Endodontic Microflora

ROOT CANAL MICROFLORA
PRESENTED BY : VISHNUJA V R NAIR
II YEAR MDS
DEPARTMENT OF CONSERVATIVE &
ENDODONTICS

CONTENTS
• Introduction
• Stages of root canal infection
• History
• Portals of entry for
Microorganisms
• Classification of
Microorganisms
• Microbial Ecosystem of the
root canal
• Microbial Virulence And
Pathogenicity
• Factors influencing the Growth
and Colonization of
Microorganisms
• Types of endodontic infections
• Biofilm
• Microbiological diagnostic
techniques
• How to combat microbes in the
endodontic therapy?
• Review of literature
• Conclusion
• Reference

Microbes virtually cause all pathoses of pulp and peri apical
tissues.
To effectively treat endodontic infections, clinicians must
recognize cause of microbial invasion.
Knowledge of microbes associated with endodontic disease
 develop basic understanding of disease process, sound
rationale for effective management of patients.
Ingle’s Endodontics 6th Edition
INTRODUCTION

Can normal flora cause disease?
• The oral microbiome is similar in all humans, although
each individual has a characteristic “fingerprint” that is
unique to himself or herself.
• This commensal flora exists in symbiotic harmony with
the host, but disease conditions supervene when the
microbial equilibrium is broken(dysbiosis).
• This transient flora, cannot get a foothold in the oral
environment because of the colonization resistance exerted
by the resident flora.
Marsh and Martin’s Oral Microbiology 6th Edition

Microbes
enter pulp
Pulp
becomes
necrotic
Pulp loses
blood supply
Reservoir of
microbes &
their end
products
CLIMAX
COMMUNITY
Stages of Root canal infection
Marsh and Martin’s Oral Microbiology 6th Edition

RATE OF BACTERIAL INVASION
Nagaoka found that rate of bacterial invasion
increased with the passage of time, and that it
was approximately 1.6 µm/day during the first
25 days and 14 µm/day by 120 days.
The deepest bacterial invasion was 3.0 mm
after 210 postoperative days.
Grossman’s Endodontic Practice 13th Edition

A HISTORIC VIEW
ANTONIE VAN LEEWENHOEK
(17th century)
• First to Describe oral microflora.
• Observed dental plaque from exposed cavity
• He reported that the root canals were stuffed with a
soft matters and the whole matter seem to be alive.
W.D. MILLER (1890)
• Father of oral microbiology.
• Wrote “Microorganisms of the human mouth”
• 1st to associate bacteria with pulpal disease.

1965: Kakehashi et al found that bacteria are the main
etiological factors in the development of pulpal and
periradicular diseases. Kakehashi et al proved that
without bacterial involvement only minor inflammation
occurred in exposed pulp.
1976: Sundqvist used different culturing techniques for
identification of both aerobic and anaerobic organisms
and concluded that root canal infections are
multibacterial.
Cohen’s pathways of pulp- 6th Edition

1904: F Billings described theory of focal infection as a
circumscribed area of tissue with pathognomic
microorganisms.
1909: EC Rosenow described theory of focal infection as
localized or generalized infection caused by bacteria
traveling via bloodstream from distant focus of infection.
1939: Fish observed four distinct zones of periapical
reaction in response to infection

PORTALS OF ENTRY FOR
MICROORGANISMS
• Under normal conditions, the pulpodentin complex is sterile
and isolated from oral microbiota by overlying enamel,
dentin, and cementum.
• The pulpodentin complex is then challenged by
microorganisms present in caries lesions, saliva bathing the
exposed area, or dental plaque formed on the exposed area.
• Microorganisms from subgingival biofilms associated with
periodontal disease may also have access to the pulp via
dentinal tubules at the cervical region of the tooth and lateral
or apical foramina.

• Whenever dentin is exposed, the pulp is put at risk of infection
as a consequence of the permeability of normal dentin dictated
by its tubular structure.
• Dentinal tubules traverse the entire width of the dentin and
have a conical conformation, with the largest diameter located
near the pulp (mean, 2.5 µm) and the smallest diameter in the
periphery, near the enamel or cementum (mean, 0.9 µm).
• The smallest tubule diameter is entirely compatible with the
cell diameter of most oral bacterial species, which usually
ranges from 0.2 to 0.7 µm.

