IN-ECrION ANI) IMMUNITY. May 1994, p.

1889-1895
0019-9567/94/$04.00+O
Copyright O 1994, American Society for Microbiology

Vol. 62, No. 5

Diversity of Cultivable and Uncultivable Oral Spirochetes from

a Patient with Severe Destructive Periodontitis
B. K. CHOI,' B. J. PASTER,2 F. E. DEWHIRST,2 AND U. B. GOBEL'*
Institlt fOir Medizinische Mikrobiologie lund Hygiene, Klinikum der Albert-Ludwigs-Universitat Freiblrg,
D-79104 Freiblrg, Germany, and Department of Moleclular Genetics, Forsyth Dental Center,
Boston, Massachusetts 021152
Received 8 July 1993/Returned for modification 1 September 1993/Accepted 14 December 1993

detectable biota in subgingival plaque from patients with acute

necrotizing ulcerative gingivitis and chronic adult periodontitis
(3, 19-21, 43). Although only four species of oral spirochetes,
Treponema denticola, Treponema pectinovorum, Treponema
socranskii, and Treponema vincentii, have been cultured so far,
ultrastructural evidence indicates that there are at least a
dozen morphotypes of oral spirochetes, some of which may
play a role in the pathogenesis of periodontal infections. In
more recent studies, unclassified, as-yet-uncultivable spirochetes, so-called pathogen-related oral treponemes, that crossreact with monoclonal antibodies raised against Treponema
pallidlum were shown to be capable of invasion (22, 34-36).
Here we describe the comparative sequence analysis of in
vitro-amplified 16S rRNA genes to study the diversity of oral
spirochetes, including those that are currently uncultivable.
This study revealed 8 to 23 clusters of both known and
previously unclassified oral treponemes in a subgingival plaque
sample from a single patient with destructive periodontitis. In
situ hybridization with a fluorescently labeled species-specific
oligodeoxynucleotide probe was used to identify one of the
uncultivable species directly in clinical samples.

In the past, analysis of the normal human flora and of

polymicrobial infections in humans was hampered by the
complexity of the bacterial community and the fastidious
nature of some of its population members. The same is true for
populations in nature, and traditionally microbial ecology was
restricted to the description of cultivable microorganisms (28).
However, only a few members of natural microbial communities were cultivable or had sufficient phenotypical traits to
allow their identification on the basis of distinct bacterial
morphotypes. A breakthrough began with the pioneering work
of Woese and collaborators, who used universally occurring
rRNA sequences for phylogenetic study (48). It then became
obvious that these macromolecules could be used for the
analysis of natural populations (30) and for the specific identification of bacteria by rRNA-based hybridization probes (2,
6, 7, 11, 14, 15). Initial limitations in identifying clones carrying
rRNA genes from recombinant libraries of bulk genomic
biomass DNA were overcome by the application of rRNAspecific primers to selectively amplify rRNA sequences before
cloning (4, 23). This molecular approach has been applied
successfully to analyze environmental samples, e.g., oligotrophic ocean water and photosynthetic microbial mats (10, 13,
45), endosymbionts (1), and uncultivable infectious agents
associated with disease, such as the Whipple's disease-associated bacterium Tropheryma whippelii and the agent causing
bacillary angiomatosis (32, 33, 47).
More than 300 bacterial species have been isolated from the
subgingival plaque of patients with severe periodontitis (25-27,
41, 42). This research was undertaken because it had been
reported that spirochetes can represent up to 50% of the

MATERIALS AND METHODS

Sample collection and nucleic acid extraction. The subgingival plaque sample (about 1 mg [wet weight]) was collected
with a calibrated curette from a deep periodontal pocket
(probing depth, >9 mm) of a 29-year-old white female patient
with severe destructive periodontitis. The material was gently
dispersed in 1 ml of prereduced broth and analyzed by
dark-field microscopy for the presence of motile spirochetes.
Approximately 20% of the bacteria were spirochetes. A 100-pl
aliquot of this suspension was frozen for subsequent in situ
hybridization analyses; the remaining part was centrifuged,
washed once in phosphate-buffered saline (PBS) (pH 7.0), and

*
Corresponding author. Present address: Universitatsklinikum
Charit6, Medizinische Fakultat der Humboldt-Universitat, Institut fur
Mikrobiologie und Hygiene. Clara-Zetkin-Str. 96, D-101 17 Berlin,
Germany. Phone: 49 030 220 24 11, ext. 280. Fax: 49 030 229 27 41.

