Vol. 7(18), pp.

1819-1826, 30 April, 2013

DOI: 10.5897/AJMR12.1731
ISSN 1996-0808 2013 Academic Journals
http://www.academicjournals.org/AJMR

African Journal of Microbiology Research

Full Length Research Paper

Microorganisms involved in endodontic infection of

permanent teeth: A systematic review
Viviane Andrade Cancio de Paula1*, Raquel dos Santos Pinheiro2, Rafael de Lima Pedro1,
Ktia Regina Netto dos Santos1, Laura Guimares Salignac Primo1 and Luciane Coplle Maia1
1

Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro,
Rio de Janeiro RJ - Brazil.
2
Department of Medical Microbiology, Institute of Microbiology, Federal University of Rio de Janeiro,
Rio de Janeiro RJ -Brazil.
Accepted 29 March, 2013

This study investigated the microbial species present in necrotic pulps of permanent teeth needing
endodontic treatment. The search for articles was conducted on the Health Science Database and
considered all articles published until October 2011, with selected human clinical studies examined,
through molecular biology the microorganisms that are present in root canals of permanent teeth
requiring root canal treatment with necrosis pulps (exposition). The selected articles were categorized
according to the methodological quality and evidence in levels A (high), B (moderate), and C (low). The
search strategy was based on PubMed, Bireme, Cochrane and OVID databases. The data extracted from
the studies were also tabulated. Eighty-four titles and abstracts were assessed and eight articles met
the inclusion criteria, showing that there is a high diversity of microorganisms involved in necrotic
permanent teeth. The microorganisms found in these articles were: Porphyromonas gingivalis (27.8%),
Porphyromonas endodontalis (42.6%), Prevotella intermedia (5.6%), Prevotella nigrescens (7.4%),
Bacteroides forsythus (21%) and Enterococcus faecalis (>50%). This systematic review found high
evidence of the polymicrobial nature of primary endodontic infections in necrotic permanent teeth, and
also showed that there is a dominance of anaerobic bacteria.
Key words: Microorganisms, endodontic infection, root canal.

INTRODUCTION
The oral cavity is potentially susceptible to a variety of
infections due to the presence of numerous proliferative
microorganisms that cause fungal, viral and bacterial
infections (Ruvire et al., 2007; Sakamoto et al., 2007).
Dental caries can be characterized as a multifactorial
infection directly related to three main factors: Microflora,
substrate and host. Some carious processes may
develop to the point where dental pulp is affected

(Barcelos et al., 2001; Siqueira, 2003). Microbial

involvement has been indicated as the main factor
associated with the etiology of endodontic diseases
(Siqueira et al., 2002, 2005).
Culture procedures have traditionally been used as the
reference in the assessment of the microbiota associated
with various infectious diseases, including infections of
endodontic origin (Siqueira, 2003). Isolation and

*Corresponding author. E-mail: vicancio@ig.com.br. Tel/ Fax: 55-21-27097777.

1820

Afr. J. Microbiol. Res.

