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J Clin Microbiol. Dec 2004; 42(12): 5523–5527.
PMCID: PMC535285

Culture-Independent Identification of Periodontitis-Associated Porphyromonas and Tannerella Populations by Targeted Molecular Analysis

Abstract

Periodontitis is the commonest bacterial disease of humans and is the major cause of adult tooth loss. About half of the oral microflora is unculturable; and 16S rRNA PCR, cloning, and sequencing techniques have demonstrated the high level of species richness of the oral microflora. In the present study, a PCR primer set specific for the genera Porphyromonas and Tannerella was designed and used to analyze the bacterial populations in subgingival plaque samples from inflamed shallow and deep sites in subjects with periodontitis and shallow sites in age- and sex-matched controls. A total of 308 clones were sequenced and found to belong to one of six Porphyromonas or Tannerella species or phylotypes, one of which, Porphyromonas P3, was novel. Tannerella forsythensis was found in significantly higher proportions in patients than in controls. Porphyromonas catoniae and Tannerella phylotype BU063 appeared to be associated with shallow sites. Targeted culture-independent molecular ecology studies have a valuable role to play in the identification of bacterial targets for further investigations of the pathogenesis of bacterial infections.

Periodontitis is a multifactorial disease resulting from the interaction between bacteria in dental plaque and the individual's inflammatory response. Ultimately, periodontitis destroys the tissues that support the teeth and can cause tooth loss. The pocket formed as a result of the disease harbors a predominantly anaerobic microflora of considerable complexity (27). Of the approximately 600 bacterial taxa present in the subgingival environment, some have been shown to be specifically related to disease in association studies. For example, the so-called red cluster of species, Porphyromonas gingivalis, Tannerella forsythensis, and Treponema denticola, have been shown to be associated with bleeding on probing, both individually and as a group (29). Longitudinal studies have indicated that P. gingivalis is associated not only with inflammation but also with progressive destructive disease and is a predictor of future periodontal breakdown in adults (10). Similarly, in a nonhuman primate model, the introduction of P. gingivalis into the oral cavity caused destructive periodontal disease in the animals (13), while prior immunization with P. gingivalis helped prevent destructive disease (28).

T. forsythensis has been shown to be one of three species associated with sites that have converted from periodontal health to disease (31). Indeed, the odds of loss of attachment in adolescents were shown to be 8.16 times greater in subjects colonized by T. forsythensis in a 3-year longitudinal study (11). In adults with a low prevalence and a low severity of periodontitis, subjects persistently colonized with T. forsythensis had 5.3 times higher odds of losing attachment at at least one site compared to the odds for those in whom the organism was not detected or only occasionally detected (33). In another longitudinal study of a similar adult group, the presence of T. forsythensis at the baseline significantly increased the likelihood of loss of attachment at follow-up 2 to 5 years later (23).

Porphyromonas endodontalis was first isolated from infected root canals (34), but its presence in other oral infections is only rarely reported from studies based on culture, perhaps because the majority of biotypes of this organism are unculturable (5). Interestingly, in a study that used a PCR-based detection method, P. endodontalis was shown to be as strongly associated with periodontitis as P. gingivalis (17).

Members of the genus Porphyromonas and its close relative, Tannerella, are clearly, then, important in periodontitis. However, these associations with disease have been derived from cultural studies of the periodontal microflora, 50% of which is now recognized to be unculturable (27). Molecular analyses based on PCR of housekeeping genes, such as the gene encoding the 16S rRNA (16S rDNA), allied with cloning and sequencing are revolutionizing the study of the composition of the oral microflora in health and disease by revealing the true extent of the bacterial communities present, free of the biases of culture (5, 16, 25, 27). The majority of these studies use universal primers in an attempt to characterize the entire community. However, a powerful modification of the technique is to design primers that are specific for a particular group of interest. For example, primers designed to be specific for a group of oral asaccharolytic Eubacterium species revealed two novel disease-associated taxa in periodontitis patients (30).

The aim of this study was to design 16S rDNA primers specific for the genera Porphyromonas and Tannerella and use them to construct clone libraries of amplified 16S rDNA genes from samples collected from patients with advanced periodontitis and controls. Although these two genera together make up only a small proportion of the subgingival microflora, the associations between particular species and disease make them worthwhile targets for study.

