Molecular analysis of oral microbiota

Abstract

The application of molecular, mainly 16S ribosomal RNA (rRNA)-based approaches enables researchers to bypass the cultivation step and has proven its usefulness in studying the microbial composition in a variety of ecosystems, including the human oral cavity. In this mini-review, we describe the impact of these culture-independent approaches on our knowledge of the ecology of the human oral cavity and provide directions for future studies that should emphasize the role of specific strains, species and groups of microbes in periodontal disease.
Recent findings are summarized to elucidate the relationship between periodontal disease and human oral microbiota, including as-yet-to-be-cultured organisms.
The real-time polymerase chain reaction (PCR) method was developed to detect and quantify periodontopathic bacteria, such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis (formerly Bacteroides forsythus) and Treponema denticola. The checkerboard DNA-DNA hybridization technique allowed enumeration of large numbers of species in very large numbers of samples. 16S rRNA gene clone library analysis revealed the diversity of human oral microbiota and the existence of as-yet-to-be-cultured organisms that are presumed periodontal pathogens. In addition, terminal restriction fragment length polymorphism (T-RFLP) analysis was applied for assessment of diversity of human oral microbiota.
Culture-independent approaches are useful for studying the microbial ecology in the human oral cavity and should be useful in the future to elucidate the etiology of periodontal disease.

Full text

Mini review
Molecular analysis of
human oral microbiota
Mitsuo Sakamoto
1
, Makoto
Umeda
2
, Yoshimi Benno
1
1
Microbe Division/Japan Collection of
Microorganisms, RIKEN BioResource Center,
Wako, Saitama and
2
Division of Periodontology,
Department of Hard Tissue Engineering,
Graduate School, Tokyo Medical and Dental
University, Bunkyo-ku, Tokyo, Japan
Periodontal disease, a polymicrobial
mixed infection, is a major oral disease.
It is caused by several microbial spe-
cies, such as Actinobacillus actin-
omycetemcomitans,Fusobacterium
nucleatum,Porphyromonas gingivalis,
Prevotella intermedia,Tannerella
forsythensis (formerly Bacteroides
forsythus) (1) and Treponema denticola.
Analysis of the human oral microbiota
has been limited by conventional cul-
ture-dependent methods; thus, more
oral bacteria remain uncultured and
uncharacterized. Consequently, studies
of causal microorganisms of oral dis-
eases including periodontal disease are,
in general, restricted to cultivable spe-
cies such as the aforementioned path-
ogens. It is therefore probable that a
large number of as-yet-to-be-cultured
organisms present in the human oral
cavity may play a role in periodontal
disease. The best model available at
present for determining microbial
diversity, without cultivation, is based
on isolation of DNA from the target
environment, polymerase chain reac-
tion (PCR) amplification of the ribo-
somal RNA (rRNA) gene, cloning the
amplicons into Escherichia coli, and
sequence analysis of the cloned 16S
rRNA gene inserts (2). These culture-
independent approaches have been
used to determine the diversity of
spirochetes in the subgingival pocket of
subjects with a range of periodontal
Sakamoto M, Umeda M, Benno Y. Molecular analysis of human oral microbiota.
J Periodont Res 2005; 40: 277–285. Blackwell Munksgaard 2005
Objectives: The application of molecular, mainly 16S ribosomal RNA (rRNA)-
based approaches enables researchers to bypass the cultivation step and has
proven its usefulness in studying the microbial composition in a variety of eco-
systems, including the human oral cavity. In this mini-review, we describe the
impact of these culture-independent approaches on our knowledge of the ecology
of the human oral cavity and provide directions for future studies that should
emphasize the role of specific strains, species and groups of microbes in perio-
dontal disease.
Materials and methods: Recent findings are summarized to elucidate the
relationship between periodontal disease and human oral microbiota, including
as-yet-to-be-cultured organisms.
