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Microbial Pathogenesis: Short Communication
Microbial Pathogenesis: Short Communication
Microbial Pathogenesis: Short Communication
Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath
Short communication
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 [4]. Using these criteria, strong evidence has been demonstrated
for Aggregatibacter (Actinobacillus) actinomycetemcomitans, Por-
Periodontal disease is a plaque biofilm- induced inflammatory phyromonas gingivalis and Tannerella forsythia, as concluded at the
disease of the supporting tissue of the tooth [1]. It can be described World Workshop in 1996 [5].
as one of the predominant polymicrobial infections of humans [2]. Aggressive periodontitis (AgP) is a specific type of periodontitis
So far, more than 700 bacterial taxa have been identified in samples with clearly identifiable clinical and laboratory findings which
taken from oral cavities as identified by cultivation or traditional make it sufficiently different from chronic periodontitis (CP) [6].
cloning and sequencing [3]. Evidence for periodontal etiology is The common features of AgP are: except for the presence of peri-
based on the fulfillment of several criteria described by Socransky 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
* Corresponding author. Department of Periodontology, Tokyo Dental College, associated with CP [8].
1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan. Tel.: þ81 43 270 3953;
fax: þ81 43 270 3955.
P. gingivalis and T. forsythia are considered as part of the red
E-mail addresses: atsaito@tdc.ac.jp, atsaito1@gmail.com (A. Saito). complex [9] and is related to CP and periodontal disease progres-
1
These authors contributed equally to this work. 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 subgingival plaque samples were collected by inserting two sterile
significantly associated with CP [11], and A. actinomycetemcomitans paper points for 30 s into the deepest area of the pocket accessible
was detected in half of a group of Japanese CP patients by culture after carefully removing the supragingival plaque with sterilized
method [12]. A microbiological diagnosis has been suggested to cotton pellets. They were immediately transferred into sampling
complement the clinical diagnosis and to be used for guidance for tubes supplied in a commercial kit (GC, Tokyo, Japan) and sent to a
more aggressive treatment such as surgery and antimicrobial microbiological testing laboratory (GC Oral Check Center, Tokyo,
therapy [13e16]. With the latest advances in molecular biology Japan) for the quantitative analysis of A. actinomycetemcomitans,
techniques, studies have shown that recognized periodontal P. gingivalis and T. forsythia. The real-time PCR was performed using
pathogens can also be recovered from healthy individuals, and new methods described previously [20,21]. Briefly, genomic DNA was
uncultivated species have been associated with periodontitis [17]. extracted from the samples by using of the QuickGene DNA tissue
Although many studies have aimed to characterize the micro- kit S (KURABO, Osaka, Japan) on the QuickGene-810 extraction
biota associated with specific disease type and extent of peri- platform according to the manufacturer’s instructions. Primers
odontal destruction, there is still lack of quantitative data on the were synthesized according to the sequences published previously
levels of these pathogens in AgP and CP. The aim of this retro- [20,21]. Quantitative PCR was performed using 7500 Fast Real-time
spective study was to evaluate the prevalence and levels of PCR System (Applied Biosystems, Foster City, CA, USA) with an
P. gingivalis, T. forsythia and A. actinomycetemcomitans in sub- initial cycle of 95 C for 20 s followed by 40 cycles of 95 C for 3 s,
gingival plaque samples of a group of Japanese patients with CP and 60 C for 30 s. The detection limit is confirmed to be 20 cells/tube.
AgP, using a real-time polymerase chain reaction (PCR) technique.
2.4. Statistical analysis
2. Materials and methods
Differences in clinical parameters among groups were sought
2.1. Study population using the KruskaleWallis and c2 test. The prevalence of bacteria
among groups was assessed by c2 test. The differences in bacterial
Study subjects were recruited from the patient population of numbers or proportion within groups were analyzed by Kruskale
Department of Periodontology, Tokyo Dental College, Chiba, Japan, Wallis test with Dunn post test. The differences in bacterial
from November 2008 to November 2012. This study was approved numbers or proportion between groups were analyzed by Manne
by the ethics committee of Tokyo Dental College (No.391). Com- Whitney U test or KurskaleWallis test with Dunn post test. The
plete medical and dental histories were obtained from all subjects. correlation between bacterial numbers and periodontal parameters
Patients were clinically diagnosed as having AgP [18] based on the at sampled sites was investigated by Spearman’s rank correlation
following criteria: systemically healthy; history of rapid attachment analysis. Statistical analysis was performed using a software pack-
loss and bone destruction as well as familial aggregation (if avail- age (InStat 3.10, GraphPad Software, La Jolla, CA, USA). P-values less
able); 39 years of age; presence of at least 3 sites on 3 different than 0.05 were considered statistically significant.
