Molecular analysis of bacteria in periodontitis: evaluation of clone libraries, novel phylotypes and putative pathogens

Gerhard Hutter1, Ulrich Schlagenhauf2, Giuseppe Valenza1, Matthias Horn3, Stefan Burgemeister2, Heike Claus1 and Ulrich Vogel1

1 Institute for Hygiene and Microbiology, University of Würzburg, Germany
2 Department of Periodontology, University of Würzburg, Germany
3 Department of Microbiology, Technische Universität, Munich, Germany

Correspondence
Ulrich Vogel
uvogel{at}hygiene.uni-wuerzburg.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Subgingival plaque samples were obtained from 26 subjects with advanced periodontal lesions. Bacterial diversity was analysed by amplification of the 16S rRNA genes with two different primer sets, and subsequent cloning and sequencing. A total of 578 sequences was analysed after the exclusion of chimeras. The authors found 148 phylotypes with the clone library 27f/519r (number of clones n=322; coverage, C=66 %) and 75 phylotypes with the clone library 515f/1525r (n=256; C=84 %). Comparative sequence analysis revealed that 70 % of all of the analysed sequences showed a similarity of at least 99 % to sequences deposited in public databases. The classes Actinobacteria, Bacilli, Bacteroidetes, Clostridia, Deferribacteres, Flavobacteria, Fusobacteria, Mollicutes, Spirochaetes and all classes of the Proteobacteria were represented. Sequences that were at least 99 % identical to Porphyromonas gingivalis, Filifactor alocis and Treponema socranskii were present in at least one-third of the patients. Libraries generated with the two PCR primer pairs differed significantly in their representation of the families Porphyromonadaceae, Prevotellaceae, Fusobacteriaceae, Eubacteriaceae, Streptococcaceae and Acidaminococcaceae. A total of 14 sequences exhibited less than 97 % identity to sequences published previously and were assigned to six different families within the phyla Bacteroidetes and Firmicutes. Twelve of 20 putative pathogens were recovered, which were recently proposed to be associated with periodontitis.

The GenBank/EMBL accession numbers for the cloned 16S rRNA gene sequences reported in this paper are AJ428412 (oral clone 10B6), AF515487 (1B13), AF515488 (2B14), AF515489 (2B16), AF515490 (3B5), AF515491 (3B9), AF515492 (3B18), AF515493 (1A13), AF515494 (1D7), AF515495 (10B11), AF515496 (21B4), AF515497 (24B14), AF515498 (28B11), AF515499 (29B17) and AF515500 (29D19).