Results: The real-time polymerase chain reaction (PCR) method was developed to
detect and quantify periodontopathic bacteria, such as Actinobacillus actino-
mycetemcomitans,Porphyromonas gingivalis,Prevotella intermedia,Tannerella
forsythensis (formerly Bacteroides forsythus) and Treponema denticola. The
checkerboard DNA–DNA hybridization technique allowed enumeration of large
numbers of species in very large numbers of samples. 16S rRNA gene clone library
analysis revealed the diversity of human oral microbiota and the existence of
as-yet-to-be-cultured organisms that are presumed periodontal pathogens. In
addition, terminal restriction fragment length polymorphism (T-RFLP) analysis
was applied for assessment of diversity of human oral microbiota.
Conclusion: Culture-independent approaches are useful for studying the microbial
ecology in the human oral cavity and should be useful in the future to elucidate the
etiology of periodontal disease.
Mitsuo Sakamoto, PhD, Microbe Division/Japan
Collection of Microorganisms, RIKEN
BioResource Center, 2-1 Hirosawa, Wako,
Saitama 351-0198, Japan
Tel: + 81 48 467 9562
Fax: + 81 48 462 4619
e-mail: sakamoto@jcm.riken.jp
Key words: checkerboard DNA–DNA hybridiza-
tion; real-time polymerase chain reaction; 16S
rRNA gene clone library; terminal restriction
fragment length polymorphism
Accepted for publication November 1, 2004
J Periodont Res 2005; 40; 277–285
All rights reserved
Copyright Blackwell Munksgaard Ltd
JOURNAL OF PERIODONTAL RESEARCH
doi: 10.1111/j.1600-0765.2005.00793.x

conditions, including two healthy, one
adult periodontitis, three acute necro-
tizing ulcerative gingivitis, eight
refractory periodontitis, and one
human immunodeficiency virus (HIV)
periodontitis, and the prevalence of
cultivable and uncultivable treponemes
in oral diseases (3). In this mini-review,
we discuss the relationships between
periodontal disease and human oral
microbiota including as-yet-to-be-
cultured organisms.
Human oral spirochetes
The bacterial flora associated with
gingivitis (4, 5) and periodontitis (6)
have been investigated. Spirochetes are
the predominant microorganisms
known to proliferate in periodontal
disease sites among the bacterial flora.
Although the relationship between
periodontitis and oral treponemes has
been emphasized clinically, cultivation
studies of oral treponemes are limited
because of the oxygen sensitivity and
unique nutritional requirements of
these microorganisms and the long
cultivation period (7, 8). The following
species of cultivable oral treponemes
have been validated: Treponema amylo-
vorum (9), T. denticola (10), T. leci-
thinolyticum (11), T. maltophilum (12),
T. medium (13, 14), T. parvum (15),
T. pectinovorum (16), T. putidum (17),
T. socranskii (18), and ÔT. vincentiiÕ.
(The last treponeme has not been val-
idated in a peer-reviewed publication.
It has been published however, in
Bergey’s Manual of Systematic Bac-
teriology (19) and is commonly used.)
These species are classified into two
groups according to the fermentation
of carbohydrates. The saccharolytic
oral treponemes contain six species
(T.amylovorum,T. lecithinolyticum,
T. maltophilum,T. parvum,T. pectino-
vorum, and T. socranskii), and the
asaccharolytic oral treponemes contain
four species (T. denticola,T. medium,
T. putidum, and ÔT. vincentiiÕ). Paster
et al. (20) reported the phylogeny of
cultivable oral treponemes isolated by
Robert Smibert (Virginia Polytechnic
Institute, Blacksburg, VA, USA). They
proposed three novel species (Trepo-
nema Smibert-2, Treponema Smibert-3,
and Treponema Smibert-5) based on
16S rRNA gene sequence comparisons.
Treponema Smibert-2 was later con-
sidered a novel species, Treponema
parvum (15). The taxonomy of oral
spirochetes has been discussed in a
mini-review article (21).