ROUTES OF ROOT CANAL INFECTION
-Caries, open cavity
-Trauma induced
cracks, fractures
-Faulty Restorations
-Wasting diseases
-Scaling
-Root planing
-Subgingival
plaque biofilms
-Apical,
Lateral,
Accessory
foramina
-Root
perforations
Anachoresis
If pulp vital  protection from bacterial invasion
If pulp necrotic  Easily infected (Host defences do not function)

MICROBIAL PENETRATION IN CANAL
DURING
TREATMENT
Remnants of biofilm,
calculus.
Caries on tooth crown
Leaking rubber dam
Contamination of
endodontic instruments,
irrigant solutions
BETWEEN
APPPOINTMENTS
Leakage
through
temporary
restoration
Fracture of
tooth
Open dressing
AFTER
OBTURATION
Leakage through
temporary/permanent
restoration
Delay in placement
of permanent
restoration
Recurrent decay

Whatever the route of bacterial access to the root
canal, necrosis of pulp tissue is a prerequisite for
the establishment of primary endodontic
infections.
cohen’s pathways of pulp- 6th Edition

SOURCES OF NUTRIENTS FOR BACTERIA
COLONIZING IN ROOT CANAL
• Necrotic pulp tissue
• Proteins and glycoproteins from tissue fluids and
exudate that seep into Root canal via apical and
lateral foramen
• Salivary components
• Products of metabolism of other bacteria

CLASSIFICATION OF MICROORGANISMS
• Microbial flora can be classified on the basis of: Gram
Stain Technique
 Gram-positive organisms, e.g. Streptococcus,
Enterococcus, Treponema, Candida, Actinomyces,
Lactobacillus, etc.
 Gram-negative organisms, e.g. Fusobacterium,
Campylobacter, Bacteroides, Veillonella, Neisseria, etc.

 Obligate aerobes: The organisms which require oxygen
for their growth, e.g. tubercle bacilli.
 Facultative anaerobes: These organisms can grow in the
presence or absence of oxygen, e.g. Staphylococcus.
 Microaerophilic: They grow in an oxygen environment
but derive their energy only from fermentative pathways
that occur in absence of oxygen, e.g. Streptococcus, etc.
 Obligate anaerobes: These bacteria can grow only in
absence of oxygen, e.g. Bacteroides, Fusobacterium

MICROBIAL ECOSYSTEM OF THE ROOT
CANAL
• Most commonly gram-positive organisms are found in the root
canals, but gram-negative and obligate anaerobes have also
been found in the root canals.
• In necrotic pulp, there is lack of circulation with compromised
host defense mechanism; this makes pulp as a reservoir for
invading microbes.

• Several putative endodontic pathogens have been shown to be
capable of penetrating dentinal tubules in vitro, including
Porphyromonas endodontalis, Porphyromonas gingivalis,
Fusobacterium nucleatum, Actinomyces israelii,
Propionibacterium acnes, Enterococcus faecalis, Candida
albicans, and streptococci.
• In their clinical study, Peters and colleagues isolated and
identified bacteria present in root dentin at different depths,
and the most common isolates belonged to the genera
Prevotella, Porphyromonas, Fusobacterium, Veillonella,
Peptostreptococcus, Eubacterium, Actinomyces, lactobacilli,
and streptococci.

The organisms most commonly encountered in endodontic
infections include
 Bacteroides  B.endodontalis, B. gingivalis
 streptococci S.milleri
 Enterococci
 Lactobacilli
 Actinomyces
 Yeasts
 Tannerella forsythia
 Fusobacterium nucleatum
Grossman’s endodontic practice 13th Edition

B.melaninogenicus Eubacterium
yurii subsps. as well as key
‘bridging oral species’ F. nucleatum
sp., Corynebacterium were highly
associated with traumatized teeth
Bacteroides in association with
Porphyromonas.endodontalis, P.
gingivalis and Prevotella
intermedius.
Microflora of traumatized but intact teeth with necrotic pulps
Microflora of acute infections of endodontic origin
Manoharan L, Brundin M, Rakhimova O, Chávez de Paz L, Romani Vestman N. New insights into the microbial profiles of infected root canals in traumatized
teeth. Journal of clinical medicine. 2020 Dec;9(12):3877.
Fouad AF, Barry J, Caimano M, Clawson M, Zhu Q, Carver R, Hazlett K, Radolf JD. PCR-based identification of bacteria associated with
endodontic infections. Journal of clinical microbiology. 2002 Sep;40(9):3223-31.

Obligate Anaerobes-
Microflora of endodontic flare-up’infections
Veillonella
Capnocytophaga
Eiknella
Bacteroides
Fusobacterium
Treponema.
Microflora on refractory endodontic cases are
E. faecalis
Candida albicans
Actinomyces israelii
Siqueira Jr JF. Microbial causes of endodontic flare‐ups. International endodontic journal. 2003 Jul;36(7):453-63.
Sunde PT, Olsen I, Debelian GJ, Tronstad L. Microbiota of periapical lesions refractory to endodontic therapy. Journal of endodontics.
2002 Apr 1;28(4):304-10.