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Culture-independent analysis of bacterial rRNA genes has provided insights into the composition of mixed microbial communities occurring in the environment or in animals (Chapelle et al., 2002; Giovannoni et al., 1995; Hugenholtz et al., 1998a; Leser et al., 2002; Ward et al., 1990; Zhu et al., 2002). Of the medically important communities analysed by 16S rRNA-based techniques, oral bacteria have been a major focus of interest (Choi et al., 1994, 1996; Dewhirst et al., 2000; Dymock et al., 1996; Harper Owen et al., 1999; Hugenholtz et al., 1998a; Jung et al., 2000; Kroes et al., 1999; Paster et al., 2001; Rolph et al., 2001; Sakamoto et al., 2000; Socransky et al., 1994; Spratt et al., 1999; Tanner et al., 1994; Wade et al., 1997; Ximenez-Fyvie et al., 2000). Several of the studies on oral bacteria have demonstrated a tremendous richness of species within the microbial community associated with periodontitis, a very common oral disease. No single species could be identified as the decisive pathogen in periodontitis; instead the disease is induced by the activity of a mixed bacterial biofilm growing under anaerobic conditions. In one patient with mild gingivitis, Kroes et al. (1999) using sequence analysis of cloned 16S rRNA gene fragments found a total of 59 different phylotypes (i.e. groups of sequence showing at least 99 % similarity) belonging to the Bacilli, Clostridia, Fusobacteria, Actinobacteria, Alpha-, Beta-, Gamma- and Epsilonproteobacteria, Bacteroidetes, Spirochaetes and Chlamydiales. Sakamoto et al. (2000) found members of the Bacilli, Clostridia, Actinobacteria, Alpha-, Beta- and Epsilonproteobacteria, Bacteroidetes and Spirochaetes in two patients with adult periodontitis and rapidly progressing periodontitis. In addition, a recent and very comprehensive analysis by Paster et al. (2001) of 31 individuals with four different forms of periodontitis or gingivitis (11 subjects with refractory periodontitis, nine with periodontitis, four with acute necrotizing ulcerative gingivitis, two with HIV-associated gingivitis and five healthy subjects) revealed the presence of members of the Deferribacteres and Mollicutes, and by the use of Spirochaetes- and Bacteroidetes-specific primers, added much to the knowledge of the diversity of Treponema spp. and Bacteroidetes in periodontitis. The authors found as many as 347 phylotypes in subgingival bacterial plaque, 40 % of which were novel. Differences in the bacterial species retrieved by 16S rRNA sequencing projects can be attributed to a variety of factors: the numbers and clinical presentations of subjects affect the representation of bacterial populations as do technical aspects such as DNA extraction, template composition, set-up of the PCR assay (i.e. selection of primers, template concentration, number of amplification cycles, annealing temperature), chimera formation and the number of recombinant clones analysed (Chandler et al., 1997; Farrelly et al., 1995; Giovannoni et al., 1995; Kopczynski et al., 1994; Kroes et al., 1999; Marchesi et al., 1998; Reysenbach et al., 1992; Shuldiner et al., 1989; Suzuki & Giovannoni, 1996).

Here we report the results of our 16S rRNA gene sequencing effort on the periodontal microflora. A total of 578 16S rRNA gene sequences were evaluated, which were obtained from subgingival samples of 26 patients suffering from aggressive periodontitis who were visiting the Department of Periodontology, Würzburg, Germany, between December 2000 and July 2001. To the best of our knowledge, this is the largest 16S rRNA gene sequencing study on periodontitis with respect to the number of patients evaluated.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Subjects.
Twenty-six patients, 15 females and 11 males, with multiple advanced periodontal lesions were included in this study. Median age was 46–49 years (range 22–73 years). The clinical classification of the disease type was mostly congruent with aggressive periodontitis. Subgingival samples were obtained by insertion of a sterile paper point into periodontal pockets. A single site was evaluated per patient. The mean pocket depth was 8·1 mm (SD=1·9; range 5–12 mm; median 8 mm). Bleeding on probing after withdrawal of the paper point was observed in all but two of the patients; suppuration from the depth of the periodontal lesion was found in 13 of the 26 patients. Thirteen of the patients were known to be regular smokers. Four of the patients had been unsuccessfully treated once 2 months earlier by subgingival scaling in conjunction with the systemic administration of amoxicillin and metronidazole or metronidazole alone. In addition, six samples obtained from six periodontally healthy individuals were analysed as controls (three females, three males; median age 37–43 years; range 23–49 years; pocket depth of sampled site <3 mm; no pus; bleeding on probing, 2–7 % of all sites; all subjects were non-smokers and received no antimicrobial therapy within 8 weeks prior to sampling). Paper points were transferred to 300 µl of 10 mM Tris/1 mM EDTA (TE) buffer, pH 8·0. The material was transported immediately to the Institute for Hygiene and Microbiology, University of Würzburg, Germany. Thereafter, the paper points in TE buffer were vortexed for 1 min. DNA was either extracted immediately or the samples were frozen at -20 °C.

