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Journal of Clinical Microbiology, September 2006, p. 3078-3085, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.00322-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Epidemiology of Oral Treponemes in Patients with Periodontitis and in Periodontitis-Resistant Subjects

Annette Moter,1* Birgit Riep,2 Vesna Haban,1 Klaus Heuner,3 Gerda Siebert,4 Moritz Berning,1 Chris Wyss,5 Benjamin Ehmke,6 Thomas F. Flemmig,7 and Ulf B. Göbel1

Institut für Mikrobiologie und Hygiene,1 Institut für Parodontologie und Synoptische Zahnmedizin,2 Institut für Medizinische Biometrie, Charité-Universitätsmedizin, Berlin, Germany,4 Institut für Molekulare Infektionsbiologie, Julius-Maximilians Universität Würzburg, Würzburg, Germany,3 Institut für Orale Biologie und Allgemeine Immunologie, Universität Zürich, Zürich, Switzerland,5 Poliklinik für Parodontologie, Westfälische Wilhelms Universität Münster, Münster, Germany,6 Department of Periodontics, School of Dentistry, University of Washington, Seattle, Washington7

Received 14 February 2006/ Returned for modification 23 April 2006/ Accepted 17 June 2006


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ABSTRACT
 
The etiologic role of oral treponemes in human periodontitis is still under debate. Although seen by dark-field microscopy in large numbers, their possible role is still unclear since they comprise some 60 different phylotypes, most of which are still uncultured. To determine their status as mere commensals or opportunistic pathogens, molecular epidemiological studies are required that include both cultured and as-yet-uncultured organisms. Here we present such data, comparing treponemal populations from chronic periodontitis (CP) or generalized aggressive periodontitis (GAP) patients. As a periodontitis-resistant (PR) control group, we included elderly volunteers with more than 20 natural teeth and no history of periodontal treatment and no or minimal clinical signs of periodontitis. Almost every treponemal phylotype was present in all three groups. For most treponemes, the proportion of subjects positive for a certain species or phylotype was higher in both periodontitis groups than in the PR group. This difference was pronounced for treponemes of the phylogenetic groups II and IV and for Treponema socranskii and Treponema lecithinolyticum. Between the periodontitis groups the only significant differences were seen for T. socranskii and T. lecithinolyticum, which were found more often in periodontal pockets of GAP patients than of CP patients. In contrast, no difference was found for Treponema denticola. Our findings, however, strengthen the hypothesis of treponemes being opportunistic pathogens. It appears that T. socranskii, T. lecithinolyticum and group II and IV treponemes may represent good indicators for periodontitis and suggest the value of the respective probes for microbiological diagnosis in periodontitis subjects.


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INTRODUCTION
 
Periodontitis is a chronic inflammatory disease of infectious origin leading to the destruction of tooth-supporting tissues. The disease is frequently associated with a number of periodontal pathogens such as Tannerella forsythensis, Porphyromonas gingivalis, and Treponema denticola (35). It is the major cause of tooth loss in the adult population. Chronic periodontitis (CP) is the most common form and is characterized by a gradual loss of clinical periodontal attachment (4). In contrast to this, a rapid destruction of periodontal tissues is evident in aggressive periodontitis. Generalized aggressive periodontitis (GAP) usually affects young adults under the age of 30 years and attachment loss occurs in pronounced episodic periods of destruction (3).

The etiologic role of oral treponemes in periodontitis has been postulated based on their high frequency in periodontal lesions (5, 13, 21, 24). In most patients with chronic periodontal disease, spirochetes were observed by dark-field microscopy as predominating bacterial morphotypes. Spirochetal cell counts have been shown to correlate well with the severity of disease (5, 18). However, their identification has thus far been limited to cultivated species by using antibodies, PCR, or anaerobic culture methods (32, 34). For a recent review of involvement of spirochetes in periodontal disease, see the study by Ellen et al. (12).

