimary infection is caused by microorganisms that initiallyinvade and colonize the necrotic tissue.Primary infections are characterized by a mixed clostridiumcomposed of 10-30species per canal.The number of bacterial cells in an infected canals varies from103-108 cells. Siqueira et al 2005 Sakamoto et al 2007 Vianna et al 2006
  42. 42. Primary endodonticinfection Anaerobic bacteriaPrevotella,Porphyromonas, Actinomyces,PeptostreptTanerella, Fusobacterium, ococcus, Eubacterium,Dialister, Camphylobactor, Filifactor,Treponema Psudoramibactor Sterptococci
  43. 43. Black pigmented bacteria Saccharolytic; Prevotella Bacteroides Asaccharolytic; Porphyromonas
  44. 44. Black pigmented bacteriaIn 1980, Griffee et al reported that B.melaninogenicus wasfound to be significantly related to pain, sinus tract formation,and foul odor. Griffee et al 1980
  45. 45. Black pigmented bacteria Prevotella  PorphyromonasP.intermedia P.endodontalisP.nigrescense P.gingivalisP.tanneraeP.multisaccharivorax They seem to play important role in etiology of both acute and chronic apical periodontitis. Siqueira et al 2001Sundqvist et al 1989Dougherty et al 1998
  46. 46. Fusobacterium nucleatum Gram negative obligate nonmotile anaerobe bacteria. Most common in symptomatic infection and abscess or excarcerbation lesion than asymptomatic infection. 5 subspecies of F.nucleatum have been found and different in genetic data but no study found that which subspecies have more virulence factor. Different type of subspecies are found in the same root canals. Moraes et al 2002 Siqueira et al 2005
  47. 47. Spirochete bacteriaAlthough spirochetes have been frequently observed in samplesby microscopy, they had never been identified to the specieslevel.The application of molecular diagnosis to identification of spiralbacteria has been overlooked the culture techniques.All oral spirochetes are genus Treponema .They can be classified in 2 group; saccharolytic and saccharolytic.T.denticola, T.sokranskii have been detected in both symptomaticand asymptomatic lesion. Dewhirst et al 2000 Baumgartner et al 2007 Rocas et al 2003
  48. 48. Secondary endodontic infectionIf microorganisms are allowed to remain at the timeof filling , there is increased risk of adverse outcomeof the endodontic treatment Sjogren et al 1997 Waltimo et al 2005
  49. 49. Secondary endodontic infection Create both excellent apical and coronal seal. Prevent bacterial nutrient regain in to the canal.
  50. 50. Microbial in root filled teethUnlike primary infection, a more restricted group ofmicrobial species has been found inpersistent/secondary infection . The prevalence of enterococci has been finding in all studies that investigated flora in root-filled teeth. “ Enterococcus feacalis “
  51. 51. Microbial in root filled teethRecently findings from molecular studies have alsosuggested that some anaerobic species commonlyfound in primary infection. T.forsythia, P.alactolyticus, F.acolis, D.pnuemosintes Siqueira et al 2004,2005
  52. 52. Microbial in root filled teethBacteria are secondary invaders that can gain entry into due to a breach in the aseptic chain duringintracanal intervention. P.aeruginosa, Staphylococcus Ranta et al 1988 Siqueira et all 2002
  53. 53. E.feacalis in secondaryinfection Facultative anaerobie, Gram positive coccus. This species has been found in low prevalence value in case primary infection and more relate in asymptomatic cases than symptomatic cases. Rocas et al 2005
  54. 54. E.feacalis in secondaryinfectionE.feacalis has been found in root filled teeth evincingpersistent apical periodontitis in prevalence valuesranging from 30-90% of the cases.This species can be inhibited by other members ofmixed bacterial consortium commonly present inprimary infection. Sedgley et al 2006
  55. 55. E.feacalis in secondaryinfectionVirulence factor Lytic enzyme, cytolysin, gelatinase, hyaluronidase, pheromone, lipotheichoic acid, adhesion molecules. But cannot clarified what factor play role in pathogenesis. Kayaoglu et al 2004
  56. 56. E.feacalis in secondaryinfection Why E.feacalis can survive in root filled teeth?
