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Institute of Oral Biology, Section of Oral Microbiology and General Immunology, University of Zürich, Plattenstrasse 11, CH-8032 Zürich, Switzerland
Correspondence
Rudolf Gmür
rudolf.gmuer{at}zzmk.uzh.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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estimated that, on a population basis, the human oral cavity might harbour some 500 bacterial species. With the emergence of culture-independent methods for the identification of microbial biota (see Amann et al., 1995 for a review) it became clear that probably less than 50 % of the oral taxa have been cultured so far (Wilson et al., 1997). During the last decade hundreds of previously unknown phylotypes and clones have been identified in studies investigating 16S rRNA diversity in different forms of dental plaque (Paster et al., 2001, 2002; Munson et al., 2004; Aas et al., 2005; Kumar et al., 2005; de Lillo et al., 2006). Among the many uncultivable human oral phylotypes two clones with an apparently supragingival habitat, BU045 and BU063, attracted some interest, because their 16S rRNA gene sequence groups them within the genus Tannerella (Paster et al., 2001; Leys et al., 2002; de Lillo et al., 2004), which otherwise contains, besides some uncultivated clones from soil, only a single species, Tannerella forsythia (also known as Tannerella forsythensis) (Tanner et al., 1986; Tanner & Izard, 2006). T. forsythia is a ‘consensus periodontal pathogen’ (Haffajee & Socransky, 2006) and associated with subgingival plaque from chronic and occasionally aggressive periodontitis (see Tanner & Izard, 2006, for review) rather than periodontal health like the two clones BU045 and BU063. Thus, although all three taxa colonize areas of the gums in the human oral cavity, current evidence indicates that they reside in quite distinct environments with markedly different nutrient sources. The aims of the present study were to morphologically identify the uncultivable Tannerella phylotypes represented by clones BU045 and BU063, and to compare with T. forsythia their prevalence and abundance in plaques collected from patients with gingivitis, necrotizing ulcerative gingivitis (NUG) and chronic progressive periodontitis. Based on the available infrastructure, experience and DNA sequence information, we opted for a microscopic approach combining fluorescent in situ hybridization (FISH), using DNA probes to phylotype-specific 16S rRNA sequences, with indirect immunofluoresence (IF) to target the Tannerella organisms.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) enriched with 5 % horse serum and 100 mg N-acetylmuramic acid l–1 or in the chemically defined medium OMIZ-W68 with 100 mg N-acetylmuramic acid l–1 (Wyss, 2007), the latter medium being superior in promoting growth of T. forsythia. Bacteria were harvested after 24–36 h from the exponential phase of growth.
Subgingival plaque samples (n=17) were obtained with paper points (Gmür et al., 1989) from the deepest periodontal pockets (
5 mm) of one or more quadrants of seven patients (five female) with a mean age of 59.1 years (range 50–71 years). The patients had been referred to our institute due to refractory chronic periodontitis (Armitage, 1999) for microbial testing at the sampled sites. Samples were processed for culture within 1 h from collection and for FISH and IF either within 1 h of collection or after storage at –80 °C and a single freezing/thawing cycle (Gmür & Thurnheer, 2002).
Pooled marginal supragingival plaque samples were derived from individual Chinese patients affected by gingivitis (n=7, four female) or NUG (n=7, four female) with a mean age of 35.9 years (range 27–53 years) and 32.6 years (range 27–52 years), respectively. They were taken from a previously described collection of frozen (liquid N2) aliquoted samples (Gmür et al., 2004) that had not been frozen/thawed more than twice before.
Preparation of multiwell slides for FISH and IF analyses.
Suspensions of cultured bacteria (Gmür & Thurnheer, 2002) were washed in 0.9 % NaCl, diluted in coating buffer (0.9 % NaCl, 0.02 % NaN3, 0.00025 % cetyltrimethylammonium bromide), spotted directly on 18- or 24-well slides (Cel-Line Associates) and air-dried. For FISH, slides were fixed by a 20 min incubation at 4 °C in 4 % paraformaldehyde/PBS; for IF, by a 2 min dip in methanol. Plaque samples were vortexed for 60 s at maximum speed and immediately diluted 1 : 8 in coating buffer. Thereafter, 10 µl plaque suspension per well was dropped onto 18- or 24-well slides, air-dried and fixed as described above. Slides, stored at room temperature, were processed for FISH or IF within 48 h from fixation.
FISH and IF procedures
FISH.
