J Periodontol December 2011

Detection and Clonal Analysis

of Anaerobic Bacteria Associated
to Endodontic-Periodontal Lesions
fling
Cassio V. Pereira,* Rafael N. Stipp, Douglas C. Fonseca, Luciano J. Pereira, and Jose F. Ho

Background: Microbial agents in root canal systems can induce periodontal inflammation. The aims of this study are to
detect anaerobic microorganisms in endodontic-periodontal
lesions, determine the genetic diversity among them, and assess the simultaneous colonization of the pulp and periodontal
microenvironments by a single clone.
Methods: Twenty-seven teeth of patients with endodonticperiodontal lesions were selected. Samples were spread on an
agar-blood medium, the detection of each species was performed using a polymerase chain reaction, and the determination of the simultaneous presence of the same species in the
microenvironments by one or more clones was determined using
arbitrarily primed PCR.
Results: Prevotella intermedia (Pi) was the most prevalent
species of the colonies in periodontal pockets, whereas Porphyromonas gingivalis (Pg) and Pi were the more prevalent in root
canals. Isolates of Pi and Pg were simultaneously identified in
root canals and periodontal pockets. Eighteen percent of teeth
exhibited the simultaneous colonization by Pg, Tannerella forsythia (previously T. forsythensis), and Porphyromonas endodontalis in the pulp and periodontal microenvironments. The
presence of these species was noted even in niches from which
no colonies were isolated. Seventeen different genotypes were
found in periodontal and pulp sites, with the majority of sites colonized by one or two different genotypes. A high degree of genotype similarity was found for samples of Pg isolated from only
one site as well as for those isolated from both microenvironments.
Conclusion: Different clones of Pi and Pg with a high intraspecific genotype similarity were found to colonize the same anatomic sites in endodontic-periodontal infections. J Periodontol
2011;82:1767-1775.
KEY WORDS
Bacteria, anaerobic; endodontics; periapical diseases;
periodontics; random amplified polymorphic DNA technique.
* Department of Microbiology and Immunology, Lavras University Center, Lavras, MG,
Brazil.
Department of Microbiology and Immunology, Piracicaba Dental School, University of
Campinas, Piracicaba, SP, Brazil.
Department of Periodontics, Lavras University Center.
Department of Physiology and Pharmacology, Federal University of Lavras, Lavras, MG,
Brazil.

acteria can harm the oral tissue

through the release of toxins, enzymes, and residual metabolic
products. Such microorganisms are inevitably correlated to the etiology of pulp and
periapical lesions, particularly proteaseproducing anaerobic bacteria, which break
down diverse polypeptides involved in the
host defense mechanism.1-3
There is a close relationship between
root canal systems (root canal treatment) and the periodontal environment.
Lateral and accessory canals can transport toxic substances and facilitate the
bacterial colonization of dental tissues.
Toxins and microbial agents in the pulp
space can induce periodontal inflammation. However, the effect of these irritants
from the periodontal space to the pulp is
less clearly defined.4 Experiments comparing polymorphisms in genomic fingerprints were conducted to clarify this
issue.5
The evidence of a microbial involvement, especially of pigmented species
such as Porphyromonas gingivalis (Pg),
Prevotella intermedia (Pi), and Prevotella
nigrescens (Pn), in the pathogenesis of
periodontal inflammation and periapical
lesions accumulated in the literature.6,7
However, a microbial species consists of
a large number of clones, and the majority of infectious bacterial diseases can be
caused by some clones of pathogenic
species.8-10
A number of studies5,11,12 that aimed to
characterize the microbial communities of
doi: 10.1902/jop.2011.110063

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Anaerobic Bacteria and Endodontic-Periodontal Lesions

