choi 1994

IN-ECrION ANI) IMMUNITY. May 1994, p. 1889-1895 Vol. 62, No. 50019-9567/94/$04.00+OCopyright ©O 1994, American Society for Microbiology

Diversity of Cultivable and Uncultivable Oral Spirochetes froma 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

To determine the genetic diversity of cultivable and uncultivable spirochetes in the gingival crevice of apatient with severe periodontitis, partial 16S rRNA genes were cloned from PCR-amplified products ofDNA andRNA extracted from a subgingival plaque sample. Approximately 500 bp were amplified in PCRs by usinguniversally conserved primers with polylinker tails. Purified PCR products were cloned into Escherichia coli byusing the plasmid vector pUC19. The resultant clone library was screened by colony hybridization with aradiolabeled, treponeme-specific oligonucleotide probe. The 16S rRNA inserts of 81 spirochetal clones were thensequenced 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 oforal treponemes from a single patient. When 98% or greater sequence similarity was used as the definition ofa species-level cluster, the clone sequences were found to represent 23 species. When 92% similarity was usedas the definition, the clones fell into eight major groups, only two of which contained named species, Treponemavincentii and Treponema denticola, while Treponema pectinovorum and Treponema socranskii were not representedin any cluster. Seven of the 81 spirochetal clones were found to contain chimeric 16S rRNA sequences. In situfluorescence hybridization with a fluorescein isothiocyanate-labeled oligonucleotide probe specific for one of thenew species representing cluster 19 was used to identify cells of the target species directly in clinical samples.

In the past, analysis of the normal human flora and ofpolymicrobial infections in humans was hampered by thecomplexity of the bacterial community and the fastidiousnature of some of its population members. The same is true forpopulations in nature, and traditionally microbial ecology wasrestricted to the description of cultivable microorganisms (28).However, only a few members of natural microbial communi-ties were cultivable or had sufficient phenotypical traits toallow their identification on the basis of distinct bacterialmorphotypes. A breakthrough began with the pioneering workof Woese and collaborators, who used universally occurringrRNA sequences for phylogenetic study (48). It then becameobvious that these macromolecules could be used for theanalysis of natural populations (30) and for the specific iden-tification of bacteria by rRNA-based hybridization probes (2,6, 7, 11, 14, 15). Initial limitations in identifying clones carryingrRNA genes from recombinant libraries of bulk genomicbiomass DNA were overcome by the application of rRNA-specific primers to selectively amplify rRNA sequences beforecloning (4, 23). This molecular approach has been appliedsuccessfully to analyze environmental samples, e.g., oligotro-phic ocean water and photosynthetic microbial mats (10, 13,45), endosymbionts (1), and uncultivable infectious agentsassociated with disease, such as the Whipple's disease-associ-ated bacterium Tropheryma whippelii and the agent causingbacillary 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 beenreported that spirochetes can represent up to 50% of the

* Corresponding author. Present address: UniversitatsklinikumCharit6, Medizinische Fakultat der Humboldt-Universitat, Institut furMikrobiologie 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.

detectable biota in subgingival plaque from patients with acutenecrotizing ulcerative gingivitis and chronic adult periodontitis(3, 19-21, 43). Although only four species of oral spirochetes,Treponema denticola, Treponema pectinovorum, Treponemasocranskii, and Treponema vincentii, have been cultured so far,ultrastructural evidence indicates that there are at least adozen morphotypes of oral spirochetes, some of which mayplay a role in the pathogenesis of periodontal infections. Inmore recent studies, unclassified, as-yet-uncultivable spiro-chetes, so-called pathogen-related oral treponemes, that cross-react with monoclonal antibodies raised against Treponemapallidlum were shown to be capable of invasion (22, 34-36).Here we describe the comparative sequence analysis of invitro-amplified 16S rRNA genes to study the diversity of oralspirochetes, including those that are currently uncultivable.This study revealed 8 to 23 clusters of both known andpreviously unclassified oral treponemes in a subgingival plaquesample from a single patient with destructive periodontitis. Insitu hybridization with a fluorescently labeled species-specificoligodeoxynucleotide probe was used to identify one of theuncultivable species directly in clinical samples.