teeth (other than incisors or first molars) with a probing depth
(PD) 6 mm and clinical attachment level (CAL) 5 mm, and
3. Results
bleeding on probing (BOP). Clinical diagnosis of generalized CP was
made when patients were presented >30% of sites with PD and/or
3.1. Patient demographics and clinical parameters
CAL 4 mm, and BOP. Systemic exclusion criteria were the pres-
ence of cardiovascular and respiratory diseases, systemic inflam-
Forty patients (20 AgP and 20 CP) who met the inclusion criteria
matory conditions, such as diabetes mellitus, or non-plaque
were included in the data analysis. As a control group, 10 peri-
induced oral inflammatory conditions, immunodeficiency, and
odontally healthy volunteers (PH) were also included. De-
current pregnancy or lactation. The patients received no medica-
mographic information and periodontal parameters of participants
tion that could affect their periodontal conditions, such as antibi-
and sampled sites were listed in Table 1. The PH group had a higher
otics or anti-inflammatory drugs, for at least 6 months prior to the
proportion of men than periodontitis group. The PH and AgP groups
microbiological testing. Periodontally healthy volunteers (PH) were
had younger individuals than CP. None of the PH group was
also asked to participate as controls.
smokers. Mean values of PD, CAL and BOP (%) at the sampled sites in
Table 2
Prevalence of A. actinomycetemcomitans, P. gingivalis and T. forsythia.
Pathogens AgP CP PH
Table 5 due to the considerably large SD values resulting from great vari-
Correlation between bacterial counts and clinical parameters at sampled sites in AgP ability between individuals, the interpretation of the bacterial
and CP patients.
counts was rather difficult.
Total bacteria A. actinomycetem- P. gingivalis T. forsythia As for the proportion within the total bacterial counts, a sig-
comitans nificant difference between periodontitis groups was found only
PD 0.406a 0.230 0.510a 0.584a with T. forsythia, showing that CP patients harbored higher pro-
(P ¼ 0.01) (P ¼ 0.16) (P < 0.01) (P < 0.01) portion of T. forsythia than AgP patients. Although studies have
CAL 0.277 0.234 0.466a 0.526a
implicated T. forsythia in the progression of periodontitis [32], its
(P ¼ 0.08) (P ¼ 0.15) (P < 0.01) (P < 0.01)
relationship with various forms of periodontitis remains under-
Spearman’s rank correlation analysis (r coefficient is provided).
studied. Our finding that bacterial counts for P. gingivalis and
PD; probing depth, CAL; clinical attachment level.
a
Significant correlation.
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
3.2.5. Smoking status
disease progression has changed from the specific plaque hypoth-
A relatively high level of A. actinomycetemcomitans (4.9%) in
esis in the 80’s over clusters and complexes to the ecological plaque
one smoker and T. forsythia (2.2%) in another was found in AgP
hypothesis [33,34], clinical relevance of assessment targeting a
group. However, no significant difference in the prevalence or
particular segment of the subgingival periodontal microbiota is
level of any of the pathogens was found between smokers and
being questioned. In a clinical setting, however, it is still premature
non-smokers.
to introduce the comprehensive microbiological assessment such
as microarray [35], mainly because of the cost involved and the
4. Discussion difficulty in interpreting profile results often complex. Decades of
research efforts have been devoted to understanding the signifi-
In the present study, we compared the subgingival microbiota of cance of selected periodontal pathogens such as A. actinomycetem
periodontally healthy individuals, AgP patients and CP patients, comitans, P. gingivalis and T. forsythia. Therefore, we feel it is still
using the quantitative PCR method. Our findings demonstrate that important to analyze the prevalence of the ‘classical’ periodontal
P. gingivalis and T. forsythia were highly prevalent in this population pathogens in relation to periodontal diagnosis and rational treat-
of Japanese periodontitis patients, corroborating the association of ment of periodontal diseases in one’s own clinical setting. Even if
these pathogens with periodontitis. However, no significant dif- microbiological testing alone cannot distinguish between AgP and
ference in the prevalence or levels of A. actinomycetemcomitans, CP, the question as to whether access to microbiological data may
P. gingivalis or T. forsythia was found between CP and AgP groups. improve the outcome of periodontal treatment remains to be
Moreover, in most periodontitis patients, these pathogens were clarified [26].