MATERIALS AND METHODS

Plaque samples were collected from five pairs of patients matched for age (±3 years) and gender, as described in a previous study (1). Briefly, one member of each pair had periodontitis with pockets of at least 6 mm at two or more sites and the other member of the pair had no pockets greater than 4 mm with minimal or no bone loss. Patients were excluded if they had medical conditions that influenced their periodontal condition or if they had received subgingival scaling or antibiotics within the previous 6 months.

Supragingival plaque was scored as clearly visible, detectable only with a probe, or absent and, when present, was carefully removed before a subgingival plaque sample was collected with a sterile curette from shallow pockets (≤4 mm) in patients and controls and from deep pockets (≥6 mm) in the patients. Plaque samples were collected in 500 μl of storage buffer (10 mM Tris HCl, 1 mM EDTA [pH 8.0]) and were stored at −70°C. Sites were probed manually; and bleeding after probing was scored as immediate (score = 2), within 30 s (score = 1), or absent (score = 0). To compare the levels of oral hygiene and marginal gingival health in the patients and controls, plaque and gingival bleeding indices were recorded for the buccal and lingual surfaces of the six Ramfjord teeth.

The two plaque samples from shallow sites were pooled, as were the two samples from deep sites, and DNA was extracted from the samples by a standard method (9).

A specific PCR primer directed against the 16S rRNA genes of the genera Porphyromonas and Tannerella was designed by visual inspection of the aligned sequences of reference strains. The primer was designated PT997R and had the following sequence: 5′-ATCTACATTCAATCCC-3′. The specificity of the primer was checked by performing PCR under the conditions described below with DNA extracted from the following strains: Porphyromonas asaccharolytica ATCC 25260T, P. gingivalis ATCC 33277T, P. endodontalis ATCC 35406T, Porphyromonas catoniae ATCC 51270T, T. forsythensis ATCC 43037T, Porphyromonas levii NCTC 11028T, and Porphyromonas macacae DSM 20710T.

Primer PT997R was used in conjunction with primer 27F, which is specific for the domain Bacteria (18). Five replicate amplification reactions were set up for each plaque sample by using Ready-to-Go PCR beads (Amersham) with 1 μl of the DNA and 10 pmol of each primer, made up to a total of 25 μl with sterile water. Amplifications were carried out with 10 cycles of 94°C for 60 s, 56°C for 30 s, and 72°C for 2 min, followed by a further 20 cycles of 92°C for 30 s, 56°C for 30 s, and 72°C for 2.5 min. A final 5-min extension at 72°C was used, and the samples were kept at 4°C until they were purified.

The five replicate PCR products were pooled and then cloned into the pGEM-T Easy vector (Promega), according to the instructions of the manufacturer. The vector was then transformed into XL1 Blue MFR′ supercompetent cells (Stratagene), according to the instructions of the manufacturer. Two hundred white colonies were then chosen at random, and the colonies were checked for the presence of the inserts by PCR with vector-specific primers SP6 and T7 under the conditions described above. Aliquots were electrophoresed on a 1% agarose gel and then stained with ethidium bromide and checked for the presence of an ~1,000-bp band.

Approximately 20 clones from each library were then partially sequenced by using the universal sequencing primer 357F (18). Sequencing was performed with a Beckman Coulter CEQ2000 automated DNA sequencer, according to the instructions of the manufacturer.

The libraries were checked for the presence of chimeras by use of the Chimera_Check program, made available by the Ribosomal Database Project II (2). Sequences were provisionally identified by interrogation of the GenBank nucleotide database with the BLAST algorithm. From the phylogenetic position indicated by the output from the search with the BLAST algorithm, related sequences were selected from sequence databases and aligned by use of the Clustal X program (32). Further analysis was performed with the PHYLIP suite of programs (6). Specifically, the DNADIST program was used to compare sequences by use of the Jukes-Cantor algorithm, and the NEIGHBOR program was used to construct phylogenetic trees, which were viewed by using TreeView software (26).

The mean plaque and gingival bleeding indices were calculated for each patient. Mann-Whitney U tests were used to compare the bleeding and plaque indices and the percentage of clones of each taxa recovered from the sampled sites in the patients and the controls. Wilcoxon sign rank tests were used to compare the plaque and bleeding indices and the microbial variables from the shallow and deep pockets in the patients with periodontitis.

Nucleotide sequence accession number.

The GenBank accession number for the 16S rRNA gene sequence for Porphyromonas P3 clone 34_5 is AY546091.