MATERIALS AND METHODS

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

1889

at Harvard Libraries on O

ctober 29, 2008 iai.asm

.orgD

ownloaded from

1890 CHOI ET AL.

recentrifuged. The final pellet was suspended in 200 ,u of PBSand homogenized by vortexing. The homogenate was divided.One half of the homogenate was used for DNA extraction bybeing mixed with an equal volume of lysis buffer (500 mMTris-HCl [pH 9.0], 20 mM EDTA, 10 mM NaCl, 1% sodiumdodecyl sulfate) containing lysozyme (Serva, Karlsruhe, Ger-many) at a final concentration of 1 mg/ml and incubated at37°C for 1 h. After proteinase K (Boehringer, Mannheim,Germany) at a final concentration of 200 jxg/ml was added,incubation was continued at 37°C overnight. DNA was purifiedby phenol-chloroform extraction and precipitated with coldabsolute ethanol. The DNA was pelleted by centrifugation,washed once with 70% ethanol, dried under a vacuum for 10min, and dissolved in 10 ,Iu of TE buffer (10 mM Tris-HCI [pH8.0], 1 mM EDTA). The other half of the homogenate wasused for extraction of bulk RNA. Lysozyme and proteinase Kwere added sequentially to final concentrations of 1 mg/ml and200 ug/ml, respectively. The mixture was incubated at 37°C for1 h after each addition. RNAzol (1 ml; Wak-Chemie) wasadded and mixed in by vortexing. After the addition of 0.1 mlof chloroform to initiate phase separation, the sample wascentrifuged. The aqueous phase was removed subsequently,mixed with an equal volume of isopropanol, and kept at - 20°Covernight to precipitate RNA. The precipitate was pelleted bycentrifugation, washed once with 75% (vol/vol) ethanol, anddried under a vacuum for 10 min. The dry pellet was dissolvedin 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 GCGGC[G/T] GCT G-3', corresponding to complementary posi-tions 519 to 536 in Escherichia coli 16S rRNA; the underlinedsequences at the 5' end represent the SmaI, BamHI, andHindlIl restriction sites) and incubated at 70°C for 10 min.Reverse transcriptase buffer (4 [lI of a 5 x buffer containing250 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 deoxynucleosidetriphosphates (dNTPs) (Pharmacia, Freiburg, Germany), and1 [LI (10 U) of Moloney murine leukemia virus reversetranscriptase (Stratagene, Heidelberg, Germany) were addedto a final volume of 20 ,ul. The mixture was incubated at 40°Cfor 30 min. The reaction was stopped by inactivating theenzyme at 95°C for 5 min.PCR amplification. (i) Amplification of DNA. 16S rRNA

sequences were amplified from subgingival plaque DNA andcDNA in parallel. PCR amplification of DNA was done byadding 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 perml), 200 ,uM dNTPs, 0.3 ,uM (each) primers RE-TPU1 (5'-CCG AAT TCG TCG ACA ACA GAG 'rITl GAT C[A/C]TGGC TCA G-3', corresponding to positions 8 to 27 in E. coli16S rRNA; underlined sequences at the 5' end representEcoRI and Sall restriction sites) and RE-RTU3 (see above),and 2.5 U of Taq polymerase (Gibco BRL) to a final reactionmixture volume of 100 [lI. After the addition of 100 plA of sterilemineral oil, amplification was started. Reactions were per-formed in an automated thermal cycler (Perkin-Elmer). DNAsamples were denatured by incubation at 95°C for 2 min andamplified by 30 cycles of 95, 55, and 72°C, for 1 min at eachtemperature. Amplicons were purified by agarose electro-phoresis and eluted by centrifugation with a small Mobicolcolumn (Mobitech, Gottingen, Germany).

(ii) Amplification of cDNA. To the reverse transcriptionmixture (20 RI) 13 plA of dNTPs (1.25 mM; Pharmacia), 8 pA oflOx PCR buffer, 1 pLI of 30 ,uM RE-TPU1 primer, 2.5 U (0.5[L) of Taq polymerase (Gibco BRL), and double-distilled

water were added to a final volume of 100 RI. Amplificationwas done as described above.

Cloning of amplified mixed-plaque 16S rRNA genes. Puri-fied amplicons were cleaved with BamHI and Sall (Pharmacia)according to the manufacturer's recommendations and puri-fied twice on a StrataClean resin (Stratagene) prior to ligationinto the multiple cloning site of precut plasmid pUC19 (Phar-macia). Ligation was done at 16°C overnight in a final volumeof 20 pA by using one-half of the amplified DNA, 50 to 100 ngof pUC19, 1 U of T4 DNA ligase (Gibco BRL), and 25 mMTris-HCl buffer (pH 7.8) containing 10 mM MgCl2, 1 mMdithiothreitol, and 1 mM ATP. Transformation was done byadding 100 jil of competent E. coli SURE cells (Stratagene) to10 ,ul of the ligation mix according to the manufacturer'sinstructions. Transformed cells were plated onto Luria-Bertaniagar containing 100 jig of ampicillin per ml, 0.004% (wt/vol)X-Gal (5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside)(Boehringer), and 0.5 mM isopropylthiogalactoside (IPTG)(Gibco BRL). Agar plates were incubated at 37°C overnight.Recombinants were selected on the basis of the white colonyphenotype.

