MICROBIOLOGY IN

ENDODONTICS
PRESENTER: P. Sravan
GUIDEd BY: PG 2nd year
Dr.P.Karunakar (Prof. & H.O.D.)
Dr. Raji viola solomon (Prof.)
Dr. P. Siddhartha
 Contents
• PART 1

• Introduction
• Terminology
• Classification
• Structure of bacterial cell
• Fish zones
• Kraunfelds mountain pass concept
• General description of microbial flora
• Bacteria associated with root canal
 PART 2

• Clinical bacteriological techniques

• Rationale for microbial sampling
• Culturing
• Specimen collection and transport
• Antimicrobial susceptibility testing
• Polymerace chain reaction
• Conclusion
• References
 Introduction
• Microorganisms cause virtually all pathoses of the pulp and
the periradicular tissues.

• To effectively treat endodontic infections, clinicians must

recognize the cause and effect of microbial invasion of the
dental pulp space and the surrounding periradicular tissues
• Knowledge of the microorganisms associated with endodontic
disease is necessary to develop a basic understanding of the
disease process and a sound rationale for effective
management of patients with endodontic infections
 HISTORY
• 1665- Robert Hooke

• Used simple lens of magnification 30X

• Observed –eye of a fly, shell of protozoan, thin slices of

cork

• Found cork was made of small boxes that he referred to

as “cells”
• 1667 Antonie Van Leeuwenhoeck
• Used lenses of magnification 300×
• Viewed –scrapings from his teeth
• First observed and described bacteria
• Named them as animal ‘cules

• FATHER OF MICROBIOLOGY
 Joseph Lister

• A surgeon concerned about post

operative infection

• Demonstrated that boiling

instruments and washing hands and
spraying phenol in air around the
patient before surgery reduces the
post operative infections.
• 1843 to 1910 – Robert Koch introduced
staining technique and culture
techniques and discovered tuberculosis
and cholera.
 Kochs postulates
• A micro organism can be accepted as the causative agent of an infectious
disease only if the following conditions are fullfilled
• 1. the organism should be consistently associated with the lesions of the
disease.
• 2. it should be possible to isolate the organism in pure culture from the
lesions of the disease.
• 3. the isolated organisms when inoculated in suitable laboratory animals
should produce a similar disease.
• 4. it should be possible to re isolate the organism in pure culture from the
lesions produced in the experimental animals.
Year
1890 W. D. Miller associated the presence of bacteria with pulpal and
periapical disease

1904 F. Billings described a “focus of infection” as a circumscribed area of

tissue infected with pathogenic organisms.

1909 E. C. Rosenow, described the “Theory of Focal Infection”.

1910 William Hunter, emphasized restorations instead of tooth extraction.

• Weston Price began a 25-year study on pulpless and endodontically
treated teeth and their association with focal infection.

• With expansion of the theory, many dentists and physicians became

“100 Percenters,” who recommended the extraction of all pulpless
and endodontically treated teeth.
 1930s- editorials and research refuted the theory of focal infection
and called for a return to constructive rather than destructive dental
treatment rationale.

The studies by Rosenow and Price were flawed by

• INADEQUATE CONTROLS,
• USE OF MASSIVE DOSES OF BACTERIA,
• BACTERIAL CONTAMINATION OF ENDODONTICALLY TREATED
TEETH DURING TOOTH EXTRACTION
• In 1939,Fish recognized four zones of reaction formed in
response to viable bacteria.

• Fish theorized that removal of the nidus of infection

would lead to resolution of the infection
• This theory became the basis for successful root canal treatment
TERMINOLOGY
 NORMAL FLORA

• Normal flora or normal

microbiota, are microorganisms
that begin to colonize the body
at birth
 Normal oral flora

is the result of a
permanent microbial colonization
in a symbiotic relationship
with the host
 Colonization
is the establishment of microbes in a
host
if appropriate biochemical and
physical conditions are
available for growth
 Although the microbes in the
normal oral flora participate in
many beneficial relationships,
they are opportunistic
pathogens if they gain
access to a normally sterile area
of the body such as the
dental pulp or periradicular
tissues and produce disease
 Pathogenicity
is a term used to describe the capacity
of a microbe to produce disease,

whereas

Virulence
describes the degree of pathogenicity
Virulence Factors
that may be associated with disease.

-pili (fimbriae),
-capsules,
-extracellular vesicles,
-lipopolysaccharides,
-enzymes,
-short-chain fatty acids,
- polyamines,
-low-molecular-
weight products such as ammonia and hydrogen
sulfide
INFECTIOUS DISEASE [INFECTION]

Results if microorganisms damage the host

and produce clinical signs and symptoms.
 ANACHORESIS

• Anachoresis is a process by which microbes may be

transported in the blood or lymph to an area of
inflammation such as a tooth with pulpitis, where they may
establish an infection
Antagonistic relationships

between bacteria may

occur.

Some metabolites (eg, ammonia) may be

either a nutrient or a toxin, depending on
the concentration.

In addition, bacteria may produce

bacteriocins, which are antibiotic-like
proteins produced by one species of
bacteria to inhibit another species of
bacteria
 Obligate aerobes Obligate anaerobes

• Require oxygen to grow • Cannot grow in presence of

oxygen
• Because their ATP- generating
• They lack either superoxide
system is dependent on oxygen dismutase or catalase or both
as the hydrogen acceptor • fusobacterium,
peptostreptococcus,
• Mycobacterium tuberculosis Eubacterium, propionbacterium,
veilonella, wolinella, prevotella,
porphyromonas,

• Dominate the root canal

microflora
Facultative anaerobes

• grow in the presence or

absence of oxygen and
• usually have the enzymes
superoxide, dismutase and
catalase
 Microaerophiles

• Grow best at a low oxygen concentration

• Derive energy from anaerobic energy pathway

• Campylobacter fetus
 PROBIOTICS
• A probiotic substance or preparation.
• A microorganism introduced into the body for its beneficial
qualities
Structure of bacterial cell
 Locomotion

Bacteria - Shape and move.mp4

CLASSIFICATION
Gram –positive Cocci Gram-negative Cocci

• Streptococcus anginosus • Capnocytophaga ochracea

• S. sanguinis • C. sputigena
• S. mitis • Veillonella parvula
• S. mutans • Campylobacter rectus C. curvus
• Enterococcus faecalis
• Peptostreptococcus micros
• P. anaerobius

Sundquist 1994, Le Goff et al. 1997

• Gram-positive Rods Gram-negative Rods

• Actinomyces Israeli Fusobacterium nucleatum

Prevotells intermedia
• A.naeslundii
P. melaninogenica
• Eubacterium alactolyticum P. denticola
• E. lentum P. buccae
• E. nodatum P. buccalis
• E. timidum P. oralis
• Propionibacterium Pophyromonas gingivalis
propionicum P. endodontalis
Bacteroides gracilis
• P. granulosum
• Lactobacillus
Distribution of endodontic pathogens according to
respective phyla
• According to a study by Siqueira(2003),the bacteria fall into 6
bacterial groups or phyla-
– Actinobacteria
– Proteobacteria
– Fusobacteria
– Bacteroidetes
– Firmicutes
– Spirochetes
Recent Taxonomic Changes for Previous
Bacteroides Species