identification of infectious agents by phenotypic traits

(culture) has some serious pitfalls, most notably the
difficulty in simulating the environmental conditions
required for most microorganisms to grow. Therefore,
non-viable or uncultivable bacteria cannot be isolated
using culture methods (Siqueira, 2003; Siqueira et al.,
2004; Ruvire et al., 2007).
Microorganism detection methods that identify
microbial DNA rather than the microorganisms
themselves have been introduced in both research and
clinical laboratories and are revolutionizing our
knowledge of infectious diseases and allowing effective
and rapid diagnosis of many diseases (Siqueira, 2003).
Molecular approaches (PCR, Checkerboard, DGGE)
have revealed some uncultivable microorganisms
involved in endodontic infections, such as Micromonas
micros, Fusobacterium nucleatum ss, Tannerella
forsythia, Treponema denticola, Veillonella parvula,
Eubacterium nodatum, Porphyromonas gingivalis,
Actinomyces odontolyticus, Streptococcus constellatus,
Synergistes phylotypes and Enterococcus faecalis
(Siqueira et al., 1998, 2002, 2004, 2005, 2007, 2008;
Munson et al., 2002; Siqueira and Ras, 2004a,b, 2005,
2007; Vianna et al., 2005; Seol et al., 2006; Sakamoto et
al., 2008).
Knowledge of molecular biology and endodontic
infections has increased significantly over recent years,
but several questions have yet to be elucidated. For
example, in primary infections, not all microorganisms
involved, neither the most virulent species have been
identified yet (Siqueira, 2003). Despite there are many
papers about endodontic microbiology, a systematic
review could be more elucidative and this study propose
add news aspects in the literature. This paper intends to
find evidence of different types of microbial species
present in necrotic pulps in permanent teeth needing
endodontic treatment.
MATERIALS AND METHODS
A specific protocol and research questions were developed for this
study whose research included observational studies that
approached the prevalence of microorganisms in root canal. The
search for potentially relevant studies was carried out through to
October 2011, in the databases PUBMED (1966-2011), BIREME
(1967-2011), OVID ALL EMB Reviews (1950-2011), COCHRANE
using the MeSH terms:
molecular techniques
AND
microorganisms,
molecular
techniques
AND
oral
microorganisms, anaerobic bacteria AND endodontic,
molecular techniques AND anaerobic bacteria and endodontic
AND root canal. Moreover, the reference lists of selected articles
and literature review articles were also manually searched.
This review selected human (participants) clinical studies that
examine, through molecular biology (PCR) the microorganisms
(outcomes) that are present in root canals of permanent teeth
requiring root canal treatment with necrosis pulps (exposition).
Additional articles of potential relevance were identified by manual
searches. Observational controlled study designs composed of
microorganism matched for root canal were included without
language restrictions. The exclusion criteria were human clinical

studies in which patients had used antibiotics 6 months prior to

material collection, studies in patients with any systemic or
periodontal disease, studies with permanent teeth that have.
undergone re-treatment or in teeth with a periodontal pocket > 3
mm, and studies with radiographic periapical bone loss
Textbooks, book chapters dissertations, case reports, case
series, review articles, and abstracts were excluded. Two
examiners (V.A.C.P. and R.S.P.) independently screened each
paper by examining the title, abstract, and keywords. If examiners
had diverging opinions, the papers were reexamined until a
consensus was reached. If relevant data were missing, the authors
of the papers in question were contacted for additional information.

Quality assessment and risk of bias

We performed a quality assessment of the remaining studies to
control for influence bias, to gain insight into potential comparisons,
and to guide interpretation of findings (Meerpohl et al., 2009). The
quality of the studies was evaluated using a checklist presented in
Table 1. The selected articles were assessed in accordance with
some modifications in the criteria of high quality observational
studies proposed by STROBE (Meerpohl et al., 2009). Researchers
classified the studies into three categories with scores A to C
according to predetermined criteria for method and performance.
For each item, there were 2 options of answer (Yes, No), only one
option could be mark for each question. None answer were unclear,
when this happened the author was contacted.
The articles were categorized as category A (9-10 points): high
methodological quality, when the study included least 9 yes of the
assessed criteria; Category B (6-8 points): moderate
methodological quality, when the study included 8 yes of the
assessed criteria; Category C (0-5 points): low methodological
quality, when the study included 5 yes or less assessed criteria.
Studies classified as B or C was excluded from this research. The
prevalence of microorganisms was recorded.
All data were analyzed by using SPSS (SPSS Inc., Chicago, IL,
USA) 16.0 software program for windows. Chi-square test was used
to compare the data. Significance was established at 5% (p < 0.05).

