Endodontic Microbiology: Review of Literature

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Review Article
Endodontic Microbiology: Review of Literature

Suhail Latoo1, Ajaz A Shah2, Irshad Ahmad3, Shazia Qadir4, Ravinder Kumar
Bhagat5. Khalid Amin Lone6 E mail: suhaillatoo@yahoo.com

1Department of Oral Pathology and Microbiology,
2,3,4 Department of Oral & Maxillofacial Surgery,
5,6 Department of Conservative dentistry & Endodontics,
Govt. Dental College, Srinagar, Jammu and Kashmir, India

International Journal of Clinical Cases and Investigations 2011. Volume 2
(Issue 6), 24:36, 6th November, 2011

Key Words: endodontic, microbiology, root canal

Abstract
Since 1890, when Miller first observed microorganisms associated with pulp tissue,
microorganisms have been implicated in infections of endodontic origin. Microbes
seeking to establish in the root canal must leave the nutritionally rich and diverse
environment of the oral cavity, breach enamel, invade dentine, overwhelm the
immune response of the pulp and settle in the remaining necrotic tissue within the
root canal. During that time they have to compete in a limited space with other
microbes for the available nutrition. It is no accident that microbes berth in a
particular environment there are ecological advantages for them to establish and
flourish if conditions are favorable. This review will highlight the recent facts and
controversies related to endodontics microbiology.

Introduction:
Microorganisms were observed in samples from teeth by Leeuwenhoek soon after he
invented the microscope in 1684. Since Babylonian times, it was believed that a
‘tooth worm’ lived in the hollow portion of the tooth and caused decay. Leeuwenhoek
challenged the ‘tooth worm’ theory of decay by identifying worm-infested cheese
that he thought may be the source of disease (Cruse & Bellizzi 1980). Leeuwenhoek
also described microorganisms that he scraped from teeth as ‘cavorting beasties’.
However, it took over 200 years before his observation was confirmed and a cause
and effect relationship was suggested by Miller (Henderson & Wilson 1998). Since

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1890, when Miller first observed microorganisms associated with pulp tissue,
microorganisms have been implicated in infections of endodontic origin.

Life is not easy for an endodontic pathogen. Microbes seeking to establish in the root
canal must leave the nutritionally rich and diverse environment of the oral cavity,
breach enamel, invade dentine, overwhelm the immune response of the pulp and
settle in the remaining necrotic tissue within the root canal. During that time they
have to compete in a limited space with other microbes for the available nutrition. It
is no accident that microbes berth in a particular environment there are ecological
advantages for them to establish and flourish if conditions are favorable. Through
genetic exchange and mutation, microbes have developed specialized systems that
facilitate their ability to find, compete and survive in these very specific
environments (Sundqvist & Figdor 2003).

Microbial invasion (routes of micro-organisms ingress):
One of the primary functions of tooth enamel is to exclude these microorganisms
from the underlying dentine–pulp complex. As long as the enamel and cementum
layers are intact, the pulp and root canal are protected from invasion, but loss of
these structures by caries, cracks or trauma opens an avenue for penetration of
bacteria through the dentinal tubules (Sundqvist 1994, Siqueira & Janeiro 2002).

Dental caries is the most common cause of pulp injury. Most authors believe that
acid-producing bacteria invade the dentinal tubules and demineralize the tubule
walls. Thereafter proteolytic bacteria follow, acting on the organic matrix, which is
denuded in the enlarged dentinal tubules. The bacteria in the front of the carious
process are the first to reach the pulp. Gram-positive bacteria predominate among
the advancing bacteria in a carious process. Some investigators have found
exclusively lactobacilli whereas others also report streptococci (S. mitis, S. milleri;
now termed S. oralis and S. anginosus), propionobacteria, Actinomyces, and some
obligatory anaerobic gram-negative and gram positive nonsporulating rods. Most of
the bacteria in the carious process are non-motile. Therefore bacterial penetration in
the dentinal tubules is slow; the acids and other metabolites and toxic products
produced by the bacteria diffuse faster. A reaction of the pulp occurs only a few
hours after experimental application of bacterial products into dentin cavities
(Samaranayake 2002).