Recently, we described a procedure to detect oral treponemes in subgingival plaque from patients with generalized aggressive periodontitis (GAP) using a culture-independent molecular genetic method (23). A set of oligonucleotide probes, specific for both cultivable species and the major oral treponemal groups I to VII according to the classification of Choi et al. (9), was used in dot blot or fluorescence in situ hybridization to assess the presence of the respective phylotypes in patient material. Parallel use of group- and species-specific probes revealed higher numbers of as-yet-uncultured treponemal phylotypes compared to the known cultivable species. There was a striking discrepancy in the detection of group I and group II treponemes compared to their cultivable representatives T. vincentii and T. denticola, respectively. Thus far, little is known about the presence of the different treponemes in healthy mouth flora. We therefore used the same approach to analyze subgingival plaque from elderly volunteers largely resistant to periodontitis, as evidenced by a minimum of 20 existing teeth with no or minimal attachment loss and not having received any periodontal therapy. These volunteers were compared to subjects classified as having chronic or generalized aggressive periodontitis.


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MATERIALS AND METHODS
 
Clinical samples. A total of 687 samples from 141 subjects were included in the present study. From those seeking periodontal treatment at the Department of Periodontology at Humboldt University of Berlin or the University of Würzburg, 67 patients with chronic periodontitis (CP) and 53 patients suffering from generalized aggressive periodontitis (GAP) were selected. All patients were previously untreated and diagnosed according to the criteria of the 1999 International Workshop for the Classification of Periodontal Disease and Conditions (4). A periodontitis-resistant (PR) group consisting of 21 elderly subjects served as a control group and was recruited from a private periodontal practice in Berlin. Inclusion criteria for this group were that the subjects had to be ≥65 years old, have at least 20 natural teeth present, have only mild periodontal disease (defined by no clinical attachment loss >2 mm or probing pocket depth [PPD] >5 mm), and have no history of periodontal therapy. The exclusion criteria for all subjects were chronic systemic disease and/or anti-inflammatory or antimicrobial therapy within the preceding 6 months; pregnant or lactating women were also excluded. The demographic data for all subjects are presented in Table 1.


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  		TABLE 1. Patient demographics

Samples were taken from the four deepest periodontal pockets and, if present, from an additional control site with a PPD of ≤3 mm by inserting three sterile paper points (ISO 35; Becht, Offenburg, Germany) into the periodontal pockets after removal of the supragingival plaque. The paper points were removed after 10 s and placed into 1 ml of reduced transport fluid (36) containing 25% glucose, transferred to the laboratory, and processed immediately.

DNA extraction and amplification. For PCR amplification, plaque samples were processed as described earlier (23). Briefly, an aliquot (100 µl) of each specimen was centrifuged at 13,000 x g in a Labofuge 400 R (Heraeus, Germany) for 10 min. Lysis buffer (100 µl) (9) was added to the pellets, and no further purification of nucleic acids was performed. Of the bulk DNA, 1 µl was used for in vitro amplification by PCR (final reaction volume of 100 µl) in a thermal cycler (Trioblock, Biometra, Germany), using 30 cycles of denaturation (1 min at 95°C), annealing (1 min at 56°C), and extension (1 min at 72°C). The broad-range bacterial primers TPU1 (5'-AGAGTT TGA TCM TGG CTC AG-3'; corresponding to positions 8 to 27 in the Escherichia coli 16S rRNA gene) (6) and RTU3 (5'-GWA TTA CCG CGG CKG CTG-3'; corresponding to complementary positions 519 to 536 in E. coli 16S rRNA) (6) were used for 16S rRNA gene amplification. Successful amplification was verified by agarose gel electrophoresis.

Oligonucleotide probes. Oligonucleotide probes TRE I to TRE VII specific for the major phylogenetic groups 1 to 7 of oral treponemes were designed according to the phylogenetic tree of Choi et al. (9) as published earlier (23). This classification was extended later by Dewhirst et al. (11), changing group 6 into T. socranskii-related phylotypes. Probes TVIN, TDEN, TMAL, TSOC, and TPEC identifying known cultivable treponemes T. vincentii, T. denticola, T. maltophilum, T. socranskii, and T. pectinovorum were described previously. To detect the recently published species T. medium (37), T. lecithinolyticum (41) and T. amylovorum (40), the probes TMED (5'-ACCCCTTATGAAGCACTGAGTGTATT-3'), TLEC (5'-CACTCT CAG AAA GGA GCA AGC TCC-3'), and TAMY (5'-CCTTCT TAG CTT CTT CTT CAT GTA TA-3') were designed accordingly. To assess hybridization specificity, probe sequences were compared to all 16S rRNA entries at the EMBL and GenBank databases accessible (as of January 2002) by using the program BLASTN of the Husar 5.0 (Heidelberg Unix Sequence Analysis Resources) program package (DKFZ, Heidelberg, Germany). All probes have been deposited in ProbeBase (22), an online resource for 16S rRNA-targeted oligonucleotide probes where probe difference alignments are available (http://www.microbial-ecology.de/probebase/index.html). Probe TSOC detects all subspecies of T. socranskii and the recently published closely related strain Smibert-5 (25). The probe EUB338 (2), complementary to a region of the 16S rRNA highly conserved in the domain Bacteria, was used as a positive control to check PCR amplification.