Colony hybridization. White colonies were transferred withsterile toothpicks to Luria-Bertani agar media supplementedwith ampicillin and then were lifted onto positively chargednylon membranes (Biodyne B; Pall, Dreieich, Germany). Bac-terial colonies were lysed according to standard procedures.Oligodeoxynucleotide probes were 5' labeled with [T-32P]ATP(5,000 Ci mmol'-; Amersham, Braunschweig, Germany) byusing T4 polynucleotide kinase (BRL) according to the sup-plier's recommendations. Free label was removed by passingthe reaction mixture over a Sephadex G-50 column (Pharma-cia). Hybridization was performed essentially as describedpreviously (6).

Characterization of clones. The following probes (5, 8) wereused (hybridization temperatures are in parentheses): PI1V3(Prevotella internedia specific), 5'-CAC GTG CCC CGC ITTTACT CCC CAA-3' (66°C) (this sequence differs from asequence published by Dix et al. [5'-CAC GTG CCC CACrriT ACT CCC CAA-3'] (8) at one position [which is under-lined]); P12V3 (Prevotella nigrescens specific), 5'-CGT GCGCCA ATT TAT TCC CAC ATA-3' (60°C); BFV3 (Bacteroidesforsythus specific), 5'-CGT ATC TCA TTT TAT TCC CCTGTA-3' (56°C); PGV324 (Porphyromonas gingivalis specific),5'-CAA TAC TCG TAT CGC CCG TTA TTC-3' (60°C); andTREP (treponeme specific), 5'-GAC TTG CAT GCT TAA(G/A)AC-3' (45°C). One oligonucleotide complementary tothe successfully ligated polylinker-16S rRNA gene (rDNA)junction was constructed to screen the clone library for truerecombinants; this was 5'-GGT CGA CAA CAG AGT TTGAT-3' (48°C) (the sequence complementary to the pUC19polylinker is underlined). The TREP probe (base positions 46to 63 according to E. coli numbering) includes three basepositions that are single-base signatures for spirochetes: U atposition 47, U at 50, and A at 52. The consensus basesignatures for other bacteria are C at 47, A at 50, and Y at 52(45). While this probe has a single mismatch with somespirochetes (A at position 53 versus G in leptospires), it hasfour to five mismatches with essentially all other bacteria (31,45).

Sequence and phylogenetic analysis. Plasmid miniprepsfrom TREP-positive recombinants were prepared as describedpreviously (38). Both insert strands were sequenced by amodified Sanger dideoxy-chain-termination protocol using Se-quenase (U.S. Biochemicals, Munich, Germany) according tothe manufacturer's suggestions. M13 reverse-sequencingprimer (5'-AAC AGC TAT GAC CAT G-3'; Pharmacia) and

INFEc-r. IMMUN.

at Harvard Libraries on O

ctober 29, 2008 iai.asm

.orgD

ownloaded from

DIVERSITY OF CULTIVABLE AND UNCULTIVABLE SPIROCHETES

TABLE 1. Proportions of probe-positive clones

Amplilica- NO. of No. of probe-positive clones (()tion clones" Pid

method TREP" PG" 1 PN" BF/'

PCR 577 40 (6.9) 115 (19.9) 0 (0) 1 (0.2) 22 (3.8)RT-PCR9 5,841 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.

universal rRNA primer TPU2 (5'-CCA [A/G]AC TCC TACGGG AGG CA-3', corresponding to positions 334 to 353 in E.coli 16S rRNA) were used as forward primers, while sequenc-ing primer -40 (5'-GTT TTC CCA GTC ACG AC-3'; U.S.Biochemicals) and universal rRNA primer RTU2 (5'-TGCCTC CCG TAG GAG T[C/T]T GG-3', complementary topositions 353 to 334 in E. coli 16S rRNA) were used as reverse

primers for sequencing. Sequences were aligned on the basis ofconserved primary sequence analysis and secondary structureanalysis. All positions were included in distance calculations.Gaps and missing data were handled by pairwise deletion indistance calculations. Multiple base changes at single positionswere corrected by the method of Jukes and Cantor (16).Dendrograms were constructed by the neighbor-joiningmethod of Saitou and Nei (37). Negative branch lengths were

set to zero. The sequences were also examined by using theprogram CHECK-CHIMERA (29).