DNA purification and amplification of 16S rRNA genes.
DNA was purified from 200 µl of the original 300 µl bacterial suspension in TE buffer using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer's instructions (elution volume 200 µl). Aliquots of the DNA solutions (10 µl) were used for partial amplification of the 16S rRNA genes. The following oligonucleotides were used for PCR: 27f (5'-AGAGTTTGATCMTGGCTCAG-3'; accession no. J01695; positions 8–27 of the 16S rRNA gene; Lane, 1991); 519r (5'-GWATTACCGCGGCKGCTG-3'; J01695; positions 536–519; Lane, 1991); 515f (synonym A19) (5'-GTGCCSGCMGCCGCGGTAA-3'; J01695; positions 515–533; Kroes et al., 1999); 1525r (synonym S17) (5'-AAGGAGGTGATCCAGCC-3'; J01695; positions 1541–1525; Kroes et al., 1999); GV1r (5'-GATCATCGACTTGGGGGTC-3'; specific to clone 10B6; accession no. AJ428412; positions 258–240, this study); GV2f (5'-ACCCCCAAGTCGATGATCG-3'; specific to clone 10B6; AJ428412; positions 241–259; this study).

The PCR conditions for the primer pairs 27f/519r and 515f/1525r were as follows: initial denaturation at 95 °C for 10 min, followed by 26 cycles of denaturation at 94 °C for 30 s, annealing for 1 min at 55 and 58 °C, respectively, and extension at 72 °C for 1·5 min, followed by a final extension at 72 °C for 20 min. A 50 µl PCR mix contained 0·4 µl (2 U) AmpliTaq Gold (Applied Biosystems), 1 µl each of the primer solutions (10 µM), 3 µl MgCl2 (25 mM), 1 µl dNTP (2·5 mM each dNTP; PeqLab Biotechnologie), 5 µl of 10 x times; PCR buffer II (Applied Biosystems) and 10 µl extracted DNA. DNA from 40 µl of the PCR products was purified using the QIAquick PCR purification kit (Qiagen) according to the manufacturer's instructions. Purified PCR product was ligated into the vector pPCR-Script Amp SK(+) using the PCR-Script Amp cloning kit (Stratagene) according to the manufacturer's instruction. DNA was then purified by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation; plasmid DNA was transformed into E. coli DH5{alpha} by electroporation using standard protocols. Cells from recombinant colonies were subjected to PCR using vector primers. PCR products were sequenced with the primers 27f and 515f, respectively. In addition, plasmids harbouring inserts with less than 97 % identity to entries in GenBank were sequenced using the primers 519r and 1525r, respectively. PCR products, which were derived from plasmids harbouring a 515f/1525r PCR product, were sequenced using an ABI prism model 377 DNA sequencer (Applied Biosystems). Due to logistic reasons related to other diagnostic procedures at the Institute for Hygiene and Microbiology, PCR products derived from plasmids harbouring a 27f/519r PCR product were sequenced using an ABI prism model 310 DNA sequencer.

Sequence analysis.
After visual inspection of the nucleotide sequence chromatograms obtained from the ABI DNA sequencers, sequence data were edited using the 373A DNA sequencer data analysis software (Applied Biosystems). DNA sequences were compared with those in the GenBank database using the BLAST server hosted by the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA). Sequences with less than 99 % similarity to GenBank database entries were screened for chimeras using the CHIMERA CHECK program of the Ribosomal Database Project II (http://rdp.cme.msu.edu/html/). A unique phylotype was defined as comprising sequences that showed at least 99 % sequence similarity with each other (Kroes et al., 1999; Paster et al., 2001). Phylotypes were determined by alignment using the CLUSTAL W algorithm provided by the Lasergene sequence analysis software (DNASTAR). Coverage (C) of the clone libraries was determined by using the equation C=[1-(nsingletons/Nclones)]x100 (Giovannoni et al., 1995), where nsingletons represents the number of phylotypes occurring only once and N is the number of clones being examined. A BLAST stand-alone server run on a Linux computer was used for sequence comparisons with the local database. The BLAST program is available from the NCBI web-site (http://www.ncbi.nlm.nih.gov/BLAST/).