Among the cultivable oral trepo-
nemes, T. denticola is frequently isola-
ted from sites of severe infection in
patients with periodontitis (22), and
many studies have attempted to
elucidate the role of T. denticola
in periodontitis (23–25). Although
T. socranskii is frequently isolated
from the subgingival plaque samples of
periodontitis patients, in addition to
T. denticola, it is difficult to cultivate
and identify (26, 27). The PCR tech-
nique can be used to detect and iden-
tify T. socranskii (28). This technique is
a rapid and reliable method for differ-
entiating T. socranskii from other cul-
tivable oral treponemes. Takeuchi
et al. (29) used this PCR technique
to identify T. socranskii in addition to
T. denticola and P. gingivalis and to
clarify the relationship between the
presence of these microorganisms and
the severity of clinical periodontal
parameters. Their findings suggest that
T. socranskii,T. denticola, and P. gin-
givalis are associated with the severity
of periodontal tissue destruction. In
addition, restriction fragment length
polymorphism (RFLP) analysis of 16S
rRNA genes amplified by PCR was
used to differentiate three subspecies of
T. socranskii (28). Recently, the rela-
tionship between T. socranskii ssp.
buccale and periodontal disease was
emphasized (30, 31). 16S rRNA gene
PCR-RFLP analysis was also used to
differentiate cultivable oral trepo-
nemes, including T. denticola,T. med-
ium,T. pectinovorum,T. socranskii,
and ÔT. vincentiiÕ(32). Furthermore,
species-specific nested PCR was used
to detect T.amylovorum,T. denticola,
T. maltophilum,T. medium,T. pecti-
novorum,T. socranskii, and ÔT. vincen-
tiiÕin dental plaques (33).
Detection and quantification of
periodontopathic bacteria
The relationship between periodontal
disease and detection frequency of
putative periodontal pathogens was
exhaustively evaluated using PCR of
the 16S rRNA genes (34–38). These
findings suggest that several species are
strongly associated with periodontitis.
Accurate quantification of perio-
dontal pathogens in clinical samples
(saliva and subgingival plaque) is need-
ed for understanding the etiologic role
of these bacteria. The conventional
PCR (endpoint PCR) method detects
the plateau phase of the reaction, but is
not suitable for quantification of the
pathogens. In contrast, the real-time
PCR method allows monitoring of the
exponential phase. This method allows
rapid detection and quantification of
the bacteria in clinical samples. Real-
time PCR using the TaqMan system
was first used to quantitate T. forsy-
thensis in subgingival plaque (39).
Subsequently, this system was used to
determine both the density of P. gin-
givalis and the total number of bac-
terial cells in plaque samples (40). In
addition, real-time PCR using SYBR
Green dye and LightCycler system
(Roche Diagnostics, Mannheim, Ger-
many) was first used to detect and
quantify periodontopathic bacteria,
such as A. actinomycetemcomitans,
P. gingivalis,T. forsythensis,T. denti-
cola, and T. socranskii, in saliva and
subgingival plaque samples (41). Using
the LightCycler system, it is possible
to determine the amount of perio-
dontopathic bacteria within 1 hour
(A. actinomycetemcomitans: 40 min,
P. gingivalis: 34 min, T. forsythensis:
40 min, T. denticola: 32 min, T. soc-
ranskii: 46 min). Maeda et al. (42)
suggested that there was no significant
difference between the TaqMan and
SYBR Green chemistry in their spe-
cificity, quantitativity, and sensitivity.
In addition, they suggested that, since
the TaqMan assay required additional
manipulation and cost for the probe,
the SYBR Green assay might be suit-
able for routine clinical examinations.
Currently, detection and quantification
of periodontopathic bacteria by real-
time PCR method are generalized in
this field and many studies have
reported the usefulness of real-time
PCR (43–52). We anticipate that the
real-time PCR method will become in
the future an indispensable method for
the diagnosis of periodontal disease,
278 Sakamoto et al.

evaluation of treatment, and prognos-
tic judgment.