  57. 57. E.feacalis in secondary infectionStudies have revealed that E.feacalis has ability topenetrate far into dentinal tubules that can escapefrom intracanal instrumentation and irrigants. Haapasalo et al 1989 Siqueira et al 1996
  58. 58. E.feacalis in secondaryinfectionE.feacalis has been shown to be able to form biofilms inroot canals and this ability can be important forbacterial resistance and persistence in theinstrumented canals. Distel et al 2002The ability to resist high pH value seems to be related tofunctional proton pump to acidfy. Which E.feacalis isresistant to calcium hydroxide. Evans et al 2002
  59. 59. E.feacalis in secondary infectionE.feacalis can enter a VBCN state which can survive inadverse environmental condition, including starvation.They has the ability to survive in environments in scarcityof nutrients and to flourish when the nutrient source isreestablished and has a capacity to recover in root canaltreated teeth for 12 months without nutrients. Figdor et al 2003 Sedgley et al 2005
  60. 60. Fungi in secondaryinfectionThe occurrence of yeasts were taken from samples thatnot responding in favorably to conventional treatment(72% of cases ). Waltimo et al 1997Candida albicans was the most common species.
  61. 61. Fungi in secondaryinfectionBy the new detection technology, C.albicans wasdetected in primary infection in 21% too.However the finding indicates that yeasts may bepresent in low number at the start of treatment, andthey may reach higher proportion during root canalprocedures . Buamgartner et al 2000
  62. 62. Fungi in secondaryinfectionIt is also possible that yeasts from oral cavity gainaccess to root canal as contaminant duringendodontic procedures. Siren et al 1997Or they can overgrow after inefficient intracanalantimicrobial procedures, which can cause imbalancein microbiota. Siqueira et al 2004
  63. 63. Fungi in secondaryinfectionVirulence factorDentino phillic microorganism due to its abilityto colonize and invade to dentine by theirhyphae.Resist to some intracanal medication such ascalcium hydroxide.Release hydrolytic enzyme and can formmonoinfection biofilm in root canals. Sen et al 1997 Waltimo et al 1999,1997 Matusow et al 1981
  64. 64. Biofilms Definition The colonization and proliferation of microorganisms at surface and solution interface; especially problematic in the small-bore water lines of dental unit. AAE,2004
  65. 65. SEM of bacterial cellsarranged in a biofilm Siquera 2001
  66. 66. 1. Surface conditioning2. Adhesion of ‘ pioneer ’ bacteria3. Secondary colonizers4. Fully functioning biofilm 1. A cooperative “consortia” of species 2. Biofilms grow and deattachment
  67. 67. A cooperative “consortia” ofspecies
  68. 68. Biofilms grow and spread
  69. 69. Cell-cell communication Communicate with one another in biofilm communities via small diffusible molecules. Adapt & survive various environment stresses. Regulate expression of gene Ability to cause disease
  70. 70. Benefits A broader habitat range for growth A more efficient metabolism Increased resistance to stress and antimicrobial agent Enhanced virulence
  71. 71. Microbial control inbiofilmBacteria in biofilm form is more resistance to anti-microbial agent than planktonic form.P.Gingivalis in biofilm is resit to amoxycillin,metronidazole and doxycyclin when compareto planktonic form. increased MIC & MBC Larsen et al 2002
  72. 72. Irrigants for microbialcontrol •Sodium hypochlorite •Combination of sodium hypochlorite and chlorhexidine •Chlorhexidine Concentration •Povidone iodine E.feacalis, P.micros, F.nucleatum, S.intermedius Spratt et al 2001
  73. 73. Er:YAG LASER Er:YAG lasers had an anti- biofilm effect at a low energy and could reduce numbers of the 6 species of biofilm- forming cells examined. Noiri et al 2008
  74. 74. Photodynamic with methylene bluePhotodynamic therapy (PDT) was developed as atherapy for cancer.Activated by light of the appropriate wavelength togenerate singlet oxygen and free radicals that arecytotoxic to cells of the target tissue.
  75. 75. Photodynamic with methylene blue Fimple et al 2008
  76. 76. Treatment of endodontics infection Debridement of the root canal system Incision and drainage Intracanal medication Analgesics and antibiotics Follow up
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