• Porphyromonas—black-pigmented (asaccharolytic
Bacteroides species)
• Porphyromonas asaccharolyticas (usually nonoral)
• Porphyromonas gingivalis*

• Porphyromonas endodontalis*
• Prevotella—black-pigmented (saccharolytic Bacteroides
species)
• Prevotella melaninogenica
• Prevotella denticola
• Prevotella loescheii
• Prevotella intermedia*
• Prevotella nigrescens†
• Prevotella corporis
• Prevotella tannerae
• Prevotella—nonpigmented (saccharolytic
Bacteroides species)
• Prevotella buccae*
• Prevotella bivia
• Prevotella oralis
• Prevotella oris
• Prevotella oulorum
• Prevotella ruminicola
Microbiology of the dentino –
pulpal complex

1-Microbiology of
dentin and pulp under
caries

2-Biology of
microbes in the root
canal
1-Microbiology of dentin
and pulp under caries

• Caries is regarded as main

source of bacteria in
infection of pulp and
periapical area
• 93%-gram(+)ve rods
• 32%-gram(+)ve cocci
• 11.6%-gram(-)ve cocci
• 5%- gram (-)ve rods
2-Biology of microbes in the
root canal
• Environmental
factor in root canal

• Host-parasite
relation
• Microbial
interactions
 Host parasite relation
In open communication-
Whole saliva and plaque flora
Invasion via carious lesion-
Streptococci, G+ facultative
and anaerobic rods
Possibilities for entrance-
Dentinal tubules
 Fractures
 Lateral canals
Hematogenous route
 Lack of collateral circulation

Necrotic tissue

Helps in growth of proteolytic bacteria

Use tissue and serum proteins for growth

Evade host defences

Capsule provides resistance to phagocytosis

Multiplication and growth

 Microbial interactions Antagonistic forces
Production of toxic metabolites H2O2,
-Initially pulp and root NH3,bacteriocins.
canal are sterile
• Symbiotic relations
-Competition starts once Consumption of O2by facultative
bacteria favors anaerobic species
microbes enter root
canal Proteolytic degradation of Igs
destroys host defence and releases
peptides which are nutrients for the
-Competition for space, bacteria
nutrients
 Periapical pathology
• According to concept of Fish zones:

A Peri-apical lesion is not an infection by

itself, but the reaction of body to
infection in the canal.

Zone of stimulation

Zone of irritation
Zone of contamination

Zone of necrosis
 Fish theory of zones of inflammation

• The reaction of the periradicular tissues to noxious products of

tissue necrosis, bacterial products, & antigenic agents from the
root canal has been described by Fish
• Experiment- jaws of guinea pigs-drilling openings in the bone-
packing in wool fibres saturated with broth culture of
microorganisms

47
• Zone of infection
Exudative(acute)zones
• Zone of contamination

Transitional area

• Zone of irritation
Proliferative(chronic)
zones
• Zone of stimulation 48
 Zone of necrosis ( zone of infection)

• Necrotic or infected root canal contents are

• Pus fluid contains dead cells, destructive components released

from phagocytes, end products of protein decomposition
( proteolysis)
• PMN
• Microorganism, exotoxins, endotoxins, antigens , bacterial
enzymes, chemotactic factors.

49
 Zone of contamination ( exudative
inflammatory zone)

Immediate response to toxic elements coming out of root

canal are
• Principal exudative defense response – vasodilatation, fluid
exudation, cellular infiltration
• Dilution of toxic elements plus antibacterial action of
inflammatory fluid.
• Principal defense cells – PMN’s, macrophages
• Cellular destruction- bacterial toxins

50
 Zone of irritation ( granulomatous zone, proliferative
inflammatory zone)

Toxicity diminishing as distance from canal foramina

increases

1. Function – defense, healing , repair.

2. Principal proliferative response – granulation tissue capillary
proliferation & fibroblastic activity)
3. Granulomatous – granulation tissue plus chronic defense cell
4. Principal Chronic defense cell – lymphocytes, plasma cells,
blood derived macrophages,tissue macrophages

51
5. Cell derived mediators of inflammation – antibodies from
plasma cells, lymphokines from sesitized T cells, histamine &
serotonin from basophils.
6. Russel bodies- enlarged plasma cells with numerous antibody
inclusion.
7. Eosinophils – attracted by mast cell ECF- A & lymphokine
ECF – A.. They modulate inflammation & allergy by
destroying certain vasoactive substances like PAF & SRS – A
8. Foam cells , cholesterol crystals , epithelial clusters & strands.
9. Favorable environment for osteoclast
52
Zone of stimulation ( zone of encapsulation
/ productive fibrosis)

• Toxicity reduced to a mild stimulant

• Peripheral orientation of collagen ( fibroblastic activity )

• Favorable environment for osteoblastic activity
• Bone apposition & reversal lines evident
• Reactive hyperostosis when lesion encroaches on the cortical
plate.

53
 Kronfeld’s Mountain Pass Concept

• According to Kronfeld: Granuloma is

not an environment in which bacteria
survive; but they are destroyed here.

Zone 1

Mountain pass

Zone 2

Zone 3
 Kronfeld’s Mountain pass concept

• Compares the bacteria in the root canal (zone 1) with an army

entrenched behind high & inaccessible mountains.
• Praposed the theory in 1939
• The foramina serve as mountain passes.
• The exudative & proliferative tissue of the granuloma
represents a mobilized army defending the plains (periapex)
from the invaders.

55
• If only a few invaders enter the plain through the mountain
pass, they are destroyed by the defenders(leucocytes).
• A mass attack of invaders results in a major battle, analogous
to acute inflammation (zone 2).

56
• Only complete elimination of the invaders from the
mountainous entrenchment will eliminate the need for a
defense force in the plains.
• Once this is accomplished, the defending army of leukocytes
withdraws & the local destruction created by the battle is
repaired(zone3) & the environment returns to the normal
pattern.

57
 Morphological localisation of bacteria

-Infection in the main and lateral canals

-Dentinal tubule invasion
-Bacteria In Periapical Tissues
-Bacteremia in connection with apical periodontitis
 Usually infection does not extend
beyond the apex and bacteria
Morphological localisation of cannot be detected outside the root.
bacteria

♣ Infection in the main and Apical flora is delineated from

periradicular tissues by PMNL’s at
lateral canals or near apical foramen
It depends on
Redox potential
Histological studies have shown
 Availability of nutrients bacterial penetration in lateral
 Host defences canals also.
 Morphological localisation of bacteria
♣ Dentinal tubule invasion

bacteria from main canal can spread into surrounding dentin via
dentinal tubules
Oguntebi, IEJ1994;27:218-222.

Mechanism of invasion not known. It occurs at random

Nair,JOE1987;13:29-39.