pulp and periradicular infections on the basis of

DNA fingerprints revealed an unexpected diversity
of microbial species. However, there are few studies using polymerase chain reaction (PCR) and arbitrarily primed PCR (AP-PCR) for the detection of
virulent clones of species that cause endodonticperiodontal infections.
The hypothesis of the present study is that there is
a genetic diversity among anaerobic microorganisms
in endodontic-periodontal lesions and that there is
a simultaneous colonization of the pulp and periodontal microenvironments by a single clone. This investigation is important because different strains from the
same bacteria species can present variations in their
virulence and resistance influencing the treatment
outcome. Thus, the aim of the present study is to perform molecular analyses for the detection of Pg, Pi,
Pn, Porphyromonas endodontalis(Pe), and Tannerella forsythia (Tf ) in patients with endodontic-periodontal lesions. After isolation in an anaerobic
culture medium and molecular characterization, the
samples were submitted to AP-PCR for the determination of genetic diversity among the microorganisms
and the assessment of the possibility of simultaneous
colonization of the pulp and periodontal microenvironments by a single clone.
MATERIALS AND METHODS
This study received approval from the Ethics Committee, Sacred Heart University, Bauru, SP, Brazil, under
process number 013/2003, and data collection was
done between 2004 and 2005 considering the difficulty in finding elective patients. The participants were
made aware of the objectives and procedures of the
study and agreed to participate by signing terms of informed consent.
Twenty-seven teeth were selected from male
(40%) and female (60%) adults (8 males and 13 females; age range: 27 to 51 years) with endodonticperiodontal lesions diagnosed through clinical and
radiographic examinations at the dental clinics of
the Sacred Heart University. None of the patients,
as reported in their files, were smokers or under the
use of any medication (including contraceptive pills
for females). Inclusion criteria included intact teeth
with necrotic pulp tissue, periapical lesions, chronic
periodontal disease, and the absence of systemic
conditions. For the diagnosis of chronic periodontal
disease, the following clinical-radiographic factors
were considered: clinical attachment levels, the presence of inflammation, probing depths (periodontal
pockets >5 mm), and bone loss.2,13 Exclusion criteria
were the use of antibiotic therapy (6 months before the
sample collection) and the clinical diagnosis of fistula
because these factors could have influenced the formation of the microbiota in these microenvironments.
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All volunteers had intact teeth or restorations on the

enamel level only.
After the removal of the supragingival biofilm, previously sterilized paper cones were used to collect
samples from the interior of the root canal system (after the aseptic opening of the crown) as well as from
the periodontal pocket of the tooth affected by endodontic-periodontal lesions. Samples were also collected from the gingival sulcus of the same tooth in
the opposing hemiarch as the control for the determination of the absence of microorganisms in regions
that did not exhibit periodontal disease. In cases of
atresic and/or curved canals that hindered the collection with paper cones, the procedure was carried out
with the aid of endodontic files.
Samples were transported to the Laboratory of
Microbiology and Immunology, Piracicaba Dental
School, University of Campinas, Piracicaba, SP, Brazil
in reduced transport fluid in 1 hour from the time of
collection and dispersed and agitated for 20 seconds.
For each sample, 100 mL of 10-1 to 10-4 dilutions were
spread on dishes with brucella agar medium enriched
with defibrinated sheep blood, hemin (10 mg/mL),
and vitamin K1 (1 mg/mL). The dishes were incubated
at 37C in anaerobic jars containing two anaerobiosis
generators,i one anaerobiosis indicator, and silica to
absorb moisture for 10 days. After this period, the
largest possible number of colonies with dark-brown
to black pigmentation were isolated from primary culture dishes and cut for the obtainment of a pure culture.
Pure cultures were also identified through Gram
staining for the confirmation of the microscopic characteristics with Gram-negative rods. Pairs of specific
primers were used for Pg, Pi, Pn, Pe, and Tf for the
PCR amplification of a specific sequence of DNA
(Table 1).10,14,15
PCR-amplification reactions were performed in a
reaction mixture containing 1 PCR buffer (10 mM
Tris-HCl, pH 9.0, 50 mM KCl, and15 mM MgCl2),
200 mM of four deoxynucleotide triphosphates,
12.5 pmol of each primer, 2.5 U Taq DNA polymerase,# and 5 mL supernatant from the samples submitted for DNA extraction at a final volume of 50 mL. The
reaction mixture was incubated in a thermocycler.
Samples were initially denatured at 94C for 5 minutes
and then submitted to 35 cycles of 60 seconds at
94C, 45 seconds at 67C, and 30 seconds at 72C.
A negative control (absence of DNA template), a positive control, and molecular mass marker (100base
pair [bp] DNA ladder) were included in each amplification set. PCR-amplification products were analyzed through electrophoresis in a 1.5% agarose gel
i Anaerogen, Oxoid Brazil, Sao Paulo, SP, Brazil.
Invitrogen Life Technology of Brazil, Sao Paulo, SP, Brazil.
# Invitrogen Life Technology of Brazil.