RESULTS
The electronic search results are presented in Figure 1.
Eighty-four titles and abstracts were assessed and eight
articles met the inclusion criteria and their full-text was
retrieved. All selected studies were cross-sectional
(Siqueira et al., 2001; Siqueira and Ras, 2004, 2006,
2007; Ras and Siqueira, 2005; Tomazinho and AvilaCampos, 2007). Checkerboard test was used only in two
articles (Siqueira et al., 2002, 2008). The main
microorganisms found in included studies were: P.
endodontalis, P. gingivalis, P. intermedia and P.
nigrescens in all selected articles for quality
assessement. P. endodontalis was the most prevalent
microorganism, with a strong evidence. The difference
between the techniques used to identify microorganisms
were not statistic significant (p>0.005). The percentages
of the microorganisms most frequently found in the
papers are shown in Figure 2. Selected articles were
categorized according to the quality of evidence, two
articles were considered B and the others C and the data
extracted from the studies were tabulated (Table 2).

de Paula et al.

1821

Table 1. Criteria for quality assessment of the studies included.

Item
1
2
3
4
5
6
7
8
9
10

Quality assessment
Representative sample size
Samples collected using strict asepsis methods
Same operator for the samples collected
Description of data collection
Cross-sectional studyif applicable, describe analytical methods taking
sampling strategy into account
Clearly define all outcomes, exposures, predictors, potential confounders,
and effect modifiers. Give diagnostic criteria, if applicable
Potential sources of bias addressed
Description of all statistical methods, including those used to control
confounding factors
Discuss limitations of the study
Cautious overall interpretation of results considering objectives, limitations,
multiplicity of analyses, results from similar studies, and other relevant
evidence

Yes
1
1
1
1
1

No
0
0
0
0
0

1
1

0
0

1
1

0
0

Positive response (Yes) was equal 1 point and negative response (NO) equal 0 point.

ELETRONIC SEARCH

Initial search (3956 studies)

2456 Pubmed, 1112 Bireme, 369 OVID, 19 Cochrane

Exclusion of 3912 studies

repeated on databases

84 Title and abstract evaluation

63 Studies excluded on basis

of title and abstract

21 studies retrieved for full text evaluation

13 studies were excluded:

9 studies included retreatment
and 4 studies did not have
sample larger than 50

08 studies considered to systematic review

Figure 1. Flow diagram of literature search.

DISCUSSION

P. endodontalis
P. gingivalis
P. intermedia
P. nigrescens

Figure 2. Microorganisms most frequently found in the

articles.

Knowledge of endodontic infections has increased

significantly over decades, but several questions still
await elucidation. This article has shown that the
microflora associated with endodontic infections is
diverse. Various microorganisms are associated with
primary endodontic infection (Siqueira and Ras, 2009,
2009). In this paper only studies where patients had a
primary infection were analyzed. Black-pigmented
anaerobic microorganisms were the most found in the
papers (Siqueira et al., 2000, 2001, 2002, 2005, 2007,
2008; Munson et al., 2002; Ras and Siqueira, 2006;
Seol et al., 2006; Sassone et al., 2008; Saito et al., 2009;
Siqueira and Ras, 2009a, b, c). The presence of each

1822

Afr. J. Microbiol. Res.

Table 2. Characteristics of studies included.

Author

Siqueira
et al.

Siqueira
et al.

Siqueira
and
Ras

Ras
and
Siqueira

Year

2001

2002

2004

2005

Category
(Points)

A(9)

A(9)

A(9)

A(9)

Country

Methods
Type of
study

Brazil

Crosssectional

Brazil

Brazil

Brazil

Crosssectional

Crosssectional

Crosssectional

species in the included articles is shown in Table

2. None of the articles elucidated the relationships
in primary endodontic disease (Siqueira et al.,
2002; Sakamoto et al., 2008). P. endodontalis, P.