RESULTS

The age, gender, and clinical condition of the patients included in the study are described in Table Table1.1. The patients with periodontitis had significantly higher supragingival plaque indices than the controls (Mann Whitney U test, Z = 2.61, P < 0.01), but there was no difference in the gingival bleeding indices between the patients and the controls. The mean probing depth of shallow pockets sampled in the controls was 2.6 mm (standard deviation [SD] = 0.42 mm). In the periodontitis patients, shallow sampled sites had a mean probing depth of 3.0 mm (SD = 0.35 mm) and deep pockets had a mean probing depth of 6.9 mm (SD = 1.02 mm). Supragingival plaque was detected at all sampled sites, and bleeding was detected after probing at all but one site.

TABLE 1.
Age, gender, and clinical conditions of healthy control subjects and patients with periodontitis and number of clones sequenced from each subject

PCR with primer pair 27F-PT997R gave amplification products of the expected sizes for all of the reference Porphyromonas and Tannerella strains tested. A total of 308 cloned 16S rRNA genes were sequenced and identified from the patient samples. The interrogation of the GenBank database with the BLAST algorithm indicated that all of the sequences belonged to the genera Porphyromonas and Tannerella. Cloned sequences were identified as belonging to a particular species or taxon if they showed greater than 98% sequence identity with the reference sequence in the GenBank database. Twenty-one (6.8%) of the clones were found to be chimeric, in that they were hybrid molecules consisting of genes from more than one source organism, and were therefore excluded from the further analysis. Nine clones, related to P. asaccharolytica, did not match any of the named species or phylotypes and were designated Porphyromonas P3 (Fig. (Fig.11).

FIG. 1.
Phylogenetic tree based on 16S rRNA gene sequence comparisons over 898 aligned bases showing the relationship between members of the genera Porphyromonas and Tannerella and novel phylotype Porphyromonas P3. The tree was constructed by the neighbor-joining ...

The distribution of taxa among the samples is shown in Fig. Fig.2.2. Clones were identified as belonging to one or other of the following species or phylotypes: T. forsythensis, P. endodontalis, P. catoniae, Tannerella BU063, P. gingivalis, and Porphyromonas P3.

FIG. 2.
Proportions of T. forsythensis (a), P. endodontalis (b), P. catoniae (c), Tannerella BU63 (d), P. gingivalis (e), and Porphyromonas P3 (f) in plaque from shallow and deep pockets in patients with periodontitis and healthy controls. The horizontal lines ...

T. forsythensis was found in all of the patients with periodontitis (Fig. (Fig.2a)2a) but only two of the healthy controls. A significantly higher percentages of clones were recovered from shallow sites in the patients with periodontitis (Fig. (Fig.2a)2a) than from similar sites in the healthy controls (Mann Whitney U test, Z = −2.64, P < 0.01). A similar pattern of detection was noted for P. endodontalis (Fig. (Fig.2b),2b), which was also found more commonly in patients with disease than in healthy controls. However, P. endodontalis was a relatively minor constituent of the microflora, and its proportion never exceeded 35% of the clones identified in any sample.

P. catoniae (Fig. (Fig.2c)2c) and Tannerella taxon BU063 (Fig. (Fig.2d)2d) were found most frequently in shallow pockets. In the patient group, there was a trend toward Tannerella BU063 (Fig. (Fig.2d)2d) comprising a higher percentage of the clones recovered from shallow pockets than from deep pockets (Wilcoxon sign rank test, Z = 1.70, P < 0.09). P. gingivalis (Fig. (Fig.2e)2e) and Porphyromonas P3 (Fig. (Fig.2f)2f) were not clearly associated with periodontitis or control patients, nor were they clearly associated with shallow or deep pockets. However, in two of the control subjects P. gingivalis accounted for more than 87% of the clones recovered, which was the highest proportion for any of the detectable taxa. Porphyromonas P3 was the least frequently detected taxon and was found in plaque samples from only three individuals.

DISCUSSION

This study has demonstrated that a wide range of taxa belonging to the genera Porphyromonas and Tannerella are found in deep and shallow periodontal pockets in subjects with periodontitis and age- and sex-matched controls. The method used was successful, in that all of 308 clones sequenced were found to belong to the target group. Representatives of all of the named species of human oral origin, P. endodontalis, P. gingivalis, P. catoniae, and T. forsythensis, were detected. Of the others, P. asaccharolytica is found at human nonoral sites (3); P. canis, P. cansulci, P. canoris, and P. cangingivalis are found in dogs (12, 22); P. salivosa and P. circumdentaria are found in cats (21); P. macacae is from macaques (3); P. levii is found in cattle (14); and P. gulae is found in a range of animal species (7). Although P. salivosa and P. macacae were originally isolated from cats and macaques, respectively, they have been shown to be the same species; the name P. macacae has precedence (20).