16S rRNA gene sequences that showed less than 97 % similarity to sequences in the public databases were added to the rRNA sequence database of the Technische Universität of Munich (encompassing about 15 000 published and unpublished homologous small-subunit rRNA primary structures) by use of the ARB package (http://www.arb-home.de). The initial alignment was performed by using the ARB automated alignment tool (FAST ALIGNER, version 1.03). Alignments were refined by visual inspection and by secondary structure analysis. Initially, trees were calculated with 16S rRNA gene sequences (>1000 bp in length only) using the neighbour-joining (Jukes–Cantor correction), maximum-parsimony and maximum-likelihood methods implemented in ARB. Partial sequences were subsequently added to the respective trees without changing their topology by use of the ARB ‘parsimony interactive’ method. To determine the robustness of the phylogenetic trees, analyses were performed with and without the application of filter sets excluding highly variable positions. Taxonomy is used in this report according to the Taxonomic Outline of the Procaryotic Genera, Bergey's Manual of Systematic Bacteriology, 2nd edn, release 1.0, April 2001 (http://www.cme.msu.edu/bergeys/).

Nucleotide sequence accession numbers.
The sequence of clone 10B6 has been assigned EMBL accession number AJ428412. The following clones were assigned GenBank accession numbers: oral clone 1B13, AF515487; 2B14, AF515488; 2B16, AF515489; 3B5, AF515490; 3B9, AF515491; 3B18, AF515492; 1A13, AF515493; 1D7, AF515494; 10B11, AF515495; 21B4, AF515496; 24B14, AF515497; 28B11, AF515498; 29B17, AF515499; 29D19, AF515500.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Evaluation of clone libraries
Twenty-six subgingival samples from patients mostly suffering with advanced aggressive periodontitis were analysed by using 16S rRNA gene libraries and clone sequencing. For each sample, two different PCR products, 27f/519r and 515f/1525r, were independently processed. Table 1 shows the characteristics of the clone populations obtained with the 27f/519r and 515f/1525r libraries, respectively. The 27f/519r libraries contained more singleton phylotypes (i.e. a group of sequences with at least 99 % sequence similarity) than the 515f/1525r libraries. Accordingly, the coverage of the 27f/519r libraries was lower despite a larger number of clones (66 % versus 84 %). This finding was confirmed by accumulation curves (Fig. 1). Both types of libraries contained sequences from a considerable number of different bacterial families (Tables 2 and 3). The representation of the families Porphyromonadaceae, Fusobacteriaceae, Prevotellaceae, Bacteroidaceae, Eubacteriaceae, Streptococcaceae and Acidaminococcaceae differed significantly between the libraries (Tables 2 and 3). The occurrence of bacterial species with an established taxonomy was analysed by BLAST comparison with GenBank applying stringent conditions (99 % identity). Thirty-four species were found more than once in the complete clone population (Fig. 2). Identities to Porphyromonas gingivalis were found 55 times in 16 of 26 patients, to Filifactor alocis 28 times in 12 patients and to Treponema socranskii 18 times in 14 patients. It should be noted that due to the technical bias inherent to PCR from multibacterial samples, the numbers given above may not reflect the actual proportions of species within the mixed bacterial populations.


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Table 1. Characteristics of clone populations

 


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Fig. 1. Number of phylotypes plotted against the number of clones sampled for libraries 27f/519r (black line) and 515f/1525r (grey line). The numbers represent accumulating clones obtained from consecutively analysed subjects.

 

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Table 2. Clone population 27f/519r

 

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Table 3. Clone population 515f/1525r

 


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Fig. 2. Number of sequences matching entries of established bacterial species in the GenBank database. A stringent similarity criterion of at least 99 % sequence identity was used to increase the accuracy of diagnosis. Solid bars represent the abundance of clones; white bars demonstrate the number of patients.

 
Novel sequences and putative pathogens
Only 14 sequences were less than 97 % identical to current GenBank entries. Each of these sequences was found in a single patient. The low number of novel sequences indicates that the 16S rRNA gene entries derived from human subjects with periodontitis are close to complete coverage. Phylogenetic analysis assigned the novel sequences to five families within the phyla Bacteroidetes and Firmicutes (Fig. 3; mean sequence length 527 bp; range 381–974). Two of these families (Flexibacteraceae and Syntrophomonadaceae) were not represented by the other phylotypes detected in this study. Another clone analysed phylogenetically was clone 10B6, which was identical to the 480 bp sequence of AP60-38 deposited in GenBank (Sakamoto et al., 2000). The almost complete 16S rRNA gene sequence (1490 bp) of oral clone 10B6 was determined in this study by PCR using the primer pairs 27f/GV1r and GV2f/1525r, and purified DNA from the sample from patient number 10 as the template. The PCR products were sequenced. The sequence of oral clone 10B6 was constructed by alignment of the 27f/GV1r sequence and the GV2f/1525r sequence. Correct assembly of the 1490 bp sequence of oral clone 10B6 was controlled by comparison to the published 480 bp sequence of AP60-38. The sequence of oral clone 10B6 represented a novel evolutionary lineage within the Alphaproteobacteria most closely related to the family Holosporaceae, which is composed exclusively of Rickettsia-related, obligate intracellular symbionts of paramecia and free-living amoebae.