In situ hybridization. In situ hybridization was done essen-

tially as described previously (7, 11, 12). Species-specific probeZM399, derived from cluster 19 sequences (5'-CCG TCACCA TCA TAC CAT TTC CTG-3'), was synthesized auto-matically and labeled at the 5' end with fluorescein isothiocya-nate. All specimens were examined under oil immersion byepifluorescence microscopy with a Neofluar (Zeiss, Oberko-chen, Germany) 100 x objective and a Zeiss Axioskop 20 (fordetails see reference 12).

RESULTS

Identification of clones by hybridization with DNA probes.A total of 6,418 randomly selected recombinants were trans-ferred to nitrocellulose membranes for hybridization with a

panel of radiolabeled oligonucleotide probes. We chose thisrather large sample size to ensure that species representing lessthan 0. I% of the population had a reasonable chance of beingrepresented by a clone. The numbers of clones hybridizing witheach probe are shown in Table 1.

Sequence and phylogenetic analysis of oral treponemes. Atotal of 95 recombinants carrying treponeme-specific 16SrRNA sequences were identified. Fourteen of these cloneswere contaminated and were excluded from further study. Theremaining 81 clones were sequenced. Although complete 16Ssequences will be necessary for a thorough analysis of thephylogeny of these new treponeme species, a comparison ofthe 500-base partial sequences was sufficient to establish theapproximate phylogenetic positions of the clones as shown inFig. 1. Each of the 81 sequenced clones was presumptivelyidentified as a spirochete with the TREP probe. Spirochetalidentity was verified by phylogenetic analysis, thus validatingthe specificity of the TREP probe. Seven of the 81 sequenceswere chimeras and are not included in the figure. Chimeras

were found by noting that for some clones, the first half and thesecond half of the sequence did not group together on thephylogenetic tree. Examination of aligned sequences showedthat chimeras were composed of spirochetal and nonspiro-chetal halves. Thus, it may be useful in the future to use asecond probe to identify the 3' ends of amplified spirochetalsequences. Lack of reactivity with this probe would indicate apossible chimera. The sequences were also examined by usingthe program CHECK-CHIMERA written by Niels Larsen(29). This program identified as chimeric the same sevensequences 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 numberof nodes represent two or more identical sequences (thenumber of identical sequences appears after the clone desig-nation). 16S rRNA similarity for strains of a species is usuallygreater than 99%. When a relaxed species threshold of approx-imately 98% similarity was used, the 74 clones were dividedinto 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 theleptospires, 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. Therewere no clones related to T pectinovorum or T. socranskii.

Direct visualization of oral treponemes by in situ fluores-cence hybridization. For future studies fluorescently labeledoligonucleotides will be instrumental in assessing the role ofthe newly identified spirochetes in the etiology of periodontalinfections. To demonstrate the feasibility of this approach, weconstructed a fluorescein isothiocyanate-labeled oligonucleo-tide probe specific for clone NZM399 (from cluster 19), whichwas used to identify this novel treponeme in subgingival plaquesamples. As shown in Fig. 2, the fluorescein isothiocyanate-labeled probe ZM399 identified only one distinct spirochetalmorphotype, presumably the target species. No other bacteriawere stained.

DISCUSSION

Since most of the spirochetes in the oral cavity are presentlyuncultivable, the nucleic acids of spirochetes from bacterialplaque were analyzed directly by using molecular biologicaltechniques. The strategy was based upon studies of molecularevolution using comparative sequence analysis of rRNAs (48).Four different experimental approaches for obtaining rRNAsequences have been described, which theoretically representwithout bias the entire population, including both cultivableand uncultivable microorganisms. These approaches are asfollows: (i) direct sequencing of 5S rRNA (30), (ii) construc-tion of cDNA libraries by using purified mixed-populationRNA as a template for primer-directed reverse transcription ofrRNAs (45), (iii) in vitro amplification of rRNA genes frombulk DNA by using the PCR and rRNA-specific primers (13,46), and (iv) construction of shotgun clone libraries from bulkDNA and identification of rDNA-containing clones with amixed-kingdom probe (39). The latter approach is consideredto introduce the least bias into a phylogenetic analysis (39, 40).