1889

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To determine the genetic diversity of cultivable and uncultivable spirochetes in the gingival crevice of a
patient with severe periodontitis, partial 16S rRNA genes were cloned from PCR-amplified products of DNA and
RNA extracted from a subgingival plaque sample. Approximately 500 bp were amplified in PCRs by using
universally conserved primers with polylinker tails. Purified PCR products were cloned into Escherichia coli by
using the plasmid vector pUC19. The resultant clone library was screened by colony hybridization with a
radiolabeled, treponeme-specific oligonucleotide probe. The 16S rRNA inserts of 81 spirochetal clones were then
sequenced by standard procedures. Sequences were compared with 16S rRNA sequences of 35 spirochetes,
including the four known cultivable oral treponeme species. The analysis revealed an unexpected diversity of
oral treponemes from a single patient. When 98% or greater sequence similarity was used as the definition of
a species-level cluster, the clone sequences were found to represent 23 species. When 92% similarity was used
as the definition, the clones fell into eight major groups, only two of which contained named species, Treponema
vincentii and Treponema denticola, while Treponema pectinovorum and Treponema socranskii were not represented
in any cluster. Seven of the 81 spirochetal clones were found to contain chimeric 16S rRNA sequences. In situ
fluorescence hybridization with a fluorescein isothiocyanate-labeled oligonucleotide probe specific for one of the
new species representing cluster 19 was used to identify cells of the target species directly in clinical samples.

1890

CHOI ET AL.

water were added to a final volume of 100 RI. Amplification

was done as described above.
Cloning of amplified mixed-plaque 16S rRNA genes. Purified amplicons were cleaved with BamHI and Sall (Pharmacia)
according to the manufacturer's recommendations and purified twice on a StrataClean resin (Stratagene) prior to ligation
into the multiple cloning site of precut plasmid pUC19 (Pharmacia). Ligation was done at 16C overnight in a final volume
of 20 pA by using one-half of the amplified DNA, 50 to 100 ng
of pUC19, 1 U of T4 DNA ligase (Gibco BRL), and 25 mM
Tris-HCl buffer (pH 7.8) containing 10 mM MgCl2, 1 mM
dithiothreitol, and 1 mM ATP. Transformation was done by
adding 100 jil of competent E. coli SURE cells (Stratagene) to
10 ,ul of the ligation mix according to the manufacturer's
instructions. Transformed cells were plated onto Luria-Bertani
agar containing 100 jig of ampicillin per ml, 0.004% (wt/vol)

