Endo microbiology/ dental implant courses

Endodontic Microbiology
INDIAN DENTAL ACADEMY
Leader in continuing Dental
Education
www.indiandentalacademy.com

Introduction
• Micro organisms cause virtually all pathoses
of the pulp and the perriadicular tissues. To
effectively treat endododontic infections,
clinicians must recognize the cause and
effect of microbial invasion of the dental pulp
space and surrounding periradicular tissues
• Although the vast majority of our knowledge
deals with bacteria, we are now aware of the
potential for endodontic disease to be
associated with fungi and viruses.

History
• Louis Pasture1822-1895. laid the foundation of
microbiology in France
• In Germany Robert Koch (1843-1910) perfected
bacteriological techniques; Staining and obtaining
bacteria from culture media
• W.D. Miller in 1894, associated the presence of
bacteria with pulpal disease.
• 1939- Fish’s theory became the basis for root canal
treatment. He recognized four zones of infection

Number of miroorganisms x virulence of miroorganism = severity of disease
resistance of host
• E.C rose 1909 –Theory of focal infection
• The true significance in endodontic disease was shown
in the classic study by Kakehashi et al in 1965.
• Hobson in 1965 first described this equation in
endodontic literature

Terminologies
• Facultative bacteria can grow in the presence or
absence of oxygen.
• Obligate aerobes require oxygen for growth
possess both superoxide dismutase and catalase
enzymes and
• “Strict anaerobes” grow only in the absence of
oxygen but vary in their sensitivity to oxygen.
missing the enzymes superoxide dismustase and
catalase.
• Microaerophilic can grow in the presence of
oxygen but derive their most of energy from
anaerobic energy pathway.www.indiandentalacademy.com

Association of microbes in pulpal and
Periradicular diseases
• Establishment of bacteria or other organisms in a
living host takes place through the phenomenon
known as: colonization”.
• Normal flora is the result of permanent
colonization of microbes in a symbiotic
relationship.These organisms are considered
opportunistic pathogens if they gain access to
sterile area of the body such as the dental pulp or
periradicular tissues and produce disease.

• Infectious disease (infection) results if organisms
damage the host and produce clinical signs and
symptoms.
• Pathogenicity is the capacity of organisms to
produce disease within a particular host.
• Virulence expresses the degree of pathogenicity in
a host under defined circumstances.

Stages in development of an endodontic
infection include
1. Microbial invasion
2. Colonization
3. Multiplication
4. Pathogenic activity

Microbial Invasion (Routes of
microoraganism Ingress)
Coronal Ingress:
1. Caries
2. Fracture – complete,
incomplete
3. Non fracture trauma
4. Anomalous tract
a.Dens invaginatus
b.Dens evaginatus
c. Palatogingival groove
5. Crown preparation and
leaking restoration

Radicular Ingress:
1. Caries
2. Retrogenic infection – periodontal pocket,
periodontal abscess
3. Hematogenic
4. External and Internal resorption

Coronal Ingress
Caries; Caries is the most common portal of
entry and most frequent initial cause of pulp
inflammation.
Fractured Crown: In healthy vital pulp, the
penetration of bacteria in tissue is relatively
slow. It is less than 2mm after 2 weeks.A
necrotic pulp is rapidly invaded and colonized
by bacteria. www.indiandentalacademy.com

• Anomalous Tract: Anomalous tooth
development, of both the crown and the root,
accounts for pulp deaths usually by bacterial
invasion.
RADICULAR INGRESS
• Root caries - a less frequent occurrence, but
it remains a bacterial source of pulp irritation

Retrogenic Infection – Periodontal Pocket
• Through the tubules, lateral or accessory canals,
furcation canals and apical foramina,
• The removal of cementum during periodontal
therapy exposes dentinal tubules to oral flora.
• Langeland et al found that changes in the pulp
occur when periodontal disease was present, but
pulpal necrosis occurs only if apical foramen was
involved.
• Periodontal Abscesswww.indiandentalacademy.com

Hematogenic infection:
• Anachoresis is a process which microbes
may be transported in the blood or lymph to
an area of inflammtion such as a tooth with
pulpitis , where they may establish an
infection.
• Anachoresis of bacteria from the vessels of
the gingival sulcus by Saglic et al,
• From a systemic transient bacteremia

Reaction of Pulp to Bacteria
The dynamics of the pulpal reaction is related to
• Virulence of the bacteria
• Host response,
• Amount of pulpal circulation
• Degree of drainage.

