Microbiology aspect in endodontics
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
Status of host defensive system
Associated anatomical structure
Host defense
PA Pathology
No.&Virulence of bacteria
Abscess formation
Entries of bacteria to the pulp system
•Caries•Periodontal disease•Trauma and restoration leakage•Anachoreasis
Dental caries
Facultative gram positive bacteriaPropionibacterium, 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 .
•Accessory canal•Furcation canal•Lateral canal•Apical foramen•Exposed dentinal tubule
Anerobic bacteria Gram negative rod, spirochete
Inflammation and local necrosis have been demonstrated in pulp adjacent to entry pathway.
Whole pulp tissue change due to periodontal disease
Immune system via circulation
Periodontal lesion involve apical foramen
Necrotic pulp
Guldenor et al 1985
Pulp exposure due to trauma give access to oral bacteria, this will cause bacterial invasion to the pulp inflammation
Pulp necrosis
Kakehashi et al 1965
Healthy exposed vital pulp, the penetration of tissue by bacteria is relative slow ≤2 mm./wk
Cvek et al 1978
Laboratory experiments indicate that bacteria can enter through even minor crack in enamel or dentine.
Dentinal tubules exposed by tooth fracture during cavity preparation or marginal leakage are the potential pathway.
Love et al 1996
Bender & Seltzer 1959Smulson & Sieraski 1989
Anachoresis
Intravenous bacterial injection into bloodstream could be demonstrated pulp that were inflamed due to deep cavity preparation and chemical irritation.
Burke & Knigton 1960Gier & Mitchell 1968
“Blood borne bacteria is preferentially localized in areas of inflammation.”
Polymicrobial
Microorganism
Pulpal and periapical disease
Endodontics failure
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 10 8
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 in endodontic Infections
ตาราง 2
Pisano and Weine 2004
Type of microorganisms found in endodontic Infections
Pisano and Weine 2004
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 predo minant microbe in canals undergoing retre atment
E . faecalis was found in 77% of cases, confi rming that this microbe is the most prevale
nt species in failed endodontic treatment
SSundqvist 1998undqvist 1998
Bacterial virulence factors
Newman, M. B., H. H. Takei, et al. (2 0 0 2 ).
Bacterial virulence factors
Bacteroides มี� polysaccharide capsule เป็�น virulence factor ที่�สำ��คั�ญ (Samaranayake 2002)
Porphyromonas endodontalis มี� capsule ที่�ที่��ให้�ต้��นที่�นต้�อ phagocytosis และยั�งมี� proteolytic capability ที่�สำ�มี�รถที่��ล�ยั immunoglobulin G และ E และ complement factors C3 และ C5 ได้� นอกจ�กน�!ยั�งมี� toxic metabolic products เช่�น butyrate และ propionate ที่�สำ�มี�รถพบได้�ในบร%เวณที่�มี� endodontic infection
Porphyromonas gingivalis ผล%ต้ collagenase gene, amplicon ซึ่*งช่�วยัเสำร%มีคัว�มีร+นแรงของก�รก�อโรคัได้� (Odell, Baumgartner et al. 1999)
Type of microorganisms found in endodontic 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
Aerobes Beta hemolytic และ non-hemolytic
streptococci ก�อโรคัได้�น�อยั peptidoglycans กระต้+�น lymphokine
เช่�น osteoclast-activating factor, prostaglandin
lipoteichoic acid กระต้+�น complementbone resorption
S.mitis from root canal infection bacterial endocarditis
Anaerobes: Enterococci
เป็�น facultative bacteria, frequency in Endodontic failure (Ingle and K.Bakland 2002)
Common in GI tract streptococcus faecalis ก��จ�ด้ออก
จ�กคัลองร�กฟั/นได้�ยั�ก(resistant to antibiotic)
ต้��นที่�นต้�อ benzylpenicillin, ampicillin, clindamycin, metronidazole และ tetracycline
Anaerobes: Enterococci
sensitive ต้�อ erythromycin และ vancomycin (Dahlen, Samuelsson et al. 2000)