Custom-synthesized oligonucleotide probes, labelled at the 5'-end with Cy3 or 6-FAM, were purchased from Microsynth. Probes were designed according to the criteria described by Manz (1999) using the ARB software (Ludwig et al., 2004) (http://www.arb-home.de) and rRNA sequence information from the Ribosomal Data Base Project II (Cole et al., 2005) (http://rdp.cme.msu.edu/) and the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). The sequences of the probes employed for the domain Bacteria, for T. forsythia and the Tannerella clones BU045 and BU063 are listed in Table 1. Probe EUB338 was used as universal positive control, but results are not further detailed below. Probes were used at final concentrations of 5 ng µl–1 (Cy3 conjugates) and 15 ng µl–1 (FAM conjugates) in the presence of 40 % formamide in the hybridization buffer. FISH was performed in 50 ml plastic centrifuge tubes at 46 °C as described by Thurnheer et al. (2001) except for the following modifications (Gmür & Lüthi-Schaller, 2007): (i) slides were not dehydrated in ethanol prior to hybridization, (ii) wells were covered for 60 min at 37 °C with Denhardt's solution (Sigma-Aldrich; diluted 1 : 50 in 0.9 % NaCl) in the presence of protectRNA RNase inhibitor (Sigma-Aldrich, diluted 1 : 500 in 0.9 % NaCl) prior to hybridization to prevent unspecific binding of the probes to the bacterial surface, (iii) reagent volumes for 4 mm wells were 3–5 µl, and (iv) hybridization was limited to 120 min.
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). mAb binding was visualized by consecutive incubations with biotinylated goat anti-mouse immunoglobulin G (Sigma) and streptavidin-FITC (Sigma), diluted 1 : 250 and 1 : 1500, respectively, in borate-buffered saline supplemented with 0.5 % skim-milk powder and 0.05 % Tween 20 (Werner-Felmayer et al., 1988). Bacteria labelled by FISH or IF were enumerated by counting positive cells in 8–24 randomly selected viewing fields. If samples contained very low numbers of stained cells, half of a well (corresponding to 165 viewing fields) was screened. This counting procedure resulted in a lower detection limit of approximately 0.3x104 ml–1.
Combined IF and FISH.
The combined application of the two assays was performed using a recently described experimental protocol (Gmür & Lüthi-Schaller, 2007). Cells were visualized with an Olympus BX60 epifluorescence microscope [Olympus Optical (Schweiz)] equipped with phase-contrast, an HBO 103 W/2 mercury photo optic lamp (Osram) and Olympus filter sets U-MNIBA (6-FAM, FITC), U-MA41007 (Cy3) and BX-DFC5 (6-FAM/FITC/Cy3). Colour micrographs were taken with a digital Olympus Camedia 3030 camera, transferred to an iMac G5 personal computer and processed using iPhoto 6.0.4 (Apple) and Photoshop 6.0 (Adobe) without qualitative changes to the raw images, except for contrast enhancement in Fig. 1(J).
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). Cell numbers present in periodontal pocket samples were determined by anaerobic cultivation for 5 days (37 °C) on Columbia blood agar (Oxoid) supplemented with 5 % haemolysed human blood. In a previous study we have shown that numbers of fluorescent cells determined by visual microscopy and by image analysis correspond well, whereas total numbers of c.f.u. are on average about a factor of 3 lower than those determined by image analysis (Gmür et al., 2000). Therefore, we multiplied the numbers of c.f.u. by 3 to generate the total cell number estimates used to calculate the phylotype proportions shown in Table 2 by a colour code.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The rods displayed a segmented structure. Quite frequently intensely stained segments were adjacent to terminal or inner segments without any FISH staining (Fig. 1E–H). Measurements with two samples from different periodontitis patients showed that the length of BU045 and B063 cells varied over a broad range (Table 3). Moreover, sample P6 (26d) contained on average longer BU045 and B063 cells than sample P5 (26d).
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) that were much shorter than BU045 and BU063 (Table 3) and frequently colonized with high density large multi-species aggregates (Fig. 1D). Double-labelling experiments with Cy3- and FAM-tagged probes showed, as expected, that the four probes for T. forsythia recognized the same cells (data not shown). Analogously, combined labelling by IF and FISH, using the T. forsythia-specific mAb 116BF1.2 in combination with any of the six Tannerella probes, demonstrated that the antibody and the DNA probes Tfor127, Tfor439, Tfor582 and Tfor997 identified the same short spindle-shaped cells (see Fig. 1I and J for a representative pair of images). We never found any 116BF1.2-positive elongated cells.