• Gram positive species (lactobacilli and streptococci) can invade dentinal tubules
more easily than gram negative species.
Orstavik,Endodont Dent Traumatol 1990;6:142-149
 ♣ Bacteria In Periapical Tissues

• In cases of persistent apical

Earlier accepted that bacteria in periodontitis bacteria can survive on
apical periodontitis are located the root surface or in periapical
tissues.
in root canal system only.
• Presence of sinus tract also indicates
permanent presence of bacteria in
Periapical Actinomyces is extra radicular area.
regarded as an exception as it is
present in the periapical area.
Haapasalo et al 1987, Transtadt et al 1987,
Walten et al 1992.
Fukushima et al.JOE1990;16:534-538,
Richard E Walton,JOE 1992;18:216-227
♣ Bacteremia in connection with apical periodontitis

Occurs through:
Tissue and lymphatic Species recovered from blood
vessel
• F.nucleatum
 Often by trauma • P.intermedia
• Peptostreptococcus prevotii
-Transient in nature • Propionibacterium acnes
-Number of bacteremia per • Actinimyces israelii
• Streptococcus intermedius
ml blood in endodontic
bacteremia is lower than in Debelian et al 1992
bacteremia during scaling
or tooth extraction.

Heimdahl et al 1990
 GENERAL DESCRIPTION OF
MICROBIAL FLORA

• Microscopic studies have shown the

presence of SPIROCHETES in
infected root canal.
Apical
periodontitis is a Dahle et al 1995, 1996
polymicrobial infection
dominated by obligately
anaerobic bacteria. • Teeth with periapical radiolucent
areas have increased incidence of
infected root canals.

Haapasalo et al 1986, 1993

 Bacterial Genera Most Frequently
Isolated Prior To RCT.
Gram –ve anaerobic rods Gram +ve anaerobic rods
Prevotella intermedia Eubacterium spp
P.nigrescens Propionibacterium acnes
P.denticola Actinomyces viscosus
Porphyromonas gingivalis A.israelii
P.endodontalis A.naeslundii
Fusobacterium nucleatum Lactobacillus
Campylobacter rectus
Selenomonas spp. Gram +ve Facultative cocci

Streptococcus sanguis
Gram –ve anaerobic cocci S.intermedius
S.mitis
Peptostreptococcus micros
 Microbiology Of Acute Periapical abscess

* Bacteria and clinical symptoms

* Bacterial species associated with symptoms
*Clinical clues of anaerobic infection
* Microorganisms in persistent infections
* Microorganisms in flareups
 Bacteria and clinical symptoms:

• Probability of pain increase when a

Include: minimum of 6 sp are present in
-pain root canal

-tenderness to percussion Positive relationship between the

presence of symptoms and black
-swelling pigmented bacteriodes
-abscess and or open sinus Sundqvist et al 1976
tract
 Bacterial sp associated with symptoms

Gram +ve anaerobic rods

Eubacterium spp

Gram –ve anaerobic

Gram +ve anaerobic cocci
rods
Peptostreptococcus magnus
P.intermedia
P.gingivalis
F.nucleatum
P.endodontalis
 Clinical clues of anaerobic infection

Tissue necrosis with

abscess formation -Failure to recover pathogens
from aerobic culture
Swelling, pain, fever
-Failure to respond to
antibiotics which are
Purulent discharge with effective against aerobes
foul smell
Microorganisms in
Microorganisms in flare ups
persistent infections
Bacteriodes melaninogenicus
E.faecalis, Enterobacter B.assacharolyticus
cloacea and Veilonella
F.nucleatum
P.aeruginosa Prevotella spp
Porphyromonas spp

Siren et al

Shibi Mathew,

J Endod 2010;36:832–836
MICROBIOLOGY OF
CHRONIC PERIAPICAL PERIODONTITIS

• Primary lesion

Root filled teeth

 Primary lesion
Gram +ve Facultative cocci
Gram –ve anaerobic rods Streptococcus sanguis
Prevotella intermedia S.intermedius
P.nigrescens S.Mitis
P.denticola
Campylobacter rectus
Selenomonas spp. Gram +ve anaerobic rods
Eubacterium spp
Gram –ve anaerobic cocci Propionibacterium acnes
Veilonella
Actinomyces viscosus
Gram -ve Facultative rod A.israelii
E.corrodens A.naeslundii
Capnocytophaga spp Lactobacillus
 Root filled teeth
Apical periodontitis in root filled teeth is
associated with high frequency of E.faecalis(77%)
Pseudoramibacter
alactolyticus (52%)
streptococci, enterococci,
Propionibacterium
Propionibacterium, Eubacterium, propionicum (52%)
anaerobic streptococci Dilaster pneumosintes
(48%)
Candida albicans (9%)
Engstrom and Moller
Based on polymerase
chain reaction

Siqueira and Rocas OOOE 2004;97:85-94

HERPES VIRUS IN PERIAPICAL
PATHOSIS

Strong association of HCMV and EBV with acute exacerbations:

Such lesions show

elevated occurrence of Anaerobic bacteria
Symptomatic
Large sized radiographic bone destruction
 Herpes virus may cause periapical pathosis either as a

Direct result of virus infection and replication

 Result of virally induced damage to host defense

• Hallmark of Herpes virus infection: Impairment and dysregulation of

macrophages and lymphocytes

• Series of interaction between Herpes virus, bacteria and host immune reaction
may cause some type of AGGRESSIVE PERIAPICAL PATHOSIS
PA flare ups have a multifactorial causation

 Herpes virus presence at periapical sites

 Activation of latent periapical herpes virus
 Inadequate antiviral cytotoxic T lymphocyte
 Presence of pathogenic bacteria
 Inefficient level of protective antibacterial antibodies.
 Extraradicular Infections

may be found in the following situations

 Acute periapical periodontitis

 Periapical actinomycosis
 In association with pieces of infected root dentin displaced into the
periapex
 Infected periapical cyst
 PERIAPICAL ACTINOMYCOSIS

-gram (+)ve anaerobic rods

-the most commonly isolated

species
from periapical tissue that not
respond to proper conventional
pulp
Actinomycosis
space therapy-
A.israelii
Biologic properties that enable these bacteria to establish in the
periapical tissues are not fully understood, but they appear to involve
the ability to build cohesive colonies that enables them to escape the
host defense system
LABORATORY DIAGNOSIS

Light microscopic feature

Intensely dark staining, gram
positive core with radiating
peripheral filaments that give the Actinomycosis
typical “ray fungus” appearance.