In addition to caries, pathways for the entry of microorganisms into the pulp space
include direct pulp exposure (e.g. trauma, and dental procedures), dentinal tubules,
lateral/accessory/furcation canals, and anachoresis (Sundqvist 1994, Siqueira &
Janeiro 2002).

Intraradicular microbiology:
Once the pulp is necrotic and the odontoblastic processes undergo autolysis, patent
dentinal tubules (dead tracts) are traversed by microorganisms and infect the root
canal system. When the pulpal tissue becomes necrotic, it loses its blood supply and
the root canal system becomes a reservoir for microorganisms and their by-products.
Because of the lack of circulation within the necrotic pulp, the root canal system

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becomes a sanctuary for microorganisms from the immune system (Sundqvist
1994).

The root canal flora of teeth with clinically intact crowns, but having necrotic pulps
and diseased periapices, is dominated (> 90%) by obligate anaerobes, usually
belonging to the genera Fusobacterium, Porphyromonas (formerly Bacteroides),
Prevotella (formerly Bacteroides), Eubacterium, and Peptostreptococcus (Sundqvist
et al. 1989). In contrast, the microbial composition even in the apical third of the
root canal of periapically affected teeth with pulp canals exposed to the oral cavity is
not only different from root canal flora of teeth with intact crowns but also less
dominated (< 70%) by strict anaerobes (Baumgartner & Falkler 1991).

All bacteria within the oral cavity share the same opportunities for invading the root
canal space; however only a restricted group of species have been identified in
infected root canals. The reason for the disproportionate ratio between potential and
actual number of species is that the root canal is a unique environment where
biological selection drives the type and course of infection. An anaerobic milieu,
interactions between microbial factors and the availability of nutrition are principal
elements that define the composition of the microbial flora (Sundqvist & Figdor
2003).

Anaerobic milieu and microbial interactions:
In 1894, WD Miller published his findings on the bacteriological investigation of
pulps. He observed many different microorganisms in the infected pulp space and
realized that some were uncultivable when compared with the full range observed by
microscopy, and that the flora was different in the coronal, middle and apical parts of
the canal system. Due to limitations of his sampling and cultivation technique, Miller
was unable to verify this observation and it was not until 1982 that this could be
shown by culturing. Differences in availability of nutrients and oxygen tension in the
apical region compared with the main root canal are important reasons for the
dominance of slow growing, obligately anaerobic bacteria in the apical region
(Sundqvist & Figdor 2003, Sundqvist 1994, Wittgow Jr & Sabiston Jr 1975).

Studies on the dynamics of root canal infections have shown that the relative
proportions of anaerobic microorganisms and bacterial cells increase with time and
that the facultatively anaerobic bacteria are outnumbered when the canals have
been infected for 3 months or more. When a combination of bacterial strains
originally isolated from an infected root canal were inoculated in equal quantities into
further canals in experimental infections, the original proportion of bacterial strains
was reproduced and anaerobic bacteria dominated again. This illustrates that
interactive mechanisms operate amongst these microorganisms, a concept further
supported by the finding that when Prevotella oralis (formerly Bacteroides oralis) was
inoculated on its own it was unable to survive, whereas when inoculated with other
bacteria it survived and dominated the established flora. These experiments have
shown that the endodontic milieu is a selective habitat that supports the

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development of specific proportions of the anaerobic microflora(Sundqvist & Figdor
2003, Sundqvist 1994). Oxygen and oxygen products play an important role as
ecological determinants in the development of specific proportions of the root canal
microflora. The consumption of oxygen and production of carbon dioxide and
hydrogen along with the development of a low reduction–oxidation potential by the
early colonizers favor the growth of anaerobic bacteria (Loesche et al.1983).

Nutrition as an ecological driver:
The type and availability of nutrients is important in establishing microbial growth.
Nutrients may be derived from the oral cavity, degenerating connective tissue (Nair
2004), dentinal tubule contents, or a serum-like fluid from periapical tissue (Rocas et
al. 2004). These factors in the root canal environment permit the growth of
anaerobic bacteria capable of fermenting amino acids and peptides, whereas bacteria
that primarily obtain energy by fermenting carbohydrates may be restricted by lack
of available nutrients. This is the likely reason why the flora is dominated by
facultatively anaerobic bacteria, such as streptococci, in the coronal section of root
canals exposed to the oral cavity, and anaerobic bacteria dominate in the apical
section. The succession of strict over facultative anaerobes with time is most likely
due to changes in available nutrition, as well as a decrease in oxygen availability.
Facultatively anaerobic bacteria grow well in anaerobiosis; however, their prime
energy source is carbohydrates. A decrease in availability of carbohydrates in the
root canal occurs when there is no direct communication with the oral cavity, which
severely limits growth opportunities for facultative anaerobes (Sundqvist 1994).

