Microbiology aspect in endodontics

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


Polymicrobial infection (facultative anaerobes

reduced oxygen Bacteriodes growth)

Sensitive to metronidazole แลิะ clindamycin

Resistance to penicillin (beta-lactamase)


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)


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


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


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


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


Why E.feacalis can survive in root filled teeth?


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 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

Education

Microbiology aspect in endodontics