Microflora of infected and untreated necrotic pulp
• Fusobacterium
• Porphyromonas
• Prevotella
• Eubacterium
• Peptostreptococcus
Gomes BP, Pinheiro ET, Gadê‐Neto CR, Sousa EL, Ferraz CC, Zaia AA, Teixeira FB, Souza‐Filho FJ. Microbiological
examination of infected dental root canals. Oral microbiology and immunology. 2004 Apr;19(2):71-6.
Endodontic flora in previously root filled teeth with
apical periodontitis
• Actinomyces
• Enterococcus
• Propionibacterium
Pinheiro ET, Gomes BP, Ferraz CC, Sousa EL, Teixeira FB, Souza-Filho FJ. Microorganisms from canals of root-filled teeth with
periapical lesions. International endodontic journal. 2003 Jan 1;36(1):1-1.

MECHANISMS OF MICROBIAL
PATHOGENICITY AND VIRULENCE FACTORS
• The ability of a microorganism to cause disease is regarded
as its pathogenicity.
• Virulence denotes the degree of pathogenicity of a
microorganism, and virulence factors are the microbial
products, structural cellular components, or strategies that
contribute to pathogenicity.
• Some microorganisms routinely cause disease in a given
host and are called primary pathogens. Other
microorganisms cause disease only when host defenses are
impaired and are called opportunistic pathogens.

VIRULENT FACTORS
• Lipopolysaccharides(LPS)- These are present on the surface
of gram- negative bacteria. LPS have nonspecific antigens
which are not neutralized by antibodies.
• Extracellular vesicles - Extracellular vesicles are produced by
gram-negative bacteria in the form of endotoxins, outer
membrane fragments or blebs.
• Enzymes -Enzymes produced by bacteria have numerous
activities like they help in spread of the infection, neutralization
of immunoglobulin and the complement components.

 Fatty acids- Various short chain fatty acids like propionic acid,
butyric acid are produced by anaerobic bacteria. These cause
neutrophil chemotaxis, degranulation, phagocytosis, and
stimulate interleukin-1 production which further causes bone
resorption and periradicular diseases
 Polyamines-These are biologically active chemicals found in
the infected canals. They help in regulation of the cell growth,
regeneration of tissues and modulation of inflammation.
 Pili-may play an important role in attachment of bacteria to
surfaces and interaction with other bacteria.
 Capsule-present in gram negative black pigmented bacteria,
enable them to avoid phagocytosis.

Intraradicular
Primary/
initial/ virgin
Secondary
Persistent /
Recurrent
Extraradicular
Sequalae of
intraradicular
Independent
of
intraradicular
TYPES OF ENDODONTIC INFECTIONS

• Primary infection, caused by microorganisms that initially
invade and colonize the necrotic pulp tissue (initial or “virgin”
infection)
• Secondary infection, caused by microorganisms not present in
the primary infection but introduced in the root canal at some
time after professional intervention (i.e., secondary to
intervention)
• Persistent infection, caused by microorganisms that were
members of a primary or secondary infection and in some way
resisted intracanal antimicrobial procedures and were able to
endure periods of nutrient deprivation in treated canals.

PRIMARY INTRARADICULAR INFECTIONS
• It is caused by micro-organisms that
initially invade and colonize the
necrotic pulp tissue.
• Characterized by a mixed consortium
dominated by anaerobic bacteria and
composed of 10 to 30 species per
canal
(Munson et al., 2002; Siqueira et al., 2004b; Siqueira and Rôças,
2005b).
• Total bacterial counts vary from 103
to 108 cells per infected canal
(Sundqvist, 1976; Vianna et al., 2006b; Sakamoto et al., 2007;
Siqueira et al., 2007d).

• Primary intraradicular infections are caused by
microorganisms that initially invade and colonize the
necrotic pulp tissue.
(a) Gram negative anaerobes such as Prevotella,
Fusobacterium, Tannerella, Dialister, Porphyromonas,
Campylobacter, and Treponema have been frequently
isolated from primary endodontic infections.
(b) Gram positive anaerobes from genera
Peptostreptococcus, Eubacterium, Actinomyces, and
facultative or microaerophilic streptococci can also be
commonly found in primary intraradicular infections.