Dot blot hybridization. Amplified DNA was spotted onto nylon membranes, and dot blot hybridization was carried out to detect cultivable treponemes, as well as phylogenetic genospecies, in individual patients as described earlier (23). Briefly, 1 µl of heat-denatured PCR product was spotted onto nylon membranes (Hybond N; Amersham, Buckinghamshire, United Kingdom) and fixed by UV cross-linking. A total of 33 amplified DNA samples from either recombinant clones retrieved from the original 16S rRNA gene library, known cultivable treponemes, or other putative periodontal pathogens were included as controls in all dot blot hybridizations. Probes were labeled with digoxigenin-ddUTP (Boehringer Mannheim, Germany) and detected by chemiluminescence according to the manufacturer's recommendations. All hybridizations were performed at 54°C. Stringency washes were optimized for each probe by varying the wash temperature (56 to 64°C) and the wash buffer (containing 5x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.2% sodium dodecyl sulfate [SDS], or 0.1x SSC-0.1% SDS). X-ray films were exposed to the membranes for 2 to 12 h. After stripping with 0.2 N NaOH-0.1% SDS (stripping buffer), identical membranes were used for multiple hybridization experiments with the probes mentioned above.

Statistical analysis. Statistical evaluation was performed for descriptive purposes. The exact chi-square test was applied to compare the presence of treponemal phylotypes between the three groups. A person was regarded as positive for a certain genospecies if the organism was detected in at least one periodontal pocket. The chi-square test was also used for determination of the differences between the groups regarding treponemes at different probing pocket depths. To examine differences in the number of positive sites per patient of approved treponemal species or as-yet-uncultured phylotypes, the Kruskal-Wallis rank test was used for descriptive analysis. For both statistical tests, P values of <0.05 were considered significant.


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RESULTS
 
PCR amplification was successful for all subgingival plaque specimens, as indicated by a strong hybridization signal with the bacterial probe EUB338. None of the negative controls showed a hybridization signal, indicating that carryover of amplified material did not occur (data not shown). Using optimized hybridization and washing conditions, only the appropriate PCR products were detected on the control membranes. No cross hybridization was observed.

Prevalence of treponemes in the patient groups. All treponemal phylotypes were found in both patients and PR subjects, except for T. pectinovorum, which has not been detected in any specimen. The number of individuals positive for a given species or phylotype varied considerably between the groups. The prevalence of most genospecies was highest in the GAP group, followed by the CP patient group and the PR group (Fig. 1). Significant differences were found for TRE II (P = 0.0010 [CP versus PR], P = 0.00009 [GAP versus PR]), TRE IV (P = 0.0001 [CP versus PR], P = 0.00001 [GAP versus PR]), and TSOC (P = 0.0001 [CP versus PR], P < 0.00001 [GAP versus PR]). For TRE I only the GAP group differed significantly from the PR (P = 0.0013 compared to P = 0.0323 for CP versus PR). T. lecithinolyticum was detected significantly more often in GAP patients than in CP patients and PR subjects (both P < 0.00001). The difference between the CP versus PR was not significant (P = 0.1152). Obvious differences were observed between the patient groups regarding signal intensities of the dot blot hybridizations, suggesting a higher number of treponemes in the GAP patients (Fig. 2). However, since PCR-amplified DNA was used for hybridization experiments, the signal intensity was not analyzed.


Figure 1
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  		FIG. 1. Presence of oral treponemes in the PR subjects, CP patients, and GAP patients as determined by dot blot hybridizations with phylogroup-specific (A) or species-specific (B) oligonucleotide probes. A patient was regarded as positive if at least one sample was positive. TRE I to TRE VII are group-specific probes; TVIN, TMED, TDEN, TMAL, TLEC, TAMY, and TSOC represent probes specific for the single treponeme species T. vincentii, T. medium, T. denticola, T. maltophilum, T. lecithinolyticum, T. amylovorum, and T. socranskii, respectively. Bars indicate significant differences between the groups.