J Periodontol December 2011

fling
Pereira, Stipp, Fonseca, Pereira, Ho

A random-amplification polymorphic DNA (RAPD) analysis was used

Primers for PCR Identification of Pg, Pi, and Pn
to determine the existence of the genetic affinity between the same speAnaerobic Bacteria
Primers
bp (n)
cies isolated from the root canals
and gingival sulcus simultaneously
Pg
59-AGGCAGCTTGCCATACTGCG-39
443
or from only one anatomic site in a
59-ACTGTTAGCAACTACCGATGT-39
single patient.
Pi
59-CGGTCTGTTAAGCGTGTTGTG-39
99
The specification of samples as Pg,
59-CACCATGAATTCCGCATACG-39
Pi, and Pn was carried out using PCR
Pn
59-CAGCCAAACACGATACCTGTTG-39
150
DNA amplification of a specific se59-TTCCATTGGACACATCAGCATT-39
quent of bp for each of the species
as described. In complement to the
Pe
59-GCTGCAGCTCAACTGTAGTC-39
672
clinical isolates, standard strains were
59-CCGCTTCATGTCACCATGTC-39
used as a reference: Pg American
Tf
59-GGGTGAGTAACGCGTATGTAACCT-39
127
Type Culture Collection 33277, Pi
59-ACCCATCCGCAACCAATAAA-39
American Type Culture Collection
25611, and Pn National Collection
of Type Cultures 9336.
Total genomic DNA was obtained
Table 2.
using additional RNase treatment.16
Number of Colonies Identified as Pg, Pi, Pn, Pe, and Tf
The concentration of DNA was calcuIsolated From Periodontal Pockets and Root Canals
lated from the measurement (260
nm) in a spectrophotometer at A260,
and the quality was estimated by the
Periodontal Pocket
Root Canal
Black-Pigmented
A260:A280 ratio, electrophoresis in
Microbial Species
n
%
n
%
an agarose gel, and comparison with
standard DNA.
Pg
7
9.6
5
10.9
The AP-PCR amplification was perPn
4
5.5
3
6.5
formed in a volume of 25 mL containing
200 mM deoxynucleotide triphosPi
18
24.7
5
10.9
phate,** 1.2 mM primer, 50 ng DNA
Pe
1
1.4
0
0.0
template, and 2.5 U Taq DNA polymerase in 1 PCR buffer, 10 mM Tris-HCl,
Tf
0
0.0
0
0.0
pH 9.0, 50 mM KCl, and 1.5 mM MgCl2.
Other black-pigmented
43
58.9
33
71.7
A negative control without DNA temspecies
plate was included in each experiment.
Total
73
100
46
100
Arbitrary primers 970 to 11 (59GTAAGGCCG-39) and 13 (59- CAGNumber of colonies identified as Pg, Pn, Pe, and Tf isolated from periodontal pockets and root canals
in teeth with endodontic-periodontal lesions.
CACCCAC-39) were selected.16 The
reaction mixture was submitted to
amplification in a thermocycler prorun in Tris-borate-EDTA buffer, stained with ethidgrammed for one cycle of denaturation at 94C for
12 minutes followed by 30 cycles at 94C for 1 minute,
ium bromide, and video documented under ultravio32C for 1 minute, and 72C for 2 minutes and a final
let light.
elongation at 72C for 7 minutes. Samples were
AP-PCR was used for the determination of virulent
cooled to 4C, and the PCR-amplification products
clones among species of anaerobic filamentous bacwere analyzed through electrophoresis in a 1.5% agateria collected from root canals and periodontal
rose gel run in Tris-borate-EDTA buffer. A standard
pockets.5 AP-PCR was also used for samples of spemolecular mass of 100 bp was included in each
cies isolated from only one of the microenvironments
(root canal systems or the periodontium). This pracgel. DNA was stained with ethidium bromide, and each
tice did not allow the determination of the simultagel was video documented under ultraviolet light.
neous colonization by clones of these species in
patients with this occurrence but contributed toward
** Invitrogen Life Technology of Brazil.
the knowledge of the genetic variability of these mi Invitrogen Life Technology of Brazil.
croorganisms in each anatomic site.
Invitrogen Life Technology of Brazil.
Table 1.