SYMPTOMATIC INFECTIONS
No strong evidence disclosing specific involvement of single
species with any particular form of endodontic disease
Factors other than mere presence of putative pathogens may play
a role in etiology of symptomatic infections (no. of bacterial cells,
environmental cues, bacterial interactions, difference in virulence
ability)
Bacterial succession occurs before symptoms rise
At a given moment in endodontic disease  Micro biota reaches
a certain degree of pathogenicity  elicits acute inflammation at
Peri apical tissues  Pain + Swelling

Asymptomatic  Symptomatic
Shift in structure of microbial community before
appearance of symptoms
(Arrival of new pathogens/ variations/rearrangement in
bacterial species)
detected by
Difference in dominant species in communities and larger
number of species in symptomatic cases
No key pathogen but occurrence of certain bacterial
combinations in infected root canals  decisive factors in
causing symptoms

Secondary Intraradicular Infections
• Secondary intraradicular infections are caused by
microorganisms that were not present in the primary
infection, but were introduced in the root canal at some
time after professional intervention (secondary to
intervention).
• Pseudomonas aeruginosa, Staphylococcus sp.,
Escherichia coli, Candida sp., and E. faecalis are
commonly found in such infections
• Microorganisms can penetrate the pulp space system
even after the completion of root filling

PERSISTERS AFTER INTRACANAL DISINFECTION
• Persistent intraradicular infections are caused by
microorganisms that resisted the intracanal antimicrobial
procedures. These microbes endure periods of nutrient
deprivation in a prepared canal.
• However, fewer species are present than primary infections.
• Higher frequencies of fungi (Candida species) are present than
in primary infections.
E.fecalis
Staphyloco
cci
Bifidobacter
ium
Actinomyces
S.mitis,
S.gordonii

ENTEROCOCCUS FECALIS
 E. faecalis is a gram-positive, facultative anaerobic coccus
that is strongly associated with endodontic infections
 Found in high percentage of root canal failures
 Able to survive in the root canal as a single organism or as a
major component of the flora
 Endures prolonged periods of nutritional deprivation.
 Binds to dentin and invades dentinal tubules
 Suppresses action of lymphocytes
 Utilizes serum as a nutritional source
 Resists intra canal medicaments
 Forms a biofilm Ingle’s Endodontics 6th Edition

• It is commonly found in a high percentage of root canal failures
and is able to survive in the root canal as a single organism or as
a major component of the flora.
• They can grow in extremely alkaline pH, salt-concentrated
environment, in a temperature range of 10–45°C, and survive a
temperature of 60°C for 30 minutes.
• The prevalence of E. faecalis is 40% in primary endodontic
infection and 24%–77% in persistent endodontic infection.
• E. faecalis biofilm is 1000 times more resistant to phagocytosis,
antibodies, and antimicrobials. (intracanal dressings like
calcium hydroxide)
• Rocas et al. have also suggested that E. faecalis was more
frequently detected in asymptomatic cases than in symptomatic
ones. Grossman’s Endodontic Practice 13th Edition

EXTRA RADICULAR INFECTIONS
If microorganisms invade the periradicular tissues overcoming the
defense mechanisms of the body, then extraradicular infection occurs.
The most common species are Actinomyces, Streptococcus, and P.
propionicum.
1. Extraradicular infection dependent on intraradicular
infection- This infection occurs if microorganisms invade the
periradicular tissues overcoming the defense mechanisms of the
body or debris extrusion due to over-instrumentation. Eg:Acute
alveolar abscess
2. Extraradicular infection independent of intraradicular
infection- Actinomyces species have been found in association
with unhealed periapical lesions e.g., periapical actinomycosis

Actinomyces
P. propionicum
P. gingivalis
T. forsythia
P. endodontalis

ENDODONTIC SYMPTOMS
Asymptomatic infection
Symptomatic infection +
Asymptomatic
Symptomatic infection
Foul odour + pain + sinus
tracts
Endodontic abscess in
children
Percussion pain
Symptomatic P/A lesions in
permanent + deciduous teeth
BACTERIA
Prevotella denticola
P. intermedia
Porphyromonas gingivalis
+ P. endodontalis
Bacteroides
Parvimonas micra
Peptostreptococcus +
Eubacterium +
Porphyromonas
Apical Periodontitis +
CMV + EBV
Ingle’s
Endodontics
6
th
Edition

BIOFILMS
• Biofilm can be defined as a sessile multi-
cellular microbial community characterized by
cells that are firmly attached to a surface and
enmeshed in a self produced matrix of
extracellular polymeric substances.
• Bacterial biofilms are very prevalent in the
apical root canals of teeth with primary and
post-treatment apical periodontitis.