Participants
Age
Sample
(Years)
54

50

50

50

Results
Disinfection

Sample collected by

Cryotubes

Molecular
methods

Microorganisms

PCR

P. endodontalis (42.6%)
P. gingivalis (27.8%)
P. intermedia (5.6%)
P. nigrescens (7.4%)

18-60 yrs

3% hydrogen
peroxide,
2.5% sodium
hypochlorite

AdultsX

3% hydrogen
peroxide,
2.5% sodium
hypochlorite
solution, 5%
sodium
thiosulfate

# 15 K-type file

1 ml of TSB

PCR and
checkerboard

A. actinomycetemcomitans
(0%)
P. gingivalis (21%)
T. denticola (8%)
P. endodontalis (16%)
B. forsythus (21%)
P. micros (0%)

18-60 yrs

3% hydrogen
peroxide,
2.5% sodium
hypochlorite
solution,5%
sodium
thiosulfate

# 15 K-type file

1 ml of TSB

PCR

C. periodontii (14%)

AdultsX

3% hydrogen
peroxide,
2.5% sodium
hypochlorite
solution, 5%
sodium
thiosulfate

# 15 K-type file

1 ml of TSB

PCR

T. parvum (52%)
T. putidum (2%)

Sterile paper point

gingivalis, P. nigrescens, P. intermedia appeared

in most of the articles found in this systematic
review (Siqueira et al., 2001, 2002, 2004; Munson
et al., 2002; Seol et al., 2006; Tomazinho and

1 ml of TSB

Avila-Campos, 2007; Sakamoto et al., 2008).

Differences between studies may occur for
several reasons, including differences in the
selection of cases and the methods used for

de Paula et al.

Table 2. Continued.

Siqueira
and
Ras

Tomazino
and
AvilaCampos

Siqueira
and
Ras

Sassone
et al.

2006

2007

2007

2008

= Not reported.

A(10)

A(9)

A(9)

A(9)

Brazil

Brazil

Brazil

Brazil

Crosssectional

Crosssectional

Crosssectional

Crosssectional

50

100

50

60

18-60 yrs

3% hydrogen
peroxide,
2.5% sodium
hypochlorite
solution,
5% sodium
thiosulfate

AdultsX

3% hydrogen
peroxide,
2.5% sodium
hypochlorite
solution,
5% sodium
thiosulfate

AdultsX

3% hydrogen
peroxide,
2.5% sodium
hypochlorite
solution,
5% sodium
thiosulfate

AdultsX

3% hydrogen
peroxide,
2.5% sodium
hypochlorite
solution,
5% sodium
thiosulfate

# 15 K-type file

1 ml of TSB

Sterile paper point

3 mL of Mllers
viability medium
and Eppendorf
tube containing
300l sterile
ultrapure water

# 15 K-type file

# 15 H-type file and

sterile paper point

1 ml of TSB

Eppendorf tube
containing 150l
TE

PCR

C. morbid (26%)
G. adiacens (14%)

PCR

P.gingivalis(43.3%)
P. endodontalis
(23.3%)
P. intermedia
(31.7%)
P. nigrescens
(43.3%)

PCR

Asymptomatic cases
(52%)
Symptomatic cases
(21%)
Synergistes bacteria
(clones W028, BA
121/P4G_18PI,
W090, BH017,
E3_33)

Checkerbo
ard

F. nucleatum ssp.
Vicentii (>50%)
V. parvula (>50%)
T. socranskii, (>50%)
E. faecalis, (>50%)
C. gracilis, (>50%)
E. saburreum,
(>50%)
N. mucosa, (>50%)
T. forsythia, (>50%)
P. acnes(>50%)

1823

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Afr. J. Microbiol. Res.