Enumeration of bacterial species in
complex microbial ecosystems using
checkerboard DNA–DNA
hybridization
It has been difficult to conduct large-
scale studies of microbiologically
complex ecosystems using conven-
tional microbiological techniques. The
real-time PCR technique mentioned
above is not particularly suitable for
the examination of large numbers of
samples for large numbers of different
species. In contrast, molecular identi-
fication techniques in new probe-target
formats, such as checkerboard DNA–
DNA hybridization, permit enumer-
ation of large numbers of species in
very large numbers of samples (53).
The checkerboard DNA–DNA
hybridization technique was first des-
cribed in 1994 by Socransky et al. (54).
Using 40 species-specific DNA–DNA
hybridization probes to detect oral
bacteria, it was revealed that subgin-
gival plaque contains bacterial species
in different complexes (55). Socransky
et al. (55) observed five major com-
plexes using cluster analysis (Table 1).
The red complex, consisting of P. gin-
givalis,T. forsythensis, and T. dentico-
la, showed the strongest relationship
with clinical measures of peridontal
disease, particularly pocket depth and
bleeding on probing. The checker-
board DNA–DNA hybridization
technique has been used to compre-
hensively examine the microbial com-
position of supra and subgingival
plaque in subjects in health and perio-
dontitis (56, 57), the salivary micro-
biota levels in relation to periodontal
status (58), the relationship of cigarette
smoking to the composition of the
subgingival microbiota (59, 60), the
differences between the subgingival
microbiota in subjects from dif-
ferent geographic locations (61), the
relationship of ethnic/racial group,
occupational and periodontal disease
status (62), and effects of different
periodontal therapies (63, 64).
Recently, it was reported that the
checkerboard DNA–DNA hybridiza-
tion technique is useful for the enu-
meration of bacterial species in
microbiologically complex systems
(53). This technique is rapid, sensitive,
and relatively inexpensive. It over-
comes many of the limitations of cul-
tivation-based approaches. Paster
et al. (65) developed a PCR-based,
reverse capture, checkerboard hybrid-
ization protocol to differentiate
between species of oral streptococci,
which are very closely related phylo-
genetically. Based on these techniques,
DNA microarray will be developed in
the near future.
Bacterial diversity in the human
oral cavity
16S rRNA gene clone library analysis
was first used in 1994 to determine the
genetic diversity of cultivable and
uncultivable spirochetes in the gingival
crevice of a patient with severe perio-
dontitis by Choi et al. (66). These
investigators found that the clones fell
into 23 clusters differing by about
1–2%. Their findings indicate an
unexpected diversity of oral trepo-
nemes from a single patient. There-
after, this method was applied to
analyze the diversity of asaccharolytic
Eubacterium species (67). In addition,
Kroes et al. (68) used this method to
characterize the breadth of bacterial
diversity within the human subgingival
crevice. Although the subject popula-
tion was small, Sakamoto et al. (69)
also used this method to compare the
oral microbiota in the saliva from two
patients with periodontitis and from a
periodontally healthy subject. There
was no clonal sequence affiliated with
periodontopathic bacteria in the saliva
from the healthy subject, whereas a
number of periodontal pathogens such
as Campylobacter rectus,P. intermedia,
P. gingivalis, and T. socranskii were
detected in the saliva from the patients
with periodontitis. In addition, a
number of previously uncharacterized
and uncultured microorganisms were
recognized. Subsequently, Paster et al.
