ENDODONTIC MICROBIOLOGY

Dr. Alok Misra

M.D.S.
Professor
Department of Conservative-dentistry & Endodontics
Faculty of Dentistry
Melaka-Manipal Medical College, Malaysia
 Introduction

Microorganisms cause virtually all pathosis of the pulp and the periradicular
tissues. To effectively treat endodontic infections the clinician must recognize
the cause and effect of microbial invasion of the pulp space and the
surrounding periradicular tissues.

Knowledge of microorganisms is necessary to develop understanding of the

disease and a sound management of endodontic infections.

Endodontic disease can be due to bacteria, virus or fungi.

A fundamental knowledge of endodontic microbiology is needed to
understand :

The role of bacteria in these diseases,

Pathways of pulpal and periradicular infections
The pulpal and periradicular responses to bacterial infection
The methods used to eradicate and control root canal infections during root
canal therapy.
 Theory of focal infection

1890 – W.D.Miller associated the presence of bacteria with pulpal and periapical
diseases. – Focal infection.
E.C.Rosenow in 1909 described the “theory of focal infection”.
localized or generalized infection caused by bacteria travelling through the blood
stream through the distant focus of infection.
1910, William Hunter stated that the restorations were ……..

“a veritable mausoleum of gold over a mass of sepsis”.

Condemned the practice which emphasized on restorations instead of tooth

extractions.
He believed that this was the cause of pale complexion , chronic dyspepsia ,
intestinal disorders, anemias and nervous complaints.

Dental literature also contained numerous testimonials reporting cures of

illness following tooth extraction.

These reports were empirical without adequate follow up.

In many cases disease returned and patient had to face additional difficulty
living with mutilated dentition.
 Terminology
Colonization is the establishment of bacteria or other microorganisms in a
living host. Colonization occurs:
1)If appropriate biochemical and physical conditions are available for growth
and
2)Inhibitory factors are inadequate to destroy the organism

Permanent colonization in a symbiotic relationship with the host results in

establishment of normal oral flora. These organisms participate in many
beneficial relationships but are considered opportunistic pathogens if they
gain access to a normally sterile area of the body and produce disease.
Infectious disease (infection) results if microorganisms damage the host and
produce clinical signs and symptoms.

Pulpal and Periradicular pathoses (diseases) result from opportunistic

pathogens infecting the pulp cavity and, Periradicular tissues.

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.
Virulence factors - pili (fimbriae), capsules, extracellular vesicles,
lipopolysaccharides, enzymes, short-chain fatty acids, polyamines, and low-
molecular-weight products such as ammonia and hydrogen sulfide.

Lipopolysaccharides are found on the surface of gram-negative bacteria and

have numerous biologic effects when released from the cell in the form of
endotoxins.

Enzymes are produced by bacteria that may be spreading factors for infections
or proteases that neutralize immunoglobulins and complement components
Gram-negative bacteria produce extracellular vesicles

Involved in hem agglutination, hemolysis, bacterial adhesion, and proteolytic

activities.

These vesicles have the same antigenic determinants on their surface as their
parent bacteria, they may protect the bacteria by combining with and neutralizing
antibodies that would have reacted.

Anaerobic bacteria commonly produce short-chain fatty acids including propionic,

butyric, and isobutyric acids.

As virulence factors, these acids may affect neutrophil chemotaxis, degranulation

chemiluminescence, and phagocytosis.
Polyamines are biologically active chemicals found in infected canals.

The amount of total polyamines and putrescine is higher in the necrotic pulps
of teeth that are painful to percussion or with spontaneous pain.

When a sinus tract was present, a significantly greater amount of cadaverine

was detected in the pulp space.
1970s - microbiological studies on root canal flora reported primarily the
presence of facultative bacteria in this system.

recent studies shows - root canal infections - multibacterial - anaerobic

organisms, namely Bacteroides species, play a significant role in clinical signs
and symptoms of pulpal and periapical disease.
 ENDODONTIC IMPLICATION
Fish Theory:-
He established experimental foci of infection in the jaws of guinea pigs
by drilling opening in the bone & packing in wool fibers saturated with
a broth culture of microorganism. Four well defined zones of reaction
are found:

1. Zone of infection.
2. Zone of contamination.
3. Zone of irritation.
4. Zone of stimulation.
FISH’S ZONES
 NECROTIC ROOT
ZONE
CANAL CONTENTS

• Zone of necrosis
• (Infection,Pus
center,Abscess)