Endodontic micro flora less diverse than oral micro flora
Persist in anatomical +geometrical complexities
(Isthmuses/deltas/apical portion of root canal system)
Shelter adhering bacteria from cleaning and shaping
procedures
Bacterial activities not confined to intracanal spaces, also
access regions beyond apical foramen
Root canal environment favours biofilm formation (Both
primary and post-treatment)
ENDODONTIC BIOFILMS

Conditioning layer
Planktonic bacterial
cell attachment
bacterial growth and
biofilm expansion
Detachment of biofilm
microorganisms
Development Of Biofilm

INTRACANAL
 Formed on root
canal dentin of
endodontically
treated tooth
 Documented by
Nair (1987)
 Present as both
loose collection and
biofilm structure
 E. fecalis resists
starvation, develops
biofilms under
different
environmental and
nutritional
conditions
EXTRARADICULAR
 Root surface
biofilms
 Formed on
cementum surface
adjacent to root
apex of
endodontically
treated teeth
 Asymptomatic
apical
periodontitis and
chronic abscess
 Clinical evidence :
Ricucci et al –
Reported presence
of calculus like
deposits on apex.
PERIAPICAL
 Isolated biofilms in
peri apical region of
endodontically
infected tooth
 Actinomyces
 P. Propionicum
 Sunde et al,
reported occurrence
of sulphur granules
in 9 refractory P/A
lesions 
 A. viscosus
 A. naeslundii
 A. israelii

Biomaterial Centered Infection
• Biomaterial centered infection (BCI) occurs when bacteria adheres to
an artificial biomaterial surface such as root canal obturating
materials and forms biofilms.
• Presence of biomaterial in close proximity to the host immune
system can increase the susceptibility to BCI.
• BCI usually reveals opportunistic invasion by nosocomial
organisms. Coagulase-negative Staphylococcus, S. aureus,
enterococci, P. aeruginosa, and fungi are commonly isolated from
infected biomaterial surfaces.
• Three phases of bacterial adhesion to biomaterial surface:
 Phase 1: Transport of bacteria to biomaterial surface.
 Phase 2: Initial, nonspecific adhesion phase.
 Phase 3: Specific adhesion phase.

METHODS TO ERADICATE BIOFILM
• Sodium Hypochlorite
 It is effective against biofilms containing P. intermedia, Peptostreptococcus
micros, Streptococcus intermedius, Fusobacterium nucleatum and E. faecalis
as it disrupts oxidative phosphorylation and inhibits DNA synthesis of
bacteria.
 Dunavant et al (2006) concluded that both 1 percent NaOCl and 6 percent
NaOCl were more efficient in eliminating E. faecalis biofilm than the other
solutions tested.
• Chlorhexidine Digluconate
 It is effective against both gram-positive and gram-negative bacteria due to
its ability to denaturate the bacterial cell wall while forming pores in the
membrane.
 It kills E. faecalis cells in 30 seconds or less in concentrations of 0.2 to 2
percent. Although in vitro studies have demonstrated the antibacterial effect
of CHX againstE. Faecalis to be superior to that of NaOCl.
Ong KS, Mawang CI, Daniel-Jambun D, Lim YY, Lee SM. Current anti-biofilm strategies and potential of antioxidants in biofilm control. Expert review of anti-infective therapy. 2018 Nov 2;16(11):855-64.

• EDTA- On direct exposure for extended time, EDTA extracts bacterial
surface proteins by combining with metal ions from the cell envelope,
which can eventually lead to bacterial death
• QMIX- Qmix consists of EDTA, chlorhexidine, and detergent. It is as
effective as 6 percent NaOCl in killing 1-day old E. faecalis but
slightly less effective against bacteria in 3-week old biofilm.
• Calcium Hydroxide-A commonly used intracanal medicament, has
been shown to be ineffective in killing E. faecalis on its own,
especially when a high pH is not maintained.
 However, combination of calcium hydroxide and camphorated
paramonochlorophenol completely eliminates E. faecalis.
 Two percent chlorhexidine gel when combined with calcium
hydroxide achieves a pH of 12.8 and can completely eliminate E.
faecalis within dentinal tubules
Ong KS, Mawang CI, Daniel-Jambun D, Lim YY, Lee SM. Current anti-biofilm strategies and potential of antioxidants in biofilm control. Expert
review of anti-infective therapy. 2018 Nov 2;16(11):855-64.

• MTAD
 MTAD has low pH and thus can act as a calcium chelator and
cause enamel and root surface demineralization. In addition,
it has been shown that it is a substantive medication (becomes
absorbed and gradually released from tooth structures such as
dentin and cementum.
• Tetraclean
 Pappen FG et al (2010) found that tetraclean is more effective
than MTAD against E. faecalis in planktonic culture and in
mixed-species in the in vitro biofilm as cetrimide in tetraclean
improved the antimicrobial properties of the solutions,
whereas Tween 80 present in MTAD seemed to have a neutral
or negative impact on their antimicrobial effectiveness
Giardino L, Ambu E, Savoldi E, Rimondini R, Cassanelli C, Debbia EA. Comparative evaluation of antimicrobial efficacy of sodium hypochlorite, MTAD,
and Tetraclean against Enterococcus faecalis biofilm. Journal of endodontics. 2007 Jul 1;33(7):852-5.