Because of the minute amounts of DNA and RNA availablefrom subgingival plaque, we decided to establish a 16S rRNAgene library by amplifying part of the 16S rRNA gene directlyfrom purified plaque DNA and from cDNA after reversetranscription of plaque RNA. There were several reasons tochoose this dual approach. If DNA had been used exclusively,there would have been a danger of amplifying silent rRNAgenes from bacteria carrying several rRNA operons. In addi-

VOL. 6)2, 1993t4 1891

at Harvard Libraries on O

ctober 29, 2008 iai.asm

.orgD

ownloaded from

1892 CHOI ET AL.

Xreeponeea vincentitNZ.M3i00 RNZM3!12 2P

,NZ3I 42 P

rNHZW397 R

_NZM360 RNZM3105 ANZM30464 2D0 RNZM30292 0

NZM3103 RNZM398 R

NZM3010 RNZM3166NZM334 R

NZM30378 0

NZM/30392 0

NZM3I4S RNZM3f47 R

NZM3020 40NZM30I D

Treponema phagedenisNZM3106 5RNZM30472 0

NZM3042 DNZM3!07 2RNZM3i08 R

reponeea dent JcolaNZM3158 R

NZM3081 0

NZM3.43 2RNZM3i44NZM3i38 RNZM3079 0

NZM30527 0

NZM30298 2D. RNZM302 17 0

NZM335 P

rreponema pall

___I SpJrochae ta stenos

Cluster Group98X 92%

2

-134

-5I= 6 I

7

II

Jii]2

13 III

- 141516

ziaum,trepta

t rhereophilic spirochete HfSpirochaeta zuelzeraeNZM304I0 3DNZM3125 PNZM3037 0NZM(3I10 2RNZM3122 3R

NZM3I 13 2RNZM30505 0

Treoonema brvantii, f ,,

NZM3i0i RNZM399 0.

NZM3i57 RNZM3I24

NZM3ii8NZM3i28

r NZM3109 2RNZM3155

NZM3104 P

NZM30495 0

:7 I

- 1B

Treponema sucrreponema socranski.i

Treponema pectino vorueyNZM30384 0

Treponema sp. CAIrreponeaa saccharopbiulu

= 22 = VII

NZM30394ill

23 - VIIIlin.r

Leptospira biflexaLeptospira 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 followingclones indicate whether the sequence was obtained from DNA (D) or RNA (R). Numbers preceding the letters indicate the numbers of identicalclone sequences obtained.

tion, there would have been a remote possibility of amplifyingDNA from dead organisms, resulting in an erroneous repre-sentation of appropriate clones within the 16S rRNA gene

library. The former possibility was controlled by amplifyingcDNAs, which by definition derive from actively transcribedrRNA genes. Hence, microheterogeneity among silent andexpressed rRNA genes would not interfere with subsequentphylogenetic analysis. We therefore screened all recombinantclones, randomly selected from the respective DNA and cDNAlibraries, by colony hybridization with radiolabeled probesspecific for P. gingivalis, P. intermedia, P. nigrescens, and B.forsythus. The frequencies of these recombinant clones forboth libraries were quite similar, regardless of the amplifica-tion scheme used. Moreover, the data were in good agreementwith 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, significantdiscrepancy was found with treponeme-specific clones, whichrepresented about 7 and 1% of the clones randomly chosenfrom 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 lowcopy number of treponemal rRNAs due to either downregulation of ribosome biosynthesis in these slowly growingorganisms, rapid degradation of RNA during sampling, or

inefficient binding of reverse transcriptase or amplificationprimers, and the presence of modified bases resulting in earlytermination 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

(% Difference)

I

INFECT. IMMUN.