Molander and Dahlen 2003 :Tx with calcium hydroxide+ erythromycin /tetracycline
Anaerobes: Bacteroides
เป็�น strictly anaerobes, short chain, gram negative rods and coccobacilli, common in dental plaque, non motile, no spore
มี� polysaccharide capsule เป็�น virulence factor
serious anaerobic infection เช่ น sepsis, abscess
ผลิ�ต endotoxin แลิะ proteases
Anaerobes: Bacteroides
Polymicrobial infection (facultative anaerobes
reduced oxygen Bacteriodes growth)
Sensitive to metronidazole แลิะ clindamycin
Resistance to penicillin (beta-lactamase)
Anaerobes: Bacteroides
2 genera คื�อ Porphyromonas แลิะ Prevotella
Porphyromonas เป็�น 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 endodontic abscess(Samaranaya
ke 2002)
Anaerobes: Bacteroides
Prevotella เป็�น saccharolytic bacteria, gram negative rods, non motile Prevotella melaninogenica (Bacteroides melaninogenica)= black pigment (melanin) พบได้�ในช่ องป็าก
P.intermedia พบได้�ใน periodontal disease (Samaranaya
ke 2002)
Anaerobes: Prevotella nigrescens ป็�จจ�บ นแยกออกจาก 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 Schilder(1975), Yamasaki
et.al.(1992), Horiba et.al.(1989))
Anaerobes: Prevotella nigrescens LPS+Peptidoglycans กระต��น hormone-
like cytokines tisssue destruction (Henderson and Wilson (1998), Matsushita et.al. (1998))
กระต��น B lymphocytes แลิะระบบ complement cascade
collagenase and interleukin (macrophage cells)
ผลิ�ต pain mediators เช่ น histamine, bradykinin แลิะ prostaglandin
Anaerobes: Actinomyces
เป็�น anerobic =microaerophilic gram-positive filamentous bacteria , ไมี ก อโรคืในช่ องป็าก
non motile, no spore Common in nasophalynx แลิะ
gingival crevice 70-80% chronic infection,
granulomatous แลิะ 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, Davies et al. 1996; Siqueira, Rocas et al. 2002)
Fungi
Candida albicans Debelian, Olsen et al. 1997 พบ
ว่ า candida สามีารถพบได้�ในผน งคืลิองรากฟั�นในร'ป็ blastospores แลิะ hyphal structures ซึ่)*งสามีารถแพร ผ าน dentinal tubule ได้�
Fusobacteria: Fusobacterium nucleatum
เป็�น 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 (ซึ่)*งมี�ได้�ทั้ ,ง เช่�,อต ว่เลิ-ก กลิาง แลิะใหญ่ ) non culturable
Common in root canal infections, pericoronitis, gingivitis แลิะ periodontitis (10% in endodontic abscesses). (Dahle, Tronstad et al. 1993)
Primary endodontic infectionPrimary infection is caused by microorganisms that initially invade and colonize the necrotic tissue.Primary infections are characterized by a mixed clostridium composed of 10-30species per canal.The number of bacterial cells in an infected canals varies from 103-108 cells.
Siqueira et al 2005Sakamoto et al 2007
Vianna et al 2006
Primary endodontic infection
Prevotella,Porphyromonas, Tanerella, Fusobacterium, Dialister, Camphylobactor, Treponema
Actinomyces,Peptostreptococcus, Eubacterium, Filifactor, Psudoramibactor
Anaerobic bacteria
Sterptococci
Black pigmented bacteria
BacteroidesSaccharolytic; Prevotella
Asaccharolytic; Porphyromonas
Black pigmented bacteria
In 1980, Griffee et al reported that B.melaninogenicus was found to be significantly related to pain, sinus tract formation, and foul odor.
Griffee et al 1980
Black pigmented bacteria
PrevotellaP.intermediaP.nigrescenseP.tanneraeP.multisaccharivorax
PorphyromonasP.endodontalisP.gingivalis
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 2002Siqueira et al 2005
Spirochete bacteriaAlthough spirochetes have been frequently observed in samples by microscopy, they had never been identified to the species level.
The application of molecular diagnosis to identification of spiral bacteria 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 symptomatic and asymptomatic lesion.
