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Journal of Bacteriology, June 2001, p. 3770-3783, Vol. 183, No. 12
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.12.3770-3783.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Bacterial Diversity in Human Subgingival
Plaque
Bruce J.
Paster,1,2,*
Susan K.
Boches,1
Jamie L.
Galvin,1
Rebecca E.
Ericson,1
Carol N.
Lau,1
Valerie A.
Levanos,1
Ashish
Sahasrabudhe,1 and
Floyd E.
Dewhirst1,2
Department of Molecular Genetics, The Forsyth
Institute,1 and
Department of Oral Biology, Harvard School
of Dental Medicine,2 Boston, Massachusetts
Received 8 November 2000/Accepted 28 March 2001
 |
ABSTRACT |
The purpose of this study was to determine the bacterial diversity
in the human subgingival plaque by using culture-independent molecular
methods as part of an ongoing effort to obtain full 16S rRNA sequences
for all cultivable and not-yet-cultivated species of human oral
bacteria. Subgingival plaque was analyzed from healthy subjects and
subjects with refractory periodontitis, adult periodontitis, human
immunodeficiency virus periodontitis, and acute necrotizing ulcerative
gingivitis. 16S ribosomal DNA (rDNA) bacterial genes from DNA isolated
from subgingival plaque samples were PCR amplified with all-bacterial
or selective primers and cloned into Escherichia coli.
The sequences of cloned 16S rDNA inserts were used to determine species
identity or closest relatives by comparison with sequences of known
species. A total of 2,522 clones were analyzed. Nearly complete
sequences of approximately 1,500 bases were obtained for putative new
species. About 60% of the clones fell into 132 known species, 70 of
which were identified from multiple subjects. About 40% of the clones
were novel phylotypes. Of the 215 novel phylotypes, 75 were identified
from multiple subjects. Known putative periodontal pathogens such as
Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola were
identified from multiple subjects, but typically as a minor component
of the plaque as seen in cultivable studies. Several phylotypes fell
into two recently described phyla previously associated with extreme
natural environments, for which there are no cultivable species. A
number of species or phylotypes were found only in subjects with
disease, and a few were found only in healthy subjects. The organisms
identified only from diseased sites deserve further study as potential
pathogens. Based on the sequence data in this study, the predominant
subgingival microbial community consisted of 347 species or phylotypes
that fall into 9 bacterial phyla. Based on the 347 species seen in our
sample of 2,522 clones, we estimate that there are 68 additional unseen
species, for a total estimate of 415 species in the subgingival plaque.
When organisms found on other oral surfaces such as the cheek, tongue,
and teeth are added to this number, the best estimate of the total
species diversity in the oral cavity is approximately 500 species, as
previously proposed.
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INTRODUCTION |
It has been previously estimated
that about 500 species of bacteria inhabit the human oral cavity
(32, 43, 52). While the majority of these organisms are
commensals, a subset of them are likely to be opportunistic pathogens
that can cause systemic disease. For example, oral bacteria have been
implicated in bacterial endocarditis (3), aspiration
pneumonia (42), osteomyelitis in children
(10), preterm low birth weight (33), and
coronary heart disease and cerebral infarction (or stroke) (2,
53). Consequently, it is important to know what microorganisms
are present in the oral cavity for the diagnosis and rational treatment of systemic as well as oral diseases.
Studies with molecular techniques have shown that the bacterial
diversity in most environments is severely underestimated in surveys
with cultivation-based techniques (1, 19). In many natural
environments, less than 1% of the organisms are cultivable (34). Because of the significant effort extended to
cultivate oral bacteria, it is thought that about 50% of oral bacteria
have been cultivated. However, any understanding of the oral
environment requires knowledge of the entire bacterial community. There
is no reason to expect that fewer pathogens exist among the
uncultivated segment of the community than among the cultivated
segment. Highly host-adapted organisms such as Treponema
pallidum and Mycoplasma pneumoniae cannot yet be grown
or are extremely difficult to grow in culture because they have lost
the ability to synthesize many essential molecules that they normally
obtain from their host. In previous work, we have found that 75% of
oral species of Treponema have not been cultivated (6,
9). The current best model for exploring microbial diversity is
based on isolating DNA from the target environment, PCR amplifying the
ribosomal DNA (rDNA), cloning the amplicons into Escherichia
coli, and sequencing the cloned 16S rDNA inserts (18,
34). These culture-independent molecular phylogenetic methods
have been used to deduce the identity of novel phylotypes from
periodontitis subjects (6, 45), from dentoalveolar
abscesses (11, 50), and from a single subject with mild
gingivitis (21). More recently, Sakamoto et al.