(5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside)
(Boehringer), and 0.5 mM isopropylthiogalactoside (IPTG)
(Gibco BRL). Agar plates were incubated at 37C overnight.
Recombinants were selected on the basis of the white colony
phenotype.
Colony hybridization. White colonies were transferred with
sterile toothpicks to Luria-Bertani agar media supplemented
with ampicillin and then were lifted onto positively charged
nylon membranes (Biodyne B; Pall, Dreieich, Germany). Bacterial colonies were lysed according to standard procedures.
Oligodeoxynucleotide probes were 5' labeled with [T-32P]ATP
(5,000 Ci mmol'-; Amersham, Braunschweig, Germany) by
using T4 polynucleotide kinase (BRL) according to the supplier's recommendations. Free label was removed by passing
the reaction mixture over a Sephadex G-50 column (Pharmacia). Hybridization was performed essentially as described
previously (6).
Characterization of clones. The following probes (5, 8) were
used (hybridization temperatures are in parentheses): PI1V3
(Prevotella internedia specific), 5'-CAC GTG CCC CGC ITTT
ACT CCC CAA-3' (66C) (this sequence differs from a
sequence published by Dix et al. [5'-CAC GTG CCC CAC
rriT ACT CCC CAA-3'] (8) at one position [which is underlined]); P12V3 (Prevotella nigrescens specific), 5'-CGT GCG
CCA ATT TAT TCC CAC ATA-3' (60C); BFV3 (Bacteroides
forsythus specific), 5'-CGT ATC TCA TTT TAT TCC CCT
GTA-3' (56C); PGV324 (Porphyromonas gingivalis specific),
5'-CAA TAC TCG TAT CGC CCG TTA TTC-3' (60C); and
TREP (treponeme specific), 5'-GAC TTG CAT GCT TAA
(G/A)AC-3' (45C). One oligonucleotide complementary to
the successfully ligated polylinker-16S rRNA gene (rDNA)
junction was constructed to screen the clone library for true
recombinants; this was 5'-GGT CGA CAA CAG AGT TTG
AT-3' (48C) (the sequence complementary to the pUC19
polylinker is underlined). The TREP probe (base positions 46
to 63 according to E. coli numbering) includes three base
positions that are single-base signatures for spirochetes: U at
position 47, U at 50, and A at 52. The consensus base
signatures for other bacteria are C at 47, A at 50, and Y at 52
(45). While this probe has a single mismatch with some
spirochetes (A at position 53 versus G in leptospires), it has
four to five mismatches with essentially all other bacteria (31,
45).
Sequence and phylogenetic analysis. Plasmid minipreps
from TREP-positive recombinants were prepared as described
previously (38). Both insert strands were sequenced by a
modified Sanger dideoxy-chain-termination protocol using Sequenase (U.S. Biochemicals, Munich, Germany) according to
the manufacturer's suggestions. M13 reverse-sequencing
primer (5'-AAC AGC TAT GAC CAT G-3'; Pharmacia) and
X-Gal

Downloaded from iai.asm.org at Harvard Libraries on October 29, 2008

recentrifuged. The final pellet was suspended in 200 ,u of PBS

and homogenized by vortexing. The homogenate was divided.
One half of the homogenate was used for DNA extraction by
being mixed with an equal volume of lysis buffer (500 mM
Tris-HCl [pH 9.0], 20 mM EDTA, 10 mM NaCl, 1% sodium
dodecyl sulfate) containing lysozyme (Serva, Karlsruhe, Germany) at a final concentration of 1 mg/ml and incubated at
37C for 1 h. After proteinase K (Boehringer, Mannheim,
Germany) at a final concentration of 200 jxg/ml was added,
incubation was continued at 37C overnight. DNA was purified
by phenol-chloroform extraction and precipitated with cold
absolute ethanol. The DNA was pelleted by centrifugation,
washed once with 70% ethanol, dried under a vacuum for 10
min, and dissolved in 10 ,Iu of TE buffer (10 mM Tris-HCI [pH
8.0], 1 mM EDTA). The other half of the homogenate was
used for extraction of bulk RNA. Lysozyme and proteinase K
were added sequentially to final concentrations of 1 mg/ml and
200 ug/ml, respectively. The mixture was incubated at 37C for
1 h after each addition. RNAzol (1 ml; Wak-Chemie) was
added and mixed in by vortexing. After the addition of 0.1 ml
of chloroform to initiate phase separation, the sample was
centrifuged. The aqueous phase was removed subsequently,
mixed with an equal volume of isopropanol, and kept at - 20C
overnight to precipitate RNA. The precipitate was pelleted by
centrifugation, washed once with 75% (vol/vol) ethanol, and
dried under a vacuum for 10 min. The dry pellet was dissolved
in 10 plI of diethylpyrocarbonate-treated water.
cDNA synthesis from 16S rRNA by reverse transcription.
RNA (10 plI) was mixed with 1 ,lI of 30 ,uM primer RE-RTU3
(5'-CCC GGG ATC CAA GCT TG[T/A] ATT ACC GCG
GC[G/T] GCT G-3', corresponding to complementary positions 519 to 536 in Escherichia coli 16S rRNA; the underlined
sequences at the 5' end represent the SmaI, BamHI, and
HindlIl restriction sites) and incubated at 70C for 10 min.
Reverse transcriptase buffer (4 [lI of a 5 x buffer containing
250 mM Tris-HCl [pH 8.3], 300 mM KCl, and 15 mM MgCl2),
1 RI of 1 M dithiothreitol, 3 plI of 1.25 mM deoxynucleoside
triphosphates (dNTPs) (Pharmacia, Freiburg, Germany), and
1 [LI (10 U) of Moloney murine leukemia virus reverse
transcriptase (Stratagene, Heidelberg, Germany) were added
to a final volume of 20 ,ul. The mixture was incubated at 40C
for 30 min. The reaction was stopped by inactivating the
enzyme at 95C for 5 min.
PCR amplification. (i) Amplification of DNA. 16S rRNA
sequences were amplified from subgingival plaque DNA and
cDNA in parallel. PCR amplification of DNA was done by
adding 1 pA of dissolved DNA to 1 x PCR buffer (50 mM KCl,
1.5 mM MgCl2, 10 mM Tris-HCl [pH 9.0], 10 jig of gelatin per
ml), 200 ,uM dNTPs, 0.3 ,uM (each) primers RE-TPU1 (5'CCG AAT TCG TCG ACA ACA GAG 'rITl GAT C[A/C]T
GGC TCA G-3', corresponding to positions 8 to 27 in E. coli
16S rRNA; underlined sequences at the 5' end represent
EcoRI and Sall restriction sites) and RE-RTU3 (see above),
and 2.5 U of Taq polymerase (Gibco BRL) to a final reaction
mixture volume of 100 [lI. After the addition of 100 plA of sterile
mineral oil, amplification was started. Reactions were performed in an automated thermal cycler (Perkin-Elmer). DNA
samples were denatured by incubation at 95C for 2 min and
amplified by 30 cycles of 95, 55, and 72C, for 1 min at each
temperature. Amplicons were purified by agarose electrophoresis and eluted by centrifugation with a small Mobicol
column (Mobitech, Gottingen, Germany).
(ii) Amplification of cDNA. To the reverse transcription
mixture (20 RI) 13 plA of dNTPs (1.25 mM; Pharmacia), 8 pA of
lOx PCR buffer, 1 pLI of 30 ,uM RE-TPU1 primer, 2.5 U (0.5
[L) of Taq polymerase (Gibco BRL), and double-distilled

