Journal of Oral Biosciences 54 (2012) 128131

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Journal of Oral Biosciences

journal homepage: www.elsevier.com/locate/job

Review

Evaluation of salivary microbiota from an ecological perspective

Toru Takeshita n, Yoshihisa Yamashita
Section of Preventive and Public Health Dentistry, Division of Oral Health, Development and Growth, Kyushu University Faculty of Dental Science, Fukuoka 812-8582, Japan

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

Article history: A variety of bacteria densely colonize the human oral cavity not as individuals, but as members of an
Received 13 January 2012 indigenous microbiota that exhibit extensive intracellular interactions. To prevent the onset of oral
Received in revised form diseases caused by the members of this microbiota, regulation of the total oral microbiota, including
26 March 2012
the surrounding bacterial environment, is required. In this review, we highlight current knowledge on
Accepted 4 April 2012
Available online 9 August 2012
the global composition of the salivary bacterial population associated with oral conditions through a
molecular ecological approach using the 16S rRNA gene. The salivary bacterial populations of Japanese
Keywords: subjects were commonly dominated by bacterial genera such as Streptococcus, Prevotella, and Neisseria,
Microbiota and their relative abundances differed according to the specic periodontal condition of the patient.
16S rRNA
This suggests that manipulation of the predominant oral microbiota may assist in the maintenance of
T-RFLP
periodontal health. A broad view of the relationship of oral health with oral microbes would provide
Saliva
novel insights into oral health and disease.
& 2012 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
2. Molecular ecological approach using the 16S ribosomal RNA gene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
3. T-RFLP analysis of the bacterial community in saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
4. Further analysis of the salivary microbiota using the clone library method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Conict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

1. Introduction of oral environmental changes, resulting in caries [5,6]. However,

Two major oral diseases, namely, dental caries and period-
ontitis are infectious diseases that are caused by bacteria. Several
specic microbes are assumed to be causal agents, and their
etiologies have been extensively pursued. Currently, little doubt
exists that mutans streptococci are associated with the initiation
of dental caries [13], and Porphyromonas gingivalis, Tannerella
forsythensis, Treponema denticola, and a complex formed by these
bacteria are prime suspects in periodontitis [4]. However, the
presence of these organisms is not required for the onset of oral
diseases. In the study of dental caries, the ecological plaque
hypothesis, an excellent hypothesis suggesting the etiology of
this disease, recently emerged; this hypothesis proposes that
dental plaque microbiota shift to an aciduric community because
n
Corresponding author.
E-mail address: taketooo@dent.kyushu-u.ac.jp (T. Takeshita).
bacteriological etiologies of these diseases, especially periodontitis,
have not yet been elucidated.
Numerous and diverse microorganisms colonize the human oral
cavity as commensals. The number of bacterial species identied
exceeds 700 [7]. These microorganisms regulate each other and
coexist within the microbial community, i.e., the indigenous micro-
biota. Oral pathogenic bacteria are also members of this complex
microbial community, and therefore, their growth and the expres-
sion of virulence factors are most likely to be affected by synergistic
or antagonistic interactions with other indigenous bacteria.
The surrounding bacterial populations, even those with no direct
virulence factors, may thus be involved in the onset of oral disease.
Recent advances in molecular ecological techniques have allowed
comprehensive surveys of complex bacterial communities, and the
overall structure of bacterial communities formed within various oral
sites has been revealed [811]. In particular, dental plaque micro-
biota associated with dental caries and periodontitis have been
investigated in detail, and many novel putative causative agents,