Figure 2
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  		FIG. 2. Dot blot hybridizations of identical membranes with probes TDEN, TRE II, and TSOC. In columns 1 to 8, PCR products of the following strains were applied as controls. Putative oral pathogens: Actinobacillus actinomycetemcomitans MCCM 02638 (A1), Capnocytophaga gingivalis MCCM 00858 (A2), Capnocytophaga ochracea MCCM 00238 (A3), Eubacterium lentum ATCC 25559T (A4), Fusobacterium nucleatum ATCC 25586T (A5), Porphyromonas gingivalis ATCC 33277 (A6), Prevotella intermedia MCCM 00407 (A7). Cultivable treponeme species: T. vincentii ATCC 35580 (B1), T. denticola ATCC 35405T (B2), T. socranskii subsp. socranskii ATCC 35536T (B3), T. socranskii subsp. buccale ATTC 35534T (B4), T. maltophilum ATCC 51939T (B5), T. phagedenis subsp. reiterii (B6), a clinical isolate (B8, highest homology to clone NZM 3142), T. pectinovorum ATTC 33768T (E1), T. lecithinolyticum OMZ 684 (D8). Recombinant clone group I: NZM3D292 (C1), NZM3D464 (C5), NZM3112 (C6; sequence 100% homologous to probe TVIN), NZM3142 (D2), NZM3147 (D4), NZM3166 (D7). Recombinant clone group II: NZM3106 (C7), NZM3158 (D6; sequence 100% homologous to probe TDEN). Recombinant clone group III: NZM3143 (D3), NZM3D298 (C3), NZM3D527 (C4). Recombinant clone group IV: NZM3122 (C8), NZM3D505 (C2). Recombinant clone group V: NZM3124 (D1), NZM3155 (D5). Recombinant clone group VI: NZM3104 (E2). Recombinant clone group VII: NZM3D384 (E3). Asterisks indicate empty fields without PCR product. In columns 9 to 13, 14 to 18, and 19 to 23, PCR products from subgingival plaque samples of PR subjects, CP patients, and GAP patients (five patients each) were applied, respectively. Each column represents four deep pockets plus one control site of one patient. Samples were considered positive if the dot was clearly visible above the background level of the negative controls.

Number of positive sites per patient. The prevalence values as used in the preceding section might underestimate quantitative differences between the groups. To quantitatively assess the presence of different treponemes, we compared the percentage of patients with one, two, three, or four positive sites. Using a statistical rank test, all three groups differed significantly for all probes investigated except for TRE VI and TVIN (data not shown). For TRE II, TRE IV, TLEC, and TSOC, the results were most pronounced (Fig. 3). The group-specific probes TRE II and TRE IV detected a higher number of positive sites in both advanced periodontitis groups compared to the controls (for both probes, P < 0.00001 [CP versus PR and GAP versus PR]). For TDEN and TMAL these differences were statistically significant only for the GAP versus PR (TDEN, P = 0.051 [CP versus PR], P = 0.011 [GAP versus PR]; TMAL, P = 0.057 [CP versus PR], P = 0.005 [GAP versus PR]). However, for TSOC and TLEC, the GAP patients differed highly significant not only from the PR subjects (both probes, P < 0.00001) but also from the CP group (both probes, P < 0.0001) (Fig. 3). Most obvious were the differences for T. lecithinolyticum, which was detected in 19, 40.3, and 83% of the PR patients, CP patients, and GAP patients, respectively, with 0, 6, and 37.7% patients with four positive sites.


Figure 3
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  		FIG. 3. Percentage of patients with one, two, three, or four of the sites of >3 mm colonized by a treponemal species or phylotype as revealed by dot blot analysis.