INFEc-r. IMMUN.

VOL. 6)2, 1993t4

DIVERSITY OF CULTIVABLE AND UNCULTIVABLE SPIROCHETES

TABLE 1. Proportions of probe-positive clones

Amplilication
method

PCR
RT-PCR9

No. of probe-positive clones (()

NO. of
clones"
TREP"
577
5,841

PG"

Pid

PN"

BF/'

40 (6.9) 115 (19.9) 0 (0)

1 (0.2) 22 (3.8)
55 (0.9) 897 (15.4) 2 (0.03) 14 (0.2) 84 (1.4)

Raindomlv selected recombinants from the lS rRNA clone libraries.

TREP trcponemes.
PG P.Pginigi'alis.
" Pl
i LlCt(/771dia.
PN, 1' tigr.scens.
BF, B. foinsothzus.
Revcrse tr inscriptase (cDNA) PCR.

RESULTS
Identification of clones by hybridization with DNA probes.
A total of 6,418 randomly selected recombinants were transferred to nitrocellulose membranes for hybridization with a
panel of radiolabeled oligonucleotide probes. We chose this
rather large sample size to ensure that species representing less
than 0. I% of the population had a reasonable chance of being
represented by a clone. The numbers of clones hybridizing with
each probe are shown in Table 1.
Sequence and phylogenetic analysis of oral treponemes. A
total of 95 recombinants carrying treponeme-specific 16S
rRNA sequences were identified. Fourteen of these clones
were contaminated and were excluded from further study. The
remaining 81 clones were sequenced. Although complete 16S
sequences will be necessary for a thorough analysis of the
phylogeny of these new treponeme species, a comparison of
the 500-base partial sequences was sufficient to establish the
approximate phylogenetic positions of the clones as shown in
Fig. 1. Each of the 81 sequenced clones was presumptively
identified as a spirochete with the TREP probe. Spirochetal
identity was verified by phylogenetic analysis, thus validating
the specificity of the TREP probe. Seven of the 81 sequences
were chimeras and are not included in the figure. Chimeras