Unexpectedly, probes Tfor127 and Tfor997 detected two further rare populations of very elongated segmented rods (Fig. 1M). These thin organisms were not stained by any other of the six Tannerella probes, suggesting that they belong neither to T. forsythia sensu stricto, nor to clones BU045 or BU063. They are designated in the following as phylotypes 127+ and 997+, whereby the numbers refer to the DNA probes used for their detection.
Prevalence and abundance in clinical samples of the detected micro-organisms
Table 2 describes with numerical data the prevalence and abundance of the so far uncultivable phylotypes BU045, BU063, 127+ and 997+, and of T. forsythia in samples from gingivitis, NUG and chronic periodontitis patients. In addition, a colour code indicates the percentages of these taxa with the estimated total cell number being set to 100 %. The gingivitis and NUG samples contained on average total cell numbers of 2.6x108 ml–1 (range 4x107–7.4x108) and 7.6x108 ml–1 (range 2.9x108–1.4x109) whereas the periodontitis samples yielded on average 1.7x107 ml–1 (range 4.4x106–5.8x107) (data not shown). Read column-by-column, Table 2 shows that clone BU045 was identified by Tan1260a in gingivitis, NUG and periodontitis samples with a prevalence of 50, 83 and 81 %, and clone BU063 (Tan1260b) with a prevalence of 100 %, 100 % and 53 %, respectively. Both clones accounted in most samples for less than 0.1 % of the total biota; in only two cases did BU063 exceed 1 %. T. forsythia was detected with a 100 % prevalence in the Chinese supragingival gingivitis and NUG plaques by three of the four rRNA probes and by the mAb. However, cell densities were consistently very low and often near the detection limit of these microscopic assays (approx. 0.3x104 ml–1). In the subgingival plaques from periodontitis patients, T. forsythia prevalence was 100 % as well, but now all but three samples contained high levels of the organism. In more than half the samples T. forsythia exceeded 10 % of the total biota. The IF assay consistently detected higher T. forsythia cell numbers than FISH. The two new phylotypes 127+ and 997+ never reached high densities. Phylotype 997+ was very rare, whereas 127+ occurred regularly in supragingival plaques, showing a prevalence of 71 %, 86 % and 12 % in gingivitis, NUG and periodontitis samples, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Leys et al., 2002; de Lillo et al., 2004) found in this study in supra- and subgingival plaques from patients with gingivitis, NUG or chronic periodontitis with prevalences between 50 and 100 %. The two other phylotypes are previously unknown taxa, which we tentatively designated 127+ and 997+ due to their sequence homology with T. forsythia in regions 127–146 and 997–1014, respectively, of the 16S rRNA gene. Their prevalence in periodontal pocket samples is very low, but at least 127+ has revealed high prevalence in the gingivitis and NUG samples. In contrast to T. forsythia, however, these uncultivable phylotypes never seem to proliferate to high densities and therefore are not likely to be important in the pathogenesis of these inflammatory periodontal diseases.
Inter-group differences in prevalence and abundance of the detected phylotypes should be interpreted cautiously since our study groups were small (n=7), and differed both in disease status and with respect to the patients' ethnical/geographical background. Nevertheless, it is noteworthy that our data on the prevalence and abundance of T. forsythia in the periodontal pocket microbiota are in agreement with earlier results from many different studies (see Tanner & Izard, 2006 for a review). With respect to supragingival plaques from gingivitis and NUG patients, the low abundance of T. forsythia had to be expected based on previous findings with supragingival plaque from different types of study groups (Gmür & Guggenheim, 1994; Paster et al., 2002; Socransky & Haffajee, 2002). However, the observed 100 % prevalence in these samples is remarkable as it shows that T. forsythia is, even supragingivally, an ubiquitous organism in people affected by severe gingival inflammation. The critical question remains, however, what causes T. forsythia to proliferate to several orders of magnitude higher cell densities when they reach the environment of the periodontal pocket? It should be mentioned that our prevalence scores for supragingival plaques are higher than those reported by Socransky & Haffajee (2002) for both healthy subjects and patients with periodontitis. However, besides studying quite different patient groups, these authors used the checkerboard assay with whole genomic probe hybridization, which may be less sensitive and has never been compared directly to our procedures. Reportedly, the checkerboard technique is optimized to detect bacteria in the range of 104–107 cells ml–1 (Socransky et al., 2004), whereas the FISH and IF single-cell assays used in this study yielded data ranging between 2x103 and 3x108 (Table 2).