at Harvard Libraries on O

ctober 29, 2008 iai.asm

.orgD

ownloaded from

DIVERSITY OF CULTIVABLE AND UNCULTIVABLE SPIROCHETES 1893

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

leptospires than to the treponemes. As only one-third of the16S rRNA gene was sequenced, the dendrogram shown in Fig.1 has minor topological differences from our previously pub-lished tree for spirochetes, which was based upon full-lengthsequences (31). However, partial sequences have been usedsuccessfully to infer phylogenetic relationships (10, 13, 17, 39,44, 45). Because the region of 16S rRNA included had morelocales of high sequence variability than are average for theentire 16S rRNA molecule, the branch lengths are approxi-mately one-third longer than those for a tree computed fromcomplete sequences. The significance of Fig. 1 is that itdemonstrates an unexpected diversity of clones from a singlepatient. On the basis of morphological criteria, we expected tofind at least a dozen species other than the four named oraltreponemes. To find clones representing nearly 20 new speciesin a single patient implies that previously used methods forisolation, cultivation, and identification have grossly underes-timated the diversity of spirochetes present in the human oralcavity. We do not believe that the diversity seen in Fig. 1 wasdue to PCR or sequencing error. Of the 81 sequences, 35 wereidentical to other clone sequences, indicating that we couldobtain the same sequence from multiple clones. Some of theseidentical sequences were obtained from amplifications of bothDNA and RNA. In collaborative projects to be reportedelsewhere, unidentified isolates of oral treponemes have beenexamined by 16S rRNA sequence analysis (9, 49) and been

found to fall into clusters 7, 17, and 19. Thus, additionalcultivable species of oral treponemes exist. Attempts at cultur-ing presently uncultivable species will be aided by precisemeans of identification, such as rRNA-based DNA probes.Since the TREP probe was based upon known spirochetesequences, it is possible that there are spirochetes with basechanges not recognized by this probe. This would make ourestimates of the number of new species lower than that numberactually may be.

Since the formation of amplification artifacts such as chime-ras or shuffle products may occur, it is important to carefullyexamine sequences obtained (13, 18, 24). Examination ofaligned sequences revealed that most chimeras changed fromspirochetes to other bacterial taxa at about positions 310 to 360(according to E. coli numbering). This is a region of near-identity in bacteria, and therefore, partially extended PCRamplicons could anneal with other bacterial DNA and beelongated. By lengthening the extension time in the PCRs,especially in the first 10 cycles, this may be avoided. Because ofthe possibility of obtaining chimeric sequences, we recommendthat any sequences obtained by amplification of 16S rRNAsequences from mixed populations be examined by using aprogram such as CHECK-CHIMERA, which is available on-line at the University of Illinois ribosomal data base project(29).A fluorescently labeled, species-specific probe was used to

VOL. 62, 1994

at Harvard Libraries on O

ctober 29, 2008 iai.asm

.orgD

ownloaded from

1894 CHOI ET AL.

determine the relative proportions of the target species in anumber of clinical samples. A complete battery of probes canbe used in more extensive clinical studies to determine thedistribution of these hitherto-unknown bacteria within subgin-gival plaque samples and biopsy samples from diseased pa-tients. These studies will be important to identify oral spiro-chetes, including those that are presently uncultivable, that areassociated with diseased sites. There is great hope that theapproach described here will assist in elucidating the role oftreponemes in the pathogenesis of periodontal infections byassociating 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.

REFERENCES1. Amann, R., N. Springer, W. Ludwig, H. D. Gortz, and K. H.

Schleifer. 1991. Identification in situ and phylogeny of unculturedbacterial endosymbionts. Nature (London) 351:161-164.

2. Amann, R. I., L. Krumholz, and D. A. Stahl. 1990. Fluorescent-oligonucleotide probing of whole cells for determinative, phyloge-netic, and environmental studies in microbiology. J. Bacteriol.172:762-770.

3. Armitage, G. C., W. R. Dickison, R. S. Jenderseck, S. M. Levine,and D. W. Chambers. 1982. Relationship between the percentageof subgingival spirochetes and severity of periodontal disease. J.Periodontol. 53:550-556.

4. Chen, K., H. Neimark, P. Rumore, and C. R. Steinman. 1989.Broad range DNA probes for detecting and amplifying eubacterialnucleic acids. FEMS Microbiol. Lett. 57:19-24.

5. Choi, B.-K., and H. Gersdorf. Unpublished results.6. Chuba, P. J., K. Pelz, G. Krekeler, T. S. De Isele, and U. Gobel.

1988. Synthetic oligodeoxynucleotide probes for the rapid detec-tion of bacteria associated with human periodontitis. J. Gen.Microbiol. 134:1931-1938.

7. DeLong, E. F., G. S. Wickham, and N. R. Pace. 1989. Phylogeneticstains: ribosomal RNA-based probes for the identification ofsingle cells. Science 243:1360-1363.

8. Dix, K., S. M. Watanabe, S. McArdle, D. I. Lee, C. Randolph, B.Moncla, and D. E. Schwartz. 1990. Species-specific oligode-oxynucleotide probes for the identification of periodontal bacteria.J. Clin. Microbiol. 28:319-323.