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Volume 82 Number 12

Table 3.

Table 4.

Molecular Detection of Pg, Tf, and Pe

Periodontal Pockets and Root Canals

Prevalence of Pg, Pe and Tf in Samples

From Periodontal Pockets and Root Canals

Periodontal Pocket

Root Canal

Molecular
Detection

Pg
%

Tf*

Tooth

Pg

Tf

Pe

Pg

Tf

Pe

Microenvironment

Periodontal pocket

+
-

25 92.6 16 59.3 20 74.1

2 7.4 11 40.7 07 25.9

Root canal

+
-

20 74.1 19 70.4 09 33.3

07 25.9 08 29.6 18 66.7

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

+ = molecular detection of the species in the root canal or periodontal

pocket microenvironments.
- = absence of molecular detection of the species in the root canal or
periodontal pocket microenvironments.

Pe
%

Prevalence of Pg, Pe and Tf in samples from periodontal pockets and root

canals in teeth with endodontic-periodontal lesions.
* P = 0.029.
+ = molecular detection of the species in the root canal or periodontal
pocket microenvironments.
- = absence of molecular detection of the species in the root canal or
periodontal pocket microenvironments.

The determination of the colonization of the periodontium or root canal alone and the simultaneous
presence of the same species in both microenvironments by one or more infecting clones was performed
through a comparison of the RAPD fingerprinting of
the multiple isolates.
The patterns of DNA amplified through AP-PCR
(arbitrary primers 970-11) were expressed numerically by their mobility values in the gel (in millimeters).
Mobility values were converted into binary values, the
representations of which corresponded to the presence and absence of bands (1 and 0, respectively).
Isolates were characterized by the combinations of
bands of high and medium intensity and were reproducible after two independent assays such that
distinct combinations of polymorphic bands were
designated electrophoretic types.17
The genetic relationship among isolates was determined by the similarity coefficient (simple matching),
which allowed for the use of data based on binary
values. Thus, the genetic similarity matrix was designed and treated by the sequential hierarchical agglomerative non-overlapping clustering technique of
the unweighted pair group method of arithmetic
mean to generate a tree with a two-dimensional classification denominated a trellis diagram.18 These
analyses were performed with the aid of the program. In all analyses, samples with electrophoretic
profiles revealing a 95% to 100% degree of similarity
were considered to be the same genotype.19
In statistical analyses of data, the x2 test was used
to compare the prevalence of species identified in different anatomic sites (the periodontal pocket and root
canal). Data were entered in a statistical software program.ii
NTSYSpc v.1.70, Applied Biostatistics, Port Jefferson, NY.
ii SPSS, v.14.0, IBM, Chicago, IL.

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J Periodontol December 2011

Figure 2.
Representative gel and trellis diagram of Pg strains obtained with OPA970 ## in samples isolated simultaneously from both sites: periodontal
pocket and root canal (tooth 17), or from only one of the microenvironments
(tooth 3). The first number denotes the tooth number in accordance to
table 3, the letter denotes the isolated site (periodontal pocket (p) or
root canal (c) and the second number denotes the sample number from
each microenviroment. MM = molecular mass.

Figure 1.
Representative gel and trellis diagram Pi strains obtained with Operon
Primer Arbitrary (OPA-970) in samples isolated simultaneously from
both sites: periodontol pocket and root canal (teeth 16 and 17), or from
only one of the microenvironments (teeth 7, 8, 15 and 18). The first
number denotes the tooth number in accordance to table 3, the letter
denotes the isolated site (periodontal pocket (p) or root canal (c) and the
second number denotes the sample number from each microenviroment.
MM = molecular mass.

RESULTS
Table 2 displays the number of colonies identified as
Pg, Pi, Pn, Pe, and Tf by PCR based on pure cultures.
Pi was the most prevalent among the clinical isolates
from periodontal pockets, with 18 colonies identified
(24.6%). Among the samples from a root canal, five
colonies were identified as Pg, and five colonies were
identified as Pi, with each species accounting for
10.8% of the total number of isolates in this anatomic
site. Pi was identified in root canals and periodontal
pockets of two teeth (patients 16 and 17), and Pg