We noted that IF leads to higher T. forsythia cell counts than FISH when the cells are present in planktonic form, whereas the opposite is the case when cells were present in dense aggregates. The reasons for this are currently not fully understood; however, it seems likely that the small FISH probes penetrate aggregates consisting of packed cells and a diffusion-limiting extracellular matrix much more easily than the antibodies of the IF sandwich assay. Gradually impaired FISH-fluorescence of planktonic cells was presumably due to rRNA degradation during the preparatory steps of FISH, since we could greatly reduce, but still not totally control, the problem by adding an RNase inhibitor to FISH assay reagents. Certainly, the beneficial incorporation of an RNA-protective reagent into the FISH procedure will be of general interest to researchers investigating clinical and environmental microbial ecologies.
The unexpected finding that cells of the four uncultivated Tannerella phylotypes are thin segmented filamentous rods is interesting. Morphologically comparable cell types have been described as segmented filamentous bacteria (SFB) (Davis & Savage, 1974). SFB live in the small intestine of arthropods, fish, birds and many mammalian species including humans, where they are anchored to the epithelial surface (Tannock et al., 1984; Smith, 1997; Meyerholz et al., 2002). They are considered to be non-cultivable, species-specific, non-pathogenic, and potent activators of the mucosal immune system. However, based on the available 16S rRNA gene sequence data, the Tannerella phylotypes and the SFB belong to quite distinct phylogenetic groups (Snel et al., 1995; Tanner & Izard, 2006) and labelling of any SFB by our FISH probes can be excluded. The length of cells from the uncultivable Tannerella phylotypes was quite variable. It is our impression that the variable number of segments per cell could explain length variation; however, this hypothesis will need to be verified by electron microscopy. In contrast, T. forsythia cells were consistently short, 2.3 µm on average, with no evidence for segmentation. Occasionally, individual segments of apparently normal cells of BU045, BU063, 127+ and 997+ were not stainable by FISH. Estimated from such unstained segments, the length of individual segments is similar to the length of a single T. forsythia cell. The biological significance of such cell segmentation in the genus Tannerella remains to be elucidated. Lack of FISH staining of a single segment of a multi-segmented organism could reflect structural differences in cell wall composition, resulting in greatly reduced probe permeability in an affected segment, or, perhaps more likely, indicate that such a segment contains no or much less rRNA than directly adjacent segments. This would imply that segments function and perhaps die as individual self-sustaining units. It would be interesting to test whether individual segments could give rise to new multi-segmented organisms; however, so far our attempts to cultivate BU045 and BU063 have not progressed beyond the stage of heterogeneous enrichment cultures (C. Wyss, personal communication).
Obtained with a new procedure developed for the purpose of this study (Gmür & Lüthi-Schaller, 2007), our combined FISH-IF experiments clearly demonstrated that the four FISH probes to T. forsythia and mAbs 116BF1.2 and 103BF1.1 (data for the latter not shown) all identify the same short rods with tapered ends. This again confirms the two antibodies' specificity, used in many studies since 1989 to enumerate T. forsythia in clinical samples (Gmür et al., 1989, 2004; Gmür & Guggenheim, 1994; Kamma et al., 2004). Notably, cells detected in plaque were usually shorter than cells derived from in vitro-grown T. forsythia strains (data not shown). The morphology of the identified T. forsythia cells is in agreement with earlier descriptions by Tanner et al. (1986) and Lai et al. (1987), but contrasts with the description by Gersdorf et al. (1993). These authors considered T. forsythia cells to be thin elongated rods, reminiscent of the uncultivable cells identified in this study. They used the 16S rRNA FISH probe BFV530 for detection, a probe that with the rRNA gene sequences available nowadays can be shown to hybridize to T. forsythia and to BU045 and BU063. Thus, most probably Gersdorf et al. (1993) had observed BU045 and/or BU063, and not T. forsythia, several years before the two phylotypes were (re)detected by partial sequencing of plaque-derived 16S rRNA clones (Paster et al., 2001).
In conclusion, this study shows the value of combined targeted FISH and IF studies for the identification and quantification of elusive micro-organisms colonizing the human oral cavity. The study confirms the omnipresence of T. forsythia in plaques associated with different inflammatory periodontal diseases and its selective capacity to expand dramatically in the environment of the subgingival pocket. It further provides first evidence that still uncultivable human Tannerella phylotypes consist of elongated filamentous and segmented rods which colonize at low density both supra- and subgingival plaques of patients with severe inflammatory periodontal diseases.