sampling, sample transport and identification. The higher

sensitivity of the PCR method compared to culture is
probably the reason for the higher prevalence of
Porphyromonas species (Siqueira et al., 2001, 2002;
Munson et al., 2002; Ras and Siqueira, 2008; Siqueira
and Ras, 2009). In addition the PCR methods allow
detection of uncultivable strains of these species (Zoletti
et al., 2006). P. endodontalis and P. gingivalis were found
by the authors of three articles, as shown in Table 2. The
Red complex" (Bacteroides forsythus, Porphyromonas
gingivalis, and Treponema denticola) has been
associated with primary endodontic infections (Ras et
al., 2001; Siqueira and Ras, 2009), and was also
reported in this systematic review. Tomazinho and AvilaCampos (2007), using PCR, analyzed 60 samples and
the DNA of the following organisms was detected: P.
gingivalis (43.3%), P. nigrescens (43.3%), P. intermedia
(31.7%) and P. endodontalis (23.3%). Saito et al. (2009)
detected P. gingivalis in 28% of the subjects analyzed
and T. forsythia was detected in 18% to 24% of the
primary endodontic infections by conventional PCR.
The PCR is used to investigate the prevalence of
bacterial species in infected root canals (Siqueira and
Ras, 2003). The major reagents to be used in PCR are
the target DNA to be amplified, single-strand
oligonucleotides (primers) complementary to known
sequences
of
the
target
DNA,
the
four
deoxyribonucleoside triphosphates (dNTPs), and a heatstable DNA polymerase. Amplification reactions are
carried out using special DNA thermocycles (Siqueira
and Ras, 2003, 2004). Nested PCR has increased
sensitivity and possibly also improved specificity when
compared with PCR (Siqueira and Ras, 2004).
Increased sensitivity is a function of the larger total
number of cycles used in nested PRC assays. In
addition, target DNA is amplified in the first round of
amplification, with subsequent dilution of other DNA and
inhibitors present in the sample(Vianna et al., 2005).
The Denaturing Gradient Gel Electrophoresis (DGGE)
technique can also be used for the identification of
microorganisms. The application of the PCR-DGGE
method in endodontic research has revealed that there
are significant differences in the predominant bacterial
composition between asymptomatic and symptomatic
cases (Siqueira et al., 2005). In spite of the fact that
PCR-DGGE has been applied to analyze microbial
communities from diverse environments this technique
has only recently been introduced in endodontic
microbiology to fingerprint bacterial communities
associated with different types of infection and to identify
some dominant members of communities (Machado de
Oliveira et al., 2007).
The Checkerboard DNA-DNA hybridization method
was introduced for hybridizing larger numbers of DNA
samples against large numbers of DNA probes on a
single support membrane. This method permits the
simultaneous determination of a multitude of bacterial

species in single or multiple clinical samples and is

particularly applicable in epidemiological research
(Siqueira et al., 2002; Sassone et al., 2008). Studies with
this molecular method have provided substantial new
information for a better understanding of the microbiology
of endodontic diseases (Siqueira et al., 2002).
This review revealed that in the literature numerous
microorganisms are involved in permanent teeth
infections, such as: Parvimonas micra, Fusobacterium
nucleatum ssp, Tannerella forsythia, Treponema
denticola, Veillonella parvula, Eubacterium nodatum,
Porphyromonas gingivalis, Actinomyces odontolyticu, T.
parvum, T. putidum, Firmicutes, Actinobacteria,
Bacteroidetes,
Proteobacteria,
Fusobacteria,
Synergistes, Flavobacteriaceae, Saprospiraceae, P.
alactolyticus, C. durum, P. aeruginosa, P. nigrescens, E.
faecalis, and Treptococcus constellatus (Munson et al.,
2002; Siqueira et al., 2002, 2004, 2005, 2007; Ras et
al., 2003, 2004; Siqueira and Ras, 2004, 2007; Ras
and Siqueira, 2005; Vianna et al., 2005; Seol et al., 2006;
Sakamoto et al., 2008; Ras and Siqueira, 2009).
Siqueira et al. (2002) in his study using two different
molecular methods (Checkerboard and PCR) confirmed
that B. forsythus and T. denticola, (two particularly
fastidious periodontal pathogens that had not been
cultured previously from endodontic infections), are
components of the endodontic microbiota and may play a
role in the pathogenesis of periapical diseases. The same
author (Siqueira and Ras, 2004) found Dialister
pneumosintes and Filifactor alocis in endodontic
infections using the PCR multiplex method. And, in
another paper, (Ras and Siqueira, 2005) were the first
authors to report the occurrence of T. parvum and T.
putidum in samples from primary endodontic infections.
The findings of these newly named Treponema species
have expanded the list of bacteria involved with infections
of endodontic origin.
These papers cited above reveal the diversity of
microorganisms involved in endodontic infections. The
clinical implications of the presence of these
microorganisms in endodontic infections is that for a
diseases with multiple microbiological causes there is not
as yet any kind of irrigants that can be used during the
root canal treatment to eliminate all the microorganisms
involved in this pathosis. For the endodontists, the
success of endodontic treatment depends on several
factors and one of the most important is the reduction or
elimination of bacterial infections. Therefore, it is important
for the clinician to be aware of such bacteria and their
ability to grow in an endodontic microenvironment. The
clinician has to be prepared for possible treatment fail,
because some microorganism(s) might help to maintain
the disease. This research is important to disclose the
species that are putative pathogens implicated in the
pathogenesis of different types of endodontic infections
and periradicular diseases. In addition to being of clear
academic interest, this knowledge has an unquestionable