was identified in both anatomic sites in one tooth (patient 17). All samples identified as Pn (n = 7) colonized
only one microenvironment in the same tooth. Among
the 119 samples isolated, only 42 samples were identified as Pg, Pi, or Pn, indicating a greater variability of
black-pigmented species as colonizers of teeth with
endodontic-periodontal lesions. None of the strains
tested was characterized as being from the species
Tf, and only one isolate from a periodontal pocket
(tooth 6) was identified as Pe. Additionally, none of
the control samples were positive for any anaerobic
bacteria tested.
Tables 3 and 4 display the results of the genomic
DNA extraction of possible bacteria on the paper
cones used for the collection of samples from microenvironments and the confirmation of the presence of
Pg, Pe, and Tf by PCR, even if the culture had been
negative for black-pigmented colonies. The electrophoretic profiles indicated the simultaneous colonization by the three species in periodontal pockets and
root canals in 18.5% of teeth (n = 5). There was a prevalence of 92.6% (n = 25), 59.3% (n = 16) and 74.1% (n =
20) in the periodontal pocket and 74.1% (n = 20),
70.4% (n = 19) and 33.3% (n = 9) in the root canal
for Pg, Pe, and Tf, respectively.
Invitrogen Life Technology of Brazil.
## Invitrogen Life Technology of Brazil.

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of which (samples 2 and 3 from

tooth 9) exhibited a greater
similarity to one another.
DISCUSSION
Pathologic alterations in the
cervical periodontium demonstrated that the mechanisms
involved in periodontal disease were similar to those involved in the pathogenesis of
periapical lesions. In the present study, all volunteers had
intact teeth or restorations on
Figure 3.
the enamel level only, with no
Representative gel and trellis diagram of Pn strains obtained with OPA-970 *** in samples isolated
indication of the contaminafrom only the pulpar microenvironment (tooth 9). The first number denotes the tooth number in accordance
to table 3, the letter denotes the isolated site (periodontal pocket (p) or root canal (c) and the second
tion of the pulp tissue through
number denotes the sample number from each microenviroment. MM = molecular mass.
the crown pathway. Thus, the
lesions were endodontic-periThe image in the gel revealed the simultaneous
odontal lesions, in which the primary infection was
presence of Pg in the samples collected from perilikely of a periodontal origin because the direct
odontal pockets and root canals of 18 of the 27 teeth
communication between the pulp and periodontium
submitted for analysis. Only two samples obtained
was limited to the apical foramen and lateral cafrom periodontal pockets and seven from root canals
nals.5
did not exhibit this species. Only Tf had a significantly
A number of microorganisms, mostly anaerobic
greater prevalence (P = 0.029) in periodontal pockets in
bacteria, may be responsible for the onset and progrescomparison to the colonization in root canals (Table 4).
sion of endodontic-periodontal disease.4,12 Episodes
PCR results for the detection of Pg, Pe, and Tf based
of disease may represent a change in the ecological
on the DNA extraction of the bacteria present on the
balance between the bacterium and host factors as
paper cones used for the sampling also indicated
a result, for instance, of an alteration in the relative
the presence of these species in microenvironments
and absolute number of these microorganisms or
in which there was no isolation of colonies.
a modulation in specific factors of the host.20
Figures 1 through 3 display the electrophoretic proThe culture method is considered the reference
files and biologic trellis diagrams resulting from the
method compared to other techniques used to detergenetic similarity analysis of species isolated simultamine the presence of microorganisms. Advantages of
neously from both microenvironments and species
the culture method include the possibility of quantiwith >1 isolate from the same anatomic site, respecfying isolated species, obtaining strains in a pure cultively. Trellis diagrams revealed the occurrence of
ture for subsequent studies (such as genotyping), and
17 different genotypes established in periodontal
the determination of virulence factors. Moreover, the
and pulp sites, with the majority of individuals coloculture method is the only manner to obtain an antibionized by one to two distinct genotypes.
gram. In contrast, the culture consists of exhaustive,
The intra-individual analysis revealed the coloniburdensome laboratory steps and also produces lower
zation by strains with a high degree of genetic similardetection values compared to methods based on moity in microenvironments of periodontal pockets and
lecular biology in which, theoretically, much smaller
root canals in the samples identified in teeth 16 and
numbers of microorganisms can be detected.14,19
17. The results also indicate a slight coincident occurThe lower detection values for culture methods comrence between genotypes from samples 1 and 4 isopared to molecular biology methods was demonstrated
lated from the periodontium of tooth 15 and samples
in the comparison of results of the identification of Pi, Pg,
1 and 2 isolated from the root canal of tooth 18 (Fig. 1).
Pn, Pe, and Tf among isolated colonies and the deTrellis diagrams of Pg samples revealed a high detection of Pg, Pe, and Tf in periodontal pockets and necgree of genotype similarity for samples isolated from
rotized root canals (Tables 2 through 4). Results indicate
a single site as well as those isolated from both microthe detection of these species through PCR analysis in
environments (Fig. 2). However, colonies identified as
anatomic sites not detected by cultures.
Pn were only isolated from the root canal, and the trellis diagrams displayed in Figure 3 indicates the colonization by three distinct genotypes in this patient, two
*** Invitrogen Life Technology of Brazil.
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J Periodontol December 2011