Microorganisms associated with
endodontic disease
• Kakehashi et al in 1965. found that no pathologic
changes occurred in the exposed pulps or periadicular
tissue in germ free rats. Thus, the presence of absence
of microbial flora was the major determinant for the
destruction or healing of exposed rodent pulps.
• In humans, Once the pulp becomes necrotic, it becomes
a reservoir for microorganism, microbial byproduct and
microbial breakdown products.

Structure of Endodontic flora
• Of the more than 350 species of bacteria
recognized as normal flora, only relatively small
group are commonly isolated from infected pulp
cavities.
• Most of the bacteria in an endodontic infection are
“strict anaerobes”.
• Prior to 1970, very few strains of strict anaerobes
were isolated and identified because of inadequate
anaerobic culturing methods.

• The number of bacterial species in root canals may
vary from 1 to more than 12 and the number of
bacterial cells colony forming units (CFU) recovered
between < 102
to > 108
.
• A correlation seems to exist between the size of
periapical lesion and the number of bacterial species
cells(CFU)
• Teeth with large lesion usually harbor more bacterial
species and have a higher density of bacteria in their
root canals than teeth with small lesions.

Conditions conducive to Growth of
Bacteria in Necrotic Dental Pulp
• Nutritional factors
• Influence of oxygen
• Bacterial interrelationsnips
• Bacteriocins
• Coaggregation

Nutrition
• Tissue fluid and the breakdown products of
necrotic pulp rich with polypeptides and amino
acids, essential for growth of the bacteria.
• Inflammatory exudates containing serum and
blood factors
• Conditions in the root canal which permit the
growth of anaerobic bacteria capable of
fermenting amino acids and peptides.

Influence of oxygen
• Extremely low availability of oxygen in infected
root canals.
• When direct oral exposure, the oxygen level
remain low, particularly in the apical portions of
the canal where low oxidation reduction potential
is produced for growth of facultative bacteria
• These bacteria initially colonize the pulpal
chamber, but due to the resulting low oxidation
reduction potential, growth of obligate anaerobic
bacteria will be favored.

• Bacterial interrelationsnips
Bacteria can be commensal or antagonistic.Some
metabolites (eg: ammonia) may be either a
nutrition or a toxin depending on the concentration.
• Bacteriocins
Antibiotic like proteins produced by one species of
bacteria to inhibit another species of bacteria.
• Coaggregation
A “symbiotic relationship” that may lead to an
increase in virulence by the organisms in that
ecosystem.

Virulence factors
• Bacterial fimbriae are important for attachment to surfaces
and to other bacteria.
• Capsules; significant resistance factor enabling bacteria
to avoid phagocytosis..
• LPS are found on the surface of gram- negative bacteria
and have biologic effects that induce periradicular pathosis.
When LPS is released from the outer membrane of gram –
negative bacteria, it is called endotoxin.
• Endotoxin activate complement and bone resorption.
Enzymes collagenase seems to be associated with the
spread of cellulites.

• Fungi in the form of yeasts were proven to be
present in the root canal system. fungal
species Candida albicans was found to be the
commonest.
• Extracellular vesicles are formed form the
outer membrane of gram – ve bacteria and
may contain enzymes or other toxic agents.
involved in haemagglutination, hemolytic,
bacterial adhesion and proteolytic action on
host tissues.

COMPOSITION OF THE MICROBIOTA
• Morphologic forms:Studied using Dark field
and phase contrast microscopy
• Cocci, rods, filaments, spirochetes have all
been identified in infected root canals.