• Dead cells,Protein
decomposition products
• PMNs
• Exotoxins,Endotoxins,Enzymes
 ZONE OF
CONTAMINATION • IMMEDIATE RESPONSE
TO TOXIC ELEMENTS
(Primary exudative zone) • Exudative defense
force,dilution of toxic
elements,antibacterial
action of infl’m fluid
• ZONE OF IRRITATION
(Granulomatous,Prolifer
TOXICITY DIMINISHING AS
ative)
DISTANCE FROM CANAL
FORAMINA INCREASES
Defense,healing,repair,capillary
proliferation,fibroblastic
activity,environment
favourable for osteoclasts.
• ZONE OF • TOXICITY REDUCED TO
STIMULATION(ZONE A MILD STIMULANT
OF • Peripheral orientation
ENCAPSULATION,ZONE of collagen
OF PRODUCTIVE
FIBROSIS) • Favourable for
osteoblastic,bone
apposition,reversal
lines.
 KRONFIELD’S MOUNTAIN PASS CONCEPT
bacteria in the root canal (Zone I) with an army entrenched “behind high and inaccessible mountains” the
foramina serving as a mountain passess.

The exudative and granulamatous tissue = mobilized army defending the plains from the invaders.

few invaders cross over, they are quickly and readily neutralized by the defenders
mass attack = major battle analogous to acute inflammation (acute or exudative forces of Zone II).

Only complete elimination of bacteria = eliminate the need for the defense force in the in the “plains”.

Destruction created by the battle is repaired – Zone of Irritation (Zone III) and the environment returns to
normal. All this is walled off by Zone IV or Zone of Encapsulation.

# Thus KRONFIELD implied that granuloma is not an area where bacteria live but one in which they are
destroyed
Kronfeld‘s Mountain Pass Concept:-

Granuloma – bacteria are destroyed.

Bacteria in root canal – army behind high & inaccessible mountain
(foramina).
Exudative & granulomatous tissue – mobilized army defending plains
( periapex)

If the contents of Zone of necrosis or infection are eliminated, the

granuloma can complete its function of healing & repair.
KRONFELD’S MOUNTAIN PASS
CONCEPT
 Torneck’s Hypothesis(1960)
Bacteria impede healing.

Canal should be cleaned & shaped with NaOCl to remove bacteria.

Oxygenate the canals by opening it or use of oxygenating agents.

Prevent entry of oral flora into the canals.

PATHWAYS OF PULPAL & PERIAPICAL INFECTIONS
Six major pathways of pulpal contamination
1. Dentinal tubules
2. Direct pulp exposures
3. Lateral & apical foramen
4. Blood borne bacteria.
5. Through a broken occlusal seal or faulty restoration of tooth
previously treated by endodontic therapy.
6. Through extension of a periapical infection from adjacent infected
teeth.
Dentinal tubules:-
Diameter of DT near pulp – 2.5μm.
at CEJ & DEJ - 1μm.
Number of DT near CDJ – 15,000 to 20,000 per square
millimeter of dentin.

Experiments in vivo & in vitro show that dentinal tubules of viable dentin
are not easily invaded by oral microorganism.

This route of infection appears to be selective for only a few bacterial

strains, mostly facultative anaerobic bacteria found in the oral flora.
Pulpal exposure:-
Contamination of pulpal tissue can also occur due to direct pulpal
exposure as a result of traumatic injuries or tooth decay.
Species involved – mixed flora mainly anaerobic flora.

Periodontal ligament:-
When the pulp is infected through the apical foramen, it appears that
impaired state of pulp is always a prerequisite for infection.
Species involved – facultative anaerobic streptococci.
Anachoresis:-
Csernyei (1939) demonstrated the anachoretic effect of periapical
inflammation in dogs.

It has been reported that dental extractions, & even tooth brushing,
can produce bacteremia. During bacteremia, circulating microorganism
can be attracted to & can be localized in inflamed or necrotic pulps.

Moller et al showed all devitalized pulps in monkeys remained sterile

for more than 6 months.
Broken occlusal seal or faulty restoration:-
Torabinejad et al. have showed that salivary contamination from
occlusal aspect can reach the periapical area in less than 6 weeks in
canals obturated with gutta-percha & sealer.

Extension of a periapical infection from adjacent infected teeth:-

Controversial matter.
MICROFLORA OF INFECTED & UNTREATED NECROTIC PULP

Root canal flora of teeth with clinically intact crowns but having
necrotic pulps & diseased periapices:-
- Obligates anaerobes (90%) belonging to genera Fusobacterium,
Porphyromonas, Prevotella, Eubacterium, & Peptostreptococcus.
- Spirochetes.