Table 1. Microbial complexes in subgingival plaque
Complex Species
Red complex Porphyromonas gingivalis
Tannerella forsythensis
Treponema denticola
Orange complex Campylobacter gracilis
Campylobacter rectus
Campylobacter showae
Eubacterium nodatum
Fusobacterium nucleatum ssp. nucleatum
Fusobacterium nucleatum ssp. polymorphum
Fusobacterium nucleatum ssp. vincentii
Fusobacterium periodonticum
Peptostreptococcus micros
Prevotella intermedia
Prevotella nigrescens
Streptococcus constellatus
Green complex Actinobacillus actinomycetemcomitans serotype a
Campylobacter concisus
Capnocytophaga gingivalis
Capnocytophaga ochracea
Capnocytophaga sputigena
Eikenella corrodens
Yellow complex Streptococcus gordonii
Streptococcus intermedius
Streptococcus mitis
Streptococcus oralis
Streptococcus sanguis
Purple complex Actinomyces odontolyticus
Veillonella parvula
Other species Actinobacillus actinomycetemcomitans serotype b
Actinomyces naeslundii genospecies 2 (A. viscosus)
Selenomonas noxia
Molecular analysis of oral microbiota 279

(31) demonstrated that the predomi-
nant subgingival bacterial community
consisted of 347 species or phylotypes,
based on analysis of 2522 16S rRNA
clones and estimated that the total
species diversity in the oral cavity is
approximately 500 species. A similar
technique was also used to compare the
bacteria found in children with severe
caries to those found in caries-free
children (70) and to determine the
prevalent species and phylotypes in
advanced lesions of children with
noma (71). According to the most
recent report (72), it is presumed that
over 700 bacterial species (phylotypes
are included) inhabit the oral cavity,
and more than half of these cannot be
cultivated.
Detection of novel oral phylotypes
associated with periodontitis
Leys et al. (73) investigated the rela-
tionship between the presence of
T. forsythensis and a novel phylotype,
oral clone BU063 identified by Paster
et al. (31), and periodontal health sta-
tus. Harper-Owen et al. (74) designed
and validated phylotype-specific PCR
primers for phylotypes PUS3.42,
PUS9.170, and PUS9.180 identified by
Dymock et al. (75) and determined
their incidences in subgingival plaque
samples from subjects with periodon-
titis and from healthy controls. In a
previous study (69), a number of novel
oral phylotypes, representing as yet
uncultured organisms, were identified.
Among these phylotypes, Sakamoto
et al. (76) designed specific PCR
primers for five phylotypes AP12,
AP21, AP24, AP50, and RP58, which
are deeply branched particularly in the
phylogenetic tree, and determined the
prevalence of these phylotypes in 45
patients with periodontitis and 18
healthy subjects. Among the phylo-
types tested, phylotype AP24, which is
closely related to oral clone DA014
(99% sequence similarity) reported
previously (31), was significantly asso-
ciated with saliva and subgingival pla-
que samples from patients with
periodontitis (p<0.01), but the dif-
ference was not statistically significant
in the presence of other phylotypes.
These data suggest that phylotype
AP24 may play an important role in
periodontal disease. It is important to
examine not only known periodonto-
pathic bacteria but also as-yet-to-
be-cultured organisms in the study
of periodontal disease. Although
attempts have been made to isolate
phylotype AP24 from subgingival pla-
que and saliva samples, such attempts
have not yet been successful. However,
novel Prevotella species were isolated
from the human oral cavity in the
process of the research (77, 78).
Recently, it was reported that
members of the uncultivated bacterial
division TM7 (79), which have been
detected in the human oral cavity (31),
might play a role in the multifactorial
process leading to periodontitis (80). In
contrast, several phylotypes were
associated with periodontal health (73,
81). Kumar et al. (81) reported that
clone W090 from the Deferribacteres
phylum and clone BU063 from the
Bacteroidetes phylum were associated
with periodontal health. In the future,
as a new index of periodontal disease,
it is expected that the relationship
between periodontal disease and other
novel phylotypes is investigated in
more detail.
Application of terminal restriction
fragment length polymorphism
analysis in periodontics
A phylogenetic approach based on
16S rRNA has been applied to
investigate the diversity of cultivable
and uncultivable species in the human
oral cavity, without requiring culti-
vation (30, 31, 68, 69). 16S rRNA
gene clone library analysis can pro-
vide direct sequence information.
However, analysis of individual 16S
rRNA clones is an expensive and
extremely inefficient approach for
comparison of a multitude of bacter-
ial communities.