The experiments of ter Steeg and van der Hoeven offer important clues about the
likely dynamics of the root canal flora. Using serum as a substrate, they studied the
succession of subgingival plaque organisms during enrichment growth. Three phases
can be distinguished during growth(Sundqvist & Figdor 2003, Sundqvist 1994):
1. Initially, rapidly growing saccharolytic bacteria consume the low levels of
carbohydrates in serum, leading to lactic and formic acid production.
2. In a second phase, proteins are hydrolyzed, some amino acid fermentation
takes place, and there is digestion of remaining carbohydrates. Carbohydrates
are split off from the serum glycoproteins. Growth during this phase are
dominated by Prevotella intermedia, Veillonella parvula, Fusobacterium
nucleatum and Eubacterium species.
3. In a final phase, there is progressive protein degradation. The predominant
species during this phase are Peptostreptococcus micros, F. nucleatum, and
eubacteria. The dominance of P. micros in cultures originating from
subgingival microbiota, when grown in serum, has also been shown in
another study. The ecological niche of P. micros may be related to its wide
range of peptidase activities, making amino acids and peptides available from
serum glycoproteins. These amino acids can be used by P. micros, but also by
other bacteria that have little or no proteolytic activity in serum.

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The
Porphyromonas gingivalis and Porphyromonas endodontalis, are proficient in
degrading serum proteins and make peptides and amino acids available for
fermentation. The degradation of native proteins by Prevotella and Porphyromonas
species enables the growth of bacteria that depend on the availability of peptides,
such as eubacteria, fusobacteria and peptostreptococci, which produce peptidases
but cannot hydrolyze intact proteins. This is also of importance for the capacity of
root canal bacteria to induce periapical abscesses(Sundqvist & Figdor 2003, Jansen
et al. 1996). Combinations of P. micros with P. intermedia or P. endodontalis have
been implicated in the induction of periapical abscesses. Abscesses harboring a
microflora that rapidly degrade serum proteins have been shown to be nearly three
times larger than abscesses with a microflora that lack the capacity for breakdown of
serum proteins (Siqueira et al. 2001).

black-pigmented anaerobic rods, Prevotella intermedia/nigrescens,
Growth of mixed bacterial populations may depend on a food chain in which the
metabolism of one species supplies essential nutrients for the growth of other
members of the population. Black pigmented anaerobic rods (Prevotella and
Porphyromonas species) are examples of bacteria that have very specific nutritional
requirements. They are dependent on vitamin K and hemin for growth(Jansen & van
der Hoeven 1997). Vitamin K can be produced by other bacteria. Hemin becomes
available when hemoglobin is broken down, but some bacteria may also produce
hemin. Another example is Campylobacter rectus which can stimulate the growth of
Porphyromonas species by producing a growth factor related to hemin. C. rectus
itself derives a source of energy from the co-inhabiting microbial species. It is strictly
dependent on a respiratory mechanism in which only formate and hydrogen can
serve as electron donors and fumarate, nitrate, or oxygen as electron acceptors. This
makes this organism dependent on bacteria producing formate or hydrogen. A wide
range of nutritional interactions is recognized among oral bacteria and these may
also influence the associations between bacteria in the root canal (Sundqvist &
Figdor 2003).

Because the nutritional supply governs the dynamics of the microbial flora, it means
that the bacteria present in the root canal will depend on the stage of the infection.
Initially, there may be no clear associations between bacteria, but strong positive
associations develop among a restricted group of the oral flora due to the type of
nutrients in the environment (Lana et al. 2001). Thus, F. nucleatum is associated
with P. micros, P. endodontalis and C. rectus. Strong positive associations exist
between P. intermedia and P. micros and P. anaerobius. There is also a positive
association between P. intermedia, and P. micros, P. anaerobius and the eubacteria.
In general, species of Eubacteria, Prevotella and Peptostreptococcus are positively
associated with one another in endodontic samples. Properties that these bacteria
have in common are that they ferment peptides and amino acids and are anaerobic,
which indicates that the main source of nutrition in root canals is tissue remnants
and a serum-like substrate (Sundqvist 1994).