Teeth that are exposed to oral cavity by caries:-

Less dominated by strict anaerobes (70%)
ENDODONTIC FLORA IN PREVIOUSLY ROOT FILLED TEETH

Bacteria – gram +ve cocci, rods & filaments.

Species – Actinomyces, Enterococcus, &

Propionibacterium, Streptococcus faecalis

Fungus – Candida albicans.

 PATHOGENICITY OF ENDODONTIC FLORA
Pathogenicity of microbes is increased by

1. Interaction with other microorganism in the root canal.

2. The release of endotoxins.
3. The synthesis of enzymes that damage host tissues.
4. The ability to interfere with & evade host defense.
Root planing – pulpitis and bacterial penetration.

Periodontal disease -pulpal necrosis (if the apical foramen was involved).

Bacteria concurrent in both areas - the sulcus or periodontal pocket is the

source of the bacteria in root canal infections.

To differentiate an abscess of periodontal origin from that of endodontic

origin, the enumeration of spirochetes has been recommended.
Vital pulp - resistant to microbial invasion.

Movement of bacteria in dentinal tubules - restricted by viable odontoblastic

processes, mineralized crystals, and various macromolecules within the tubules.
Acid-producing bacteria (with gram-positive organisms predominating) invade the
tubules and demineralize the walls.

↓
Proteolytic species follow, acting on the organic matrix, which is denuded in the
enlarged dentinal tubules.
↓
The acids and other metabolites and toxic products diffuse faster than the bacteria,
so that the odontoblasts are affected, possibly leading to their breakdown.
If the advancing bacteria are eliminated, healing may occur.

Caries - the most common portal of entry for bacteria and bacterial byproducts
into the pulpal space.

Following trauma and direct exposure of the pulp, inflammation, necrosis, and
bacterial penetration are no more than 2 mm into the pulp after 2 weeks.

In contrast, a necrotic pulp is rapidly invaded and colonized.

Peritubular and reparative dentin impede the progress of bacteria.

Dead tracts, direct exposure (restorative procedures, trauma), pathways

associated with anomalous tooth development- passage for microbes to the
pulp.
irritants
↓

Infected root canal system tubules, lateral or accessory canals, furcation

canals, and the apical foramina
↓
Surrounding attachment apparatus.
Abscesses of periodontal origin contained 30 to 58% spirochetes, whereas
those of endodontic origin were 0 to 10% spirochetes.

Pulpal exposure – caries -Alpha-hemolytic streptococci, enterococci, and

lactobacilli are most often found.

Other facultative anaerobic organisms are present in smaller numbers.

As the depth of the necrotic pulp increases, more species of obligate

anaerobic bacteria become established.

anaerobic gram-positive cocci and gram-negative rods.

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.

localization of blood borne bacteria in instrumented but unfilled canals could

not be demonstrated

Infection of unfilled canals was possible only with over instrumentation during
the bacteremia to allow bleeding into the canals.

Anachoresis may be the mechanism through which traumatized teeth with

intact crowns become infected.
 FLORA OF PULPAL INFECTION

The importance of anaerobic bacteria in pulpal and periapical pathoses has

been revealed with the development of anaerobic culturing methods.

Multibacterial

Most of the bacteria in an endodontic infection are strict anaerobes.

They function at low oxidation-reduction potentials and generally lack the

enzymes superoxide dismutase and catalase.
no absolute correlation has been made between any species of bacteria and
severity of endodontic infections.

BPB, Peptostreptococcus, Peptococcus, Eubacterium, Fusobacterium, and

Actinomyces.

The black-pigmented Bacteroides species have gained special prominence in

the search for pathogens of endodontic infections.

This genus, a rather heterogeneous group of microorganisms, is now divided

into eight separate species, including B. asaccharolyticus, B. corporis, B.
denticola, B, endodontalis, B. gingivalis, B. intermedins, B. melanoninogenicus,
and B. loeschei.
Studies of endodontically treated teeth requiring retreatment have shown a
prevalence of facultative bacteria, especially Streptococcus faecalis, instead of
strict anaerobes.

In addition, fungi have been shown to be associated with failed root canal
treatment.