de Paula et al.

clinical importance as it provides clues for the search for

effective antimicrobial strategies to reach and eliminate
the components of the microbiota located in all
irregularities of the root canal system.
Given the small number of studies that were included in
this review, the meta-analyses on the effects of several
prognostic factors could be considered to be
compromised by lack of statistical power to demonstrate
a significant influence. Also it is important to highlight that
this diversity is not only due to the methodology used
since all studies included were A, but this is, probably,
also related to the kind of infection (primary or not) and
the symptoms involved (abscess, pocket >3mm, pain,
teeth mobility) (Zoletti et al., 2006, 2010; Sassone et al.,
2008; Siqueira and Roas, 2008). Some articles lost
points in quality assessment because they did not have a
representative sample (Saito et al., 2009), did not
describe if the samples were collected using strict
asepsis methods (Saito et al., 2009), whether the
samples were collected by the same operator
(Tomazinho and Avila-Campos, 2007), did not have a
description of the data collection (Munson et al., 2002),
and other points analyzed based on quality (Munson et
al., 2002; Saito et al., 2009).
Black-pigmented anaerobic bacteria may be commonly
associated with symptomatic endodontic infections
(Sakamoto et al., 2008). That is why A.
actinomycetemcommitans and E. faecalis, high virulent
microorganisms, were not found in a greater number of
papers (Siqueira et al., 2001, 2002; Siqueira and Ras,
2004; Ras and Siqueira, 2005). E. faecalis was much
more likely to be found in cases of failed endodontic
therapy than in primary infections in the Brazilian
population (Seol et al., 2006; Zoletti et al., 2006, 2010;
Sakamoto et al., 2008; Siqueira and Roas, 2008).
Therefore, the negative results regarding the occurrence
of these bacteria in root-canal infections when assessed
by culture, or another method, does not necessarily imply
that they were absent, but it reflects the inherent
limitations of the phenotype-based identification
approaches used.
Molecular methods may be successfully used to
overcome such limitations and have added further
important information with regard to the composition of
the endodontic microbiota (Siqueira, 2003). However,
much more studies are necessary to identify which
microorganisms are involved, their virulence, the
association with clinical symptoms and the refractory
infections. These aspects are essential to the
understanding of the disease process and to the
development of more effective and predictable
therapeutic measures to deal with endodontic diseases.
Little is known about the virulence traits and
mechanisms of the pathogenicity of these species
involved in endodontic infections. Further studies are
thereby warranted in order to confirm involvement with
endodontic disease causation and to look for specific

1825

virulence factors. This is an important step in the

understanding of the disease process and for the
development of more effective and predictable
therapeutic measures to deal with endodontic diseases.
Conclusion
This review found high evidence to confirm the
polymicrobial nature of primary endodontic infections and
the dominance of anaerobic bacteria (Porphyromonas
gingivalis, Porphyromonas endodontalis, Bacteroides
forsythus, Prevotella intermedia and Prevotella
nigrescens). Future clinical studies will be important to
confirm the results observed here in permanent and
deciduous teeth.
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Siqueira JF Jr., Ras IN, Oliveira JC, Santos KR (2001). Molecular

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