1349-0079/$ - see front matter & 2012 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.job.2012.04.003
 T. Takeshita, Y. Yamashita / Journal of Oral Biosciences 54 (2012) 128131
including not-yet-cultivated microorganisms, have been identied
[1216].
On the other hand, dental plaque microbiota are continuously
bathed in saliva and are therefore always in contact with the
bacterial population in saliva. Although the salivary bacterial
population is a mixture of bacterial communities that exist at
various sites in the oral cavity, its composition is more similar to
the microbiota on mucosal surfaces, which may act as a reservoir
for dental pathogens, compared to dental plaque [17]. We hope
that the observation of salivary bacterial populations may provide
further insights into the involvement of oral microbiota in the
oral environment, affecting the onset of oral diseases.
In this review, we introduce our progress regarding the
relationship between the overall structure of the salivary bacterial
population and oral health and disease.
2. Molecular ecological approach using the 16S ribosomal
RNA gene
In the past, comprehensive characterization and comparison of
multiple complex bacterial communities, such as dental plaque
and saliva, have been regarded as unrealistic because conven-
tional methods rely on the cultivation of bacteria and are
laborious, time-consuming, and expensive. In addition, noncul-
turable bacteria can be missed. A molecular ecological approach
using DNA extracted from the microbial community overcomes
these limitations. Use of the 16S ribosomal RNA (rRNA) gene in
combination with polymerase chain reaction (PCR) has become
common in analyses of bacterial communities. The 16S rRNA gene
(total length: approximately 1500 bp) is present in almost all
bacterial species. This gene has remained relatively conserved
throughout evolution, but contains 9 variable regions that can be
utilized for classifying bacteria at the species level [18]. PCR using
a primer set that targets a conserved region of the 16S rRNA gene
allows us to collect genes from nearly all members of the bacterial
community.
Two major approaches are used to analyze PCR amplicon
sequence information: sequencing and ngerprinting. In clone
library analysis (the sequencing approach), amplicons are cloned
into a plasmid vector, and the resultant plasmids are transformed
into Escherichia coli. The inserts are sequenced individually and
compared to sequences of known origin deposited in a public
database. Members of bacterial communities can be identied
individually. Using this method, previous studies have documen-
ted the overall composition of various oral bacterial communities,
such as supragingival plaques of subjects with periodontitis and
periodontally healthy subjects [9], tongue coatings of subjects
with and without halitosis [10], dentoalveolar abscesses [11], and
root canal microbiota associated with treatment failures [19].
In addition, a database limited to oral bacteria was constructed [20],
and it can be used to facilitate analysis of the oral bacterial
community.
However, while the cloning and sequencing method does generate
accurate phylogenic information, is somewhat laborious for identi-
cation of the overall microbiota structure because of the need to
sequence many clones. Fingerprinting approaches, such as terminal
restriction fragment length polymorphism (T-RFLP) [21] and denatur-
ing gradient gel electrophoresis [22], are more convenient and allow
rapid assessment and comparison of a large number of bacterial
community samples. For example, in T-RFLP analysis, 16S rRNA genes
are amplied using uorescent-labeled primers, and the amplicons
are digested with a restriction enzyme. The resultant fragments are
separated by capillary electrophoresis, and only the uorescent-
labeled 5-terminal-labeled fragments are detected. Labeled frag-
ments from differing bacterial origins were discriminated based on
129
their size, and their uorescent intensities reect the abundance of
that bacterium in the microbiota. The peak pattern represents an
overview of the microbial community structure that can be easily
compared with other samples, although additional work is required
for phylogenetic interpretation of a peak pattern. This method is
especially powerful for monitoring shifts in microbiota and has been
adopted for characterizing these oral microbiota shifts [23].
Both sequencing and ngerprinting have their advantages and
disadvantages, and thus, a combination of these 2 methods
is required to compare large numbers of complex bacterial
community samples. To characterize the relationship between
salivary bacterial population structure and oral health, we primarily
utilized the T-RFLP method. Our efforts to improve fragment size