Ultra structurally centre of the • Several layers of PMNL’s

colony consists of a dense surround an actinomycotic
aggregation of branching colony
filamentous organisms held
together by an extacellular
matrix.
 •
ENTEROCOCCUS FAECALIS

gram (+)ve cocci

facultative anaerobes
-Plays a major role in the
-Prevalence rates
etiology of persistent In primary infection- 9-18%
periradicular lesions after In persistent lesions- 24-67%
pulp space therapy Rocas JOE 2004

-It is associated with -In many cases it has been isolated as

primary endodontic and the only organism in root filled teeth
persistent infections. with periradicular lesions.
OOOE 1998;85:86-93

JOE 2004;30:315-320.
 ENTEROCOCCUS FAECALIS
Methods to detect
Culturing techniques
Polymerase chain reaction
Optical spectroscopy

Survival and virulence factors

-Endures prolonged periods of nutritional
deprivation
-Binds to dentin and proficiently invades dentinal
tubules
-Alters host responses
-Suppresses action of lymphocytes
-Possesses lytic enzymes, cytolysin, aggregation
substances, pheromones, lipoteichoic acid.
• Utilises serum as nutritional source
• Resists intracanal medicaments
• Competes with other cells
• Forms a biofilm- 1000 times more resistant to phagocytosis, antibodies,
antimicrobials
JOE 2002; 28:689-93.
METHODS TO ERADICATE E. FAECALIS

• Good aseptic technique

• Pretreatment CHX rinse
• Disinfection of tooth and rubber dam
• Disinfection of gutta percha with NaOCl
• Adequate instrumentation
• Canal irrigation: 6% NaOCl, 17%EDTA, 2% CHX
• Intracanal Medicaments – 2%CHX gel
• AH plus sealer
• Well sealed coronal restoration
FUNGI

Fungi in primary endodontic infections

Siqueira et al 2002–prevalence rate 2% by PCR
Siqueira et al 2002–prevalence rate 7% by SEM
These studies indicate that fungi are not common members of
microbiota associated with primary endodontic infections.
• Fungi in persistent or secondary
endodontic infections

Pinheiro et al 2003 in a culture found

prevalence rate 4%
Siqueira and Rocas 2004 in a PCR found
prevalence rate 9%
• Fungi are more common in failed RC Candida albicans
• C.albicans was the most common isolate –
Nair et al.
• Fungi can gain entry in to root canal during
endodontic therapy and can be involved in
the etiology of recalcitrant periradicular
lesions
• Mechanism of Fungal pathogenicity

-Adaptability to a variety of environmental conditions (pH<7)

-Adhesion to a variety of surfaces (collagen type I,IV; coaggregation with S.sanguis,
S.gordonii, S.oralis).
-Production of hydrolytic enzymes- degradation of extracellular matrix (collagenase,
aminopeptidase).
-

Siqueira et al, OOOEndod 2004;97,632-41

Morphologic transition- C.albicans is polymorphic
fungus(blastospores,germ tube, true hyphae,
pseudohyphae,chamydospores) depending
on environmental condition.

C.Albicans
-Biofilm formation: C.albicans growing in a biofilm is 100-fold more
resistant antifungal fluconazole and 20-30 fold more resistant to
antifungal amphotericin B
• Evasion and immunomodulation of the host
defenses – various mechanisms of evasion
of host defenses by C.albicans.

• Blocks functions of PMN cells

• Produces proteinases(degrade complement factors and
immunoglobulins)
• Cell wall components involved in pathogenesis of periradicular
lesions(glucan, chitin, mannoprotein)
• Capable of stimulating pro-inflammatory cytokine synthesis and
release
• Can activate complement system
Dentin Colonisation by Fungi

Leads to persistent root canal infections

C.albicans is a dentinophillic microorganism due to invasive affinity to
dentin (Thigmotropism).
Enamel and Cementum are readily colonised by C.albicans
Presence of smear layer increased the adhesion of C.albicans to
dentin.
Sen et al 1997
Susceptibility to antimicrobial endodontic medicaments
C.albicans is more resistant in the presence of smear layer.

EDTA has the most effective antifungal action.

• It reduces adhesive properties of C.albicans.
• Removes calcium ions from cell walls
• Inhibits enzymatic reactions
• It has anti-growth chelating properties that chelate to
calcium, magnesium, manganese and zinc ions that are required
for growth and morphogenesis of C.albicans

Sen et al 1999
Calcium hydroxide provides calcium required
for growth and morphogenesis of fungi
(Waltimo et al 1999).

However, paste of calcium hydroxide in camphorated

paramonochlorophenol/ glycerin has pronounced antifungal
activity (Siqueira et al 2004)

NaOCl, CHX, H2O2 are effective against C.albicans even

if it is significantly diluted (Fergusson et al 2002).
Predominant oral microflora Predominant root canal flora
Streptococcus spp. Streptococcus spp.
(mutans,sanguis,pyogenes) (gordinii,anginosus,
intermedius
Porphyromonas spp
Actinomyces (commensals) A.radicidentis, A.israeli,
A.naeslundii

S.gordinii, S.mutans, P.gingivslis

 T. Denticola
• The numbers of Treponema species/phylotypes per selected
positive sample ranged from 2 to 12. Species-specific nested
PCR detected T. denticola, T. socranskii, and T. maltophilum
in 59 (66%), 33 (37%), and 26 (29%) of the 90 cases of
primary endodontic infections

• Species-specific nested PCR demonstrated that T. denticola, T.

socranskii, and T. maltophilum, in decreasing order of
prevalence, are frequently found in different forms of apical
periodontitis

J Clin Microbiol. 2009 May; 47(5): 1352–1357

 Biofilm
• Biofilm- A biofilm (Costerton et al. 2003) is a community of
microorganisms embedded in an exopolysaccharide matrix
that adheres onto a moist surface

Nair PNR. On the causes of persistent apical

periodontitis: a review. Int Endod J 2006;39:249–81
• Caldwell et al., a biofilm has the following attributes:
1. Autopoiesis : ability to self-organize
2. Homeostasis : ability to resist environmental disturbances
3. Synergy : effective in association with fellow microrganisms
than in isolation
4. Communality: respond to environmental challenges as a
combined unit
• Intracanal microbial biofilms: radicular dentin-
endodontically infected tooth
E.faecalis- most therapy resistant,prevalent

• Extra radicular microbial biofilms: cemental surface

around the root apex - endodontically infected tooth

• Periapical microbial biofilms: isolated ; independent of the

above two
Actinomyces & P.propionicum
 Biofilm formation

How bacteria form a biofilm.mp4

 MICROBIOLOGY IN
ENDODONTICS
PRESENTER: P. Sravan
GUIDEd BY: PG 2nd year
Dr. P.Karunakar (Prof. & H.O.D.)
Dr. Raji viola solomon (Prof.)
Dr. P. Siddhartha (Sr. lecturer)
• PART 1 Contents
• Introduction
• Terminology
• Classification
• Structure of bacterial cell
• Fish zones
• Kraunfelds mountain pass concept
• General description of microbial flora
• Bacteria associated with root canal
• Biofilms
 PART 2
• Clinical bacteriological techniques
• Rational for microbial sampling
• Specimen collection and transport
• Culturing
• Antimicrobial susceptibility testing
• Polymerace chain reaction
• Clinical correlations
• Conclusion
• References
 CLINICAL BACTERIOLOGICAL
TECHNIQUES

Bacteria present in root canal system can be evaluated by

1. Staining
2. Culturing
3. Immunological techniques
4. DNA probes
 GRAM STAINING
• Introduced by Christian Gram in 1884

• Gram (+)ve –purple (hamatoxyline)

• Gram (-)ve – pink (eosin)
Advantages:
Rapid, Relatively easy and Inexpensive.