The results of the present study were interesting, in that T. forsythensis was found to be associated with periodontitis, as was P. endodontalis, although to a lesser extent. These findings are consistent with those presented in previous reports (10, 17, 19). By contrast, P. catoniae and Tannerella BU063 appeared to be associated with shallow sites unaffected by destructive disease. Indeed, others (17, 19) have found Tannerella BU063 to be more strongly related to periodontal health than disease, and P. catoniae has been found in the mouths of infants before eruption of their teeth (15). P. gingivalis, perhaps the most studied of the periodontal pathogens, was found in two patients and three control subjects. Although many workers have associated P. gingivalis with destructive periodontal disease, it has also been associated with gingivitis and shallow pockets (4, 24); and in this study, all but one of the sampled sites were inflamed. Also, no attempt was made in the present work to separate the more pathogenic strains of P. gingivalis from those considered less pathogenic (8).

There are clearly some additional limitations to this study. The results show the distribution of taxa within two genera, but there is no possibility of finite quantitation. Thus, although P. gingivalis accounted for the highest proportion of detectable clones in two of the control samples, it is possible that the total numbers of the species were very low compared to those seen at the diseased sites in the periodontitis patients. However, the results do indicate that P. gingivalis was present at these sites, which is perhaps not surprising, as all but one of the sites included in this study were inflamed and it is known that the presence of hemin stimulates the growth of this organism. The other limitation was the relatively small number of subjects studied. At present, this is inevitable for this type of study because of the time-consuming nature of the PCR, cloning, and sequencing procedure. In addition, we did not simply rely on estimates of sequence identity for identification but manually inspected the alignments of all test sequences with those from reference strains in order to exclude errors arising from poor sequence quality and/or misalignments. Studies in the future will be able to take advantage of robotic automation, which will enable the number of samples studied to be greatly increased, together with improved software tools for identification, which are in development. Nevertheless, this type of study is still extremely valuable and should be regarded as the first step in the culture-independent assessment of bacterium-host interactions in complex diseases associated with commensal microflora. For example, having identified the taxa present in these samples, including one previously undescribed group, Porphyromonas P3, it will be possible to use other techniques such as reverse capture hybridization or real-time PCR to determine the numbers of these taxa in periodontal health and disease.

It is intriguing that this study has confirmed the association of Tannerella BU063 with health described previously (17). Furthermore, the fact that the closest phylogenetic relative of this organism is T. forsythensis makes this pair of organisms an ideal target for comparative genomic studies with the aim of identifying genes responsible for virulence in T. forsythensis. The major obstacle to this is that, at present, Tannerella BU063 has yet to be cultured; work to determine its growth requirements must be an urgent priority.

A level of 98% sequence identity was chosen as the cutoff for the assignation of sequences to individual species. At this level, with partial sequences of about 500 bp being used for identification, all sequences with the exception of those identified as Porphyromonas P3 and Tannerella BU063 were found to match named species within the target genera. The full sequence was obtained for a representative clone of Porphyromonas P3, although this was limited to 916 bases (when the primer sequences were removed) because they were obtained with a reverse primer that annealed at position 997. Figure Figure11 was therefore constructed with 898 unambiguously aligned bases. The effect of constructing the tree with partial sequences is demonstrated, for example, by Porphyromonas P1 appearing to be closely related to P. endodontalis, whereas these taxa share only 97% sequence identity over their full 16S rRNA genes. Nevertheless, Fig. Fig.11 shows that a large number of phylotypes that are distinct from, but closely related to, the named species have been identified. Interestingly, these could be seen to be clustered around P. catoniae and P. endodontalis, in particular, but none that were related to P. gingivalis were seen. Discussion of the possible reasons for this is beyond the scope of this report, but this observation is worthy of further investigation.

In conclusion, this study has demonstrated the value of targeted molecular ecology studies for the identification of associations between bacterial species and specific disease states. Although the technique does not allow quantitation, this can be addressed in subsequent focused studies.

Acknowledgments

This study was supported by a grant (grant 061118) from the Wellcome Trust.