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Extraradicular microbiology:
The development of periradicular lesions creates a barrier with in the body to prevent
further spread of microorganisms. Bone tissue is resorbed and substituted by a
granulation tissue containing defense elements, such as cells (phagocytes) and
molecules (antibodies and complement molecules). A dense wall composed of
polymorphonuclear leucocytes, or less frequently an epithelial plug, is usually
present at the apical foramen, blocking the egress of microorganisms into the
periradicular tissues. Very few endodontopathogens can advance through such
barriers. However, microbial products can diffuse through these defence barriers and
are able to induce or perpetuate periradicular pathosis (Vigil et al.1997).

Ever since Miller demonstrated the presence of bacteria in diseased dental pulp,
microorganisms have been suspected to play a causative role in periapical
periodontis. As a result, voluminous literature exists to collaborate or disprove the
presence of bacteria in apical periodontic lesion(Vigil et al.1997).

Over years there has been conflicting reports regarding the presence and role of
microorganisms in periapical lesions. Many histobacterialogic studies have been
conducted on periapical lesions removed in toto, with complete connective tissue
encapsulation and firm attachment. Stewart in 1947 and later supported by Hedman
in 1951 proposed that bacteria were present in periapical lesions (Stewart 1947).
Hedman’s study which was accomplished prior to present techniques for culturing
anaerobic bacteria, bacteria were reported in 68% of 82 periapical lesions(Hedman
1951). Winkler et al using a modified gram stain for periapical tissue, were able to
demonstrate the presence of bacteria in 87% or 13 out of 15 cases examined under
a light microscope (Winkler et al. 1972).

Many investigators have challenged the concept of bacteria in periapical region. In a
good number of studies, it was not possible to demonstrate the presence of bacteria
in periapical lesions. Based on classic histology (Harndt 1926), many investigators
therefore believe that ‘solid granuloma’ may not harbor infectious agents within the
inflamed periapical tissue, but that micro-organisms are consistently present in the
periapical tissue of cases with clinical signs of exacerbation, abscesses, and draining
sinuses (Nair 2004). Quoting Kronfeld, Grossman said that “a tooth with a
granuloma may have infected root canal, but a sterile periapical tissue in gram
stained sections through infected pulpless teeth in situ that were examined; bacteria
in abundance were always found within the root canal but granulation tissue and
cysts attached to the apices of teeth were often free from microorganisms” and that
“granuloma is not an area in which bacteria live, but in which they are destroyed”
(Kronfeld 1974). Shindell reported 60 out of 63 specimens were negative for
periapical region of endodontically
histopathological and histobacterialogic study of 35 periapical endodontic surgical
specimens, it was observed that although bacteria were identified in five specimens;
in only one case were the bacteria located in the disintegrating tissue of root canal
and periapical tissue (Laaangeland et al. 1977). Many recent investigations using
electron microscopy supports granulomas being bacteria-free (Oguntebi et al.
1982).

treated tooth(Shindell 1961). In a

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One of the challenging aspects for determining bacteriological status in periapical
tissue is microbial contamination of periapical samples. Microbial contamination of
periapical samples is sometimes viewed as happening from the oral cavity and other
extraneous sources. Many researchers believe that even if such ‘extraneous
contaminations’ are avoided, contamination of periapical tissue samples with
microbes from the infected root canal remains a problem. This is because micro-
organisms generally live at the apical foramen of teeth affected in both primary and
post-treatment apical periodontitis. Here, microbes can be easily dislodged during
surgery and the sampling procedures. Tissue samples contaminated with
intraradicular microbes may be therefore reported as positive for the presence of an
extraradicular infection (Nair 2004).