accuracy [24] have allowed for more accurate predictions of
bacterial compositions from a T-RFLP peak pattern, and the applica-
tion of this method has been specialized for the analysis of oral
microbiota [25]. The microbiota structures characterized by T-RFLP
analysis were conrmed using a clone library analysis of some
representative samples.
3. T-RFLP analysis of the bacterial community in saliva
To explore the relationship between the salivary bacterial
population and oral health, we examined the oral condition of
200 Japanese subjects, ages 1540 years, and collected their saliva
[26]. The bacterial composition and communities in the saliva of
these 200 subjects was investigated using T-RFLP analysis of the
16S rRNA gene, and data were displayed as peak patterns.
We used forward and reverse primers labeled with the uorescent
dyes 6-carboxyuorescein (6-FAM) and hexachloro uorescein
(HEX), respectively; thus 2 peak patterns per sample were
obtained in each electrophoretic run. T-RFLP proles of the 200
subjects contained 110 distinct peaks, 69 in the 6-FAM proles
(F1F69) and 41 in the HEX proles (R1R41). No peaks were
specically detected in subjects with many decayed teeth, no
caries, severe periodontitis, and healthy gingiva. In contrast, large
peaks were detected at the same position in almost all T-RFLP
proles, suggesting that the dominant indigenous bacteria in
saliva were common to all subjects. In addition, their relative
abundances among subjects differed considerably.
To correlate the T-RFLP peak patterns with oral health condi-
tions, we classied subjects based on their T-RFLP proles and
compared the oral condition of subjects in each group. Three
major patterns were identied from the 200 proles using the
partitioning around medoids analysis (Fig. 1). The relative abun-
dances of dominant peaks were different in each of the 3 clusters.
For example, peaks F19 and F33 were characteristically more
predominant in the 6-FAM T-RFLP proles of cluster I compared
Fig. 1. Peak patterns of 6-carboxyuorescein-labeled terminal restriction frag-
ment length polymorphisms showing the bacterial composition of the saliva of
200 subjects displayed as a 3-dimensional plot. Peaks (x-axis), subjects sorted into
each cluster (41, 74, and 85 subjects in clusters I, II, and III, respectively; y-axis),
and area proportion of each peak (z-axis) are shown.
130 T. Takeshita, Y. Yamashita / Journal of Oral Biosciences 54 (2012) 128131
Table 1
Comparison of oral conditions among 3 clusters of subjects classied based on terminal restriction fragment length polymorphism proles.
Parameters Cluster I
(n 41)
Teeth condition
Number of missing teeth (mean 7 SD) 0.27 0.6
Number of DMFT (mean 7 SD) 9.47 6.4
Periodontal condition
Percentage of sites bleeding on probing (mean 7SD) 27.6 7 18.1
Percentage of sites with periodontal pockets 10.17 15.4
(mean 7 SD)
(pocket depth 44 mm)
Number of subjects without periodontal pockets (%) 10 (24.3)
Number of subjects with periodontal pockets 420% (%) 9 (21.9)
Abbreviations: DMFT, decayed, missing, lled teeth; SD, standard deviation; NS, not signicant.
a
Statistical differences were evaluated among all 3 clusters by the Steel-Dwass test. A P valueo 0.05 was considered statistically signicant.
b
Statistical differences were evaluated by Fishers exact test.
Fig. 2. Principal components analysis biplot showing the relationships among 200
terminal restriction fragment length polymorphism proles classied into 3 clus-
ters (3 types of dots) and each peak (arrows). Only 12 peaks with large loadings on
the rst 2 principal components of 110 peaks were selected and shown. Candidate
bacterial genera corresponding to peaks, as determined by fragment size, are
indicated.
with the other clusters. The gingival health conditions of subjects
in these 3 clusters were signicantly different (Table 1). Clusters I
and II had a higher percentage of sites in periodontal pockets
greater than 4 mm, and cluster I most often contained sites that
exhibited bleeding on probing. Signicant differences among the
clusters were also observed in a number of subjects without
periodontal pockets and with periodontal pockets greater than
20%. No signicant differences were observed for other clinical
parameters, including dental caries history.
T-RFLP proles were further subjected to principal compo-
nents analysis (PCA) to characterize the bacterial community
structures of each cluster (Fig. 2). Subjects within each cluster
were localized differently in the PCA plot of the rst 2 compo-
nents. Subjects in cluster I were localized in the lower left region,
and peaks F19, F33, R16, and R22 had large loading in this
direction. Based on fragment size, Prevotella species, including