Terminal restriction fragment length
polymorphism (T-RFLP) is an alter-
native molecular approach that allows
the assessment of a diversity of com-
plex bacterial communities and rapid
comparison of the community struc-
ture and diversity of different ecosys-
tems (82). This technique has been used
for assessing the diversity and structure
of complex bacterial communities in
various environments (83–90) and has
been evaluated in separate review arti-
cles (91, 92). In addition, the T-RFLP
analysis program (TAP) has been
developed and published on the
worldwide web (http://rdp.cme.msu.
edu/html/analyses.html) (93).
Sakamoto et al. (94) used T-RFLP
analysis to characterize and compare
oral microbiota present in saliva sam-
ples of 18 healthy subjects and 18
patients with periodontitis. They pre-
sented the first report on characteriza-
tion of oral microbiota based on
T-RFLP patterns. Their study indica-
ted that T-RFLP analysis is useful for
the assessment of diversity of oral
microbiota and rapid comparison of
the community structure between sub-
jects with and without periodontitis. In
contrast, two groups (95, 96) used
denaturing gradient gel electrophoresis
(DGGE) analysis to study bacterial
community structure in pockets of
periodontitis patients. However, it is
difficult to create a database from the
band profiles obtained by DGGE
analysis compared with the terminal
restriction fragment (T-RF) profiles
obtained by T-RFLP analysis. T-RF
lengths can be predicted from known
16S rRNA gene sequences. Multiple
species can be predicted for the same
T-RF length, but it is possible to
identify bacterial species by analysis
of digests with multiple restriction
enzymes. Changes in the subgingival
microbiota in adult Down’s syndrome
patients with periodontitis (63), and
adult periodontitis patients after sca-
ling and root planing (97) or antibiotic
(amoxicillin or metronidazole) therapy
combined with scaling and root pla-
ning (62) have been investigated using
checkerboard DNA–DNA hybridiza-
tion or PCR techniques. However,
these studies report changes in only a
limited part, which represents the cul-
tivable known species, of the subgin-
gival microbiota. Recently, Zijnge
et al. (96) used DGGE analysis, which
takes into account the presence of
unidentified and hard-to-cultivate spe-
cies present in the subgingival plaque
(like T-RFLP analysis), to study
shifts in the subgingival microbiota
before, 1 day after and 3 months after
280 Sakamoto et al.

treatment. Although the subject popu-
lation was small, Sakamoto et al. (98)
used T-RFLP analysis to study the
change of oral microbiota in saliva and
subgingival plaque samples of patients
with periodontitis before and 3 months
after periodontal treatment. Significant
changes in the T-RFLP patterns of
subgingival plaque samples of the
patients were noted after 3 months of
improved oral hygiene, and full mouth
supra- and subgingival scaling and
root planing (Fig. 1). Although the
proportions of T-RFs larger than
1000 bp were notable in the T-RFLP
patterns generated after digestion with
HhaI of the samples from the patients
before treatment, the proportions of
0 100 200 300 400 500 600 700 800 900 1000 11000 100 200 300 400 500 600 700 800 900 1000 1100
Peptostreptococcus sp.
(clone AJ062, AP24, BS044 & FG014)
S. anginosus,
S. intermedius,
V. dispar
Fi. alocis
E. saphenum,
S. cristatus,
S. mitis,
S. salivalius,
S. sanguinis
Fu. nucleatum
Po. gingivalis
Pr. intermedia
S. anginosus,
S. gordonii,
S. intermedius,
V.atypica,
V.dispar,
V.parvula
S. cristatus,
S. mitis,
S. salivalius,
S. sanguinis
‘T. aromaticivorans’
S. mutans
Fu. nucleatum N. pharyngis
Terminal restriction fragment (bp)
Terminal restriction fragment (bp)
Relative fluorescence units
Relative fluorescence units
Before
After
0 100 200 300 400 500 600 7000 100 200 300 400 500 600 700
Before
After
S. anginosus,
S. intermedius
S. cristatus,
S. mitis,
S. salivalius,
S. sanguinis
Fi. alocis
Eubacterium
sp.