Disadvantages:
Clinical usefulness is questionable.
Can reveal only morphology of organism.
Identification at genus or species level is practically impossible.
Limited sensitivity and specificity.
PHASE CONTRAST MICROSCOPY / DARK FIELD MICROSCOPY

Easy and quick to perform

Requires more experience than Gram staining
Cell boundaries appear sharp.

Motility of microbes can be seen in

fresh samples.
Spirochetes can be detected- Trope et al
 Immunological methods
Based on specificity of the antigen antibody reaction.
Most commonly used methods
ELISA
Direct/indirect immunofluorescence
Advantages:
Takes less time to identify a microbial
species.
Can detect dead organisms
Can be easily standardized
 DNA probes
• In molecular biology, a
hybridization probe is a fragment
of DNA or RNA of variable length
(usually 100–1000 bases long)
which can be radioactively labeled.
It can then be used in DNA or
RNA samples to detect the
presence of nucleotide sequences
(the DNA target) that are
complementary to the sequence in
the probe.
DNA Probes & Hybridisation.mp4
 Rationale for microbial sampling:

Diagnostic sampling
provides information as to whether or not infection is present.
This is useful in necrotic pulps with acute symptoms which may require
antimicrobial treatment.

Sampling during treatment indicates treatment progress.

Sampling at the end is used to disclose a bacteria- free root canal before a
permanent filling is made.
The principles and practices of bacterial cultivation, have three main purposes:

• To grow and isolate all bacteria present in an infection.

• To determine which of the bacteria that grow are most likely to cause infection and
which are likely contaminants or colonisers.
• To obtain sufficient growth of clinically relevant bacteria to allow identification
and characterization.
 R IN G
U LT U
C
Specimen collection and transport
The following general rules apply to all specimens:

1. A sufficientquantity of specimen must be provided to permit

thorough study.
2. The sample should be representative of the infectious process.
3. Only sterile equipment and aseptic techniques should be used to
prevent specimen contamination.
4. The specimen must be transported to the laboratory and examined
promptly. Special transport media may be helpful or required (i.e.,
anaerobes).
5. The specimen should be accompanied by a request form indicating
the genera suspected. Separate samples must be submitted for
aerobic and anaerobic cultures.
 Steps in taking a culture

• The dressing from previous visit is removed and discarded

• A sterile absorbent paper point is inserted in to the canal, with wiping
motion, to cleanse the canal of any trace of medicament
• If there is drainage collect the sample using sterila needle (16-20
gauge)syringe
• The paper point is removed and discarded
• A fresh, sterile absorbent point is inserted and allowed to remain in
canal for at least 1 min, so that tip of absorbent point will be moist
• Remove the absorbent point and examine the tip
• unscrew the cap of the test tube by wrapping the little finger of the right
hand around it and turning the culture tube counter-clockwise with left
hand
• The lip of test tube is flamed under bunsen burner
• Absorbent point dropped in to the culture tube and replace the cap
• Culture tube incubated properly
 FALSE POSITIVE & FALSE NEGATIVE SAMPLES

• The main reasons for false positive results is sampling through a non-
sterile operation field.
• Leakage at the rubber dam border or through cracks and fillings may cause
false positive results.
• Polysaccharide producing streptococci such as lactobacillus,
corynebacterium, neisseria or vielonella are most common in
contaminated samples.
• False negative samples may be avoided by leaving the canal empty but
sealed for 2-7 days prior to sampling.

• This is particularly relevant for clinical testing of medicaments.

 Limitations:

1. Costly
2. Can take several weeks or days to identify bacteria
3. Low sensitivity and specificity
4. Dependence on mode of transport
5. laborious.
6. Impossibility of cultivating a large number of bacterial species as well as
difficulties in identifying many cultivable species.
• General concepts of culture media :

 If nutrients added to medium to meet a bacterial cells growth requirements ,that cell
will multiply to sufficient numbers to allow visualization by unaided eye.

 Certain bacteria need exceptional media components for growth –fastidious.

 Nutritional needs of most clinically important bacteria are relatively basic –

nonfastidious.
 Functions of the media:
• Support the growth of bacteria.
• Aid in recognition ,selection and isolation of particular groups.
• Facilitate the study of properties or activities of bacteria.
• Combine more than one of the above functions.
INDICATIONS OF CULTURING
1. Isolate bacteria in pure culture,
2. Demonstrate their properties,
3. Obtain sufficient growth for preparation of antigens.
5. Determine sensitivity to antibiotics,
6. Estimate viable counts, and
7. Maintain stock cultures.
 Factors in the media which support
bacterial growth:
• Nutrition: sources of carbon, nitrogen, minerals, essential metabolites, vitamins or
a combination of these.

• Factors controlling environmental conditions: pH or oxidation reduction

potential

• Sources of energy: more exacting organisms which require all their amino acids
preformed may use carbohydrates mainly as energy source.
TYPES OF MEDIA
 Media based on chemical content :

 Synthetic or defined
 Semidefined
 Nonsynthetic

• Based on consistency :

 Solid
 Liquid
 Semisolid
Media based on laboratory use :

 All purpose (basal media)

 Enriched
 Enrichment
 Selective
 Differential
 Transport
 Sugar media
• Based on environmental conditions :
 Aerobic
 Anaerobic
• Also classified into :
 Simple
 Complex
 Special
• Depending on the selectivity :
 Selective
 Nonselective
• Synthetic or defined :

Media in which exact chemical composition is known as called synthetic media.

 May be simple salt solution with a carbon source or it may include many organic
components .

 Used for various special studies such as metabolic requirements.

• Non synthetic :

Media in which exact chemical composition is not known is called nonsynthetic

media. It is primarily composed of tissue extracts or infusions.
Eg : Nutrient broth which contains
 Peptone – a partially digested protein which serves as a source of carbon , nitrogen
and energy.
 Beef extract ,a mixture of vitamins and minerals.
 Sodium chloride ,a salt that regulates the osmotic properties of the medium.
• Semidefined media :

Media whose composition is approximately known is called as semi defined media.

Eg: simple peptone water medium:1% peptone with 0.5% sodium chloride in water.
• Solid media:

Solidified to consistency of a jelly with agar, a

setting agent that allows sterilization when
liquid at 900c but does not set until 400, so that
heat sensitive components such as blood can be
added.
Different agar media according to the nutritive
components of the medium-

eg: sheep blood agar, bile esculin agar, xylose-

lysine-desoxycholate agar.
Solid media are more useful than liquid media as they facilitate :

 Discrete colony formation, allowing single pure colonies to be picked from

primary plate for subculture on secondary plate. This can then be used for
identification of the organism.
 Observation of colonial characteristics helpful for identification of organism.
 Quantification of organism as colony forming units (CFU). eg: a mixed saliva
sample with more than 106 CFU/ml of streptococcus mutans indicates high
cariogenic activity.
Liquid media :

• Promote growth of small numbers of bacteria present in

specimens contaminated with antibiotics (antibiotic is
diluted in the fluid medium thereby promoting growth of
organism)
• Test biochemical activities of bacteria for identification
purposes.
• For obtaining bacterial growth from blood or water when
large volumes have to be tested, and for preparing bulk
cultures for preparation of antigens or vaccines.
• Stored in tubes or screw capped bottles.
• Growth is recognized by turbidity.
• Eg: peptone water or nutrient broth.