REFERENCES

1. Booth, V., J. Downes, J. van den Berg, and W. G. Wade. 2004. Gram-positive anaerobic bacilli in human periodontal disease. J. Periodontal Res. 39:213-220. [PubMed]
2. Cole, J. R., B. Chai, T. L. Marsh, R. J. Farris, Q. Wang, S. A. Kulam, S. Chandra, D. M. McGarrell, T. M. Schmidt, G. M. Garrity, and J. M. Tiedje. 2003. The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res. 31:442-443. [PMC free article] [PubMed]
3. Coykendall, A. L., F. S. Kaczmarek, and J. Slots. 1980. Genetic heterogeneity in Bacteroides asaccharolyticus (Holdeman and Moore 1970) Finegold and Barnes 1977 (Approved Lists, 1980) and proposal of Bacteroides gingivalis sp. nov. and Bacteroides macacae (Slots and Genco) comb. nov. Int. J. Syst. Bacteriol. 30:559-564.
4. Dahlen, G., F. Manji, V. Baelum, and O. Fejerskov. 1989. Black-pigmented Bacteroides species and Actinobacillus actinomycetemcomitans in subgingival plaque of adult Kenyans. J. Clin. Periodontol. 16:305-310. [PubMed]
5. Dymock, D., A. J. Weightman, C. Scully, and W. G. Wade. 1996. Molecular analysis of microflora associated with dentoalveolar abscesses. J. Clin. Microbiol. 34:537-542. [PMC free article] [PubMed]
6. Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package), version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle.
7. Fournier, D., C. Mouton, P. Lapierre, T. Kato, K. Okuda, and C. Menard. 2001. Porphyromonas gulae sp. nov., an anaerobic, gram-negative coccobacillus from the gingival sulcus of various animal hosts. Int. J. Syst. Evol. Microbiol. 51:1179-1189. [PubMed]
8. Griffen, A. L., S. R. Lyons, M. R. Becker, M. L. Moeschberger, and E. J. Leys. 1999. Porphyromonas gingivalis strain variability and periodontitis. J. Clin. Microbiol. 37:4028-4033. [PMC free article] [PubMed]
9. Grimont, F., and P. A. D. Grimont. 1991. DNA fingerprinting, p. 249-276. In E. Stackebrandt and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons Ltd., Chichester, United Kingdom.
10. Grossi, S. G., R. J. Genco, E. E. Machtei, A. W. Ho, G. Koch, R. Dunford, J. J. Zambon, and E. Hausmann. 1995. Assessment of risk for periodontal disease. II. Risk indicators for alveolar bone loss. J. Periodontol. 66:23-29. [PubMed]
11. Hamlet, S., R. Ellwood, M. Cullinan, H. Worthington, J. Palmer, P. Bird, D. Narayanan, R. Davies, and G. Seymour. 2004. Persistent colonization with Tannerella forsythensis and loss of attachment in adolescents. J. Dent. Res. 83:232-235. [PubMed]
12. Hirasawa, M., and K. Takada. 1994. Porphyromonas gingivicanis sp. nov. and Porphyromonas crevioricanis sp. nov., isolated from beagles. Int. J. Syst. Bacteriol. 44:637-640. [PubMed]
13. Holt, S. C., J. Ebersole, J. Felton, M. Brunsvold, and K. S. Kornman. 1988. Implantation of Bacteroides gingivalis in nonhuman primates initiates progression of periodontitis. Science 239:55-57. [PubMed]
14. Johnson, J. L., and L. V. Holdeman. 1983. Bacteroides intermedius comb. nov. and descriptions of Bacteroides corporis sp. nov. and Bacteroides levii sp. nov. Int. J. Syst. Bacteriol. 33:15-25.
15. Kononen, E., A. Kanervo, A. Takala, S. Asikainen, and H. Jousimies-Somer. 1999. Establishment of oral anaerobes during the first year of life. J. Dent. Res. 78:1634-1639. [PubMed]
16. Kroes, I., P. W. Lepp, and D. A. Relman. 1999. Bacterial diversity within the human subgingival crevice. Proc. Natl. Acad. Sci. USA 96:14547-14552. [PMC free article] [PubMed]
17. Kumar, P. S., A. L. Griffen, J. A. Barton, B. J. Paster, M. L. Moeschberger, and E. J. Leys. 2003. New bacterial species associated with chronic periodontitis. J. Dent. Res. 82:338-344. [PubMed]
18. Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115-175. In E. Stackebrandt and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons Ltd., Chichester, United Kingdom.
19. Leys, E. J., S. R. Lyons, M. L. Moeschberger, R. W. Rumpf, and A. L. Griffen. 2002. Association of Bacteroides forsythus and a novel Bacteroides phylotype with periodontitis. J. Clin. Microbiol. 40:821-825. [PMC free article] [PubMed]
20. Love, D. N. 1995. Porphyromonas macacae comb. nov., a consequence of Bacteroides macacae being a senior synonym of Porphyromonas salivosa. Int. J. Syst. Bacteriol. 45:90-92.
21. Love, D. N., G. D. Bailey, S. Collings, and D. A. Briscoe. 1992. Description of Porphyromonas circumdentaria sp. nov. and reassignment of Bacteroides salivosus (Love, Johnson, Jones, and Calverley 1987) as Porphyromonas (Shah and Collins 1988) salivosa comb. nov. Int. J. Syst. Bacteriol. 42:434-438. [PubMed]
22. Love, D. N., J. Karjalainen, A. Kanervo, B. Forsblom, E. Sarkiala, G. D. Bailey, D. I. Wigney, and H. Jousimies-Somer. 1994. Porphyromonas canoris sp. nov., an asaccharolytic, black-pigmented species from the gingival sulcus of dogs. Int. J. Syst. Bacteriol. 44:204-208. [PubMed]
23. Machtei, E. E., E. Hausmann, R. Dunford, S. Grossi, A. Ho, G. Davis, J. Chandler, J. Zambon, and R. J. Genco. 1999. Longitudinal study of predictive factors for periodontal disease and tooth loss. J. Clin. Periodontol. 26:374-380. [PubMed]
24. McNabb, H., A. Mombelli, R. Gmur, S. Mathey-Dinc, and N. P. Lang. 1992. Periodontal pathogens in the shallow pockets of immigrants from developing countries. Oral Microbiol. Immunol. 7:267-272. [PubMed]
25. Munson, M. A., T. Pitt-Ford, B. Chong, A. Weightman, and W. G. Wade. 2002. Molecular and cultural analysis of the microflora associated with endodontic infections. J. Dent. Res. 81:761-766. [PubMed]
26. Page, R. D. M. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357-358. [PubMed]
27. Paster, B. J., S. K. Boches, J. L. Galvin, R. E. Ericson, C. N. Lau, V. A. Levanos, A. Sahasrabudhe, and F. E. Dewhirst. 2001. Bacterial diversity in human subgingival plaque. J. Bacteriol. 183:3770-3783. [PMC free article] [PubMed]
28. Persson, G. R., D. Engel, C. Whitney, R. Darveau, A. Weinberg, M. Brunsvold, and R. C. Page. 1994. Immunization against Porphyromonas gingivalis inhibits progression of experimental periodontitis in nonhuman primates. Infect. Immun. 62:1026-1031. [PMC free article] [PubMed]
29. Socransky, S. S., A. D. Haffajee, M. A. Cugini, C. Smith, and R. L. Kent, Jr. 1998. Microbial complexes in subgingival plaque. J. Clin. Periodontol. 25:134-144. [PubMed]
30. Spratt, D. A., A. J. Weightman, and W. G. Wade. 1999. Diversity of oral asaccharolytic Eubacterium species in periodontitis—identification of novel phylotypes representing uncultivated taxa. Oral Microbiol. Immunol. 14:56-59. [PubMed]
31. Tanner, A., M. Maiden, P. Macuch, L. Murray, and R. L. Kent, Jr. 1998. Microbiota of health, gingivitis and initial periodontitis. J. Clin. Periodontol. 25:85-98. [PubMed]
32. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876-4882. [PMC free article] [PubMed]
33. Tran, S. D., J. D. Rudney, B. S. Sparks, and J. S. Hodges. 2001. Persistent presence of Bacteroides forsythus as a risk factor for attachment loss in a population with low prevalence and severity of adult periodontitis. J. Periodontol. 72:1-10. [PubMed]
34. van Steenbergen, T. J. M., A. J. van Winkelhoff, D. Mayrand, D. Grenier, and J. de Graaff. 1984. Bacteroides endodontalis sp. nov., an asaccharolytic black-pigmented Bacteroides species from infected dental root canals. Int. J. Syst. Bacteriol. 34:118-120.

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