(41) used these methods to compare the bacterial species
and phylotypes in saliva from a healthy subject and two periodontitis
subjects. In preliminary studies with similar methods, we identified
known species and novel phylotypes in subgingival plaque from subjects with refractory periodontitis (6, 22), adult periodontitis (6, 24), and acute necrotizing ulcerative gingivitis
(ANUG) (6, 8); in advanced lesions of noma or facial
gangrene (B. J. Paster, W. A. Falkler, Jr., C. O. Enwonwu, E. O. Idigbe, K. O. Savage, V. A. Levanos,
M. A. Tamer, R. L. Ericson, C. N. Lau, and F. E. Dewhirst, Abstr. 98th Gen. Meet. Am. Soc. Microbiol., p. 480, 1998); on
or in epithelial cells of the tongue dorsum of healthy subjects and
subjects with halitosis (4); on or in crevicular
epithelial cells from healthy and diseased subjects (25);
and in dental plaque from children with early childhood caries (M. R. Becker, A. L. Griffen, E. J. Leys, S. G. Kenyon, S. K. Boches, J. L. Galvin, F. E. Dewhirst, and B. J. Paster, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., p. 244, 2000).
The primary purpose of this study was to determine the diversity of
bacteria in human subgingival plaque by using culture-independent methods. The secondary purpose was to obtain qualitative data on the
diversity of bacteria in different periodontal disease states and
periodontal health. This study reports on the analysis of 2,522 16S
rRNA sequences, thus making it an order of magnitude larger than
previous 16S rRNA clonal analyses of oral bacteria (6, 11, 21,
41).
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MATERIALS AND METHODS |
Subject populations. (i) Refractory periodontitis.
Refractory periodontitis is defined as failure to respond to at least
three forms of therapy, including scaling and root planing, periodontal
surgery, and systemically administered tetracycline antibiotic. A poor
treatment response was defined as a mean periodontal attachment loss
computed on a whole-mouth basis or attachment loss of >2.5 mm at >3
periodontal sites occurring within 1 year posttherapy. Attachment
refers to the periodontal connective tissue surrounding the root of a
tooth that separates it from and attaches it to the alveolar bone. All
subjects had at least 20 teeth.
(ii) Periodontally healthy.
Periodontally healthy subjects
had no pockets >3 mm and no attachment loss >2 mm at any site in the
mouth. The subjects had less than 15% of sites with bleeding on
probing or with redness.
(iii) Periodontitis.
All periodontitis subjects had at least
20 teeth, at least eight sites with pocket depths of >4 mm, and six
sites with attachment level of >3 mm. Subjects had received no
systemic antibiotics in the previous 6 months and no prior periodontal
therapy. Sites may or may not have shown signs of gingival inflammation
or suppuration.
(iv) HIV periodontitis.
The human immunodeficiency virus
(HIV) periodontitis patients were classified according to the criteria
used above, except that subjects tested positive for HIV. Stanley C. Holt (presently at The Forsyth Institute) kindly provided these samples.
(v) ANUG.
ANUG subjects had no known systemic abnormality.
The sites sampled were necrotic and ulcerative at the tips of the
interdental papillae.
Microbiological sampling
After removal of
supragingival plaque with a sterile Gracey curette, a subgingival
plaque sample was removed from the four deepest or most diseased sites
with individual sterile Gracey curettes.
Sample lysis.
Samples were directly suspended in 50 µl of
a mixture of 50 mM Tris buffer (pH 7.6), 1 mM EDTA (pH 8), 0.5% Tween
20, and 200 µg of proteinase K per ml. The samples were heated at
55°C for 2 h. Proteinase K was then inactivated by heating at
95°C for 5 min.
Amplification of 16S rRNA cistrons by PCR and purification of PCR
products.