Disadvantages:
• Bacteria growing in liquid media may not exhibit specific characteristics for their
identification.

• It is also difficult to isolate different types of bacteria from mixed populations.

 Semisolid media:
• If the concentration of agar is reduced to 0.2-0.5%
semisolid or sloppy agar is obtained which enables
motile organisms to spread.

• Increasing the concentration of agar to 6% prevents

spreading or swarming by organisms such as
proteus.
All Purpose Media (basal media/simple):

Is designed to support growth of most microorganisms.

eg: Nutrient broth ,Nutrient yeast and Trypticase soy agar (TSB).
TSB is most frequently used for maintenance of microbial populations.
Nutrient Agar made by adding 2% agar to nutrient broth is simplest and
commonest medium used in routine diagnostic laboratories.
 Special media
• Enriched media
• Enrichment media
• Selective media
• Differential media
• Indicator media
• Transport media
• Sugar media
 Enriched media

• In these media ,substances such as blood,

serum ,egg are added to a basal medium.

Eg: blood agar, chocolate agar and egg

media.
Enrichment media:

Fluids which encourage the preferential growth of

a particular bacterium and contains inhibitors
for contaminants.

eg: Tetrathionate broth where the tetrathionate

inhibits coliforms while allowing typhoid
paratyphoid organisms to grow freely.
 Selective media:
Are solid media that contain ingredients (dyes
like crystal violet or antibiotics) to inhibit
unwanted contaminants (eg: from normal
flora) but which allow certain pathogens to
grow .

eg: Desoxycholate citrate medium for

dysentry bacilli.
Rogosas Medium For Lactobacilli - Contains
Acetate And Other salts to suppress other
bacteria and enrichment for lactobacilli.
• MacConkeys agar contain crystal violet –inhibits gram +ve
organisms.
• Helpful for separating mixed cultures.
• Other selective factors that have been employed
are :
 Nutrient manipulation : Addition of specific carbon and energy
sources can produce selective agents.
 PH Adjustments : Maximum growth in range of 6.5-7.2.
 Some organisms prefer an acidic or basic medium eg: fungi pH
(4 and 6 ).
• Osmotic adjustments : Osmotic properties of a medium can be increased through
addition of nonmetabolizable compounds such as sodium chloride.

• Oxygen tension adjustments : Adjusting the oxygen and carbon dioxide tension is
important in selecting aerobes and anaerobes.
 Differential media (indicator ):

• Is designed to distinguish between various genera and species of

microorganisms.
• Often the differentiation is on the basis of colour differences of the
isolated colonies.
• A medium which has substances incorporated in it , enabling it
to bring out differing characteristics of bacteria and thus
helping to distinguish between them is called “differential
medium”.

• Eg :MacConkeys medium which consists of peptone, lactose,

agar, neutral red and taurocholate shows up lactose fermenters
as
• Pink colonies, non lactose fermenters as colourless or pale .

• Another example is blood agar showing colonies of streptococcus pyogenes and

staphylococcus aureus.
 Transport media:

• To preserve delicate pathogens during

transit to the laboratory or to prevent
overgrowth by nonpathogens.

• Eg :Stuart medium a semisolid

non nutrient agar with thioglycolic acid
(as reducing agent), electrolytes, and
sometimes pieces of charcoal to neutralise
certain bacterial inhibitors- for gonococci.
 Sugar media:
• They may be:
1.monosaccharides- a) pentoses
eg: arabinose, xylose.
b)hexoses eg: dextrose , mannose
2.Disaccharides eg: saccharose, lactose.
3.Polysaccharides eg: starch, inulin
4.Trisaccharides eg: raffinose.
5.Alcohols eg: glycerol, sorbitol.
6.Glucosides eg: salicin , aesculin
7.Noncarbohydrate substance eg: inositol.
 Complex media:

• These have added ingredients for special purposes or for bringing out certain
characteristics or providing special nutrients required for the growth of bacteria
under study.
 Anaerobic media
• Used for culture of anaerobic bacteria
• Cooked meat broth
• Thioglycollate broth
 Media for maintenance of cultures
 These are simple and should not encourage luxurious growth . Eg: nutrient agar
(commonest)
 For preservation of serological characters

eg: Dorset egg – medium of choice.

 Robertson's cooked meat medium- all round maintenance medium especially for
clostridia.
 Media for determining nutritional
requirements
• When nutritional requirements of an organism are not known omission of one
component at a time and substitution of another may be used to identify which
substrate are essential for growth and which can be replaced by others.

• Time consuming and not used in routine work.

• Simplified form- kosers citrate medium-a test for ability to use citrate as a source of
carbon is a useful characterizing test for enteric bacteria.
 Media for bacterial characterization
• Consists of simple but nutritionally adequate base to which the substrate under
test has been added .

• Reagents are added after a specified period of incubation to show that reactions
have occurred.
 Screening media:

• Intended to show at a glance the reactions obtained with several substrates hence the
term multitest media.
• eg: Knox plate (1949)

(can be relied upon only when pure cultures are obtained-hence used only for
subcultures)
 Media for microbiological assay of
vitamins and amino acids
• Need stringent control during preparation to ensure freedom from impurities.
 ANTIMICROBIAL SUSCEPTIBILITY TESTING

• Essential roles of a clinical microbiology are the isolation of clinically

significant microorganism from clinical specimens and the assessment of
their response to antimicrobial agent.
• Can be of two types

• Quantitative

• Qualitative
• Quantitative test- minimum amount of antimicrobial agent that inhibits
the visible growth of a bacterial isolate or minimal inhibitory concentration
(MIC) is determined.

• Qualitative test- categorize a bacterial isolate as susceptible, intermediate,

or resistant to a particular antimicrobial agent.
 Indications
A number of factors must be considered when determining whether
antimicrobial susceptibility tests are necessary.

• Predictability of susceptibility to the DRUG OF CHOICE

• Body site from where the organism was isolated
• Quantity of organism present
• Presence of other organism and quantitation of each other
• Presence of any unique host factor
• Methods – most commonly used methods in clinical laboratories are

• Disk diffusion
• Agar dilution
• Macrobroth dilution
• Microbroth dilution
 DISK DIFFUSION

• In this test bacteria are

spread over surface of an
agar plate, and then paper
disk to which antimicrobial
agents have been added are
placed on the agar surface.

• The plates are incubated at

35°c and the zones of
inhibition are examined.
 AGAR DILUTION TEST

• In this test, varying

concentration of
antimicrobial agents are
incorporated in cooled,
molten agar.

• This is poured into Petri

plates
• Following cooling and solidification of the agar, a
standardized number of bacteria are “spot inoculated” on to
agar containing antimicrobial agent and also one or more
control plates without any drug.