Presence of treponemes depending on probing pocket depths. To check whether there was a correlation between certain phylotypes and the severity of disease, we compared the number of positive samples from pockets of different probing depths. This parameter differed significantly for the patient groups versus the PR group for PPD from 4 to 6 mm using group-specific probes TRE II (Fig. 4) and TRE IV (data not shown). At sites with PPD from 7 to 9 mm, differences were statistically significant for TRE II (Fig. 4), TRE III, and TRE VII (data not shown) between the CP and GAP patients. Although T. denticola, a group II treponeme, was detected slightly more often in GAP patients at sites with PPDs greater than 3 mm, none of the results was statistically significant (Fig. 4). The same was true for T. maltophilum, a group IV treponeme (data not shown). In contrast, T. lecithinolyticum (another group IV treponeme) was detected significantly more often in GAP samples at all pocket depths from 1 to 3 mm, 4 to 6 mm, 7 to 9 mm, and ≥10 mm (P = 0.036, P < 0.00001, P < 0.00001, and P = 0.013, respectively) (Fig. 4).


Figure 4
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  		FIG. 4. Percentages of positive samples at different probing pocket depths. For statistical accuracy, only one positive sample was counted for each individual at each pocket depth. At pocket depths of 7 to 9 mm and ≥10 mm, only the two periodontitis groups were compared, since the PR subjects did not have deep pockets. Asterisks indicate significant differences.


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DISCUSSION
 
The association between oral treponemes and periodontitis has been described by many authors in the past (19-21, 27, 31). By dark-field microscopy a quantitative correlation between the number of spirochetes in subgingival plaque and the severity of disease has been observed (5). Due to the lack of adequate methods, epidemiological investigations have been mainly restricted to the few cultivable species, and analyses regarding virulence determinants have been performed especially for T. denticola (14). Recently, we showed that the number of not cultured treponemes in GAP was underestimated by far. In the present study we analyzed the prevalence of cultivable and not-yet-cultured oral treponemes in three subject groups. We compared patients with GAP to patients with CP, the most prevalent periodontal disease, along with a group of PR patients. The latter group served as a control group since the volunteers were at least 65 years of age and had still most of their teeth (≥20 teeth) and only localized slight periodontitis with no history of periodontal therapy. These subjects can therefore be considered resistant to periodontal disease.

All investigated treponemal species (except T. pectinovorum) and phylogroups were detected in all three patient groups in at least one sample. This strengthens the view that in periodontitis as a mixed bacterial infection not a single species is responsible for the severity of disease alone. Furthermore, host factors such as the innate immune system have been shown to influence the course of disease (33).

The percentage of positive patients and the intensity of dot blot signals showed quantitative differences for most treponemal phylotypes between the PR subjects versus the diseased (CP or GAP) patients. To analyze these differences more accurately, we analyzed the site-specific data by comparing the number of positive sites per patient between the three patient groups. This analysis revealed profound differences between GAP and CP versus the PR patients for treponemes of groups I, II, and IV: T. socranskii, T. maltophilum, and T. lecithinolyticum. As seen in Fig. 3, GAP patients were also more likely to harbor a certain phylotype in every subgingival pocket investigated compared to CP patients. This difference was significant for T. socranskii and T. lecithinolyticum but not for T. denticola or T. maltophilum (Fig. 3). These findings are in contrast to a previous report using nested or seminested PCR with specific primers for the detection of cultivable species in periodontitis patients and a healthy population (39). The authors of that study describe a significant difference between the groups for T. pectinovorum and T. vincentii but not for T. socranskii. These discrepancies are likely to be due to methodology. Nested PCR with subsequent agarose gel electrophoresis is more sensitive than single eubacterial PCR but might not be as specific without hybridization, especially since closely related treponemes were not used as negative controls in their study. This might be particularly true for T. vincentii and T. denticola, representing members of the larger phylogenetic groups I and II, respectively. Accordingly, immunological assays used for identification of T. denticola (34) and formerly called pathogen-related oral spirochetes (PROS) (26, 29, 30) carry the risk of cross-reactivity that cannot be controlled due to the high number of uncultivable treponemes (10, 28). Dealing with a variety of closely related and not-yet-cultured organisms, we favored specificity rather than sensitivity. However, one could argue that in a deep periodontal pocket more bacterial mass is taken by the paper points and treponemes would be easier above the detectable level. In this case the higher percentage of treponemes in GAP patients would just reflect the higher number of deeper pockets. We therefore analyzed the site-specific data depending on probing pocket depths. For most genospecies and probing pocket depths, most samples were positive in the GAP group, followed by the CP patients and the PR subjects. The differences, however, were only marginal and not always statistically significant due to low numbers in the test groups. Significant differences between the periodontitis groups and the PR group were detected for TRE II and TRE IV at pockets between 4 to 6 mm in depth. For pockets from 7 to 9 mm, statistically significant differences between GAP and CP patients were observed for the treponemes of groups II, III, and VII. The most pronounced differences between GAP and CP patients were seen for T. lecithinolyticum, which was found >2-fold more often in GAP patients at any probing pocket depth. This is in line with some recent studies suggesting an involvement of T. lecithinolyticum in bone and tissue destruction by induction of osteoclast differentiation, activation of matrix metalloproteinase-2 and upregulation of intercellular adhesion molecule 1 and proinflammatory cytokines (7, 8, 17). In contrast, there were no significant differences regarding T. denticola and T. maltophilum. Although prospective studies with higher sample numbers are needed, these species, as well as T. amylovorum and T. pectinovorum, do not seem to have specific value as diagnostic markers. The presented data indicate that group II and IV treponemes, as well as T. socranskii and T. lecithinolyticum, could serve as diagnostic markers for the progressing periodontal disease, and probe TLEC might also be used as a prognostic marker for the diagnosis of GAP. Although TRE III and TRE VII showed a significant difference between GAP versus CP patients, these two probes would not be useful as diagnostic markers since the prevalence of those phylotypes was not very high.