Association of Bacteria with
Periaradicualr infections:
• Bacterial byproducts alone are capable of
causing periradicular pathosis.
• Infection beyond the apex depends on the
number and virulence of the organisms, host
resistance, and anatomic structures
associated with the infection

• The microorganisms identified in periradicular
infections( abscesses ) of endodonntic origin are similar to
bacteria isolated form the root canal system.
• In mixed culture with F. Nucleatum, the BPB Prevoltella
intermedia and Porphyromanas gingivalis were significantly
more abscessogenic than F. nucleatum in pure culture.
• Prophyromonas gingivalis has been shown to produce
severe rapidly spreading abscess while prevotella
intermedius and Prophyromonas endodontalis cause
localized abscesses.
• P. gingivalis has also been shown to express “collagenase”
as a potential “ virulence factor”

• Whether asymptomatic chronic apical
periodontitis lesions (periapical granulomas) are
sterile has been controversial since the beginning
of 1900s
• study by Nair; Species of Actinomyces and
Proprinibacterium have been shown to be able to
persist inflammatory tissue
• Actinomyces israelli is a spices of bacteria
isolated from periapical tissues that does not
always respond to conventional endodontic
therapy.

• sodium, hypochlorite and calcium hydroxide have been
shown to be highly effective in killing A. israelli
• endodontic surgery apparently is an effective way to
remove A. israelli from the periapex and seems to provide
a high success rate without antibiotics
• Studies identified the presence of fungi .Waltimo et al
showed that strains of Candida albicans which required
incubation with a saturated solution of calcium hydroxide
for about 16 hours kill 99.9% of the fungi.

• In 1989 Michael Glick and other examine
dental pulp tissue from a patient with (AIDS)
to determine the presence of human
immunodeficiency virus (HIV).
• Ebstein Bar viruses have also been isolated
in other cases.

• several species have been implicated with some
clinical signs and symptoms.
BPB (Black Pigmented Bacterodies)
Peptostrptococcus, Peptococcus
Eubacterium, Fustobacterium
Actinomyces.
Recent Taxonomic changes for previous
Bacteroides species; ‘Goran Sundquist’
Three saccharolytic species of BPB have been
reclassified into a separate genus named
Porphyromonas.
Six species of saccharolytic BPB have been
reclassified into a separate genus named
Prevotella. www.indiandentalacademy.com

• Porphyromonas gingivalis has been shown
to produce severe rapidly spreading abscess
• Prevotella intermedias and prophyrmonas
endodontalis cause localized abscesses.
• Prevotella intermedia was found in both
Symptomatic and asymptomatic infections
• In 1992, isolates previously thought to be, P.
intermedia ar now known as P. nigrescens
• P. nigrescens is actually the BPB most
commonly isolated from both root canals and
Periradicular abscesses of endodontic origin.

Factors responsible for non responsive
Endodontic cases
• Faulty temporary seal
• Additional canals
• canal configuration
• Inaccurate length determination
• Incomplete canal disinfection
• Fractures
• Refractory failure
• Post space failure

• Endodontically treated teeth requiring
“retreatment” show prevalence of
facultative bacteria, especially
streptococcus faecalis, instead of strict
anaerobes.
• In Teeth with persisting periapical
lesions flora differs from that of
untreated necrotic dental pulps.

• The microbial flora detected in root- filled
canals would be characterized as “
monoinfections of predominantly gram-
positive microorganisms”, with approximately
equal proportions of facultative and obligate
anerobes.
• This differs from that seen in infections of the
untreated canal, which have a polymicrobial
flora with approximately equal proportion of
gram- negative and gram positive bacteria
and are dominated by obligate anerobes.

• Enterococus faecalis highly resistant to the
medicaments used during treatment
• one of the few micro organisms that has been shown
in vitro to resist the antibacterial effect of calcium
hydroxide.
• survive the antibacterial treatment, and persist after
obturation.
• in low numbers can usually be eliminated, once
established in the root canal it is difficult to eradicate.
• Have an ability to survive in root canals as single
organism without the support of other bacteria.
• usually enters the canal “ during treatment

• Bacteria can also prevent the healing of
periapical lesions by establishing
themselves in periapial tissue.
• A Israeli and Propionibacterium propionicum,
have the ability to establish extraradicular
infections.