In general, there is a consensus of opinion among most of the researchers that
chronic periapical lesion may harbor microorganisms if it is infected or it is an
abscessed lesion (Cohen & Burns 2002). The periapical abscess is dominated (>
90%) by obligate anaerobes, usually belonging to the genera Bacteroides group,
Peptostreptococcus spp., Peptococcus spp., and Fusobacterium spp (Goumas et al.
1997; Lewis et al. 1988; Siqueira Jr. et al. 2001). Lewis et al in their study on
dentoalveolar abscess reported that anaerobic gram-negative bacilli are major
pathogens in acute dentoalveolar abscesses (Lewis et al.1988). Among these
organisms there is high prevalence of P. endodontalis and P. Gingivalis(Siqueira Jr.
et al. 2001). Studies have shown that suggests that P. endodontalis and P. gingivalis
play an important role in the pathogenesis of periradicular diseases (Dymock et al.
1996).

Resurgence of concept of extraradicular microbiology:
In recent years there is resurgence of the idea of extraradicular microbes in apical
periodontis lesions with implied, controversial suggestion that extraradicular infection
is the cause of many failed endodontic treatment (Vigil et al. 1997). Tronstad et al
investigated the presence of periapical microbial flora of eight cases which had not
healed with non surgical endodontic treatment. In all eight cases bacterial growth
was evident. Three samples from each case were cultured and Tronstad et al. stated
that their study clearly showed that anaerobic bacteria are able to survive in
periapical tissue (Tronstrad et al. 1987). Haapasalo et al treated a case in which non
surgical endodontics, calcium hydroxide, systemic erythromycin, and finally, a
regimen of systemic metronidazole failed to resolve the draining fistula associated
with a maxillary lateral incisor.Following periapical surgery, the lesion resolved the
lesion. A mixed infection of anaerobic and facultative anaerobic microorganism was
cultured from the root canal and the periapical lesion (Haapasalo et al. 1987).
Presence of several species of bacteria have been reported at extraradicular sites of
lesions described as “symptomatic periapical inflammatory lesion…..refractory to
endodontic treatment,” with the declaration that “….these findings clearly end the
erra of sterile periapical granuloma”( Cohen & Burns 2002). Iwu et al studied 16
periapical granuloma that were collected “during normal periapical curettage,
apecectomy, or retrograde filling”. It was seen that most of the organism cultured
were Veillonella species, Streptococcus milleri, Streptococcus sanguis, Actinomycetes

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naeslundii, Propionibacterium acnes and Bacteroides species (Iwu & Wallace 1990).
In majority of the recent studies on bacteriological and histological evaluation of
periapical lesions, commonly isolated organisms were Staphylococcus epidermidis,
Fusobacterium species, Propionibacterium acnes, Peptostreptococcus micococcus and
Bacteriodes gracilis. Among these organisms, gram-negative bacteria like
Propionibacterium acnes and Bacteriodes gracilis were the most commonly isolated
ones (Wayman et al. 1992; Kiryu et al. 1994; Abou-Rass & Bogen 1998).

Black-pigmented bacteria in periapical lesions:
Black pigmented anaerobic gram negative bacteria are part of normal microbiota at
various sites of human body and are often isolated from mixed infection sites. They
belong to the family Bacteroides and are included in the genera Prevotella and
Porphyromonas (Kiryu et al.1994; Trowbridge & Stevens 1992).

In majority of studies on bacteriological studies on periapical lesions, it has been
observed that Porphyromonas and Prevotella had a higher prevalence in periradicular
lesions associated with pain, purulent abscess and teeth refractory to conventional
root canal treatment (Kiryu et al. 1994; Trowbridge & Stevens 1992; Bogen & Slots
1999; Yamasaki et al. 1998). Moreover, some black pigmented anaerobic rods are
major pathogen in destructive periodontal diseases. It has been therefore proposed
that these organisms may play an important role in the pathogenesis of periradicular
diseases(Bogen & Slots 1999).

Enterococcus Faecalis in periapical lesions:
Enterococcus Faecalis is a normal human commensals adapted to the nutrient-
enriched, oxygen-depleted, ecologically complex environments of the oral cavity,
gastrointestinal tract, and vaginal vault. It is a nonspore-forming, fermentative,
facultatively anaerobic, Gram-positive coccus.

E. faecalis is significantly more associated with asymptomatic cases of primary
endodontic infections than with symptomatic ones. Furthermore, E. faecalis was
much more likely to be found in cases of failed endodontic therapy than in primary
infections (Pinheiro et al. 2003; Rocas et al. 2004).