According to these data, the majority of treponemes seems to be rather opportunistic. However, they may be necessary for maintaining the disease, potentially being a major player in subgingival biofilm architecture, rather than causing the clinical differences between CP and GAP (15, 38).

Similar to our earlier study, more samples were positive for the group-specific probes TRE I, TRE II, and TRE IV than for the probes for the respective cultivable members of the groups T. vincentii, T. medium (both group I and PROS), T. denticola (group II), and T. maltophilum and T. lecithinolyticum (both group IV), supporting data that many treponemal species still have not been cultured. This is in accordance with other molecular studies showing a high number of not-yet-cultured and therefore not-investigated oral species (1, 16). For analysis of virulence factors, it seems to be necessary to strengthen the efforts to cultivate these organisms. Regarding treponemes, this would especially be important for group II organisms since these were most prevalent in sites with PPDs of 4 to 6 mm for GAP or CP patients rather than for PR subjects, whereas T. denticola was not. Therefore, virulent species of group II might have been missed thus far. Also, group I treponemes would be of interest since there is a great discrepancy between the number of TRE I- and T. vincentii- or T. medium-positive samples, and they may also enclose the formerly so-called PROS.

In summary, oral treponemes were detected in all subject groups. Most species and phylogroups were detected more often in GAP patients than in CP patients and considerably less often in the PR subjects. There are still many oral treponemes uncultured. Regarding probing pocket depths there were significant differences between GAP and CP patients and PR subjects for phylogroups II and IV. Between GAP and CP patients significant differences were seen for TRE II, III, and VII for pockets deeper than 6 mm and for T. lecithinolyticum at any probing pocket depth. Therefore, we suggest TRE II, TRE IV, and TLEC as diagnostic marker-probes rather than the detection of T. denticola, which has been used most frequently thus far.


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ACKNOWLEDGMENTS
 
We thank Cindy Hefenbrock and Jörg Schneiderheinze for excellent technical assistance. Strains A. actinomycetemcomitans MCCM 02638, C. gingivalis MCCM 00858, C. ochracea MCCM 00238, E. lentum ATCC 25559T, F. nucleatum ATCC 25586T, P. gingivalis ATCC 33277, and P. intermedia MCCM 00407 were provided by R. Mutters, Marburg, Germany. T. maltophilum ATCC 51939T and the clinical isolate in Fig. 2 was provided by C. Wyss, Zurich, Switzerland. T. phagedenis subsp. reiterii was provided by B. Wilske, Munich, Germany.

This study has been supported by a grant (01KI9318) from the Bundesministerium für Bildung und Forschung (U.B.G.) and a Körber European Research Award (U.B.G.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Mikrobiologie und Hygiene, Charité-Universitätsmedizin Berlin, Dorotheen-Str. 96, D-10117 Berlin, Germany. Phone: 49 30 450524226. Fax: 49 30 450524902. E-mail: annette.moter{at}charite.de. Back


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Journal of Clinical Microbiology, September 2006, p. 3078-3085, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.00322-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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