9. Fraser, G. J., E. C. S. Chan, R. Siboo, F. E. Dewhirst, and B. J.Paster. Phylogenetic position of new oral treponemes. J. Dent.Res., in press.

10. Fuhrman, J. A., K. McCallum, and A. A. Davis. 1992. Novel majorarchaebacterial group from marine plankton. Nature (London)356:148-149.

11. Gersdorf, H., A. Meissner, K. Pelz, G. Krekeler, and U. B. Gobel.1993. Identification of Bacteroides forsythus in subgingival plaquefrom patients with advanced periodontitis. J. Clin. Microbiol.31:941-946.

12. Gersdorf, H., K. Pelz, and U. B. Gobel. 1993. Fluorescence in situhybridization for direct visualization of gram-negative anaerobesin subgingival plaque samples. FEMS Immunol. Med. Microbiol.6:109-114.

13. Giovannoni, S. J., T. B. Britschgi, C. L. Moyer, and K. G. Field.1990. Genetic diversity in Sargasso Sea bacterioplankton. Nature(London) 345:60-63.

14. Giovannoni, S. J., E. F. DeLong, G. J. Olsen, and N. R. Pace. 1988.Phylogenetic group-specific oligodeoxynucleotide probes for iden-tification of single microbial cells. J. Bacteriol. 170:720-726.

15. Gobel, U. B., A. Geiser, and E. J. Stanbridge. 1987. Oligonucleo-tide probes complementary to variable regions of ribosomal RNAdiscriminate between Mycoplasma species. J. Gen. Microbiol.133:1969-1974.

16. Jukes, T. H., and C. R. Cantor. 1969. Evolution of proteinmolecules, p. 21-132. In H. N. Munro (ed.), Mammalian proteinmetabolism, vol. 3. Academic Press, Inc., New York.

17. Liesack, W., and E. Stackebrandt. 1992. Occurrence of novelgroups of the domain Bacteria as revealed by analysis of geneticmaterial isolated from an Australian terrestrial environment. J.Bacteriol. 174:5072-5078.

18. Liesack, W., H. Weyland, and E. Stackebrandt. 1991. Potentialrisks of gene amplification by PCR as determined by 16S rDNAanalysis of a mixed culture of strict barophilic bacteria. Microb.Ecol. 21:191-198.

19. Listgarten, M. A. 1976. Structure of the microbial flora associatedwith periodontal health and disease in man. A light and electronmicroscopic study. J. Periodontol. 47:1-18.

20. Listgarten, M. A., and L. Hellden. 1978. Relative distribution ofbacteria at clinically healthy and peridontally diseased sites inhumans. J. Clin. Periodontol. 5:115-132.

21. Loesche, W. J. 1988. The role of spirochetes in periodontal disease.Adv. Dent. Res. 2:275-283.

22. Lukehart, S. A., M. R. Tam, J. Hom, S. A. Baker-Zander, K. K.Holmes, and R. Nowinski. 1985. Characterization of monoclonalantibodies to Treponema pallidum. J. Immunol. 134:585-592.

23. Medlin, L., H. J. Elwood, S. Stickel, and M. L. Sogin. 1988. Thecharacterization of enzymatically amplified eukaryotic 16S-likerRNA-coding regions. Gene 71:491-499.

24. Meyerhans, A., J. P. Vartanian, and S. Wain-Hobson. 1990. DNArecombination during PCR. Nucleic Acids Res. 18:1687-1691.

25. Moore, W. E. C. 1987. Microbiology of periodontal disease. J.Periodontal Res. 22:335-341.

26. Moore, W. E. C., L. V. Holdeman, E. P. Cato, R. M. Smibert, J. A.Burmeister, and R. R. Ranney. 1983. Bacteriology of moderate(chronic) periodontitis in mature adult humans. Infect. Immun.42:510-515.

27. Moore, W. E. C., L. V. Holdeman, R. M. Smibert, D. E. Hash, J. A.Burmeister, and R. R. Ranney. 1982. Bacteriology of severeperiodontitis in young adult humans. Infect. Immun. 38:1137-1148.

28. Olsen, G. J. 1990. Variation among the masses. Nature (London)345:20.

29. Olsen, G. J., R. Overbeek, N. Larsen, T. L. Marsh, M. J.McCaughey, M. A. Maciukenas, W.-M. Kuan, T. J. Make, Y. Xing,and C. R. Woese. 1992. The ribosomal database project. NucleicAcids Res. 20:2199-2200.