Some sites had Tf and Pe, as detected by PCR, but

there was no growth of Tf and Pe on agar plates. This
result implies that Tf and Pe were under the culture detection levels. Thus, the non-detection of certain species using the culture method did not necessarily
mean the absence of these species but, rather, indicated that the number of microorganisms was below
the detection threshold of the method. The absence
of pure cultures justified the non-use of PCR for the
detection of Pi and Pn. In contrast, the clinical significance of the detection of pathogens that represented
a tiny portion of the oral microbiota in terms of the colonization consistency seemed questionable, as did
whether such low levels were capable of offering sufficient virulence to cause the destruction of dental support tissues.16 These statements appeared to explain
the results of the present study and complemented
those of previous studies1,10,21 that demonstrated the
frequent isolation of black-pigmented anaerobic bacteria of the genera Porphyromonas and Prevotella in
root canal infections, whereas the culture of these microorganisms in the laboratory was variable. According to the authors cited,1,10,21 these variations were
due to differences in the selection of cases and the collection, transportation, cultivation, and identification
methodologies.
Values displayed in Table 2 indicate the greater
prevalence of Pi among the clinical isolates from periodontal pockets (24.6%) compared to the other species identified. Previous studies1,10,16 that involved
the identification of pathogens of endodontic and periodontal infections using a similar methodology reported values close to those of the present study.
The greater prevalence of Pi in these microenvironments was directly related to its virulence because
its high degree of hemolytic activity determined its
pathogenicity and the progression of the disease.
The hemoglobin and hemin that resulted from the
hemolytic action on red blood cells was a strong exogenous source of iron that favored the growth of blackpigmented bacteria, including Pi.10 However, there
was no significant difference between the different microenvironments in the prevalence of Pg, Pn, and Pi.
The low prevalence of Pn in the present study is
compatible with that reported in previous studies,7,22
which indicated that that this species accounted for
the majority of the genus Prevotella in healthy individuals, whereas active, healthy sites in patients with
periodontal infection were mainly colonized by Pi.
The colonization of Pi appeared to be more stable in
sites with periodontal destruction compared to in
healthy sites.23 In contrast, one study24 reported that
both species were often found in sites with and without
periodontal disease.
Among clinical isolates, a significant number of
samples were not identified as belonging to any of