found in combination with one another. These data were consistent As experienced by many clinicians, there is an overlapping
with the report by Socransky and Haffajee [10] showing that certain ‘grey area’ in which definition of AgP and CP (according to the
bacterial complexes are observed together more frequently than 1999 definition) does not allow for a clear-cut diagnosis and
others in subgingival plaque. therefore raises the question as to how different these two entities
Numerous reports aimed to characterize the microbiota associ- really are [36]. Large-scale comparative studies to establish spe-
ated with AgP. Some studies have associated the species cific risk factors for either of the two disease entities are
A. actinomycetemcomitans with AgP in different populations [7,23e necessary.
25]. On the contrary, lack of association has also been reported There were relevant limitations to this study. The patient sample
[26], and several studies found this pathogen to be prevalent in in- is small and may not represent subjects with AgP and CP in Japan.
dividuals with CP [12,27e29]. In the present study, no significant Another limitation is that the subgingival plaque sampling was
difference in the prevalence of A. actinomycetemcomitans was performed from a single site (the deepest site) per individual.
observed between AgP and CP groups. Furthermore, both bacterial Although sampling of the deepest sites would result in successful
counts and proportions for A. actinomycetemcomitans were low recovery of A. actinomycetemcomitans, P. gingivalis and Prevotella
when compared to those for P. gingivalis or T. forsythia, even in AgP intermedia in subjects who harbored these species [37], it seems
patients. In Japanese AgP patients, it was reported that P. gingivalis, T. possible to see a different bacterial profile when multiple sampling
forsythia, Campylobacter rectus and Treponema denticola were the was utilized. And our sampling method does not guarantee that
predominant periodontopathic bacteria [30]. In their study, the these patients are not colonized by the 3 species tested. Finally, we
prevalence of A. actinomycetemcomitans in AgP subjects was much could not include other important periodontal pathogens in the
lower than that of P. gingivalis. A recent study by Heller et al. [29], commercial real-time PCR testing. Despite these limitations, the
showed that very few subgingival species differed in prevalence and/ present study adds to the existing literature by providing salient
or levels between AgP and CP in a sample population in Brazil. The information regarding the prevalence of selected periodontal
findings from these studies as well as our study are consistent with pathogens in Japanese patients with periodontitis. Given the pol-
the view that the presence or absence of A. actinomycetemcomitans, ymicrobial nature of periodontitis, a further large-scale study is
P. gingivalis or T. forsythia cannot discriminate between subjects with needed so as to fully unveil the microbiological profile in patients
AgP from those with CP. with AgP and CP and its significance in the pathogenesis and pro-
It has been reported that the main difference between clinical gression of two disease entities.
groups may not be the prevalence but rather the amount of path-
ogens found in positive samples [31]. Therefore, we assessed the 5. Conclusions
bacterial counts and proportions of A. actinomycetemcomitans,
P. gingivalis or T. forsythia in the subgingival plaque samples. The Our data showed that P. gingivalis and T. forsythia were highly
statistical analysis showed no significant difference in bacterial prevalent in both AgP and CP subjects. No significant differences in
counts of A. actinomycetemcomitans, P. gingivalis and T. forsythia the prevalence and level of the pathogens were found between
between AgP and CP subjects. It is, however, important to note that periodontitis groups, although the proportion of T. forsythia was
S. Tomita et al. / Microbial Pathogenesis 61-62 (2013) 11e15 15
significantly higher in CP subjects than that in AgP subjects. The [17] Shaddox LM, Huang H, Lin T, Hou W, Harrison PL, Aukhil I, et al. Microbio-
logical characterization in children with aggressive periodontitis. J Dent Res
finding that bacterial counts for P. gingivalis and T. forsythia were
2012;91:927e33.
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Acknowledgments Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. J Clin
Microbiol 2003;41:863e6.
[21] Suzuki N, Yoshida A, Saito T, Kawada M, Nakano Y. Quantitative microbio-
This work was supported in part by Grants-in-Aid for Scientific
logical study of subgingival plaque by real-time PCR shows correlation be-
Research (C) 22592317 (to AS) and for Young Scientists (B) tween levels of Tannerella forsythensis and Fusobacterium spp. J Clin Microbiol
24792339 (to ST) from Japan Society for Promotion of Science. The 2004;42:2255e7.
authors have no conflicts of interest to disclose. [22] Japanese Society of Periodontology. Guideline for antimicrobial treatment in
patients with periodontal disease 2010. Tokyo: Japanese Society of Peri-
odontology; 201180 [in Japanese].
[23] Leung WK, Ngai VKS, Yau JYY, Cheung BPK, Tsang PWK, Corbet EF. Charac-
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