Microbial Pathogenesis 61-62 (2013) 11e15

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Microbial Pathogenesis
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Short communication

Prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas

gingivalis and Tannerella forsythia in Japanese patients with
generalized chronic and aggressive periodontitis
Sachiyo Tomita a,1, Akiyo Komiya-Ito a,1, Kentaro Imamura a, b, Daichi Kita a, Koki Ota a,
Saori Takayama a, Asako Makino-Oi a, Takashi Kinumatsu a, Mikio Ota a, Atsushi Saito a, b, *
a
Department of Periodontology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan
b
Oral Health Science Center, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to investigate the prevalence and levels of major periodontal pathogens, Aggregati-
Received 2 February 2013 bacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in subgingival plaque
Received in revised form samples of a group of Japanese patients with aggressive periodontitis (AgP) and chronic periodontitis
3 April 2013
(CP). A total of 40 patients with clinical diagnosis of AgP or CP and 10 periodontally healthy volunteers
Accepted 9 April 2013
were subjected to clinical and microbiological analysis. Subgingival plaque samples were analyzed for
Available online 20 April 2013
A. actinomycetemcomitans, P. gingivalis and T. forsythia with a real-time polymerase chain reaction (PCR)
technique. The prevalence of P. gingivalis and T. forsythia was relatively high in patients with peri-
Keywords:
Aggressive periodontitis
odontitis: over 60% of AgP or CP patients harbored these pathogens whereas they were not detected in
Chronic periodontitis the subgingival plaque samples from periodontally healthy individuals. P. gingivalis and T. forsythia were
Aggregatibacter actinomycetemcomitans relatively frequently detected together in AgP and CP patients. No significant differences in the preva-
Porphyromonas gingivalis lence or level of the 3 pathogens were found between periodontitis groups. The proportion of T. forsythia
Tannerella forsythia was approximately 4-fold higher in CP group than in AgP group (P ¼ 0.02). In periodontitis patients, a
significant positive correlation was found between periodontal parameters (probing depth and clinical
attachment level) and the numbers of total bacteria, P. gingivalis and T. forsythia. No distinct pattern of the
subgingival profile of these pathogens was discerned between the two disease entities, except for the
difference in the proportion of T. forsythia. The red complex bacteria, P. gingivalis and T. forsythia were
highly prevalent in this population of Japanese AgP and CP patients, collaborating their roles in
periodontitis.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction
Periodontal disease is a plaque biofilm- induced inflammatory
disease of the supporting tissue of the tooth [1]. It can be described
as one of the predominant polymicrobial infections of humans [2].
So far, more than 700 bacterial taxa have been identified in samples
taken from oral cavities as identified by cultivation or traditional
cloning and sequencing [3]. Evidence for periodontal etiology is
based on the fulfillment of several criteria described by Socransky
* Corresponding author. Department of Periodontology, Tokyo Dental College,
1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan. Tel.: þ81 43 270 3953;
fax: þ81 43 270 3955.
E-mail addresses: atsaito@tdc.ac.jp, atsaito1@gmail.com (A. Saito).
1
These authors contributed equally to this work.
[4]. Using these criteria, strong evidence has been demonstrated
for Aggregatibacter (Actinobacillus) actinomycetemcomitans, Por-
phyromonas gingivalis and Tannerella forsythia, as concluded at the
World Workshop in 1996 [5].
Aggressive periodontitis (AgP) is a specific type of periodontitis
with clearly identifiable clinical and laboratory findings which
make it sufficiently different from chronic periodontitis (CP) [6].
The common features of AgP are: except for the presence of peri-
odontitis, patients are otherwise clinically healthy, rapid attach-
ment loss and bone destruction, familial aggregation. Among
periodontal pathogens, Aggregatibacter actinomycetemcomitans has
been implicated in the etiology of AgP [7], but it has also been
associated with CP [8].
P. gingivalis and T. forsythia are considered as part of the red
complex [9] and is related to CP and periodontal disease progres-
sion [10]. In previous studies from our research group, detection of