• Antibacterial nanoparticles-bind to negatively charged surfaces
and have excellent antimicrobial and antifungal activities.
 Studies have also shown that the treatment of root dentin with ZnO
nanoparticles, Chitosan-layer-ZnO nanoparticles, or Chitosan
nanoparticles produces an 80 to 95 percent reduction in the
adherence of E. faecalis to dentin.
• Ultrasonically Activated Irrigation
 Bhuva B et al (2010) found that use of ultrasonically activated
irrigation using 1 percent sodium hypochlorite, followed by root
canal cleaning and shaping improves canal and isthmus cleanliness
in terms of necrotic debris/biofilm removal.
Wolfmeier H, Pletzer D, Mansour SC, Hancock RE. New perspectives in biofilm eradication. ACS infectious diseases. 2018 Feb 9;4(2):93-106.

Culture Microscopy Immunology
Molecular methods
Microbiological diagnostic
techniques

• Microbial sampling from the root canal
Types
1. Initial or diagnostic
2. Intermediate
3. Final
 Methods
1. Through the canal

Through the canal
Isolate tooth
with rubber
dam
Surface of
tooth
disinfected
Access to canal
with sterile
burs
If drainage
present,collect
with sterile
paper points
Air vented
from syringe
Aspirate
placed in
anaerobic
medium

Microscopic examination-Gram stained smears
• It was developed by Christian Gram in 1884.
• It helps in differentiating bacterias in gram-positive
and gram-negative organisms.

Microscopy
 Rarely provides definitive evidence of infection by a particular
species
 Microscopy has limited sensitivity and specificity
• Phase contrast and dark field microscopy
 The sample is taken in a similar way as for gram staining. After the
sample has been suspended in a water drop on the thin cover slip to
avoid drying and studied under the microscope.
 The cell boundaries appear sharper both in phase contrast
microscopy and dark microscopy than in gram staining.
 Dark field microscopy has been used for the detection of spirochetes
in root canal samples.

Culture
 In microbial culture, samples are collected and
transported to the lab in a viability preserving,
anaerobic medium
 Then distributed into various types of agar media and
cultured under aerobic or anaerobic condition
 Colombia agar plate was incubated at 37°C for 2 days
under aerobic conditions. Sabouraud’s agar plate was
kept at room temperature for 5 days (Gajan et al)
 Individual colonies are sub cultured and identified on
the basis of gram staining pattern, oxygen tolerance and
metabolic end product analysis .

SABOURAUD’S AGAR PLATE
COLOMBIA AGAR PLATE
5% Colombia agar and fibrin
bovine blood for aerobes
Sabouraud’s agar with chloramphenicol
for spores

Immunological methods
• Employ antibodies that recognize specific
microbial antigens to directly detect target
species
• These methods may require the use of
monoclonal antibodies to assure high
specificity

Molecular genetic method
•Molecular approaches for microbial identification rely on certain
genes that contain revealing information about the microbial identity.

• Of the several genes that have been chosen as targets
for bacterial identification
 The 16S rRNA gene (or 16S rDNA) has been the most
widely used because it is universally distributed among
bacteria, is long enough to be highly informative and
short enough to be easily sequenced.
 Similarly, the 18S rRNA gene of fungi and other
eukaryotes has also been used extensively to identify
these organisms

Why 16S rRNA?
• Present in all organisms
• Sequence is sufficiently
conserved
• Is of sufficient size
• Long enough to contain
information for identification
and analysis and short
enough to be easily
sequenced.
• affords reliability for
inferring phylogenetic
relationships.
Several genes have been chosen as target for bacterial identification

Advantages of molecular methods
• They are helpful in detection of both cultivable and uncultivable
microbial species
• They are more sensitive tests
• Molecular methods have greater specificity
• They are less time consuming
• Do not need special control for anaerobic bacteria
• Are useful when a large number of samples are needed to be
analyzed for epidemiologic studies
• They do not require cultivation
• They can be identified even when they are viable
• They can be used during antimicrobial treatment
• Large number of samples can be stored at low temperature and
surveyed at once

Polymerase chain reaction
Application of PCR for detection of microbial pathogens- most
widespread advance in clinical diagnostic technology.
•Possible to isolate essentially
any gene from any organisms
using PCR.
•At least 10 to 100 times more
sensitive than the other more
sensitive identification
method.
 Polymerase chain reaction (PCR) method involves in vitro replication of
DNA, therefore it is also called as “genetic xeroxing” method.
 Multiple copies of specific region of DNA are made by repeated cycles
or heating and cooling

How to combat microbes in the endodontic
therapy?
 Thorough cleaning and shaping of the root canal system with three
dimensional obturation of the root canals have shown to produce
complete healing of periradicular tissue .
 Complete debridement of canal should be done with adjunctive use of
irrigants like sodium hypochlorite which efficiently removes bacteria as
well as their substrate from irregularities of canal system where instruments
cannot reach such as fins, indentations, culde-sacs, etc.
 Oxygenating a canal simply by opening it is detrimental to anaerobes. Use
of oxygenating agents as glyoxide can be of great help but care should be
taken to avoid inoculation of these oxygenating agents into periapical
tissues.