(clone AP54)
V. dispar
E. nodatum,
Peptostreptococcus
sp.
(clone AJ062, AP24, BS044 & FG014)
Fu. nucleatum
E. saphenum
Eubacterium
sp.
(clone BP1-27 & PUS9.170)
Po. gingivalis Pr. intermedia
S. anginosus,
S. gordonii,
S. intermedius,
S. mutans
S. cristatus,
S. mitis,
S. salivalius,
S. sanguinis
N. pharyngis
‘T. aromaticivorans ’
V. atypica,
V. dispar,
V. parvula
Fu. nucleatum
(a)
(b)
Fig. 1. Terminal restriction fragment length polymorphism patterns of 16S rRNA genes from subgingival plaque samples of a patient with
periodontitis taken before treatment and after treatment generated after digestion with HhaI (a) and MspI (b). 16S rRNA genes were
amplified with universal primers 27F and 1492R. Almost all the terminal restriction fragments were presumed to be species or phylotypes
detected by the 16S rRNA gene clone library analysis. E., Eubacterium;Fi., Filifactor;Fu., Fusobacterium;N., Neisseria;Po., Porphyromonas;
Pr., Prevotella;S., Streptococcus;T., ÔTerrahaemophilusÕ;V., Veillonella. Reproduced with permission from Sakamoto et al. (98). (2004)
Society for General Microbiology.
Molecular analysis of oral microbiota 281

these T-RFs were significantly reduced
or not detected after treatment. T-RFs
larger than 1000 bp comprised several
phylotypes of Peptostreptococcus spe-
cies, including phylotype AP24 (76).
Peptostreptococcus species are mem-
bers of the normal commensal flora of
humans and animals, but some species
are associated with anaerobic infec-
tions, including gingivitis and perio-
dontitis. Peptostreptococcus micros has
been associated with periodontal dis-
ease (99). Consequently, monitoring of
the proportion of T-RFs larger than
1000 bp in the T-RFLP pattern may be
useful for the evaluation of the prog-
nosis of periodontal disease. T-RFLP
analysis data were in agreement with
real-time PCR and 16S rRNA gene
clone library analysis data. After
3 months, the P. gingivalis population
was markedly reduced (3.1 ·10
)3
%),
although the proportion of P. gingiva-
lis before treatment was 7.6%. The
proportion of the T-RF presumed to
be P. gingivalis was 5.9%, and became
undetected after 3 months. In addition,
T-RF presumed to be P. intermedia,
which is an important periodontal
pathogen, could be differentiated from
the T-RF presumed to be P. gingivalis
by being 2-bp larger. Before treatment,
the proportion of the T-RF presumed
to be P. intermedia was 2.8%. After
3 months, this T-RF was not detected.
These findings indicate that T-RFLP
analysis is useful for evaluation of the
effects of medical treatment of perio-
dontitis. However, further analyzes of
digests with multiple restriction
enzymes (four or five) are necessary
because multiple species, which belong
to different genera, were detected from
the same T-RF.
Conclusion
With the advancement of molecular
biology in recent years, the initiation
and progression mechanisms of perio-
dontitis are becoming clearer gradually.
As the culture-independent approaches
have revealed the diversity of human
oral microbiota and the existence of a
large number of as-yet-to-be-cultured
organisms which are presumed as per-
iodontal pathogens, the researches on
periodontal disease and human oral
microbiota are coming to a new turn.
Using the sequence information of 16S
rRNA gene obtained, it is possible to
detect not only known oral species but
also the newly identified uncultivated
species (phylotypes) directly in clinical
samples. At present, a Human Oral
Microbe Identification Microarray
(HOMIM) slide system for the identi-
fication of essentially all of the more
than 600 species encountered in the oral
cavity is under development (100). This
microarray should be extremely useful
in clinical studies to simultaneously
examine the roles of all bacterial species
present at sites of oral diseases. The
culture-independent approaches des-
cribed in this mini-review will become
indispensable in the future to elucidate
the etiology of periodontal disease.
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