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ACKNOWLEDGEMENTS |
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Edited by: M. Kilian
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Amann, R. I., Ludwig, W. & Schleifer, K.-H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59, 143–169.
Armitage, G. C. (1999). Development of a classification system for periodontal diseases and conditions. Ann Periodontol 4, 1–6.[CrossRef][Medline]
Cole, J. R., Chai, B., Farris, R. J., Wang, Q., Kulam, S. A., Mcgarrell, D. M., Garrity, G. M. & Tiedje, J. M. (2005). The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res 33, D294–D296.
Daims, H., Brühl, A., Amann, R., Schleifer, K.-H. & Wagner, M. (1999). The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22, 434–444.[Medline]
Davis, C. P. & Savage, D. C. (1974). Habitat, succession, attachment, and morphology of segmented, filamentous microbes indigenous to the murine gastrointestinal tract. Infect Immun 10, 948–956.
de Lillo, A., Booth, V., Kyriacou, L., Weightman, A. J. & Wade, W. G. (2004). Culture-independent identification of periodontitis-associated Porphyromonas and Tannerella populations by targeted molecular analysis. J Clin Microbiol 42, 5523–5527.
de Lillo, A., Ashley, F. P., Palmer, R. M., Munson, M. A., Kyriacou, L., Weightman, A. & Wade, W. G. (2006). Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets. Oral Microbiol Immunol 21, 61–68.[Medline]
Gersdorf, H., Meissner, A., Pelz, K., Krekeler, G. & Göbel, U. B. (1993). Identification of Bacteroides forsythus in subgingival plaque from patients with advanced periodontitis. J Clin Microbiol 31, 941–946.
Gmür, R. & Guggenheim, B. (1983). Antigenic heterogeneity of Bacteroides intermedius as recognized by monoclonal antibodies. Infect Immun 42, 459–470.
Gmür, R. & Guggenheim, B. (1994). Interdental supragingival plaque – a natural habitat of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, and Prevotella nigrescens. J Dent Res 73, 1421–1428.
Gmür, R. & Lüthi-Schaller, H. (2007). A combined immunofluorescence and fluorescent in situ hybridization assay for single cell analyses of dental plaque microorganisms. J Microbiol Methods 69, 402–405.[CrossRef][Medline]
Gmür, R. & Thurnheer, T. (2002). Direct quantitative differentiation between Prevotella intermedia and Prevotella nigrescens in clinical samples. Microbiology 148, 1379–1387.
Gmür, R., Strub, J. R. & Guggenheim, B. (1989). Prevalence of Bacteroides forsythus and Bacteroides gingivalis in subgingival plaque of prosthodontically treated patients on short recall. J Periodontal Res 24, 113–120.[CrossRef][Medline]
Gmür, R., Guggenheim, B., Giertsen, E. & Thurnheer, T. (2000). Automated immunofluorescence for enumeration of selected taxa in supragingival dental plaque. Eur J Oral Sci 108, 393–402.[CrossRef][Medline]
Gmür, R., Wyss, C., Xue, Y., Thurnheer, T. & Guggenheim, B. (2004). Gingival crevice microbiota from Chinese patients with gingivitis or necrotizing ulcerative gingivitis. Eur J Oral Sci 112, 33–41.[CrossRef][Medline]
Haffajee, A. D. & Socransky, S. S. (2006). Introduction to microbial aspects of periodontal biofilm communities, development and treatment. Periodontol 2000 42, 7–12.[CrossRef]
Kamma, J. J., Nakou, M., Gmür, R. & Baehni, P. C. (2004). Microbiological profile of early onset/aggressive periodontitis patients. Oral Microbiol Immunol 19, 314–321.[CrossRef][Medline]
Kumar, P. S., Griffen, A. L., Moeschberger, M. L. & Leys, E. J. (2005). Identification of candidate periodontal pathogens and beneficial species by quantitative 16S clonal analysis. J Clin Microbiol 43, 3944–3955.
Lai, C.-H., Listgarten, M. A., Shirakawa, M. & Slots, J. (1987). Bacteroides forsythus in adult gingivitis and periodontitis. Oral Microbiol Immunol 2, 152–157.[Medline]
Leys, E. J., Lyons, S. R., Moeschberger, M. L., Rumpf, R. W. & Griffen, A. L. (2002). Association of Bacteroides forsythus and a novel Bacteroides phylotype with periodontitis. J Clin Microbiol 40, 821–825.
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S. & other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32, 1363–1371.