The 16S rRNA genes were amplified under standard
conditions with three different primer sets
all-bacterial selective,
Spirochaetes selective, and Bacteroidetes
selective. The sequences of the primers are shown in Table
1. PCR was performed in thin-walled tubes with a Perkin-Elmer 9700 Thermocycler. One microliter of the DNA template was added to a reaction mixture (50-µl final volume) containing 20 pmol of each primer, 40 nmol of deoxynucleoside triphosphates (dNTPs), and 1 U of Taq 2000 polymerase
(Stratagene, La Jolla, Calif.) in buffer containing Taqstart antibody
(Sigma Chemical Co.). In a hot start protocol, samples were preheated at 95°C for 8 min followed by amplification under the following conditions: denaturation at 95°C for 45 s, annealing at 60°C
for 45 s, and elongation for 1.5 min with an additional 5 s
for each cycle. A total of 30 cycles were performed, which was followed by a final elongation step at 72°C for 10 min. The results of PCR
amplification were examined by electrophoresis in a 1% agarose gel.
DNA was stained with ethidium bromide and visualized under short-wavelength UV light.
Cloning procedures.
Cloning of PCR-amplified DNA was
performed with a Zero Blunt Cloning kit, TOPO TA Cloning kit
(Invitrogen, San Diego, Calif.), or Prime PCR Cloner cloning system
(5'-3', Inc., Boulder, Colo.) according to the manufacturers'
instructions. Transformation was done with competent E. coli
TOP10 cells provided by the manufacturer. The transformed cells were
then plated onto Luria-Bertani agar plates supplemented with
kanamycin and incubated overnight at 37°C. Colonies were then placed
into 40 µl of 10 mM Tris. One microliter was used as the template to
determine the correct sizes of inserts in PCR with an M13 (
40)
forward primer and an M13 reverse primer. The size of inserts
(approximately 1,500 bp) was determined by PCR with flanking vector
primers followed by electrophoresis on a 1% agarose gel.
16S rRNA sequencing.
Purified DNA from PCR was sequenced
with an ABI Prism cycle-sequencing kit (BigDye Terminator Cycle
Sequencing kit with AmpliTaq DNA polymerase FS; Perkin-Elmer). The
primers in Table 1 were used for sequencing. Quarter dye chemistry was
performed with 80 µM primers and 1.5 µl of PCR product in a final
volume of 20 µl. Cycle sequencing was performed with an ABI 9700 sequencer with 25 cycles of denaturation at 96°C for 10 s and
annealing and extension at 60°C for 4 min. Sequencing reactions were
run on an ABI 377 DNA sequencer.
16S rRNA sequencing and data analysis of unrecognized
inserts.
A total of 2,522 clones with the correct size insert of
approximately 1,500 bases were analyzedapproximately 50 to 100 per subject. In these studies, approximately 500 bases were obtained first
to determine the identity or approximate phylogenetic position. Full
sequences (about 1,500 bases with five to six additional sequencing
primers) (Table 1) were obtained for most of the novel species. For
identification of the closest relatives, the sequences of the
unrecognized inserts were compared to the 16S rRNA gene sequences of
over 4,000 microorganisms in our database and the 16,000 sequences in
the Ribosomal Database Project (RDP) (28) and GenBank.
Programs for data entry, editing, sequence alignment, secondary
structure comparison, similarity matrix generation, and phylogenetic
tree construction were written by F. E. Dewhirst (36). The similarity matrices were corrected for multiple
base changes at single positions by the method of Jukes and Cantor (20). Similarity matrices were constructed from the
aligned sequences by using only those sequence positions for which 90% of the strains had data. Phylogenetic trees were constructed by the
neighbor-joining method of Saitou and Nei (40). TREECON, a
software package for the Microsoft Windows environment, was used for
the construction and drawing of evolutionary trees (47). Two hundred bootstrap trees were generated, and bootstrap confidence levels were determined with the TREECON program.
We are aware of the potential creation of 16S rDNA chimera molecules
assembled during the PCR (
26). The percentage of chimeric
inserts in 16S rRNA libraries ranged from 1 to 15%. Chimeric sequences
were identified by using the Chimera Check program in RDP, by
treeing
analysis, or by base signature analysis. Species identification
of
chimeras was obtained, but the sequences were not examined
for
phylogenetic
analysis.
Estimation of unseen species.
In 1943, Fisher et al.