were found by noting that for some clones, the first half and the
second half of the sequence did not group together on the
phylogenetic tree. Examination of aligned sequences showed
that chimeras were composed of spirochetal and nonspirochetal halves. Thus, it may be useful in the future to use a
second probe to identify the 3' ends of amplified spirochetal
sequences. Lack of reactivity with this probe would indicate a
possible chimera. The sequences were also examined by using
the program CHECK-CHIMERA written by Niels Larsen
(29). This program identified as chimeric the same seven
sequences we had identified as chimeric.
The remaining 74 clones, derived by amplifying either DNA
or RNA, are shown as 53 nodes on the tree because a number
of nodes represent two or more identical sequences (the
number of identical sequences appears after the clone designation). 16S rRNA similarity for strains of a species is usually
greater than 99%. When a relaxed species threshold of approximately 98% similarity was used, the 74 clones were divided
into 23 clusters. When a threshold of approximately 92%
similarity was used, the 23 clusters fell into 8 major groups.
With the exception of cluster 23, which was related to the
leptospires, the clusters fell into the genus Treponema. T.
vincentii was represented by three clones in cluster 1, and T.
denticola was represented by two clones in cluster 11. There
were no clones related to T pectinovorum or T. socranskii.
Direct visualization of oral treponemes by in situ fluorescence hybridization. For future studies fluorescently labeled
oligonucleotides will be instrumental in assessing the role of
the newly identified spirochetes in the etiology of periodontal
infections. To demonstrate the feasibility of this approach, we
constructed a fluorescein isothiocyanate-labeled oligonucleotide probe specific for clone NZM399 (from cluster 19), which
was used to identify this novel treponeme in subgingival plaque
samples. As shown in Fig. 2, the fluorescein isothiocyanatelabeled probe ZM399 identified only one distinct spirochetal
morphotype, presumably the target species. No other bacteria
were stained.

DISCUSSION
Since most of the spirochetes in the oral cavity are presently
uncultivable, the nucleic acids of spirochetes from bacterial
plaque were analyzed directly by using molecular biological
techniques. The strategy was based upon studies of molecular
evolution using comparative sequence analysis of rRNAs (48).
Four different experimental approaches for obtaining rRNA
sequences have been described, which theoretically represent
without bias the entire population, including both cultivable
and uncultivable microorganisms. These approaches are as
follows: (i) direct sequencing of 5S rRNA (30), (ii) construction of cDNA libraries by using purified mixed-population
RNA as a template for primer-directed reverse transcription of
rRNAs (45), (iii) in vitro amplification of rRNA genes from
bulk DNA by using the PCR and rRNA-specific primers (13,
46), and (iv) construction of shotgun clone libraries from bulk
DNA and identification of rDNA-containing clones with a
mixed-kingdom probe (39). The latter approach is considered
to introduce the least bias into a phylogenetic analysis (39, 40).
Because of the minute amounts of DNA and RNA available
from subgingival plaque, we decided to establish a 16S rRNA
gene library by amplifying part of the 16S rRNA gene directly
from purified plaque DNA and from cDNA after reverse
transcription of plaque RNA. There were several reasons to
choose this dual approach. If DNA had been used exclusively,
there would have been a danger of amplifying silent rRNA
genes from bacteria carrying several rRNA operons. In addi-