Manz, W. (1999). In situ analysis of microbial biofilms by rRNA-targeted oligonucleotide probing. Methods Enzymol 310, 79–91.[Medline]
Meyerholz, D. K., Stabel, T. J. & Cheville, N. F. (2002). Segmented filamentous bacteria interact with intraepithelial mononuclear cells. Infect Immun 70, 3277–3280.
Moore, W. E. C. & Moore, L. V. H. (1994). The bacteria of periodontal diseases. Periodontol 2000 5, 66–77.[CrossRef]
Munson, M. A., Banerjee, A., Watson, T. F. & Wade, W. G. (2004). Molecular analysis of the microflora associated with dental caries. J Clin Microbiol 42, 3023–3029.
Paster, B. J., Boches, S. K., Galvin, J. L., Ericson, R. E., Lau, C. N., Levanos, V. A., Sahasrabudhe, A. & Dewhirst, F. E. (2001). Bacterial diversity in human subgingival plaque. J Bacteriol 183, 3770–3783.
Paster, B. J., Russell, M. K., Alpagot, T., Lee, A. M., Boches, S. K., Galvin, J. L. & Dewhirst, F. E. (2002). Bacterial diversity in necrotizing ulcerative periodontitis in HIV-positive subjects. Ann Periodontol 7, 8–16.[CrossRef][Medline]
Smith, T. M. (1997). Segmented filamentous bacteria in the bovine small intestine. J Comp Pathol 117, 185–190.[CrossRef][Medline]
Snel, J., Heinen, P. P., Blok, H. J., Carman, R. J., Duncan, A. J., Allen, P. C. & Collins, M. D. (1995). Comparison of 16S rRNA sequences of segmented filamentous bacteria isolated from mice, rats, and chickens and proposal of "Candidatus Arthromitus". Int J Syst Bacteriol 45, 780–782.[CrossRef][Medline]
Socransky, S. S. & Haffajee, A. D. (2002). Dental biofilms: difficult therapeutic targets. Periodontol 2000 28, 12–55.[CrossRef]
Socransky, S. S., Haffajee, A. D., Smith, C., Martin, L., Haffajee, J. A., Uzel, N. G. & Goodson, J. M. (2004). Use of checkerboard DNA-DNA hybridization to study complex microbial ecosystems. Oral Microbiol Immunol 19, 352–362.[CrossRef][Medline]
Sunde, P. T., Olsen, I., Göbel, U. B., Theegarten, D., Winter, S., Debelian, G. J., Tronstad, L. & Moter, A. (2003). Fluorescence in situ hybridization (FISH) for direct visualization of bacteria in periapical lesions of asymptomatic root-filled teeth. Microbiology 149, 1095–1102.
Tanner, A. C. R. & Izard, J. (2006). Tannerella forsythia, a periodontal pathogen entering the genomic era. Periodontol 2000 42, 88–113.[CrossRef]
Tanner, A. C. R., Listgarten, M. A., Ebersole, J. L. & Strzempko, M. N. (1986). Bacteroides forsythus sp. nov., a slow-growing, fusiform Bacteroides sp. from the human oral cavity. Int J Syst Bacteriol 36, 213–221.[CrossRef]
Tannock, G. W., Miller, J. R. & Savage, D. C. (1984). Host specificity of filamentous, segmented microorgansims adherent to the small bowel epithelium in mice and rats. Appl Environ Microbiol 47, 441–442.
Thurnheer, T., Gmür, R., Giertsen, E. & Guggenheim, B. (2001). Automated fluorescent in situ hybridization for the specific detection and quantification of oral streptococci in dental plaque. J Microbiol Methods 44, 39–47.[CrossRef][Medline]
Werner-Felmayer, G., Guggenheim, B. & Gmür, R. (1988). Production and characterization of monoclonal antibodies against Bacteroides forsythus and Wolinella recta. J Dent Res 67, 548–553.
Wilson, M., Weightman, A. & Wade, W. G. (1997). Applications of molecular ecology in the characterization of uncultured microorganisms associated with human disease. Rev Med Microbiol 8, 91–101.
Wyss, C. (2007). Fatty acids synthesied by oral treponemes in chemically defined media. FEMS Microbiol Lett 269, 70–76.[CrossRef][Medline]
Received 21 July 2007;
revised 14 August 2007;
accepted 16 August 2007.
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0.1 % of all bacteria; green, between 0.1 and 1 % of all bacteria; yellow, 1–10 % of all bacteria; red, >10 % of all bacteria.