0882-4010/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.micpath.2013.04.006
12 S. Tomita et al. / Microbial Pathogenesis 61-62 (2013) 11e15
P. gingivalis and T. forsythia by immunofluorescence technique was
significantly associated with CP [11], and A. actinomycetemcomitans
was detected in half of a group of Japanese CP patients by culture
method [12]. A microbiological diagnosis has been suggested to
complement the clinical diagnosis and to be used for guidance for
more aggressive treatment such as surgery and antimicrobial
therapy [13e16]. With the latest advances in molecular biology
techniques, studies have shown that recognized periodontal
pathogens can also be recovered from healthy individuals, and new
uncultivated species have been associated with periodontitis [17].
Although many studies have aimed to characterize the micro-
biota associated with specific disease type and extent of peri-
odontal destruction, there is still lack of quantitative data on the
levels of these pathogens in AgP and CP. The aim of this retro-
spective study was to evaluate the prevalence and levels of
P. gingivalis, T. forsythia and A. actinomycetemcomitans in sub-
gingival plaque samples of a group of Japanese patients with CP and
AgP, using a real-time polymerase chain reaction (PCR) technique.
2. Materials and methods
2.1. Study population
Study subjects were recruited from the patient population of
Department of Periodontology, Tokyo Dental College, Chiba, Japan,
from November 2008 to November 2012. This study was approved
by the ethics committee of Tokyo Dental College (No.391). Com-
plete medical and dental histories were obtained from all subjects.
Patients were clinically diagnosed as having AgP [18] based on the
following criteria: systemically healthy; history of rapid attachment
loss and bone destruction as well as familial aggregation (if avail-
able); 39 years of age; presence of at least 3 sites on 3 different
teeth (other than incisors or first molars) with a probing depth
(PD)  6 mm and clinical attachment level (CAL)  5 mm, and
bleeding on probing (BOP). Clinical diagnosis of generalized CP was
made when patients were presented >30% of sites with PD and/or
CAL  4 mm, and BOP. Systemic exclusion criteria were the pres-
ence of cardiovascular and respiratory diseases, systemic inflam-
matory conditions, such as diabetes mellitus, or non-plaque
induced oral inflammatory conditions, immunodeficiency, and
current pregnancy or lactation. The patients received no medica-
tion that could affect their periodontal conditions, such as antibi-
otics or anti-inflammatory drugs, for at least 6 months prior to the
microbiological testing. Periodontally healthy volunteers (PH) were
also asked to participate as controls.
2.2. Clinical examination
After collection of full medical and dental histories, a peri-
odontal examination was carried out. The following baseline clin-
ical parameters were recorded: PD was measured using a Williams
probe (#2, YDM, Higashi Matsuyama, Japan) with an approximate
force of 0.2e0.25 N and rounded to the nearest millimeter. CAL was
measured from the cemento-enamel junction to the apical depth of
periodontal probe penetration. BOP was recorded as the presence
or absence of bleeding following measurement of PD. The presence
or absence of supragingival dental plaque at gingival margin was
recorded by the O’Leary Plaque Control Record [19].
2.3. Assessment of subgingival plaque samples
The participants were asked to avoid oral hygiene measures,
eating, drinking for 2 h before collection of subgingival plaque
samples. Samples were obtained from a site with the deepest PD