Periapical actinomycosis:
Actinomycosis is a chronic, granulomatous, infectious disease in man and animals
caused by the genera Actinomyces and Propionibacterium (that normally colonize the
mouth, colon, and vagina). They are non-acid, fast, non-motile, Gram-positive
organisms revealing characteristic branching filaments that end in clubs or hyphae.
The intertwining filamentous colonies are often called "sulphur granules" because of
their appearance as yellow specks in exudate. Four clinical forms of actinomycosis
account for most of these human infections: the cervicofacial, thoracic,
abdominopelvic, and cerebral forms. Cervicofacial actinomycosis presents as a
chronic, slowly evolving induration in the mandibular preauricular region, often
accompanied by fistular tracts to the skin that discharge typical sulfur granules.

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Invasion of the microorganism is usually a result of the disruption of the mucosal
barrier after trauma or dental manipulation (Nair 2004).

The endodontic infections of actinomyces are a sequel to caries and are caused by
Actinomyces israelii and Propionibacterium propionicum, commensals of the oral
cavity. Because of the ability of the actinomycotic organisms to establish
extraradicularly, they can perpetuate the inflammation at the periapex, even after
orthograde root canal treatment. Therefore, periapical actinomycosis is important in
endodontics. A. israelii and P. proprionicum are consistently isolated and
characterized from the periapical tissue of teeth which did not respond to proper
conventional endodontic treatment(Hirshberg et al.2003). A strain of A. israelii,
isolated from a case of failed endodontic treatment and grown in pure culture, was
inoculated into subcutaneously implanted tissue chambers in experimental animals.
Typical actinomycotic colonies were formed within the experimental host tissue. This
would implicate A. israelii as a potential etiological factor of failed endodontic
treatment. The properties that enable these bacteria to establish in the periapical
tissues are not fully understood, but appear to involve their ability to build cohesive
colonies that enable them to escape the host defense system(Nair 2004). Periapical
actinomycosis is thought to be rare. Nevertheless, it is assumed to be more frequent
than is commonly believed. There are only limited data on the frequency of periapical
actinomycosis among periapical lesions or on the correlation between periapical and
cervicofacial actinomycosis (Hirshberg et al.2003).

Viruses in periapical lesions:
Most recently, a series of publications appeared in various journals from one
research group that reported the presence of certain viruses in inflamed periapical
tissues and suggested an ‘etiopathogenic relationship’ to apical periodontitis. The
reported viruses are present in almost all humans in latent form from previous
primary infections(Nair 2004).

One of these studies has investigated the occurrence of herpes viruses in periapical
granulomas. cDNA identification of genes transcribed late during the infectious cycle
of herpes viruses was used to indicate an active herpes virus infection (Sabeti et al.
2003). It was proposed that herpes viruses may cause periapical pathosis as a direct
result of virus infection and replication or as a result of virally induced damage to the
host defense(Slots et al. 2003).

A strong association of human cytomegalovirus and Epstein- Barr virus with the
acute exacerbation of periapical lesions has been reported(Sabeti, Simon & Slots
2003). Periapical lesions harboring dual cytomegalovirus–Epstein-Barr virus infection
tended to exhibit elevated occurrence of anaerobic bacteria, be symptomatic, and
show large size radiographic bone destruction. Cytomegalovirus and Epstein-Barr
virus, in cooperation with specific bacterial species, have also been associated with
various types of advanced marginal periodontitis and several non oral infectious
diseases(Sabeti et al 2003; Sabeti, Simon & Slots 2003). (Figure 1)

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Fungi in periapical lesion:
Fungi have also isolated in periapical lesions but in far rear instances. Among fungi,
Candida is most frequently found, although other fungi have been cultivated. It has
been detected in therapy resistant periapical lesions(Takahashi 1998).

Conclusion:
Microbes seeking to establish in the root canal must leave the nutritionally rich and
diverse environment of the oral cavity, breach enamel, invade dentine, overwhelm
the immune response of the pulp and settle in the remaining necrotic tissue within
the root canal. During that time they have to compete in a limited space with other
microbes for the available nutrition.