(13) described a model for estimating the number of unseen
species in a population. In addition to ecological studies of
biological diversity, similar methods have been used to estimate the
number of words known but not used by Shakespeare and other authors
(12). In this study, the number of unseen species that
were missed was calculated with an improved estimator as proposed by
Boneh et al. (5). The estimator is based on a continuous
time model of parallel independent Poisson processes. The estimator is:
where
(
t) is the number of new species
expected to be observed over time zero to
t when the
original observations were
over time
1 to 0.
k is the
number of times a species is seen.
Nk is
the number of species seen
k times. Bias correction and
other details of the estimator are beyond the scope of the current
discussion, and the reader is referred to the publication of Boneh
et
al. (
5).
Nucleotide sequence accession number.
The complete 16S rRNA
gene sequences of clones representing novel phylotypes defined in this
study, sequences of known species not previously reported, and
published sequences are available for electronic retrieval from the
EMBL, GenBank, and DDBJ nucleotide sequence databases under the
accession numbers shown in Fig. 1 through 7.
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RESULTS |
Partial sequences of about 500 bp were obtained for 2,522 16S rRNA
clones in order to identify the predominant bacterial species present
in the subgingival plaque of healthy and diseased subjects. Approximately 60% of the 2,522 clones had greater than 99% sequence similarity to one of 132 known species. The remaining 40% of the clones fell into 215 previously unrecognized clusters, termed phylotypes. Full 1,500-base sequences were determined for
representatives of each novel phylotype. The term "phylotype" is
used for clusters of clone sequences that differed from known species
by approximately 30 bases (or 2%) and were at least 99% similar to
members of their cluster. The diversity of known species and novel
phylotypes with respect to periodontal health status is shown in Table
2. Approximately 40 to 60% of clones
analyzed in each category of healthy or diseased subjects were novel
phylotypes. Overall, we detected 347 species or phylotypes in
subgingival plaque.
The intent of using different PCR primer sets was to obtain the widest
spectrum of phylogenetic groups. In previous studies, results obtained
with the Spirochaetes-selective primers indicate that 85%
of the clones have spirochetal inserts (6). Our results using the Bacteroidetes-selective primers were not as
selective but yielded a wide range of bacterial typesincluding novel
taxa at the phylum level. In designing the "universal" bacterial
primer set, a "G" was added to the 5' end of the reverse primer
(E94) to enhance cloning of amplicons with the TOPO-TA cloning kit. Despite presenting a mismatch at this position to most 16S rRNA sequences in the RDP, a continuous stretch of 20 matching bases were
available for optimal PCR. Consequently, a wide range of phylogenetic
types was obtained by using this "universal" primer set.
Estimation of unseen species.
By using the estimator of Boneh
et al. (5) and applying the suggested bias reduction
procedure, we calculated that there are 68 unseen species in addition
to the 347 found, for a total estimate of 415 subgingival species.
Table 3 shows the number of additional
species that one would expect to identify by examining various numbers
of additional clones. As shown in Table 3, fewer new species were
predicted to be found for each thousand additional clones analyzed. The
model suggests that by examining 10,000 additional clones, we would
find all but one of the estimated 415 species.
As shown in Fig.
1, the oral bacteria
identified in this study fell into nine bacterial groups or phyla as
follows: Obsidian
Pool OP11 (
19), TM7 (
38);
Deferribacteres,
Spirochaetes,
Fusobacteria,
Actinobacteria,
Firmicutes,
Proteobacteria, and
Bacteroidetes (the latter seven phyla are listed in the
current taxonomic outline
for
Bergey's Manual of Systematic
Bacteriology, 2nd ed., vol.
2, at
http://www.cme.msu.edu/bergeys/). The distribution of known
species and
novel phylotypes within each of these phyla is shown
in Table
4. In Fig.
1 through
7, the phylogenetic
diversity within
each phylum is shown and is discussed in detail below.
The information
presented includes bacterial species or phylotype;
strain or clone
identification, sequence accession number, total number
of retrieved
clones, and number of subjects in each health status
category
in which each species was identified (color- and
shape-coordinated
symbols). Prevalent species or phylotypes are defined
as those
identified from four or more subjects and are noted in bold
and
underlined in each dendrogram.
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FIG. 1.
Bacterial phyla identified from subgingival plaque. Nine
bacterial phyla were represented from clonal analysis. As shown, the
first two phyla, Obsidian Pool OP11 and TM7, have no cultivable
representatives. Oral members of the phylum
Deferribacteres form a cluster consisting of only
not-yet-cultivated phylotypes. The information presented in Fig.