fling
Pereira, Stipp, Fonseca, Pereira, Ho

the species tested. This result suggested that endodontic-periodontal lesions were multicolonized by
a considerable variety of microbial agents.4,9,12,19
The greater prevalence of species from the genus
Prevotella in the results of the isolation and identification of black-pigmented bacteria may have been
explained by their greater ease of culture because
these species seemed to tolerate minimal levels of
oxygen. Similar behavior seems not to occur with
species of the genus Porphyromonas, which are considered more fastidious and therefore, may be
underestimated by culture methods such as those
applied in the present study.1,25
Tables 3 and 4 indicate that there was the simultaneous colonization in the pulp and periodontal anatomic sites by all species tested. Pg was the most
prevalent, followed by Pe, which is the same prevalence order reported in the literature,4 with values of
72% and 56%, respectively. Different bacterial patterns were evidenced between the periodontium and
root canals, which thereby confirmed the need for additional studies that can demonstrate the degrees of
colonization of each species and their importance to
the etiology of the disease and the colonizing communities of these microenvironments.12,26,27
There was a significant difference in the detection of
Tf between the root canal (33%) and periodontium
(74.1%). Previous studies10,28 that performed PCRs
report similar values (79%) regarding the prevalence
of this species in periodontal disease. The comparison
of the results in the different sites suggested the existence of modulating factors, such as immunologic
mechanisms of the host, genetic predisposition, systemic alterations, and smoking habits, as determinants of the microbial colonization and succession
in oral microenvironments, favoring the colonization
and growth of this microorganism in the periodontal
environment.4
The analysis of the genetic diversity of the Pi isolates (Figs. 1 through 3) revealed that the majority
of individuals had one to two different clones, which
corroborated the findings of previous studies7,23,29-31
that used similar methodologies. In two teeth (16
and 17), colonies of Pi were simultaneously isolated
from the periodontal pocket and root canal. The genotyping of these microorganisms indicated an electrophoretic profile that was similar to the 80% level
between these colonies (Fig. 1), thereby confirming
that the pulp-infection pathway originated from a primary periodontal infection.32,33 One study30 on the
clonality of other bacterial species involved in the etiopathogenesis of periodontal disease also reveals the
detection of some clone types per patient or site.
Pg was identified in colonies isolated from the pulp
and periodontal microenvironments of tooth 17. The
trellis diagram in Figure 2 demonstrates a nearly
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Anaerobic Bacteria and Endodontic-Periodontal Lesions

90% similarity between isolates and suggested that

the colonization of both anatomic sites was performed
by the same clone of this species, as demonstrated
elsewhere.16
Only one tooth exhibited >1 colony as Pn from
the same microenvironment (9C1 through 9C3).
The genotyping indicated a high degree of similarity
between two of the samples (Fig. 3). Previous studies
detected multiple clone types of Pn in 43% of individuals,16 whereas other studies reported only one clone
type of the species colonizing periodontal and endodontic sites in the same oral cavity.30 Studies on the
genetic diversity of periodontal pathogens revealed a
broad heterogeneity within the species7,16,23,24,29,30
and the absence of predominant clone types associated with the disease and demonstrated the stability
of clones even after periodontal treatment.23,24,30
These findings suggested that all clones of a species
are equally effective at colonizing periodontal sites
because the potential for virulence is not restricted
to a particular clone, in contrast with other truly pathogenic species.
AP-PCR was selected for this study because nested
PCR may be useful for distinguishing alleles but not for
genotyping. Furthermore, multiplex PCR is a modification of PCR intended to rapidly detect deletions or
duplications in a large gene. Although it can be useful
for genetic discrimination, the number of produced
PCR bands is frequently lower than that obtained with
AP-PCR. Thus, multiplex PCR can be considered less
efficient for genetic discrimination. AP-PCR (also
known as RAPD) is the technique of choice for rapid,
cheap, and confident genetic discrimination or for
tracing microorganisms in the environment. Because
many amplicons are generated, differences, if they
exist, are easier detected.5,11
CONCLUSIONS
The present study demonstrates that Pi and Pg were
the most frequently identified species among the colonies isolated from periodontal pockets and root canals, respectively. Pn was less prevalent in patients
with endodontic-periodontal lesions, whereas Pg had
high rates of simultaneous detection in the pulp and
periodontal microenvironments. Different clones of
Pi and Pg colonized the same anatomic sites, the periodontium and/or pulp, in the endodontic-periodontal
infection, with these clones exhibiting a high degree of
intraspecific genotype similarity.
ACKNOWLEDGMENTS
The authors are grateful to the Brazilian fostering
agency Research Support Foundation of the State
of Sao Paulo, Sao Paulo, SP, Brazil, for supporting this
study (grant process number 2003/10423-4) and researchers Vanessa Pardi, PhD, from Sacred Heart
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University, and Daniel Saito, PhD, from State University of Campinas, Piracicaba, SP, Brazil, for statistical
analyses and technical assistance, respectively. Dr.
Pereira acknowledges a scholarship from the National Council for Scientific and Technological Development (Brasilia, Federal District, Brazil). The
authors report no conflicts of interest related to this
study.
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Correspondence: Dr. Luciano Jose Pereira, Department of

Physiology and Pharmacology, Federal University of
Lavras, Caixa Postal 3037, CEP 37200-000, Lavras, MG,
Brazil. E-mail: lucianojosepereira@dmv.ufla.br.
Submitted January 31, 2011; accepted for publication
March 11, 2011.

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