(excluding teeth with hopeless prognosis) per individual. The
subgingival plaque samples were collected by inserting two sterile
paper points for 30 s into the deepest area of the pocket accessible
after carefully removing the supragingival plaque with sterilized
cotton pellets. They were immediately transferred into sampling
tubes supplied in a commercial kit (GC, Tokyo, Japan) and sent to a
microbiological testing laboratory (GC Oral Check Center, Tokyo,
Japan) for the quantitative analysis of A. actinomycetemcomitans,
P. gingivalis and T. forsythia. The real-time PCR was performed using
methods described previously [20,21]. Briefly, genomic DNA was
extracted from the samples by using of the QuickGene DNA tissue
kit S (KURABO, Osaka, Japan) on the QuickGene-810 extraction
platform according to the manufacturer’s instructions. Primers
were synthesized according to the sequences published previously
[20,21]. Quantitative PCR was performed using 7500 Fast Real-time
PCR System (Applied Biosystems, Foster City, CA, USA) with an
initial cycle of 95  C for 20 s followed by 40 cycles of 95  C for 3 s,
60  C for 30 s. The detection limit is confirmed to be 20 cells/tube.
2.4. Statistical analysis
Differences in clinical parameters among groups were sought
using the KruskaleWallis and c2 test. The prevalence of bacteria
among groups was assessed by c2 test. The differences in bacterial
numbers or proportion within groups were analyzed by Kruskale
Wallis test with Dunn post test. The differences in bacterial
numbers or proportion between groups were analyzed by Manne
Whitney U test or KurskaleWallis test with Dunn post test. The
correlation between bacterial numbers and periodontal parameters
at sampled sites was investigated by Spearman’s rank correlation
analysis. Statistical analysis was performed using a software pack-
age (InStat 3.10, GraphPad Software, La Jolla, CA, USA). P-values less
than 0.05 were considered statistically significant.
3. Results
3.1. Patient demographics and clinical parameters
Forty patients (20 AgP and 20 CP) who met the inclusion criteria
were included in the data analysis. As a control group, 10 peri-
odontally healthy volunteers (PH) were also included. De-
mographic information and periodontal parameters of participants
and sampled sites were listed in Table 1. The PH group had a higher
proportion of men than periodontitis group. The PH and AgP groups
had younger individuals than CP. None of the PH group was
smokers. Mean values of PD, CAL and BOP (%) at the sampled sites in
Table 1
Demographic characteristics and periodontal parameters of participants.
AgP (n ¼ 20) CP (n ¼ 20) PH (n ¼ 10)
a
Men (%) 8 (40) 5 (25) 8 (80)
Age (years; mean  SD)b 33.3  8.1 43.6  11.1 28.7  3.2
Smokers (%) 5 (25) 3(16) 0 (0)
Parameters at sampled sites
PD (mm; mean  SD)c 7.4  1.4 7.2  1.4 2.6  0.5
CAL (mm; mean  SD)c 8.1  2.2 7.8  1.4 2.6  0.5
BOP (% positive sites)d 55.0 52.6 8.3
Full-mouth parameters
PCR (%; mean  SD)e 55.9  16.5 45.3  16.4 32.9  13.1
Number of teeth (n; mean  SD) 27.5  3.4 27.6  2.4 28.7  3.2
a: P < 0.05; c2 test，b: P < 0.0001; KruskaleWallis test (P < 0.05 for AgP vs. CP,
P < 0.001 for CP vs. PH by Dunn post test), c: P < 0.0001; KruskaleWallis test
(P < 0.001 for AgP vs. PH, CP vs. PH by Dunn post test), d: P < 0.0001; c2 test, e:
P ¼ 0.001; KruskaleWallis test (P < 0.001 for AgP vs. PH by Dunn post test).
AgP; aggressive periodontitis, CP; chronic periodontitis, PH; periodontally healthy,
PD; probing depth, CAL; clinical attachment level, BOP; bleeding on probing, PCR;
O’Leary plaque control record.
 S. Tomita et al. / Microbial Pathogenesis 61-62 (2013) 11e15 13