Dewhirst et al 2000Baumgartner et al 2007
Rocas et al 2003
Secondary endodontic infectionIf microorganisms are allowed to remain at the time of filling , there is increased risk of adverse outcome of the endodontic treatment
Sjogren et al 1997Waltimo 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 of microbial species has been found in persistent/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 teeth
Recently findings from molecular studies have also suggested that some anaerobic species commonly found in primary infection.
T.forsythia, P.alactolyticus, F.acolis, D.pnuemosintes
Siqueira et al 2004,2005
Microbial in root filled teeth
Bacteria are secondary invaders that can gain entry in to due to a breach in the aseptic chain during intracanal intervention.
P.aeruginosa, Staphylococcus
Ranta et al 1988Siqueira et all 2002
E.feacalis in secondary infection
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 secondary infectionE.feacalis has been found in root filled teeth evincing persistent apical periodontitis in prevalence values ranging from 30-90% of the cases.
This species can be inhibited by other members of mixed bacterial consortium commonly present in primary infection.
Sedgley et al 2006
E.feacalis in secondary infection
Virulence 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 secondary infection
Why E.feacalis can survive in root filled teeth?
E.feacalis in secondary infection
Studies have revealed that E.feacalis has ability to penetrate far into dentinal tubules that can escape from intracanal instrumentation and irrigants.
Haapasalo et al 1989Siqueira et al 1996
E.feacalis in secondary infectionE.feacalis has been shown to be able to form biofilms in root canals and this ability can be important for bacterial resistance and persistence in the instrumented canals.
Distel et al 2002
The ability to resist high pH value seems to be related to functional proton pump to acidfy. Which E.feacalis is resistant to calcium hydroxide.
Evans et al 2002
E.feacalis in secondary infection
E.feacalis can enter a VBCN state which can survive in adverse environmental condition, including starvation.
They has the ability to survive in environments in scarcity of nutrients and to flourish when the nutrient source is reestablished and has a capacity to recover in root canal treated teeth for 12 months without nutrients.
Figdor et al 2003Sedgley et al 2005
Fungi in secondary infectionThe occurrence of yeasts were taken from samples that not responding in favorably to conventional treatment (72% of cases ).
Waltimo et al 1997
Candida albicans was the most common species.
Fungi in secondary infectionBy the new detection technology, C.albicans was detected in primary infection in 21% too.
However the finding indicates that yeasts may be present in low number at the start of treatment, and they may reach higher proportion during root canal procedures .
Buamgartner et al 2000
Fungi in secondary infectionIt is also possible that yeasts from oral cavity gain access to root canal as contaminant during endodontic procedures.
Siren et al 1997
Or they can overgrow after inefficient intracanal antimicrobial procedures, which can cause imbalance in microbiota.
Siqueira et al 2004
Fungi in secondary infection
Virulence factor
Dentino phillic microorganism due to its ability to colonize and invade to dentine by their hyphae.Resist to some intracanal medication such as calcium hydroxide.Release hydrolytic enzyme and can form monoinfection biofilm in root canals.
Sen et al 1997Waltimo 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 cells arranged in a biofilm
SSiquera 2001iquera 2001
1. Surface conditioning2. Adhesion of ‘ pioneer ’ bacteria3. Secondary colonizers4. Fully functioning biofilm
1. A cooperative “consortia” of species
2. Biofilms grow and deattachment
A cooperative “consortia” of species
Biofilms grow and spread
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 in biofilm
Bacteria 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 compare to planktonic form. increased MIC & MBC
Larsen et al 2002
Irrigants for microbial control
•Sodium hypochlorite•Combination of sodium hypochlorite and chlorhexidine•Chlorhexidine•Povidone iodine
E.feacalis, P.micros, F.nucleatum, S.intermedius
Spratt et al 2001
Concentration
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.
Er:YAG LASER
Noiri et al 2008
Photodynamic with methylene blue
Photodynamic therapy (PDT) was developed as a therapy for cancer.Activated by light of the appropriate wavelength to generate singlet oxygen and free radicals that are cytotoxic 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