Downloaded from iai.asm.org at Harvard Libraries on October 29, 2008

universal rRNA primer TPU2 (5'-CCA [A/G]AC TCC TAC

GGG AGG CA-3', corresponding to positions 334 to 353 in E.
coli 16S rRNA) were used as forward primers, while sequencing primer -40 (5'-GTT TTC CCA GTC ACG AC-3'; U.S.
Biochemicals) and universal rRNA primer RTU2 (5'-TGC
CTC CCG TAG GAG T[C/T]T GG-3', complementary to
positions 353 to 334 in E. coli 16S rRNA) were used as reverse
primers for sequencing. Sequences were aligned on the basis of
conserved primary sequence analysis and secondary structure
analysis. All positions were included in distance calculations.
Gaps and missing data were handled by pairwise deletion in
distance calculations. Multiple base changes at single positions
were corrected by the method of Jukes and Cantor (16).
Dendrograms were constructed by the neighbor-joining
method of Saitou and Nei (37). Negative branch lengths were
set to zero. The sequences were also examined by using the
program CHECK-CHIMERA (29).
In situ hybridization. In situ hybridization was done essentially as described previously (7, 11, 12). Species-specific probe
ZM399, derived from cluster 19 sequences (5'-CCG TCA
CCA TCA TAC CAT TTC CTG-3'), was synthesized automatically and labeled at the 5' end with fluorescein isothiocyanate. All specimens were examined under oil immersion by
epifluorescence microscopy with a Neofluar (Zeiss, Oberkochen, Germany) 100 x objective and a Zeiss Axioskop 20 (for
details see reference 12).

1891

1892

INFECT. IMMUN.

CHOI ET AL.

Cluster Group
92%
98X

(% Difference)
Xreeponeea vincentit
NZ.M3i00 R
NZM3!12 2P
,NZ3I 42 P
rNHZW397 R
_NZM360 R
NZM3105 A
NZM30464 2D0 R
NZM30292 0
NZM3103 R
NZM398 R
NZM3010 R
NZM3166

-13
4
-5

= 6

NZM334 R
NZM30378 0
NZM/30392 0
NZM3I4S R
NZM3f47 R
NZM3020 40
NZM30I
D

Treponema phagedenis

5R

NZM3106

NZM30472

NZM3042

II

NZM3!07 2R
NZM3i08 R

reponeea dent Jcola

Jii

NZM3079

]2

III

13

NZM30527 0
NZM30298 2D. R
NZM302 17 0
NZM335 P
rreponema pall
Sp Jr oc hae ta steno s ,trepta
___I

ziaum

14
15
16

rhereophilic spirochete Hf

Spirochaeta zuelzerae

NZM304I0 3D
NZM3125 P
NZM3037 0
NZM(3I10 2R3R
NZM3122
NZM3I 13 2R

:7
-

NZM30505 0

1B

Treoonema brvantii
f
,,

NZM3i0i
NZM399

NZM3i57

0.

NZM3I24

NZM3ii8
NZM3i28

r NZM3109
NZM3155

2R

NZM3104

NZM30495

Treponema suc

rreponema socranski.i
Treponema pectino voruey

NZM30384

NZM30394

22

Treponema sp. CA
Irreponeaa saccharopbiulu

23

VII

VIII

ill lin.r
Leptospira biflexa
Leptospira Pnterrogans

FIG. 1. Phylogenetic tree of oral treponemes based on the comparison of partial 16S rRNA sequences. The scale bar represents a 10%
difference in nucleotide sequences as determined by measuring the lengths of the horizontal lines connecting two species. The letters following
clones indicate whether the sequence was obtained from DNA (D) or RNA (R). Numbers preceding the letters indicate the numbers of identical
clone sequences obtained.

tion, there would have been a remote possibility of amplifying

DNA from dead organisms, resulting in an erroneous representation of appropriate clones within the 16S rRNA gene
library. The former possibility was controlled by amplifying
cDNAs, which by definition derive from actively transcribed
rRNA genes. Hence, microheterogeneity among silent and
expressed rRNA genes would not interfere with subsequent
phylogenetic analysis. We therefore screened all recombinant
clones, randomly selected from the respective DNA and cDNA
libraries, by colony hybridization with radiolabeled probes
specific for P. gingivalis, P. intermedia, P. nigrescens, and B.
forsythus. The frequencies of these recombinant clones for
both libraries were quite similar, regardless of the amplification scheme used. Moreover, the data were in good agreement
with culture studies, in which P. intermedia, P. gingivalis, and B.
forsythus represented about 5, 21, and 11% of the total number

of cultivable bacteria, respectively (12). However, significant

discrepancy was found with treponeme-specific clones, which
represented about 7 and 1% of the clones randomly chosen
from the DNA and cDNA libraries, respectively (Table 1).
Approximately 20% of the total number of bacteria were
spirochetes, as determined by phase-contrast light microscopy.
Many reasons may account for this phenomenon, such as a low
copy number of treponemal rRNAs due to either down
regulation of ribosome biosynthesis in these slowly growing
organisms, rapid degradation of RNA during sampling, or
inefficient binding of reverse transcriptase or amplification
primers, and the presence of modified bases resulting in early
termination of the reverse transcription (45).
The clones represented approximately 20 new species of
Treponema. One of the clones, NZM3D394 (from cluster 23),
may represent a new genus related more closely to the