periodontitis groups were significantly greater than those in PH Table 3

group. No significant difference in these periodontal parameters Bacterial counts (104) of total bacteria, A. actinomycetemcomitans, P. gingivalis and
T. forsythia.
was noted between periodontitis groups. Mean value of PCR was
lower in PH group than that in AgP group, although no significant
difference was found between periodontitis groups. All participants
had at least 27 teeth in their mouth.

3.2. Microbiological findings

3.2.1. Prevalence of microorganisms

Prevalence of A. actinomycetemcomitans, P. gingivalis and
T. forsythia is shown in Table 2. None of the target bacteria was
detected in the PH group. Among the pathogens, T. forsythia was
most frequently detected in the subgingival plaque samples from
both AgP (75%) and CP (85%) patients. No statistically significant
differences in the detection of those pathogens were noted be-
tween periodontitis groups. Among subjects with AgP, 6 subjects
harbored only one of the 3 pathogens tested; A. actinomycetem
comitans in one subject, P. gingivalis in one and T. forsythia in 4 (data
not shown). Among those with CP, 4 subjects were found to harbor
T. forsythia only.
When the prevalence was analyzed in terms of the combination
of 3 pathogens, P. gingivalis and T. forsythia were relatively
frequently detected together in periodontitis patients; 7 of 20 pa-
tients in AgP, and 10 of 20 patients in CP. Twenty percent of patients
in AgP and 25% of CP harbored all three pathogens in their peri-
odontal pockets.
3.2.2. Bacterial counts
When the bacterial counts were compared, there was a great
variability between subjects (Table 3). The mean value for total
bacteria in PH group was significantly lower than that in AgP or CP
group. Although the mean values for total bacteria,
A. actinomycetemcomitans, P. gingivalis and T. forsythia appeared to
be greater in AgP group than in CP group, the difference was not
statistically significant.
When the differences in bacterial counts of the 3 pathogens
were analyzed within each periodontitis group, a significant dif-
ference was observed only in CP groups (P ¼ 0.0002 by Kruskale
Wallis test): the bacterial counts for P. gingivalis and T. forsythia was
significantly higher than those for A. actinomycetemcomitans
(P < 0.001 and P < 0.01, respectively, by KruskaleWallis test with
Dunn post test).
a; P < 0.001 vs P. gingivalis, b; P < 0.01 vs T. forsythia within CP group by Kruskal-
Wallis with Dunn post test
*P < 0.001; Kruskal-Wallis test between AgP and PH or CP and PH groups.
difference in proportion of the pathogens was also noted in CP
group (P ¼ 0.0002, KruskaleWallis test): the percentage of
P. gingivalis or T. forsythia was significantly greater than that of
A. actinomycetemcomitans.
No significant differences in the proportion of A. actinomycetem
comitans or P. gingivalis were found between periodontitis groups
(Table 4). However, the proportion of T. forsythia was approximately
4-fold higher in CP group than that in AgP group, a difference which
was statistically significant (P ¼ 0.02).
When the individual risk was assessed according to the refer-
ence value for A. actinomycetemcomitans proposed by the Japanese
Society of Periodontology [22], 3 AgP and 4 CP patients were
considered to be at high risk (A. actinomycetemcomitans  0.1%). As
for P. gingivalis, 10 AgP and 13 CP patients were at high risk
(P. gingivalis  1%).
3.2.4. Correlation of periodontal parameters with the numbers of
pathogens
When correlation analyses were performed between PD at
sampled sites and the bacterial counts in AgP and CP groups, a
significant positive correlation was found with total bacteria,
P. gingivalis and T. forsythia (Table 5). As for CAL, a significant cor-
relation was found with P. gingivalis and T. forsythia. Mean
A. actinomycetamcomitans counts showed no significant correlation
with PD or CAL.

3.2.3. Proportions of A. actinomycetemcomitans, P. gingivalis and

Table 4
T. forsythia
Proportions (%; mean  SD) of A. actinomycetemcomitans, P. gingivalis and T. forsythia
Next, the percentage of the 3 pathogens within the total bac- within the total bacterial counts.
terial population was assessed. (Table 4). In AgP group, a significant
difference in proportion of the pathogens was observed (P ¼ 0.021,
KruskaleWallis test): the percentage of P. gingivalis was signifi-
cantly greater than that of A. actinomycetemcomitans. A significant

Table 2
Prevalence of A. actinomycetemcomitans, P. gingivalis and T. forsythia.