Recent studies have demonstrated that P. nigrescens is actually the BPB most
commonly isolated from both root canals and periradicular abscesses of
endodontic origin.
Bacteria Cultured and Identified from the Root Canals of Teeth with Apical Radiolucencies
Bacteria Incidence (%)
Fusobacterium nucleatum 48
Streptococcus sp 40
Bacteroides sp* 35
Prevotella intermedia 34
Peptostreptococcus micros 34
Eubacterium alactolyticum 34
Peptostreptococcus anaerobius
31
Lactobacillus sp 32
Selenomonas sputigena 9
Veillonella parvula 9
Eubacterium lentum 31
Porphyromonas endodontalis
Fusobacterium sp 29
9
Campylobacter sp 25
Prevotella buccae 9
Peptostreptococcus sp 15
Prevotella oralis
Actinomyces sp 15
Proprionibacterium
Eubacterium timidum 11
propionicum 8
Capnocytophaga ochracea 11
Prevotella denticola 6
Eubacterium brachy 9
Prevotella loescheii 6
Eubacterium nodatum 6
 Pulpal and Periradicular Response
The degree of pulpal and periradicular response to bacterial irritants varies from slight tissue
inflammation to complete pulpal necrosis or acute periradicular osteomyelitis with systemic
signs and symptoms of severe infection.

As a result of the presence of microorganisms in the dentin, a variety of immunocompetent

cells can be recruited to the dental pulp.

It is initially infiltrated by chronic inflammatory cells, such as macrophages, lymphocytes, and

plasma cells.

The concentration of these cells increases as the decay progresses toward the pulp.

Polymorphonuclear leukocytes are the predominant cells at the site of pulp exposure
As a result of the interaction of microorganisms and their by-products, various
mediators of inflammation, such as neuropeptides, vasoactive amines, kinins,
complement components, arachidonic acid metabolites, and I cytokines are
released.
Neuropeptides :
Neuropeptides are proteins generated from somatosensory and autonomic
nerve fibers following injury.
They include substrate P, calcitonin gene-related peptides, and neurokinins
originating from the sensory nerve fibers, as well as dopamine B-hydrolase and
neuropeptide Y originating from sympathetic a nerve fibers.
These substances, which participate in the process of inflammation and pain
transmission, were recently demonstrated in intrapulpal nerve fibers using
immunohistochemistry.
Vasoactive amines:
Histamine like substances were found in the walls of the blood vessels.
High concentrations of kinins have been found in symptomatic human periapical
abscesses.
Kinins are considered the main mediators of the pain associated with inflammatory
responses.

The complement system, when activated, causes enhanced phagocytosis, increased

vascular permeability, and lysis of cellular antigens.

Arachidonic acid is released from the phospholipids of cell membranes as a result of

cell damage.

When metabolized through the cyclooxygenase or lipoxygenase pathway, various

prostaglandins, thromboxane's, and leukotrienes are produced.
In addition to well-defined mediators of inflammation such as neuropeptides,
fibrinolytic peptides, kinins, lysosomal enzymes, complement components, and
arachidonic acid metabolites, a number of less well-characterized substances
are released by a variety of cells.

These soluble substances, which are capable of activating other cells, are
referred to collectively as cytokines.
Lymphocytes – lymphokines.

Monocytes – monokines.

Interlukines mediate communication between leukocytes.

Cytokines are glycoproteins secreted as a result of cellular stimulation and have low
molecular weights.
They are not stored within cells, are produced locally, have very short half-lives, and
are extremely potent.
They interact with cell surface receptors, which leads to a change in the patterns of
cellular RNA and protein synthesis and cell behavior.

The major cytokines include interleukins, interferons, tumor necrosis factor, and
colony-stimulating factors.
Among interleukins, IL-1 and IL-6 are pro inflammatory and chemotactic for
inflammatory cells.
Bone resorptive activity of IL-1 is probably due to its effect on osteoclast
differentiation from hematopoietic progenitor cells and formation of osteoclast-like
giant cells in cultured bone marrow
Presence of dendritic cells in the pulp that activate T lymphocytes, which, in
turn, direct other immunocompetent cells to mount a local immune response.

Immunoglobulin (Ig)G specific for bacteria in deep caries.

An increase in the ratio of T helper lymphocytes and B lymphocytes to T

suppressor cells in response to approaching caries.
PERIRADICULAR INFECTIONS:

Odontogenic infections, beyond the tooth socket, are much more common as a
result of endodontic infections than as a result of periodontal disease.

An abscess is a cavity containing pus (purulent exudate) consisting of bacteria,

bacterial by-products, inflammatory cells, numerous lysed cells, and the
contents of those cells.

Cellulitis is a diffuse, erythematous, mucosal, or cutaneous infection that may

rapidly spread into deep facial spaces and become life threatening.
Endodontic abscesses are invariably polymicrobial, and several strains of bacteria are
cultured from each infection.