• A tooth with serous or purulent or hemorrhagic exudate should be
allowed to drain with rubber dam in place for a time under
supervision.
• An abscess which is a potent irritant, has an elevated osmotic pressure.
This attracts more tissue fluid and thus more edema and pain.
Drainage by canal or by soft tissues decrease discomfort caused by
inflammatory mediators.
• Antibiotics should also be considered as adjunctive in severe
infections. The choice of antibiotic agent should be done on the
knowledge of microorganisms associated with the endodontic
infections.
• Intracanal medicaments play an important role in combating the
microorganisms. Use of calcium hydroxide in canals with necrotic
pulps after instrumentation have shown to provide the beneficial

INFLUENCE OF MICROBIOLOGY ON ENDODONTIC
FAILURE. LITERATURE REVIEW
• Most authors highlight E. faecalis as the main
microorganism associated with endodontic
failure, nevertheless there are recent studies
that isolate, to a greater extent, other bacteria
such as Fusobacterium nucleatum and
Propionibacterium.
Jhajharia K. Microbiology of endodontic diseases: A review article. Int. J. Appl. Dent. Sci. 2019;5(4).

Assessment of microbiota in root canals with
pulp necrosis by means of Gram test
• The aim of this study was to evaluate the type of microbiota present
in root canals with pulp necrosis, with and without periapical lesion.
• Absorbent paper cones were introduced in the canal for 20 s. Then,
the contaminated paper cones were inoculated in a Brain Heart
Infusion Agar-BHI culture medium and incubated in an oven for 48
h at 37°C.
• The results revealed that there was no statistically significant
difference between the amount of Gram-positive and negative
bacteria in endodontic infections, indicating that the microbiota of
these endodontic infections is mixed
de Mendonça Cavalcante A, Soares NM, Santos IC, de Azevedo Ximenes EA, da Silva MA, Junior KA. Assessment of microbiota in root
canals with pulp necrosis by means of Gram test. African Journal of Microbiology Research. 2018 Jun 14;12(22):508-11.

Evaluation of antibacterial efficacy of Chitosan, Chlorhexidine,
Propolis and Sodium hypochlorite on Enterococcus
faecalis biofilm : An in vitro study
• Chlorhexidine is equally efficacious as combination of 1%
chitosan + 1% chlorhexidine against E. faecalis biofilm.
• NaOCl performed equally well as that of 2% chitosan + 2%
chlorhexidine.
• Propolis also exhibited significant antimicrobial activity.
• Thus, from the results of the study, it can be suggested that all
these three combinations of irrigating solutions i.e 1%
chitosan+1% chlorhexidine, 0.2 chitosan+2% chlorhexidine
and 2% chitosan+ 2% chlorhexidine could be used as an
alternative to NaOCl for endodontic infections
Jaiswal N, Sinha DJ, Singh UP, Singh K, Jandial UA, Goel S. Evaluation of antibacterial efficacy of Chitosan, Chlorhexidine, Propolis and
Sodium hypochlorite on Enterococcus faecalis biofilm: An in vitro study. Journal of clinical and experimental dentistry. 2017
Sep;9(9):e1066.

Relationship of Biofilm Formation and gelE Gene
Expression in Enterococcus faecalis Recovered from
Root Canals in Patients Requiring Endodontic
Retreatment
• Wang et al. analysed the relationship of biofilm formation and
gelE gene expression in E. faecalis recovered from root canals
and found higher expression in cases of apical radiolucency
and in biofilm forming isolates. These authors suggested that
high expression of gelE may contribute to the development of
apical periodontitis.
Wang L, Dong M, Zheng J, Song Q, Yin W, Li J, Niu W. Relationship of biofilm formation and gelE gene expression in
Enterococcus faecalis recovered from root canals in patients requiring endodontic retreatment. Journal of endodontics. 2011
May 1;37(5):631-6.