Pathogens AgP CP PH

A. actinomycetemcomitans 7/20 (35%) 5/20 (25%) 0/10 (0%)

P. gingivalis 12/20 (60%) 15/20 (75%) 0/10 (0%)
T. forsythia 15/20 (75%) 17/20 (85%) 0/10 (0%)
Aa þ Pg 0/20 (0%) 1/20 (5%) 0/10 (0%)
Aa þ Tf 2/20 (10%) 0/20 (0%) 0/10 (0%)
Pg þ Tf 7/20 (35%) 10/20 (50%) 0/10 (0%)
Aa þ Pg þ Tf 4/20 (20%) 5/20 (25%) 0/10 (0%)
a; P < 0.05 vs P. gingivalis within AgP group by Kruskal-Wallis with Dunn post test
Not significant (ns) by c2 test between AgP and CP groups. b; P < 0.001 vs P. gingivalis, c; P < 0.01 vs T. forsythia within CP group by Kruskal-
Aa; Aggregatibacter actinomycetemcomitans, Pg; Porphyromonas gingivalis, Tf; Tan- Wallis with Dunn post test
nerella forsythia. * P ¼ 0.02 by Mann-Whiney U test
14 S. Tomita et al. / Microbial Pathogenesis 61-62 (2013) 11e15
Table 5
Correlation between bacterial counts and clinical parameters at sampled sites in AgP
and CP patients.
Total bacteria A. actinomycetem- P. gingivalis T. forsythia
comitans
PD 0.406a 0.230 0.510a 0.584a
(P ¼ 0.01) (P ¼ 0.16) (P < 0.01) (P < 0.01)
CAL 0.277 0.234 0.466a 0.526a
(P ¼ 0.08) (P ¼ 0.15) (P < 0.01) (P < 0.01)
Spearman’s rank correlation analysis (r coefficient is provided).
PD; probing depth, CAL; clinical attachment level.
a
Significant correlation.
3.2.5. Smoking status
A relatively high level of A. actinomycetemcomitans (4.9%) in
one smoker and T. forsythia (2.2%) in another was found in AgP
group. However, no significant difference in the prevalence or
level of any of the pathogens was found between smokers and
non-smokers.
4. Discussion
In the present study, we compared the subgingival microbiota of
periodontally healthy individuals, AgP patients and CP patients,
using the quantitative PCR method. Our findings demonstrate that
P. gingivalis and T. forsythia were highly prevalent in this population
of Japanese periodontitis patients, corroborating the association of
these pathogens with periodontitis. However, no significant dif-
ference in the prevalence or levels of A. actinomycetemcomitans,
P. gingivalis or T. forsythia was found between CP and AgP groups.
Moreover, in most periodontitis patients, these pathogens were
found in combination with one another. These data were consistent
with the report by Socransky and Haffajee [10] showing that certain
bacterial complexes are observed together more frequently than
others in subgingival plaque.
Numerous reports aimed to characterize the microbiota associ-
ated with AgP. Some studies have associated the species
A. actinomycetemcomitans with AgP in different populations [7,23e
25]. On the contrary, lack of association has also been reported
[26], and several studies found this pathogen to be prevalent in in-
dividuals with CP [12,27e29]. In the present study, no significant
difference in the prevalence of A. actinomycetemcomitans was
observed between AgP and CP groups. Furthermore, both bacterial
counts and proportions for A. actinomycetemcomitans were low
when compared to those for P. gingivalis or T. forsythia, even in AgP
patients. In Japanese AgP patients, it was reported that P. gingivalis, T.
forsythia, Campylobacter rectus and Treponema denticola were the
predominant periodontopathic bacteria [30]. In their study, the
prevalence of A. actinomycetemcomitans in AgP subjects was much
lower than that of P. gingivalis. A recent study by Heller et al. [29],
showed that very few subgingival species differed in prevalence and/
or levels between AgP and CP in a sample population in Brazil. The
findings from these studies as well as our study are consistent with
the view that the presence or absence of A. actinomycetemcomitans,
P. gingivalis or T. forsythia cannot discriminate between subjects with
AgP from those with CP.
It has been reported that the main difference between clinical
groups may not be the prevalence but rather the amount of path-
ogens found in positive samples [31]. Therefore, we assessed the
bacterial counts and proportions of A. actinomycetemcomitans,
P. gingivalis or T. forsythia in the subgingival plaque samples. The
statistical analysis showed no significant difference in bacterial
counts of A. actinomycetemcomitans, P. gingivalis and T. forsythia
between AgP and CP subjects. It is, however, important to note that
due to the considerably large SD values resulting from great vari-
ability between individuals, the interpretation of the bacterial