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Fig. 3. 16S rRNA-based neighbour-joining tree showing the phylogenetic affiliation of (i) clone 10B6 and (ii) all recovered sequences displaying less than 97 % similarity with previously recognized rRNA gene sequences. Relevant parsimony bootstrap values (ARB upper bootstrap limit method) above 80 % ({circ}) and 90 % ({bullet}) are shown. Bar, 10 % estimated evolutionary distance. Clones recovered in this study are indicated in bold type. The length of each sequence was as follows: 1B13, 485 bp; 2B14, 440 bp; 2B16, 441 bp; 3B5, 456 bp; 3B9, 479 bp; 3B18, 489 bp; 1A13, 430 bp; 1D7, 974 bp; 10B6, 1490 bp; 10B11, 470 bp; 21B4, 469 bp; 24B14, 444 bp; 28B11, 445 bp; 29B17, 381 bp; 29D19, 970 bp.

 
Most of the bacterial families covered by Paster et al. (2001) were also found in this study, with the exception of the divisions obsedian pool (OP) 11 and TM7 comprising sequences first described for hot-springs, soil and deep-sea sediments (Hugenholtz et al., 1998b; see also GenBank accession no. X97111; Hugenholtz et al., 2001). Although the PCR protocols differed between the two studies, this discrepancy might also be explained by the fact that only one of seven OP11 and TM7 clones was found in a subject with periodontitis by Paster et al. (2001), whereas the others were associated with acute necrotizing ulcerative gingivitis and refractory periodontitis.

Paster et al. (2001) described 20 phylotypes/species that were found in periodontitis but not in healthy subjects. The authors suggested that these phylotypes were therefore putative pathogens (Table 4). We compared the sequences listed in Table 4 with the local database using a stand-alone BLAST server and applying a sequence identity of at least 99 % as a cut-off. Twelve of the 20 putative pathogens were also identified in one to 12 subjects analysed in this study (Table 4).To determine whether these sequences were also found in healthy subjects, we obtained 113 sequences from six periodontally healthy individuals (52 sequences with the clone libraries 27f/519r; 60 sequences with the clone libraries 515f/1525r). A large proportion of the sequences (73 %) obtained from the periodontally healthy individuals belonged to the families Streptococcaceae, Staphylococcaceae, Neisseriaceae and Acidaminococcaceae, whereas these families comprised only 28 % of the clones obtained from periodontitis patients. We compared the sequences listed in Table 4 with the local database of the healthy subjects again using a stand-alone BLAST server and applying a sequence identity of at least 99 % as a cut-off. In a single periodontally healthy individual a sequence 99 % identical to Granulicatella adiacens and a sequence 99 % identical to Porphyromonas gingivalis were detected. All other putative pathogens were not found in the healthy individuals. The data provided in Table 4 suggest that at least Treponema socranskii subsp. buccale, Filifactor alocis, Dialister pneumosintes, Porphyromonas gingivalis and Porphyromonas endodontalis are likely to be found in periodontitis, but not regularly in periodontally healthy individuals.