Prevotella melaninogenica, and Veillonella species were selected
from our database (containing 655 oral bacterial species) as
corresponding to the combination of these peaks, suggesting that
these bacteria were more predominant in cluster I than in the
Cluster II Cluster III Statistical
(n 74) (n 85) difference
0.4 7 1.0 0.27 0.7 NSa
10.1 7 7.1 7.87 5.4 NSa
18.9 7 19.8 14.67 13.5 I 4II, I 4IIIa
9.0 7 15.1 3.77 6.4 I 4III, II4IIIa
36 (48.6) 55 (64.7) P o0.001b
14 (18.9) 2 (2.3) P o0.001b
other clusters. Peaks with large loading in the direction of cluster II
were F47 and R32, and Streptococcus species such as Streptococcus
mitis and S. salivarius were assigned to these peaks. Peaks with
large loading in the direction of cluster III were F17, F65, R12,
and R24, which corresponded to Neisseria, Haemophilus, Aggrega-
tibacter, and Porphyromonas species, excluding P. gingivalis. All
peaks with large loading were detected in every subject with a
large peak area, and the candidates corresponding to each peak
were common bacteria in human saliva.
4. Further analysis of the salivary microbiota using the clone
library method
To conrm the bacterial diversity of the salivary microbiota,
we selected 1 subject from each cluster and investigated their
microbiota using clone library analysis. The T-RFLP proles of
these representatives corresponded to the cluster center, imply-
ing that their microbiota structures were the most typical in each
cluster. As expected, common oral bacteria such as S. salivarius,
P. melaninogenica, and Veillonella dispar predominated in the
saliva of all subjects, consistent with bacterial prediction based
on the T-RFLP proles. In addition, the relative abundance of each
bacterial genus differed among subjects, also consistent with the
T-RFLP proles (Fig. 3).
The results of this investigation suggest that the global
composition of the oral indigenous microbiota reected gingival
health. A microbiota dominated by Prevotella and Veillonella was
implicated in periodontal disease, while that dominated by
Neisseria, Haemophilus, Aggregatibacter, and Porphyromonas was
associated with gingival health. This result is noteworthy because
periodontitis is local inammation of the gingival tissue, which is
thought to be an infectious disease caused by pathogenic bacteria
inhabiting the gingival crevice. Although periodontal pathogens
are also detectable in saliva [27,28], it is difcult for saliva to
access the inside of periodontal pockets compared with other oral
surfaces. Most importantly, differences in microbiota structures
were determined by the relative abundance of the dominant
indigenous bacteria; recognized pathogens, such as P. gingivalis,
were not included in the T-RFLP peaks characteristic of each
cluster. Both the local emergence of pathogens and structural
differences in the entire oral microbiota are associated with
gingival disorders. These characteristic dominant indigenous
bacteria may be nonparticipating observers that prefer the oral
environment during periodontitis or periodontal health. However,
numerous interactions occur among pathogenic and other indi-
genous bacteria within the oral microbiota. This study likely
characterized the bacterial environment surrounding a specic
 T. Takeshita, Y. Yamashita / Journal of Oral Biosciences 54 (2012) 128131
Fig. 3. Relative proportions of 6 dominant bacterial genera in the salivary
microbiota of representative samples of each cluster, as determined by clone
library analysis. The number of clones whose nucleotide sequences were deter-
mined was 159, 175, and 171 in clusters I, II, and III, respectively.
potential pathogen, and the characteristic bacterial environment
may affect gingival health through such interactions. Although a
methodology for altering oral microbiota remains to be developed,
manipulation of the dominant oral microbiota (i.e., suppression or
promotion of specic bacterial populations) represents another
approach to oral health maintenance.
5. Conclusions
In this review, we describe the relationship between the oral
microbiota as a whole, obtained using a molecular ecological
approach, and oral health. Next-generation sequencing generates
vast amounts of sequence data at a much lower cost than
conventional techniques, and detailed phylogenetic information
on a large number of bacterial community samples is accumulat-
ing. Using this novel approach, the relationship between the
structure of the salivary microbiota and dental caries was
reported [29]. A broad ecological view of our relationship with
oral microbes will provide novel insight into the most effective
methods of maintaining oral and, subsequently, systemic health.
Conict of interest
No potential conicts of interest are disclosed.
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