counts was rather difficult.
As for the proportion within the total bacterial counts, a sig-
nificant difference between periodontitis groups was found only
with T. forsythia, showing that CP patients harbored higher pro-
portion of T. forsythia than AgP patients. Although studies have
implicated T. forsythia in the progression of periodontitis [32], its
relationship with various forms of periodontitis remains under-
studied. Our finding that bacterial counts for P. gingivalis and
T. forsythia were positively correlated with PD and CAL further
implicates the role of these red-complex pathogens in the pro-
gression of periodontitis.
As the importance of various microorganisms in periodontal
disease progression has changed from the specific plaque hypoth-
esis in the 80’s over clusters and complexes to the ecological plaque
hypothesis [33,34], clinical relevance of assessment targeting a
particular segment of the subgingival periodontal microbiota is
being questioned. In a clinical setting, however, it is still premature
to introduce the comprehensive microbiological assessment such
as microarray [35], mainly because of the cost involved and the
difficulty in interpreting profile results often complex. Decades of
research efforts have been devoted to understanding the signifi-
cance of selected periodontal pathogens such as A. actinomycetem
comitans, P. gingivalis and T. forsythia. Therefore, we feel it is still
important to analyze the prevalence of the ‘classical’ periodontal
pathogens in relation to periodontal diagnosis and rational treat-
ment of periodontal diseases in one’s own clinical setting. Even if
microbiological testing alone cannot distinguish between AgP and
CP, the question as to whether access to microbiological data may
improve the outcome of periodontal treatment remains to be
clarified [26].
As experienced by many clinicians, there is an overlapping
‘grey area’ in which definition of AgP and CP (according to the
1999 definition) does not allow for a clear-cut diagnosis and
therefore raises the question as to how different these two entities
really are [36]. Large-scale comparative studies to establish spe-
cific risk factors for either of the two disease entities are
necessary.
There were relevant limitations to this study. The patient sample
is small and may not represent subjects with AgP and CP in Japan.
Another limitation is that the subgingival plaque sampling was
performed from a single site (the deepest site) per individual.
Although sampling of the deepest sites would result in successful
recovery of A. actinomycetemcomitans, P. gingivalis and Prevotella
intermedia in subjects who harbored these species [37], it seems
possible to see a different bacterial profile when multiple sampling
was utilized. And our sampling method does not guarantee that
these patients are not colonized by the 3 species tested. Finally, we
could not include other important periodontal pathogens in the
commercial real-time PCR testing. Despite these limitations, the
present study adds to the existing literature by providing salient
information regarding the prevalence of selected periodontal
pathogens in Japanese patients with periodontitis. Given the pol-
ymicrobial nature of periodontitis, a further large-scale study is
needed so as to fully unveil the microbiological profile in patients
with AgP and CP and its significance in the pathogenesis and pro-
gression of two disease entities.
5. Conclusions
Our data showed that P. gingivalis and T. forsythia were highly
prevalent in both AgP and CP subjects. No significant differences in
the prevalence and level of the pathogens were found between
periodontitis groups, although the proportion of T. forsythia was
 S. Tomita et al. / Microbial Pathogenesis 61-62 (2013) 11e15
significantly higher in CP subjects than that in AgP subjects. The
finding that bacterial counts for P. gingivalis and T. forsythia were
positively correlated with PD and CAL in periodontitis groups
further implicates the role of these red-complex pathogens in
periodontitis.
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific
Research (C) 22592317 (to AS) and for Young Scientists (B)
24792339 (to ST) from Japan Society for Promotion of Science. The
authors have no conflicts of interest to disclose.
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