Microbiology aspectinendodontic Thalerngsak Samaksamarn Department of restorative dentistry Faculty of dentistry. KKU
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
State of disease Spread of infection No.&Virulence of bacteria Associated anatomical structure Abscess formation PA Pathology Host defense Status of host defensive system
•Caries•Periodontal disease•Trauma and restoration leakage•Anachoreasis
Dental caries Facultative gram positive bacteria Propionibacterium, Eubacteruim, Arachnia, Lactobacilli, Bifidobacterium , Actinomyces Some of gram negative bacterial such as Bacteroides Hoshino et al 1985
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
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.
Inflammation and local necrosis have beendemonstrated in pulp adjacent to entry pathway. Immune system via circulation Whole pulp tissue change due to periodontal disease
Necrotic pulpPeriodontal lesion involve apical foramen Guldenor et al 1985
Pulp exposure due to trauma give access tooral bacteria, this will cause bacterial invasionto the pulp inflammation Pulp necrosis Kakehashi et al 1965
Healthy exposed vital pulp, the penetration oftissue by bacteria is relative slow ≤2 mm./wk Cvek et al 1978
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
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.
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
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
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
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.
Type of microorganisms found inendodontic Infections ตาราง 2 Pisano and
Type of microorganisms found inendodontic Infections Pisano and
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
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,
Anaerobes: Enterococci sensitive ต่อ erythromycin และ vancomycin (Dahlen, Samuelsson et al. 2000) Molander and Dahlen 2003 :Tx with calcium hydroxide+ erythromycin / tetracycline
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
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
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)
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,
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)
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)
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
Black pigmented bacteria Saccharolytic; Prevotella Bacteroides Asaccharolytic; Porphyromonas
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
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
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
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
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
Secondary endodontic infection Create both excellent apical and coronal seal. Prevent bacterial nutrient regain in to the canal.
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 “
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
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
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
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
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
E.feacalis in secondaryinfection Why E.feacalis can survive in root filled teeth?
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
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
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
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.
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
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
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
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
SEM of bacterial cellsarranged in a biofilm Siquera 2001
1. Surface conditioning2. Adhesion of ‘ pioneer ’ bacteria3. Secondary colonizers4. Fully functioning biofilm 1. A cooperative “consortia” of species 2. Biofilms grow and deattachment
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
Benefits A broader habitat range for growth A more efficient metabolism Increased resistance to stress and antimicrobial agent Enhanced virulence
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
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
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
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.
Photodynamic with methylene blue Fimple et al 2008
Treatment of endodontics infection Debridement of the root canal system Incision and drainage Intracanal medication Analgesics and antibiotics Follow up
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