1 to 7 includes bacterial species or phylotype, strain or clone
identification, sequence accession number, total number of retrieved
clones, and number of subjects in each health status category in which
each species was identified (as indicated by color-coordinated
symbols). Novel phylotypes are defined as those taxa that are <98.5 to
99% similar in sequence comparisons to the phylotype's closest
relative. Prevalent species are defined as those phylotypes or species
identified in four or more subjects and are noted in boldface and
underlined in each dendrogram. Two hundred bootstrap trees were
generated, and bootstrap confidence levels as percentages (only values
over 40%) are shown at tree nodes.
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Obsidian Pool OB11.
As shown in Fig. 1, oral clone X112
represents the sole oral member of this phylum. Although only one
phylotype was identified, it was found in multiple subjects. Distant
relatives include phylotypes identified from sludge and deep-sea
sediments. There are no cultivable representatives of this phylum.
TM7.
Five oral phylotypes of the TM7 phylum were identified as
shown in Fig. 1. Distant relatives of this phylum have been identified in 16S rRNA clones derived from DNA isolated from soil and deep-sea sediments. The oral phylotypes were initially identified with Bacteroidetes-selective primers, but later identified with
all-bacterial-selective primers, which indicates that these
phylotypes are relatively common in subgingival plaque. There are no
cultivable representatives of this phylum.
Deferribacteres.
In the
Deferribacteres phylum, the oral species formed a coherent
cluster of seven separate phylotypes, four of which were prevalent in
diseased subjects, especially in the ANUG and refractory periodontitis
subjects. Consequently, the latter phylotypes may be good candidates as
putative pathogens. Members of this oral cluster are distantly related
to Synergistes jonesii, a cultivable, bovine ruminal
bacterium able to degrade the pyridinediol toxin in the plant
Leucaena leucocephala (27, 30). One oral clone, BA121, fell within the S. jonesii cluster rather than the
oral cluster.
Spirochaetes.
The human periodontal pocket
harbors a highly diverse and numerous spirochetal community (6,
9). All species identified thus far fall in the genus
Treponema, and presently there are about 60 oral treponemal
species or phylotypes (6) (Fig.
2). As shown in Fig. 2, several prevalent
species or phylotypes were identified. The predominant cultivable known
Treponema species included T. medium (but not the
closely related "T. vincentii"), T. denticola
(which was one of the most commonly identified species in both disease
and health), T. maltophilum, and two subspecies of T. socranskii. There were at least four predominant novel phylotypes, one of which, Treponema sp. 1:G:T21, was found only
in diseased sites. The latter phylotype appears to be a good candidate
as a putative pathogen.
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FIG. 2.
Phylogenetic tree of oral treponemes identified from
clone libraries. All species identified thus far fall in the genus
Treponema, and presently there are about 60 oral
treponemal species or phylotypes (6). The marker bar
represents a 10% difference in nucleotide sequences.
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It should be noted that the treponemal clones had been specifically
sought by constructing 16S rRNA libraries with spirochete-selective
primers in the initial PCR amplification. Microscopic examination
of
plaque samples from diseased sites, especially those from ANUG
subjects, revealed that 20 to 50% of the members of the bacterial
community were spirochetes (
6). Subsequently, spirochetes
were
also detected in 16S rRNA libraries when all-bacterial primers
were used, albeit at a lower
frequency.
Fusobacteria.
As shown in Fig.
3, the members of the oral
Fusobacteria identified include species of the genera
Fusobacterium and Leptotrichia. Most noteworthy
was that Fusobacterium naviforme (earlier referred to as
Fusobacterium nucleatum subsp. vincentii)
was often identified, especially in periodontitis subjects (eight of
nine subjects). In six of these eight periodontitis subjects,
Fusobacterium naviforme represented from 50 to 80% of the
clones. Other species and phylotypes of Fusobacterium and
Leptotrichia buccalis from healthy and diseased subjects
were also commonly identified. Fusobacterium animalis was
detected only in diseased subjects but not in healthy subjects.
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FIG. 3.
Phylogenetic trees of the phyla
Fusobacteria and Actinobacteria
identified from clone libraries. The members of the oral
Fusobacteria identified include species of the genera
Fusobacterium and Leptotrichia. The
marker bar represents a 5% difference in nucleotide sequences.