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R
NZM3081
NZM3.43 2R
NZM3i44
NZM3i38 R

NZM3158

VOL. 62, 1994

DIVERSITY OF CULTIVABLE AND UNCULTIVABLE SPIROCHETES

1893

leptospires than to the treponemes. As only one-third of the

16S rRNA gene was sequenced, the dendrogram shown in Fig.
1 has minor topological differences from our previously published tree for spirochetes, which was based upon full-length
sequences (31). However, partial sequences have been used
successfully to infer phylogenetic relationships (10, 13, 17, 39,
44, 45). Because the region of 16S rRNA included had more
locales of high sequence variability than are average for the
entire 16S rRNA molecule, the branch lengths are approximately one-third longer than those for a tree computed from
complete sequences. The significance of Fig. 1 is that it
demonstrates an unexpected diversity of clones from a single
patient. On the basis of morphological criteria, we expected to
find at least a dozen species other than the four named oral
treponemes. To find clones representing nearly 20 new species
in a single patient implies that previously used methods for
isolation, cultivation, and identification have grossly underestimated the diversity of spirochetes present in the human oral
cavity. We do not believe that the diversity seen in Fig. 1 was
due to PCR or sequencing error. Of the 81 sequences, 35 were
identical to other clone sequences, indicating that we could
obtain the same sequence from multiple clones. Some of these
identical sequences were obtained from amplifications of both
DNA and RNA. In collaborative projects to be reported
elsewhere, unidentified isolates of oral treponemes have been
examined by 16S rRNA sequence analysis (9, 49) and been

found to fall into clusters 7, 17, and 19. Thus, additional

cultivable species of oral treponemes exist. Attempts at culturing presently uncultivable species will be aided by precise
means of identification, such as rRNA-based DNA probes.
Since the TREP probe was based upon known spirochete
sequences, it is possible that there are spirochetes with base
changes not recognized by this probe. This would make our
estimates of the number of new species lower than that number
actually may be.
Since the formation of amplification artifacts such as chimeras or shuffle products may occur, it is important to carefully
examine sequences obtained (13, 18, 24). Examination of
aligned sequences revealed that most chimeras changed from
spirochetes to other bacterial taxa at about positions 310 to 360
(according to E. coli numbering). This is a region of nearidentity in bacteria, and therefore, partially extended PCR
amplicons could anneal with other bacterial DNA and be
elongated. By lengthening the extension time in the PCRs,
especially in the first 10 cycles, this may be avoided. Because of
the possibility of obtaining chimeric sequences, we recommend
that any sequences obtained by amplification of 16S rRNA
sequences from mixed populations be examined by using a
program such as CHECK-CHIMERA, which is available online at the University of Illinois ribosomal data base project

(29).
A fluorescently labeled, species-specific probe was used to

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FIG. 2. In situ fluorescence hybridization with fluorescein isothiocyanate-labeled probe ZM399 for direct detection of the cluster 19 target
species in smears of formaldehyde-fixed subgingival plaque. Phase-contrast (A) and epifluorescence (B) microscopy results are shown.

1894

CHOI ET AL.

determine the relative proportions of the target species in a

number of clinical samples. A complete battery of probes can
be used in more extensive clinical studies to determine the
distribution of these hitherto-unknown bacteria within subgingival plaque samples and biopsy samples from diseased patients. These studies will be important to identify oral spirochetes, including those that are presently uncultivable, that are
associated with diseased sites. There is great hope that the
approach described here will assist in elucidating the role of
treponemes in the pathogenesis of periodontal infections by
associating defined species with healthy or diseased sites.
ACKNOWLEDGMENTS

This work was supported in part by a grant from the ITI-Foundation

(6-91/029) and grant DE 10374 from the National Institutes of Health.
We are indebted to G. Krekeler for providing patient samples.

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