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Table 4. Number of subjects harbouring phylotypes/species found in periodontitis, but not in healthy subjects

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this report, we summarize our 16S rRNA gene sequencing effort on the microflora associated with advanced aggressive periodontitis in humans. To increase the bacterial diversity recovered from the samples, we included a high number of patients in the study (n=26). This approach, however, reduced the number of clones that could be sequenced per patient to 22 on average. Therefore, lack of representation per patient was compensated for by the use of two PCR primer pairs, which differed in their clone representation. A large number of the clones sequenced could be related to one of the numerous entries in GenBank submitted mostly by Paster et al. (2001) during the course of their comprehensive sequencing effort of 2522 clones. Our results, therefore, show that there is a high coverage of 16S rRNA sequences of the microflora of periodontal lesions in public databases despite differences of the study groups with regard to genetic background, geography and nutrition (Kroes et al., 1999; Paster et al., 2001; Sakamoto et al., 2000).

In the present study, a satisfying degree of diversity was disclosed. First of all, this can be attributed to the choice of primer pairs. PCRs using the primer pairs 27f/519r and 515f/1525r differed significantly in their profiles of species representation. A higher degree of diversity was seen with 27f/519r than with 515f/1525r. This might be due to the differing discriminatory power of the respective parts of the 16S rRNA gene. Primers 27f and 519r have been used for 16S rRNA gene amplification (27f) and sequencing (519r) for many years (Lane, 1991). To our knowledge, in molecular analyses on periodontal bacteria, their combination as a PCR primer pair has been used in two studies (Choi et al., 1994; Jung et al., 2000). In both studies, however, the authors did not randomly sequence inserts of their clone libraries. Choi et al. (1994) screened a 27f/519r library with probes specific to Treponema spp., Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Tannerella forsythensis. They found a representation of these species that was comparable to the one presented in this report. A disadvantage of the primer pair 27f/519r is that the sequences generated are too short to disclose phylogenetic positions if no close match is present in the databases. However, we show that the discriminative power of this primer pair can be exploited if sufficient data are available in the databases, as is now the case for oral bacteria.

It was obvious from our results that Actinomyces spp. were only been detected once in both clone populations and that Actinobacillus actinomycetemcomitans was not identified. The primers 515f/1525r have been shown to amplify Actinomyces spp. 16S rRNA genes (Kroes et al., 1999). Therefore, we initially suggested that DNA was not efficiently released from Actinomyces spp. by the method used for DNA extraction. However, consecutive analysis of the periodontal flora of patients suffering from hypophosphatasia (H. Gierschick, U. Vogel, G. Valenza, U. Schlagenhauf & S. Burgemeister, unpublished data) revealed that Actinomyces spp. could be readily detected using the same protocol. We therefore assume that only spurious amounts of Actinomyces DNA were present in the samples analysed in this study. Analysis of an Actinobacillus actinomycetemcomitans 16S rRNA gene sequence (GenBank accession no. M75035) showed that the priming sites for the primers 27f, 515f and 519r were conserved (the entry lacks the 3' end of the sequence; therefore, the priming site of 1525r could not be confirmed). Actinobacillus actinomycetemcomitans may have been missed due to the relatively low abundance of this bacterium in periodontic lesions. Ximenez-Fyvie et al. (2000) using checkerboard DNA–DNA hybridizations found low levels of hybridization suggesting that Actinobacillus actinomycetemcomitans DNA is present in small amounts in periodontal samples. Accordingly, Paster et al. (2001) found the species only once; Sakamoto et al. (2000) did not find it at all in their clone populations. Furthermore, culture studies of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans showed that Actinobacillus actinomycetemcomitans occurs in lower proportions than Porphyromonas gingivalis (see review by Asikainen & Chen, 1999).

The libraries generated with the primer pair 27f/519r differed substantially from those generated with 519f/1525r with respect to their representation of the Porphyromonadaceae, Fusobacteriaceae, Prevotellaceae, Bacteroidaceae, Eubacteriaceae, Streptococcaceae and Acidaminococcaceae. In a previous report, Marchesi et al. (1998) systematically analysed and compared the characteristics of the primer pairs 27f/1392r and 63f/1387r and demonstrated in detail how sequence differences might result in differential amplification of species within polymicrobial mixtures. However, in practice the use of primer pairs 27f/1392r and 63f/1387r resulted in only a moderate bias with regard to the amplification of Alphaproteobacteria and Gram-positive bacteria (O'Sullivan et al., 2002). Kroes et al. (1999) reported differential amplification of Capnocytophaga and Actinomyces spp. by the different PCR primer pairs used in their study on periodontal bacteria. Sequence analysis of the microflora of the caecum of broiler chicken revealed remarkable differences in the amplification of the Selenomonas ruminantium subgroup if primer 515FPL was used instead of 8FPL (Zhu et al., 2002). These reports and our own recommend the use of different primer pairs for sequencing efforts on mixed bacterial populations in order to achieve the best possible representation of species within the clone libraries.