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Actinobacteria.
The Actinobacteria
phylum was previously referred to as the high-G+C-content,
gram-positive phylum. As shown in Fig. 3, oral genera of the phylum
Actinobacteria that were detected include Actinomyces, Atopobium, Bifidobacterium,
Corynebacterium, Propionibacterium, and
Rothia. Within these genera, Actinomyces
naeslundii II, Corynebacterium matruchotii, and
Rothia dentocariosa were commonly detected in health and
disease. Corynebacterium matruchotii appeared to be more associated with "healthy" subgingival plaque. Two species of
Atopobium were prevalent in diseased but not healthy subjects.
Firmicutes.
Most of the members of the
Firmicutes phylum were originally classified as belonging to
the low-G+C-content, gram-positive phylum. The Firmicutes
are presently divided into three classesnamely the proposed
"Bacilli," the Mollicutes, and the proposed
"Clostridia." The phylogenetic position of oral members
of the "Bacilli " and Mollicutes is shown in
Fig. 4. In the class
"Bacilli," the predominant species included the
streptococci Streptococcus oralis or S. mitis, S. mitis biovar 2, S. sanguis, S. intermedius, S. constellatus, and S. anginosus; Abiotrophia adiacens; and two species of
Gemella. Since it was difficult to differentiate S. oralis and S. mitis by 16S rRNA comparisons, they were
considered as one species in this study. Of the predominant species,
S. constellatus and Gemella haemolysans were most
associated with diseased sites, as they were not detected in
subgingival plaque from any of the healthy subjects. Although most of
the other predominant species were detected in both subjects with
disease and those with health, many of the Streptococcus
spp. were commonly identified from the healthy subjects.
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FIG. 4.
Phylogenetic tree of the phylum
Firmicutes identified from clone libraries. The phylum
Firmicutes is presently divided into three classes:
"Bacilli," Mollicutes, and
"Clostridia." This phylum represented the largest
group detected; 115 species or novel phylotypes were identified (Table
4). Streptococcus oralis and S. mitis could not
be differentiated based on sequence comparisons. Consequently, the two
species are grouped together. The marker bar represents a 5%
difference in nucleotide sequences.
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Not many members of the class
Mollicutes were detected: two
species of
Mycoplasma;
Solobacterium moorei, a
microorganism isolated
from feces that is related to species of
Erysipelothrix; and a
misclassified
Lactobacillus
species, [
L.]
catenaforme. Infections
by
species of
Erysipelothrix have been linked to endocarditis
(
16). It was encouraging to detect the purported
hard-to-lyse
mycoplasmas (
23), since the results
demonstrated that our procedures
do indeed recover a broad spectrum of
bacterial
species.
Members of the class "
Clostridia" represented one of the
largest groups detected, as shown in Fig.
5. Oral members included
species within
the genera
Catonella,
Dialister,
Eubacterium, Megasphaera,
Peptostreptococcus,
Selenomonas, and
Veillonella. There were several
good candidates as additional putative pathogens, i.e., those
species
or phylotypes that were commonly detected in disease,
but not (or
rarely) in health
namely
Eubacterium saphenum, clone
PUS9.170,
Filifactor alocis (previously
Fusobacterium
alocis)
, Catonella morbi,
Megasphaera sp.
oral clone BB166,
Dialister sp.
strain GBA27, and
Selenomonas sputigena.
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FIG. 5.
Phylogenetic tree of the class
"Clostridia" of the phylum Firmicutes
identified from clone libraries. The marker bar represents a 5%
difference in nucleotide sequences.
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Catonella morbi has been associated with periodontitis
(
31), clone PUS9.170 and
Filifactor alocis have
been associated with
dentoalveolar abscesses (
11,
50), and
Selenomonas sputigena has been associated with periodontitis
and, in rare cases, has
been involved in fatal septicemias
(
51).
Eubacterium saphenum and species of
Megasphaera have not been considered as putative
periodontal
pathogens (
44).
Oral isolate GBA27 is a slow-growing
Dialister species that
was commonly detected in clones from all disease categories and
from
one healthy subject (Fig.
6). Since
strain GBA27 cells form
only pinpoint colonies on agar medium (A. Tanner, The Forsyth
Institute, personal communication), it is likely
that this species
has eluded detection in predominant cultivable
studies over the
years. The closely related
Dialister
pneumosintes has been associated
with gingivitis (
31)
and human bite wounds (
15).