References:
1. Abou-Rass M, Bogen G(1998) Microorganisms in closed periapical lesions.
International Endodontic Journal, 1 :39-47
2. Baumgartner JC, Falkler WA(1991) Bacteria in the apical 5 mm of infected
root canals. Journal of Endodontics, 17:380–383.
3. Bogen G, Slots J(1999) Black-pigmented anaerobic rods in closed periapical
lesions. International Endodontic Journal, 3:204-10.
4. Cohen S, Burns RC (2002) Pathways of the pulp, ed 8. Harcourt Asia Co.,
India,: 457-500.
5. Cruse WP, Bellizzi R (1980) A historic review of endodontics, 1689–1963,
part 1. Jornal of Endodontics, 6: 495–499.

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6. Dymock, D; Weightman, A J; Scully, C; Wade, W G (1996) Molecular
analysis of microflora associated with dentoalveolar abscesses. Journal of
Clinical Microbiology, 34 : 537-542
7. Goumas PD, Naxakis SS, Papavasiliou DA, Moschovakis ED, Tsintsos SJ,
Skoutelis A (1997) Periapical abscesses: causal bacteria and antibiotic
sensitivity. : Journal of Chemotherapy, 6:415-9.
8. G. Sundqvist & David Figdor (2003) Life as an endodontic pathogen,
Ecological differences between the untreated and root-filled root canals.
Endodontic Topics, 6: 3–28.
9. Haapasalo M, Ranta K, Ranta H (1987) Mixed anaerobic periapical infection
with sinus tract. Endodontics & dental traumatology, 3; 83-5.
10. HedmanWJ (1951) An investigation into residual periapical infection after
pulp therapy. Oral Surgery Oral Medicine Oral Pathology, 4 : 1173-9.
11. Henderson B, Wilson M (1998) Commensal communism and the oral cavity.
Journal of Dental Research, 77 : 1674–1683.
12. Hirshberg A, Tsesis I, Metzger Z, Kaplan I, Aviv T. Israel (2003) Periapical
actinomycosis: A clinicopathologic study. Oral Surgery Oral Medicine Oral
Pathology Oral Radiology Endodontics, 95:614-20.
13. Iwu C, Wallace T (1990) The microbiology of periapical granuloma. Oral
Surgery Oral Medicine Oral Pathology, 69: 502-5.
14. Jansen H-J, van der Hoeven JS (1997) Protein degradation by Prevotella
intermedia and Actinomyces meyeri supports the growth of non-protein-
cleaving oral bacteria in serum. Journal of Clinical Periodontology, 24: 346–
353.
15. Jansen H-J, van der Hoeven JS, Walji S, Goertz JH, Bakkeren JA (1996) The
importance of immunoglobulin breakdown supporting the growth of bacteria
in oral abscesses. Journal of Clinical Periodontology, 23: 717–723.
16. Kiryu T, Hoshino E, Iwaku M (1994) Bacteria invading periapical cementum.
Bacteria invading periapical cementum. Journal of Endodontics,4:169-72.
17. Kronfeld, R (1974) Histopathology of teeth and their surrounding structures,
ed 2. Philadelphia, Lee & Febiger : p 86.
18. Laaangeland K, Block RM, Grossman LI (1977) A histopathological and
histobacterialogic study of 35 periapical endodontic surgical specimens.
Journal of Endodontics, 3: 123-124
19. Lana MA, Ribeiro-Sobrinho AP, Stehling R, Garcia GD et. al (2001)
Microorganisms isolated from root canals presenting necrotic pulp and their
drug susceptibility in vitro. Oral Microbiology & Immunology, 16: 100–105.
20. Lewis MA, MacFarlane TW, McGowan DA, MacDonald DG (1988) Assessment
of the pathogenicity of bacterial species isolated from acute dentoalveolar
abscesses. Journal of Medical Microbiology, 2:109-16.
21. Loesche WJ, Gusberti F, Mettraux G, Higgins T, Syed S (1983) Relationship
between oxygen tension and subgingival bacterial flora in untreated human
periodontal pockets. Infection & Immunity, 42: 659–667.
22. Nair PNR(2004) Pathogenesis of Apical Periodontitis and the Causes of
Endodontic Failures. Critical Reviews in Oral Biology & Medicine, 6:348-381
23. Oguntebi B, Slee AM, Tanzer TJM, Langeland K (1982) Predominant
Microflora Associated with Human Dental Periapical Abscesses. Journal of
Clinical Microbiology,15: 964-966.

