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Microbiolog

y aspect in
endodontic
s
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

Spread of infection

State of disease

Associated anatomical structure

No.&Virulence of bacteria
Abscess
formation

PA Pathology

Host defense

Status of host defensive system

Entries of bacteria
to the pulp 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 m in diameter,
bacteria are less than 1 m 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.
Immune system via circulation

Whole pulp tissue change


due to periodontal disease

Necrotic
pulp
Periodontal lesion involve apical
foramen
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.

Love et al
1996
Dentinal tubules exposed by tooth
fracture during cavity preparation or
marginal leakage are 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.

Polymicrobi
al
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 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 in


endodontic Infections

Pisano and

Type of microorganisms found in


endodontic 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 r


etreatment
E. faecalis was found in 77% of cases,
confirming that this microbe is the most prev
alent species in failed endodontic treatment

Sundqvist 1998

Bacterial virulence factors

Newman, M. B., H. H.

Bacterial virulence factors


Bacteroides polysaccharide capsule

virulence factor (Samaranayake 2002)


Porphyromonas endodontalis capsule
phagocytosis proteolytic
capability immunoglobulin G
E complement factors C3 C5

toxic metabolic products
butyrate propionate
endodontic infection
Porphyromonas gingivalis collagenase
gene, amplicon
(Odell, Baumgartner et al. 1999)

Type of microorganisms found


endodontic
Infections
Aerobes
Streptococcus sp.: S.mitis, S.salivarius

in

Anaerobic streptococci : Peptostreptococcus


Enterococci: E. faecalis
Bacteroides sp.: Porphyromonas, Prevotella
Provotella nigrescens

Actinomyces

Fungi: Candida albicans


Fusobacteria
Spirochetes

Biofilm

Aerobes

Beta hemolytic non-hemolytic

streptococci

peptidoglycans lymphokine

osteoclast-activating factor, prostaglandin


lipoteichoic acid
complementbone resorption

S.mitis from root canal infection

bacterial endocarditis

Anaerobes: Enterococci
facultative bacteria, frequency in

Endodontic failure (Ingle and K.Bakland 2002)


Common in GI tract
streptococcus faecalis
(resistant to
antibiotic)
benzylpenicillin, ampicillin,
clindamycin, metronidazole
tetracycline

Anaerobes: Enterococci
sensitive erythromycin

vancomycin (Dahlen, Samuelsson


et al. 2000)
Molander and Dahlen 2003 :Tx
with calcium hydroxide+
erythromycin / tetracycline

Anaerobes: Bacteroides
strictly anaerobes, short chain,

gram negative rods and coccobacilli,


common in dental plaque, non motile,
no spore
polysaccharide capsule
virulence factor
serious anaerobic infection
sepsis, abscess

Anaerobes: Bacteroides
Polymicrobial infection

(facultative anaerobes reduced


oxygen Bacteriodes growth)
Sensitive to metronidazole
clindamycin
Resistance to penicillin (betalactamase)

Anaerobes: Bacteroides
2 genera Porphyromonas

Prevotella
Porphyromonas 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
(Samaranayak

Anaerobes: Bacteroides
Prevotella saccharolytic

bacteria, gram negative rods, non


motile
Prevotella melaninogenica

(Bacteroides melaninogenica)= black


pigment (melanin)
P.intermedia periodontal
disease
(Samaranayak

Anaerobes: Prevotella nigrescens


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 hormone-

like cytokines tisssue destruction

(Henderson and Wilson (1998), Matsushita et.al.


(1998))

B lymphocytes

complement cascade
collagenase and interleukin (macrophage
cells)
pain mediators histamine,
bradykinin prostaglandin

Anaerobes: Actinomyces
anerobic =microaerophilic gram-

positive filamentous bacteria,

non motile, no spore


Common in nasophalynx gingival
crevice
70-80% chronic infection,
granulomatous 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

candida
blastospores
hyphal structures
dentinal tubule

Fusobacteria: Fusobacterium
nucleatum
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 (

) non
culturable
Common in root canal infections,
pericoronitis, gingivitis
periodontitis (10% in endodontic
abscesses). (Dahle, Tronstad et al.
1993)

Primary endodontic
infection
Primary 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 2005
Sakamoto et al 2007
Vianna et al 2006

Primary endodontic
infection
Anaerobic bacteria

Prevotella,Porphyromona
s, Tanerella,
Fusobacterium, Dialister,
Camphylobactor,
Treponema

Actinomyces,Peptost
reptococcus,
Eubacterium,
Filifactor,
Psudoramibactor

Sterptococci

Black pigmented bacteria

Bacteroides

Saccharolytic;
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


Prevotella

Porphyromonas

P.intermedia
P.nigrescense
P.tannerae
P.multisaccharivorax

P.endodontalis
P.gingivalis

Siqueira et al 2001
Sundqvist et al 1989
Dougherty et al 1998

They seem to play important


role in etiology of both acute
and chronic apical
periodontitis.

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
Moraes
et al 2002
same root canals.
Siqueira et al 2005

Spirochete bacteria
Although 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 2000
Baumgartner et al 2007
Rocas et al 2003

Secondary endodontic
infection
If microorganisms are allowed to remain at the time
of filling , there is increased risk of adverse outcome
of 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


teeth
Unlike 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 1988
Siqueira 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
infection
E.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 1989
Siqueira et al 1996

E.feacalis in secondary
infection
E.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 2003
Sedgley et al 2005

Fungi in secondary
infection

The 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
infection

By 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
infection

It 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 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 cells


arranged in a biofilm

Siquera 2001

1. Surface conditioning
2. Adhesion of pioneer bacteria
3. Secondary colonizers
4. 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 antimicrobial 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
Concentration
Povidone iodine

E.feacalis, P.micros,
F.nucleatum,
S.intermedius
Spratt et al 2001

Er:YAG LASER

Er:YAG lasers had an antibiofilm effect at a low energy


and could reduce numbers of
the 6 species of biofilmforming cells examined.

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