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FIG. 6.
Phylogenetic tree of the phylum
Proteobacteria identified from clone libraries. The
marker bar represents a 5% difference in nucleotide sequences.
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Proteobacteria.
Although there were 28 known
species and 23 novel phylotypes of Proteobacteria detected
(Table 4), most were only sporadically identified, as shown in Fig. 6.
Those species or phylotypes detected that were most associated with
disease include Haemophilus parainfluenzae, oral clone R004,
and Campylobacter rectus (Fig. 6). Haemophilus parainfluenzae, mostly detected in refractory periodontitis sites, has been isolated from the sputa of patients with chronic obstructive lung disease (29) and may be involved in endocarditis
(3). Oral clone R004, detected in all disease categories
(except HIV periodontitis), likely represents a novel sulfate-reducing
bacterial species belonging to the genus Desulfobulbus.
Sulfate-reducing bacteria, which have been reported in the periodontal
pocket (48), are routinely isolated from the feces of
patients with ulcerative colitis. Sulfide, an end product of sulfate
reduction, may be involved in the pathogenesis of the disease
(7). Campylobacter rectus has long been
associated with periodontal disease (44). Species or
phylotypes commonly detected in both diseased and healthy subjects
include Neisseria mucosa and two additional species of Campylobacter, C. gracilis and C. concisus.
Bacteroidetes.
There were 21 known species and
37 novel phylotypes of Bacteroidetes (previously referred to
as the
Capnocytophaga/Flavobacterium/Bacteroides phylum) detected in 16S rRNA libraries (Table 4). As anticipated, P. gingivalis and B. forsythus, two species
typically associated with periodontitis, were detected, although not as
frequently as some other species or phylotypes (Fig.
7). Other species or phylotypes that were
commonly detected include Prevotella denticola, Prevotella oris, Prevotella tannerae,
Porphyromonas endodontalis, oral clone AU126,
Capnocytophaga ochracea, and Capnocytophaga gingivalis
(Fig. 7). Porphyromonas endodontalis has been associated with endodontal infections (49), but it was often detected
in diseased subjects and not in any of the healthy subjects (Fig. 7).
Prevotella oris has been associated with dentoalveolar
abscesses (11). The remaining cultivable species of this
phylum have not been typically considered periodontal pathogens
(44).
View larger version (73K):
[in this window]
[in a new window]
|
FIG. 7.
Phylogenetic tree of the phylum
Bacteroidetes identified from clone libraries. Oral
species identified generally fall within the genera
Porphyromonas, Prevotella, and
Capnocytophaga. An asterisk denotes sequence of only
about 1,200 bp. The marker bar represents a 5% difference in
nucleotide sequences.
|
|
| |
DISCUSSION |
Human subgingival plaque harbors a highly diverse bacterial
community, as demonstrated by our detection of 347 species or phylotypes by clonal analysis. Using the estimator of Boneh et al.
(5), we predicted that approximately 68 additional unseen species were present and could be detected by examining 10,000 additional clones. This estimate is a rough approximation because the
clones came from libraries constructed from subjects with different
health and disease categories, violating the assumption of sampling
with replacement from a homogeneous population. However, the model
suggests that we have already identified 85% of the species and makes
testable predictions as to how many unseen species should be found by
examining additional clones (Table 3). Examination of 179 additional
clones, not included in this data set, from subgingival plaque from
sites of necrotizing ulcerative periodontitis (29) yielded
six novel species where the model would have predicted five. Thus, the
model and predictions appear quite accurate. When organisms found in
other studies (11, 21, 41, 45, 50) and on cheek, tongue,
and other oral tissues are added to our estimate of 415 subgingival
species, the best estimate of the total species diversity in the oral
cavity is between 500 and 600 species, as has been previously proposed.
A list of known human oral species and phylotypes can be obtained from
the corresponding author.
Of the 215 phylotypes identified in this study, 33 were cultivable
strains that have not yet been characterized. The remaining 182 were
represented only by clones. It is likely that the vast majority of
these phylotypes cannot be cultivated with standard anaerobic media and
techniques. It is also likely that many of these phylotypes are
"hidden" within culture collections, since the strains could not be
differentiated from closely related known species. In this regard, we
are currently examining isolates of over 100 unnamed taxa from the oral
anaerobic collection of W. E. C. Moore and L. V. Moore
(Virginia Polytechnic Institute, Blacksburg, Va.). It will be
interesting to see how many of Moore's groups represent cultivable
examples of groups previously represented only by clone phylotypes.