Fourteen novel sequences found in this study were deposited in the GenBank database. Phylogenetic analysis assigned these sequences to five families within the phyla Bacteroidetes and Firmicutes. More sensitive techniques will be applied to study the distribution of the novel phylotypes among patients and healthy subjects, because there are currently no data confirming their pathogenic role in periodontitis. We report the complete 16S rRNA gene sequence of clone 10B6, which initially matched a partial 16S rRNA gene sequence (480 bp) deposited in GenBank, whose phylogeny could not be resolved unambiguously (Sakamoto et al., 2000). The complete sequence of clone 10B6 was now shown to represent a novel evolutionary lineage within the Alphaproteobacteria, which was most closely related to the family Holosporaceae. The family Holosporaceae exclusively comprises Rickettsia-related, obligate intracellular symbionts of paramecia and free-living amoebae (Horn et al., 1999; Springer et al., 1993). Since amoebae have been observed in periodontal lesions, we will test in future investigations whether 10B6 is an endosymbiont of oral amoebae, e.g. Entamoeba gingivalis (Dao et al., 1983; Gottlieb & Miller, 1971; Keller et al., 1967; Lucht et al., 1998; Yamamoto et al., 1995). Although endosymbionts have not been described for Entamoeba gingivalis, data available in the literature do not rule out their possible existence.

Paster et al. (2001) recently proposed 20 phylotypes to be associated with periodontitis, but not with health. The proposal was made on the basis of nine subjects with periodontitis and five healthy subjects. Among the 20 phylotypes proposed by Paster and colleagues were several species that had been found in periodontitis patients by other authors, i.e. Porphyromonas gingivalis, Filifactor alocis, Treponema socranskii, Dialister pneumosintes, Campylobacter rectus, Tannerella forsythensis and Streptococcus constellatus (Albandar et al., 1997; Contreras et al., 2000; Dewhirst et al., 2000; Macuch & Tanner, 2000; Moore & Moore, 1994; Ximenez-Fyvie et al., 2000). However, Abiotrophia adiacens has been described to occur in the oral cavity of healthy individuals (Sato et al., 1999). Gemella haemolysans and Campylobacter rectus were present in the clone libraries derived from a healthy subject with only mild gingivitis (Kroes et al., 1999). Some species like Porphyromonas endodontalis or PUS9.170 had been found initially in oral diseases other than periodontitis (Sundqvist, 1992; Wade et al., 1997). Based on the number of periodontitis patients and healthy subjects harbouring the putative pathogens proposed by Paster et al. (2001), we suggest that in our study Treponema socranskii subsp. buccale, Filifactor alocis, Dialister pneumosintes, Porphyromonas gingivalis and Porphyromonas endodontalis were associated with periodontitis but not with health.


   ACKNOWLEDGEMENTS
 
This work was supported by Graduate College 520 (Immunmodulation) of the Deutsche Forschungsgemeinschaft, and by the German Ministry for Education and Research via the IZKF Würzburg (01 KS 9603). The Graduate College supported the participation of G. H. in the ARB course (Bremen, 2001). We thank Matthias Frosch for continuous support. The expert technical assistance of Gabi Heinze, Carmen Roldan, Marion Patzke-Oechsner, Angelika Hansen and Susanne Ebner is gratefully acknowledged. We thank Annette Moter, Berlin, for helpful discussions. The NCBI is gratefully acknowledged for providing access to the BLAST program.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 4 June 2002; revised 28 September 2002; accepted 4 October 2002.