30. Pace, N. R., D. A. Stahl, D. J. Lane, and G. J. Olsen. 1986. Theanalysis of natural microbial populations by ribosomal RNAsequences. Adv. Microb. Ecol. 9:1-55.

31. Paster, B. J., F. E. Dewhirst, W. G. Weisburg, L. A. Tordoff, G. J.Fraser, R. B. Hespell, T. B. Stanton, L. Zablen, L. Mandelco, andC. R. Woese. 1991. Phylogenetic analysis of the spirochetes. J.Bacteriol. 173:6101-6109.

32. Relman, D. A., J. S. Loutit, T. M. Schmidt, S. Falkow, and L. S.Tompkins. 1990. The agent of bacillary angiomatosis-an ap-proach to the identification of uncultured pathogens. N. Engl. J.Med. 323:1573-1580.

33. Relman, D. A., T. M. Schmidt, R. P. MacDermott, and S. Falkow.1992. Identification of the uncultured bacillus of Whipple's dis-ease. N. Engl. J. Med. 327:293-301.

34. Riviere, G. R., M. A. Wagoner, S. A. Baker-Zander, K. S. Weisz,D. F. Adams, L. Simonson, and S. A. Lukehart. 1991. Identifica-tion of spirochetes related to Treponema pallidum in necrotizingulcerative gingivitis and chronic periodontitis. N. Engl. J. Med.325:539-543.

35. Riviere, G. R., K. S. Weisz, D. F. Adams, and D. D. Thomas. 1991.Pathogen-related oral spirochetes from dental plaque are invasive.Infect. Immun. 59:3377-3380.

36. Riviere, G. R., K. S. Weisz, L. G. Simonson, and S. A. Lukehart.1991. Pathogen-related spirochetes identified within gingival tissuefrom patients with acute necrotizing ulcerative gingivitis. Infect.Immun. 59:2653-2657.

37. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a newmethod for reconstructing phylogenetic trees. Mol. Biol. Evol.4:406-425.

38. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring Harbor Labo-ratory, Cold Spring Harbor, N.Y.

39. Schmidt, T. M., E. F. DeLong, and N. R. Pace. 1991. Analysis of a

INFEC-F. IMMUN.

at Harvard Libraries on O

ctober 29, 2008 iai.asm

.orgD

ownloaded from

DIVERSITY OF CULTIVABLE AND UNCULTIVABLE SPIROCHETES 1895

marine picoplankton community by 16S rRNA gene cloning andsequencing. J. Bacteriol. 173:4371-4378.

40. Schmidt, T. M., E. F. DeLong, and N. R. Pace. 1991. Phylogeneticidentification of uncultivated microorganisms in natural habitats,p. 37-46. In A. Vaheri, R. C. Tilton, and A. Balows (ed.), Rapidmethods and automation in microbiology and immunology.Springer Verlag, Berlin.

41. Slots, J. 1979. Subgingival microflora and periodontal disease. J.Clin. Periodontol. 6:351-382.

42. Socransky, S. S., and A. D. Haffajee. 1988. Microbiology (plaque),p. 147-197. In D. A. Grant, I. B. Stern, and M. A. Listgarten (ed.),Periodontics in the tradition of Gottlieb and Orban. Mosby, St.Louis.

43. Soder, P. O., L. Frithiof, and B. Soder. 1990. Spirochetes andgranulocytes at sites involved in periodontal disease. Arch. OralBiol. 35:197S-200S.

44. Stackebrandt, E., W. Liesack, and B. M. Goebel. 1993. Bacterialdiversity in a soil sample from a subtropical Australian environ-ment as determined by 16S rDNA analysis. FASEB J. 7:232-236.

45. Ward, D. M., R. Weller, and M. M. Bateson. 1990. 16S rRNAsequences reveal numerous uncultured microorganisms in a nat-

ural community. Nature (London) 344:63-65.46. Weisburg, W. G., S. M. Barns, D. A. Pelletier, and D. J. Lane.

1991. 16S ribosomal DNA amplification for phylogenetic study. J.Bacteriol. 173:697-703.

47. Wilson, K. H., R. Blitchington, R. Frothingham, and J. A. P.Wilson. 1991. Phylogeny of the Whipple's-disease-associated bac-terium. Lancet 338:474-475.

48. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221-271.

49. Wyss, C., B.-K. Choi, and U. B. Gobel. Submitted for publication.

VOL. 62, 1994

at Harvard Libraries on O

ctober 29, 2008 iai.asm

.orgD

ownloaded from