Page 12

35

24. Pinheiro ET, Gomes BP, Ferraz CC, Sousa EL, Teixeira FB, Souza-Filho FJ
(2003) Microorganisms from canals of root-filled teeth with periapical
lesions. International Endodontic Journal, 1:1-11.
25. Rocas IN, Siqueira JF Jr, Santos KR (2004) Association of Enterococcus
faecalis with different forms of periradicular diseases. Journal of
Endodontics,5:315-20.
26. Sabeti M, Simon JH, Slots J (2003) Cytomegalovirus and Epstein-Barr virus
are associated with symptomatic periapical pathosis. Oral Microbiology &
Immunology,18:327-8.
27. Sabeti M, Valles Y, Nowzari H, Simon JH, Kermani-Arab V, Slots J (2003)
Cytomegalovirus and Epstein-Barr virus DNA transcription in endodontic
symptomatic lesions. Oral Microbiology Immunology,18:104-8.
28. Samaranayake LP (2002) Essentials of microbiology for dentistry, ed 2,
Churchill Livingstone, Philadelphia, 205 - 252
29. Shindell WJ (1961) A study of some periapical radiolucencies and their
significance. Oral Surgery Oral Medicine Oral Pathology, 14 : 1057-65
30. Siqueira JF, Janeiro R (2002) Endodontic infections: Concepts, paradigms,
and perspectives. Oral Surgery Oral Medicine Oral Pathology Oral Radiology
Endodontics, 94:281-93.
31. Siqueira JF Jr, Rocas IN, Oliveira JC, Santos KR (2001) Detection of putative
oral pathogens in acute periradicular abscesses by 16S rDNA-directed
polymerase chain reaction. Journal of Endodontics, 3:164-7.
32. Siqueira JF Jr, Rocas IN, Souto R, Uzeda M, Colombo AP (2001)
Microbiological evaluation of acute periradicular abscesses by DNA-DNA
hybridization. Oral Surgery Oral Medicine Oral Pathology Oral Radiology
Endodontics, 4:451-7.
33. Slots J, Sabeti M, Simon JH (2003) Herpesviruses in periapical pathosis: An
etiopathogenic relationship? Oral Surgery Oral Medicine Oral Pathology Oral
Radiology Endododontics, 96:327-31.
34. Stewart, G.G (1947) A study of bacteria found in root canal of anterior teeth
and probable mode of ingress. Journal of Endodontics, 2:8.
35. Sundqvist G, Johansson E, Sjögren U(1989) Prevalence of black pigmented
Bacteroides species in root canal infections. Journal of Endodontics,15:13–
19.
36. Sundqvist G (1994) Taxonomy, ecology, and pathogenicity of the root canal
flora. Oral Surgery Oral Medicine Oral Pathology, 78: 522–530.
37. Takahashi K (1998) Microbiological,
immunological and molecular biological aspects of periradicular disease.
International Endodontic Journal, 5:311-25.
38. Tronstrad L, Barnett F, Risco K, Slotz J (1987) Extraradicular endodontic
infections. Endodontics & Dental Traumatology, 3 ; 83-5.
39. Trowbridge HO, Stevens BH (1992) Microbiologic and pathologic aspects of
pulpal and periapical disease. Current Opinion in Dentistry,2: 85-92.
40. Vigil GV, Wayman BE, Dazey SE, Fowler CB, Bradley DV Jr (1997)
Identification and antibiotic sensitivity of bacteria isolated from periapical
lesions. Journal of Endodontics, 2:110-4.
pathological, inflammatory,

Page 13

36

41. Wayman BE, Murata S, Almeida RJ,Fowler CB (1992) A Bacteriological and
Histological Evaluation of 58 Periapical Lesions. Journal of Endodontics, 4:
152-5.
42. Winkler TF, Mitchell DF, Healey HJ(1972) A bacterial study of human
periapical pathosis employing a modified Gram tissue stain. Oral Surgery
Oral Medicine Oral Pathology, 1:109-16.
43. Wittgow WC Jr, Sabiston CB Jr (1975) Microorganisms from pulpal
chambers of intact teeth with necrotic pulps. Journal of Endodontics, 1:
168–171
44. Yamasaki M, Nakata K, Imaizumi I, Iwama A, Nakane A, Nakamura H
(1998) Black-pigmented anaerobic rods appear to be infrequent inhabitants
of the closed periapical lesion. Journal of Endodontics, 8:534-9.