• Following overnight incubation , the MIC is read as the lowest

concentration of antimicrobial agent that inhibits the visible
growth of test bacterium.
 MACROBROTH DILUTION MIC TEST

• 1 to 2 ml of serial two fold dilutions of antimicrobial agent are inoculated

with standardized suspension of test bacteria to obtain a final concentration
of 5×105 CFU/ml.

• Following overnight incubation, the MIC is read as the lowest

concentration that visually inhibites growth as evidenced by the absence of
turbidity.
 MICROBROTH DILUTION TEST

• Polystyrene trays, which usually contain 80-100 wells, are filled with small
volumes (0.1ml) of serial two fold dilutions of Antimicrobial agents.
• These agents are diluted in Mueller-Hinton broth
• A standardized number of bacteria are inoculated into each
well simultaneously with the use of a multipronged
inoculating device.

• A growth control well (broth plus inoculum) and a sterility

control well (broth only) are included in each panel and MICs
are determined following overnight incubation.
 MICROSCOPES
• Light
• Flourescent
• Electron
• X ray
• LIGHT
• The most basic light scope, a dissecting or
stereomicroscope, allows viewing of a whole
organism at once while showing details like the
antennae of a butterfly at 100x to 150x
magnification. 

• FLUORESCENT
• The fluorescent or confocal microscope uses
ultraviolet light as its light source. When ultraviolet
light hits an object it excites the electrons of the
object, emitting light in various colors, which can
help identify bacteria inside an organism.
• ELECTRON
• The energy source used in the electron microscope is a beam of electrons.
The beam has an exceptionally short wavelength, and increases the
resolution of the image significantly over light microscopy.

• Whole objects are coated in gold or palladium, which deflects the electron
beam, creating dark and light areas as 3-D images viewed on a monitor.
Details like the intricate silica shells of marine diatoms and surface details
of viruses can be captured.

• Both transmission electron microscopes (TEM) and the newer scanning

electron microscopes (SEM) fall in this specialized category of
microscopy.
• X-RAY MICROSCOPES
• As the name suggests, these microscopes use a beam of X-rays to create an
image. Unlike visible light, X-rays do not reflect or refract easily, and they
are invisible to the human eye.

• The image resolution of an X-ray microscope falls between that of an

optical microscope and that of an electron microscope, and is sensitive
enough to determine the individual placement of atoms within molecules
of a crystal. In contrast to electron microscopy, wherein the object is dried
and fixed, these highly specialized microscopes are capable of showing
living cells.
IDENTIFICATION OF BACTERIA
 Determining the number of bacterial cells
• STANDARD PLATE COUNTS
• TURBIDITY
• DIRECT MICROSCOPE COUNTS
 TURBIDITY
• Instrument used is spectrophotometer
Grid
 Antibiogram
•  A collection of data usually in the form of a table
summarizing the percent of individual bacterial pathogens
susceptible to different antimicrobial agents

• Note: An antibiogram is generated after bacteria are isolated

(as from a patient's tissues or body fluids) and subjected to
laboratory testing.
• Antibiogram Uses

• Antibiograms help guide the clinician and pharmacist in

selecting the best empiric antimicrobial treatment in the event
of pending microbiology culture and susceptibility results.
 MOLECULAR GENETIC
MECHANISMS
It includes the
Polymerase chain reaction
 Nested PCR
 RT PCR
 Multiplex PCR
 Real time PCR

DNA-DNA hybridization technique

 Procedure of PCR

1. Denaturation of DNA to single strands

2. Annealing of primers to DNA
3. Extension by polymerase
4. Repeat 30-35 times

This is done on an automated cycler, which can

heat and cool the tubes with the reaction mixture
in a very short time.
Denaturation
• DNA heated to 94-96°C- breaks hydrogen bonds
• Time: 1-2 minutes.

5’ 3’
Target DNA
3’ 5’

95oC
Annealing

• Lower temperature 45-60ºC– Primers attach themselves to the single DNA strands.
• Temperature of this stage depends on the primers and is usually 5°C below their
melting temperature
A wrong temperature during the annealing step can result in primers not binding to
the template DNA at all, or binding at random
• Time: 1-2 minutes
 3’
5’
Target DNA

3’ 5’
A
B
primers
~55oC

5’ 3’

B
5’
5’ A

3’ 5’
Extension
• DNA Polymerase makes copy the DNA strands.
• It starts at the annealed primer and works its way along the DNA strand.

• The time for this step depends both on the DNA-Polymerase itself and on the
length of the DNA fragment to be amplified
 3’

5’
Target DNA B
5’
5’ A 5’

3’

72oC

5’ 3’

Taq polymerase 5’
5’

3’ 5’
 5’ 3’

5’

Taq polymerase
5’

3’ 5’
72oC

5’ 3’

5’
5’

3’ 5’
 The reaction products are separated by gel electrophoresis.

 The reaction products can be visualised directly by staining with

ethidium bromide, methylene blue, or Carolina Blue DNA stain or by
means of radioisotopes and autoradiography.
PCR - Polymerase Chain Reaction (IQOG-CSIC).mp4
 Uses of PCR
• Genetic fingerprinting
• Paternity testing
• Detection of hereditary diseases
• Detection of Viral diseases
• Cloning genes
• Mutagenesis
 Derivatives of PCR
• Nested PCR:

• Nested PCR uses the product of primary PCR amplification as template in a

second PCR reaction and was devised mainly to have increased sensitivity.

• The first PCR products are subjected to a second round of PCR

amplification with a separate primer set, which anneals internally to the
first products.
 Reverse Transcriptase PCR (RT-PCR)

• RT-PCR was developed to amplify RNA targets and exploits the use of the
enzyme reverse transcriptase, which can synthesize a strand of
complementary DNA (cDNA) from an RNA template
 Multiplex PCR
In multiplex PCR, two or more sets of primers specific for different targets are
introduced in the same reaction tube.

more than one unique target sequence in a clinical specimen can be

amplified at the same time,

multiplex PCR assays permit the simultaneous detection of different microbial

species.
 Real –Time PCR
• Conventional PCR assays are qualitative or can be adjusted to be semi-
quantitative. One exception is the real-time PCR, which is characterized by
the continuous measurement of amplification products throughout the
reaction.
DNA-DNA hybridization technique
Employs DNA probes which entails segments of single stranded DNA,
labeled with an enzyme, radioactive isotope or a chemiluminescence
reporter, that can locate and bind to their complimentary nucleic acid
sequence.
Socransky introduced the Checker board DNA –DNA hybrisization method.
This allows simultaneous determination of a multitude of bacterial
species.
 Uses of PCR in endodontics
• Molecular method, particularly PCR are more specific, accurate, sensitive
and rapid than culture and can detect uncultivable and fastidious
microorganisms (for instance Tannerella forysthesis, Trepenoma denticoli,
other Trepenoma species, Dialister pneumositis, Prevotella tanneria were
detected in the infected root canal for the first time by using PCR
analysis ).