Treponema amylovorum and Treponema
lecithinolyticum were identified first by clone data before the
cultivable strains were characterized (6; this study). The
phylogenetic position of not-yet-cultivated novel phylotypes,
especially if they are closely related to cultivable species, may
provide insight into their cultivation. For example, physiological
properties or antibiotic resistance can be inferred to some degree. DNA
probes specific for the phylotype can be used to monitor
enrichment efforts and to positively identify an isolate.
The definition of a species is controversial, particularly when only
molecular sequence data exist (14, 46). Therefore, we have
used the term "phylotype" in place of species for referring to
novel clusters of clone sequences. In most cases, a 2% difference in
16S rRNA sequences does indicate separate species status, but there are
exceptions. Formal naming of a species also requires a full description
of the phenotypic characteristics of an organism. It is probable that
the majority of the phylotypes identified in this report will
eventually be validated as species. In the meantime, DNA probes can be
designed to identify phylotypes and to assess their roles in disease or
health. If a phylotype proves to be associated with disease, then
efforts can be made to isolate and characterize the new species.
In general, the first 500 bases of the 16S rRNA gene are the most
informative for identifying an organism, because there are several
variable regions. However, for constructing phylogenetic trees, it is
essential that nearly complete sequences of 1,500 bases be used. This
is a deficiency of some previous studies. We have also obtained full
sequences so that the data are most useful to databases such as GenBank
or the RDP (28).
A total of 72 species or novel phylotypes were identified in the
analysis of 268 clones from healthy subjects (Table 2). Based on the
overall total of 347, there are 275 species or novel phylotypes that
were identified in diseased subjects. Part of the explanation for the
discrepancy between the number of species found in healthy subjects
versus that found in diseased subjects is that only 10.6% of the
clones analyzed were from healthy subjects. However, even with this
bias, a number of species and phylotypes were found only in multiple
(
4) diseased subjects and not in any healthy subjects. These
often-identified disease-associated species and phylotypes are shown in
Table 5 and are clearly candidates for
further study. Eighteen of the 29 species are named species, including
Porphyromonas gingivalis and B. forsythus. One
represents a cultivable, but unnamed species. Ten of the phylotypes are
represented only by clones, and thus may represent currently
not-yet-cultivated or unrecognized potential pathogens. Additional
putative pathogens are likely, since there were several species or
phylotypes that were often identified from diseased sites, but only
rarely from health: e.g., Dialister sp. strain GBA27 (Fig.
5), other species or phylotypes of Fusobacterium (Fig. 3),
or oral clone AU126 (Fig. 7). There were also several species that were
more associated with health, namely Actinobaculum sp. clone
EL030 and Corynebacterium matruchotii (Fig. 3). However, to
rigorously assess the association of specific species or phylotypes
with certain periodontal diseases or periodontal health, it will be
necessary to analyze large numbers of clinical samples for the levels
of all oral bacteria in well-controlled clinical studies. The
definition of five subgingival plaque bacterial complexes by Socransky
et al. (44) was based on analysis of the microbial
community of over 13,000 plaque samples from 185 subjects by using DNA
probes in checkerboard hybridization assays. We are currently
developing DNA probes for approximately all 500 known species and novel
phylotypes for use in large clinical studies. These DNA probes are
being developed for use in the checkerboard hybridization assay
(35) and DNA microarray formats. Based upon knowledge of
the full bacterial diversity, and using molecular techniques to
identify these bacteria, future studies will be able to associate a
number of additional oral bacteria with various oral and systemic
diseases.
| |
ACKNOWLEDGMENTS |
This study was supported by NIH grants DE-11443 and DE-10374 from
the National Institute of Dental and Craniofacial Research.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: The Forsyth
Institute, 140 Fenway, Boston, MA 02115. Phone: (617) 262-5200, ext.
288. Fax: (617) 262-4021. E-mail: bpaster{at}forsyth.org.
| |
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Journal of Bacteriology, June 2001, p. 3770-3783, Vol. 183, No. 12
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.12.3770-3783.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