Fusobacterium nucleatum, Peptostreptococcus anaerobius, and Veillonella parvula, but not

any strains of BPB could produce abscesses

In mixed culture with F. nucleatum, the BPB Prevotella intermedia and Porphyromonas
gingivalis were significantly more abscess forming than F. nucleatum in pure culture.

This supports the concept of synergistic relationships between bacteria in an endodontic

infection.
PERIADICULAR PATHOGENESIS
Enzyme-linked immunosorbent assay (ELISA), radio-immunosorbent tests, and
radial immunodiffusion assays have detected IgG, IgA, IgM, or IgE in fluids of
explant (tissue) cultures of endodontic periapical lesions.

P. intermedia - endodontic-periodontal disease.

symptomatic periapical lesions were shown to contain higher concentrations of

β-glucuronidase and interleukin-1β

Severe involvement had higher IgG.

Those with a sinus tract or swelling contained higher concentrations of IgM.
Periapical lesions associated with untreated teeth have a denser inflammatory
cell infiltrate than periapical lesions associated with treated teeth.

majority of lymphocytes in periapical lesions associated with untreated teeth

are T cells.

Inflammatory lesions associated with endodontically treated teeth had more B

than T cells.

Interleukin-1 and prostaglandins have been especially associated with

periapical bone resorption.

Prostanoids, kinins, and neuropeptides are endogenous mediators responsible

for intermediate-type responses that include vasodilatation, increased vascular
permeability, and leukocyte extravasation.
 CULTURING AND IDENTIFICATION TECHNIQUES
Occasionally, conventional root canal therapy does not eliminate symptoms of
endodontic infection.
If unusual pathogens are present, culturing along with antibiotic-sensitivity
testing may be the only way to determine accurately their presence and to
select the most appropriate antibiotic.
A number of patients today may require more precise bacteriologic monitoring.
These include patients who are:
(1) undergoing immunosuppressive therapy.
(2) at high risk for endocarditis.
(3) have heart valve prostheses and may require a sampling at an early
appointment to determine whether an antimicrobial susceptibility test may be
needed.
 COLLECTION OF A MICROBIAL SAMPLE
First isolate the tooth with a rubber dam.

Disinfect the tooth surface and rubber dam with sodium hypochlorite or other disinfectant.

Sterile burs and instruments must be used to gain access to the root canal system.

If there is drainage from the canal, it may be sampled with a sterile paper point or aspirated
into a syringe with a sterile 18- to 25-gauge needle.

Any aspirated air should be vented from the syringe into a sterile gauze.

The aspirate should either be taken immediately to a microbiology laboratory in the syringe or
injected into pre-reduced transport media.
To sample a dry root canal, a sterile syringe should be used to place some pre-
reduced transport medium into the canal.
A sterile endodontic instrument is then used to scrape the walls of the canal to
suspend microorganisms in the medium.
To prevent contamination by “normal oral flora,” a microbial sample from a soft
tissue swelling should be obtained before making an incision for drainage.
A sterile 16- to 20-gauge needle is then used to aspirate the exudate.
Once the sample is collected, it is important to get it to the microbiology
laboratory as quickly as possible to ensure successful culturing results.
• Final results are usually available in 4 to 7 days.
• Computer-based data systems that identify bacterial isolates often produce
results in less than 24 hours.
 CONCLUSION
The apparent presence of microorganism does not ensure endodontic failure
nor does the apparent absence of microbes guarantee success. Therefore, the
control of microbes and possible substrate must be an objective in every
endodontic case.

It is important that all clinicians understand the close relationship between the
presence of microorganisms and endodontic disease process to develop an
effective rationale for treatment.
 REFERENCES
• Endodontic therapy-weine,6th edition
• Pathways of pulp-cohen,9th edition
• Endodontology-Gunnar Bergenholtz
• Endodontology-seltzer
• Endodontics-Gulabivala
• Apical Periodontitis-Pittford
• Focal infection: a new perspective on an old theory. Brett et al., General dentistry; July-august 2004,357-
361

• Autoaggregation and coaggregation of bacteria associated with acute endodontic infections. Saenguesa k. et
al., J Endo 2006;32:312-318.

• Elimination of candida albicans infection of radicular dentin by intracanal medications. Jose FS. Et al., J Endo
2003;29(8):501-504.

• Microbiota of periapical lesions refractory to endodontic therapy. pia TS. Et al.,J Endo 2002;28(4):304-310