Comparative efficacy of endodontic medicaments
and sodium hypochlorite against Enterococcus
faecalis biofilms
• The aim of this study was to compare the efficacy of commonly used
medicaments against E. faecalis cultured as a biofilm on dentine substrate.
• Sodium hypochlorite achieved total bacterial elimination. Ledermix and
Odontopaste had no significant effect on the E. faecalis biofilm. Ca(OH)2
and 50:50 combinations of Ca(OH)2/Ledermix or Ca(OH)2/Odontopaste
reduced the viability by more than 99% while 0.2% chlorhexidine reduced
bacterial numbers by 97%.
• Sodium hypochlorite remains the gold standard for bacterial elimination in
root canal therapy. However, Ca(OH)2 in isolation and combined with
Ledermix, and Odontopaste was highly effective in reducing bacterial
viability.
Plutzer B, Zilm P, Ratnayake J, Cathro P. Comparative efficacy of endodontic medicaments and sodium
hypochlorite against Enterococcus faecalis biofilms. Australian dental journal. 2018 Jun;63(2):208-16.

ROLE OF TRIPLE ANTIBIOTIC
PASTE
• William Windley observed a statistically significant
reduction in bacteria, following the irrigation and
antibiotic paste protocol. 90% of the bacteria remained
positive following irrigation with 10 ml 1.25% sodium
hypochlorite. However, this dropped to 30% following
the application of the TAP for 2 weeks.
• TAP seems to be promising medicament in the
sterilization and revascularization.

Other Treatment options….
• Both probiotics and prebiotics would strengthen beneficial
microflora so the body naturally fight off disease-causing
agents.
• Immunization proposed for both dental caries and
periodontitis.
• In case of dental caries, protein involved in colonization of
teeth by S.mutans can produce antibodies that inhibit the
cariogenic process.
• Periodontal vaccines are less well-developed, but some
antigenic targets have been identified.

Association of endodontic signs and symptoms with
root canal pathogens: A clinical comparative study
•There was a strong association between S. mitis and E. faecalis
with specific endodontic signs and symptoms.
•The pain was associated with S. mitis in primary infections.
• There was a significant correlation between tenderness on
percussion and E. faecalis in secondary infections.
•Association was noted between larger periapical lesion size (>5
mm) and S. mitis in primary and secondary infections.
Vineet RV, Nayak M, Kotigadde S. Association of endodontic signs and symptoms with root canal pathogens: A
clinical comparative study. Saudi Endodontic Journal. 2016 May 1;6(2):82.

ORAL FLORA – friend or foe?
Although micro-organisms have been living as
commensals in the oral cavity, any change in the
environment may render them pathogenic
Therefore, one must maintain the ecology of both the
microbes and the host to smoothly continue with this
commensalism
CONCLUSION

• Cohen’s pathways of pulp
• Ingle’s Endodontics 6th Edition
• Marsh and Martin’s Oral Microbiology 6th Edition
• The oral microbiome – an update for oral healthcare professionals
M.Kilian 2016, British Dental Journal
• L Lakshmi Narayanan , C Vaishnavi : Endodontic Microbiology; J
Conserv Dent 2010
• Newman MG, Takei HH, Carranza FA, Clinical
Periodontology, 9th ed. W B Saunders Company
Philadelphia.2000.
• Fejerskov O, Kidd E, Dental Caries The Disease and its Clinical
Management, 2ne ed. Blackwelll Munksgaard. Singapore.2008.
• Vineet R V, Nayak M, Kotigadde S. Association of endodontic
signs and symptoms with root canal pathogens: A clinical
comparative study. Saudi Endod J 2016;6:82-6
REFERENCES

• Samaranayake, L., & Matsubara, V. H. (2017). Normal Oral Flora and the
Oral Ecosystem. Dental Clinics of North America, 61(2), 199–215.
doi:10.1016/j.cden.2016.11.002
• Siqueira, J. F., & Rôças, I. N. (2009). Diversity of Endodontic Microbiota
Revisited. Journal of Dental Research, 88(11), 969–
981.doi:10.1177/0022034509346549
• Munson et al., 2002; Siqueira et al., 2004b; Siqueira and Rôças, 2005b.
• Sundqvist, 1976; Vianna et al., 2006b; Sakamoto et al., 2007; Siqueira et
al., 2007d.
• Fukushima, H., Yamamoto, K., Hirohata, K., Sagawa, H., Leung, K.-P., &
Walker, C. B. (1990). Localization and identification of root canal bacteria
in clinically asymptomatic periapical pathosis. Journal of Endodontics,
16(11), 534–538. doi:10.1016/s0099-
• Pathways of pulp 9th edition-Stephen Cohen
• Endodontic therapy 5th edition-Franklin S Weine
• Vineet R V, Nayak M, Kotigadde S. Association of endodontic signs and
symptoms with root canal pathogens: A clinical comparative study. Saudi
Endod J 2016;6:82-6
• Siqueira Jr JF. Microbial causes of endodontic flare‐ups. International
Endodontic Journal. 2003 Jul;36(7):453-63.

Endodontic Microflora

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Endodontic Microflora

Similar to Endodontic Microflora (20)

Recently uploaded

Recently uploaded (20)

Endodontic Microflora

Editor's Notes