• Despite of great advance and revolutions bought about by molecular

diagnostic method in diverse medical discipline, endodontic microbiota is
still undergoing shift from cultural era to molecular era, proper exploiting
this molecular diagnostic method in endodontics enhance high success
rate in endodontic treatment
 Clinical correlation
• Condition of RCT to take culture
• Microbes and unsuccessful endodontic treatment
• Micro organisms and endodontic flare ups
• Role of single vs multiple visit
• Antibiotic resistance
• Methods to eradicate biofilms
 When to take culture from a rootcanal

• for testing the microbiota in primary infections.

• To test the organisms responsible for persitent endodontic
infections.
• At the time of obturation to evaluate complete disinfection of
the canals.
 Microbes and unsuccessful
endodontic treatment
• Microbiota associated with the failed root
canal treatment differs from that associated
with the primary root canal infections.
• Secondary are dominated by gram positive
facultative anaerobes principally by
Enterococcus sp.
• Prevalence is about one third of the failed
rootcanal therapies.
• Reasons for survival are :
• 1. has the capacity to survive under various
stressful environmental conditions including
intracellular survival in macrophages.
• 2. has the capacity to survive more than 90 days on
hospital used fabrics and up to 4 months in water.
• 3. capable of entering and recovering from the
viable but non culturable state.
• 4. displays cell wall alerations provide rotection
under unfavourable environmental conditions.
• 5. capable of producing a variety of stress protiens when
exposed to adverse factors such as sodium hypochlorite , salts.
Bile salts, acid and heat, alkaline stress, glucose starvation,
elevetaed temperatures.
• 6. low sensitivity to antimicrobial agents.
• 7. ability to resist hgh ph
• Love postulated that e. foecalis can invade dentinal tubules for
protection againts intracanal medicaments and has the better
bonding capaccity to collagen.
• 8.co aggregation with other species.
 Endodontic Flare ups
• Acc. to AAE flare up is defined as an acute exacerbation of
an asymptomatic pulpal or periapical pathosis after the
initiation or continuaution of root canal treatment.
• Chavez de paz et al reported that F. nucleatum is associated
with the most severe flare up pain and swelling.
• Peciuline et al reported that F. nucleatum as an responible
organism for flare up from a root filled tooth with chronic
apical periodontitis undergoing retreament after first
apppointment.
• The combination of F. nucleatum, prevotella species and
porphyromonas sp. may provide risk factor for endodontic
flare up by actine in synergy to incraese the intensity of peri
apical inflammatory reaction.
 Role of single vs multiple RCT
Single Multiple
RCT is generally completed in single RCT is generally completed In 3-4
appointment. appointments

In pateints with apical periodontitis high Low survival rate of bacteria due to inter
chances of survival of bacteria. appointment medicaments.

Acc. To literature predictable disinfection of the root canal system is only

achieved after
Proper anti microbial medicaments are placed in the canal and left there
in between the appointments
 Antibiotic Resistance

• Plasmids for bacteria provide anti bacterial resistance with this

antibiotics lose their effctiveneess aganist such bacteria.

• In addition to antibiotics certain range of bacterial products

such as cytotoxins, adhesions and certain metabolic enzymes
provide high virulence to the bacteria.
 Methods to eradiacte biofilms
• Microbial ecosystem will persist untill source of irriatation is
removed.
• Main factor for successful treatment for pulp and peri
radicular inflammation is complete removal of infection.
 By means of
• Sodium hypochlorite (disrupts oxidative posphorylation and
inhibits DNA synthesis of bacteria)
• Chlorhexidine gluconate (denatures bacterial cellwall
forming pores in the membrane )
• Qmix (EDTA ,chlorhexidine and detergent)
• Iodine (penetrates into micro organisms attacks
protiens,nucleotides, fatty acids)
• EDTA (extracts bacterial surface protiens combining with
metal ions from cell envelope eventually leads to cell death)
• MTAD (has low ph acts as a calcium chelator and acts as
substantive medication )
• Tetra clean (doxycycline hyclate. more effective against E.
foecalis)
• Calcium hydroxide (along with parachlorophenol completely
eliminats E. foecalis )
• Ultrasonically activated irrigation (improves isthmus
cleanliness interms of biofilm removal)
• ozone/ ozonated water (0.1 to 0.3 ppm completely elimiates
bacteria )
• Lasers (induce thermal effect producing an alteration in the
bacterial cell wall leading to changes in osmotic gradient and
cell death )
• Plasma dental probe (uses atomic oxygen as active agent for
the bactericidal effect)
• Photo activated disinfection (combination of photosensitizer
solution along with laser light )
• Anti microbial nanoparticles (bind to negatively charged
surface and have excellent antimicrobial and antifungal
activity e.g zno nanoparticles , chitosan layerzno, produces 95
% reduction in the adherence of e. foecalis to dentin )

• Endoactivator system (able to debride in to deep lateral

anatomy , dislodge simulated biofilm clumps within curved
canals )
• In their study, Nair et al. showed that 88% of root canal–treated
mandibular molars showed residual infection of mesial roots after
instrumentation, irrigation with NaOCl, and obturation in a one-visit
treatment. BioPure MTAD (Dentsply Tulsa Dental, Johnson City, TN,
USA) has been described as a universal irrigating solution.

• Torabinejad et al. have shown that MTAD removes the smear layer
safely; also, it is effective against E. faecalis and it can eliminate bacteria
in human root canals that had been infected by whole saliva.

• A new irrigant, Tetraclean, which is mixture of doxycycline hyclate

present at a lower concentration than MTAD, an acid, and detergents, has
the ability to eliminate microorganisms and smear layer in dentinal tubules
of infected root canals with a final 4-min rinse.
 CONCLUSION
• It is important that clinicians
understand the close
relationship of microorganism
with endodontic disease to
develop an effective treatment
rationale.

• Therefore the control of

microorganisms and possible
substrate must be an objective
in every endodontic case
 References
• Endodontic therapy –Franklin S. Weine, Sixth Edition

• Endodontics- John I. Ingle, Leif K. Bakland, Fifth edition, 6th edn

• Essentials of diagnostic microbiology,Lisa Anne
• Endodontic Practice – Louis I.Grossman, Seymour Oliet, Carlos E.
Del Rio , Eleventh Edition

• Pathways of the pulp – Stephen Cohen, Richard C. Burns, Eigth

Edition
 References
• J Clin Microbiol. 2009 May; 47(5): 1352–1357Diversity of Spirochetes in
Endodontic Infections
Mitsuo Sakamoto,1 José F. Siqueira, Jr.,2* Isabela N. Rôças,2 and Yoshimi Benno

• Debelian GF,Olsen I, Tronstad L. Bacteremia in conjunction with endodontic

therapy. Endod Dent Traumatol 1995;11:142.

• Heimdahl A, Hall G, Hedberg M, Sandberg H. Detection and quantitation by

lysis-filtration of bacteremia after different oral surgical procedures. J Clin
Microbiol 1990;28:2205.
• Sundqvist G. Taxonomy, ecology, and pathogenicity of the root canal flora. Oral
Surg 1994;78:522.
• Sundqvist G. Associations between microbial species in dental root canal
infections. Oral Microbiol Immunol 1992;7:257
 References
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Thank you!!!