Bateriologic Examination
Microbiological identification techniques
• Anaerobic culturing
• Indirect immunofluoresene; Porphyromonas &
Prevotella
• Sodium dodecyl sulfate polyacrylamide gel phoresis
(SDS-PAGE) Prevotella nigrescens from Prevotella
intermedia
• Polymerase chain ration- DNA method applies
genetic code analysis
• DNA analysis with biochemical enzyme analysiswww.indiandentalacademy.com

Culture media
• single organism is sufficient for growth in culture medium
and maximum ten microorganisms are needed.
• Brain heart infusion broth, Trypticase dexrose broth ,serum
dextrose broth were not suitable for anaerobic organisms.
Addition of small amount of agar, serum,blood or ascitic fluid
facilitate their growth.
• Trypticase soy agar broth with 0.1% agar
• Thioglycolate and glucose ascits broth
• Viability medium for growth
• Stuart’s transport mediumwww.indiandentalacademy.com

Steps in taking a culture
1. Wipe away residual
antibiotic or antiseptic
from surface of root
canal
2. Insert the absorbent
point and let remain in the
root anal for atleast 1
minute

3. Tip of the absorbent
point is will be moist
when removed from the
canal
4. Flame the lip of
the culture tube

5. Drop the absorbent
point into the culture
tube and replace the
cap.
Anaerobic culturing
•Requires special equipment in temperature controlled
oxygen free environment
•Meticulous care must be taken to prevent contact of
the specimen with atmospheric oxygen.
• canal orifice must b flushed with nitrogen gas during
sampling to maintain Anaerobiosis.

Periradicular sample
• Insert the sterile needle of leur lock syringe
into periradicular space
• Aspirate fluid
• Eject any air inside the syringe barrel
immediately
• Insert the needle through the rubber septum
stopper of anaport vial and eject the fluid
• Vial should be transported to anaerobic
culture depot with in 4 hours after taking a
sample

Root canal sample
• Aseptically prepare an ass cavity into the pulp
chamber.
• Inject few drops of prereduced anaerobicaly
sterilized medium (chopped meat glucose
broth) into the chamber.
• Pump the medium into the root anal with
sterile endodontic file.
• Aspirate fluid with leur lock syringe into
periradicular space.
• eject any air inside the syringe barrel
immediately.
• transported to anaerobic culture depot with in
4 hours after taking a sample.

Culture reversal
• Negative culture become positive by the time
of obturation.
• Advised to allow more than 48 hours between
taking culture and obturation(96 or more
hours)
• Culture tube should be reexamined
immidiately before obturating a canal so that
no evidence of growth is present.

Root canal debridement and intracanal
medication
Crown down technique with rotary instruments
and abundant irrigation.
• Debris is not extruded apically
• Enlarges coronal portion of canal to allow
large reservoir for irrigants
• Sodium hypochlorite(.5%-5.25%)
• Sodium hypochlorite an EDTA(15%) is more
effective.

Intracanal asepsis
Calcium hydroxide-
• mixed with saline or glycerin
• Carried with amalgam carrier in pulp
chamber.
• Carried down in the canal using plugger,
lentulospiral.
• Can be removed easily in next appointment.
Calcium hydroxide and sodium hypochlorite
(increases ability of NaOcl)

• Good temporary coronal seal-3-4mm thick
• Camphorated choloroxylenol-liquid medication(ED84)- new
root canal medicament ,effective for two days.
Infection control
• Patient screening
• Presonal protection
• Equipment asepsis
• Instrument sterilization
• Surface disinfection
• Aseptic technique

Endodontic Flareups
Is a true complication characterized by development of
pain swelling or both, which commences with in few
hours or days after root canal preparation.
Require an unscheduled visit for emergency treatment
Microbial mechanisms in inductuion of flare up
• Apical extrusion of infected debris
• Changes in endodontic microbiota or in
enviornmental conditions
• Secondary infections
• Increase of the oxidation reduction potential.

Preventive measures
• Selection of instrumentation technique.
• Completion of chemomechanical procedure
in single visit.
• Use of antimicrobial intracanal medication
between appointments.
• Not leaving teeth open for drainage.
• Maintain aseptic chain during intra canal
procedure

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