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
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ABSTRACT |
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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).
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INTRODUCTION |
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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.
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METHODS |
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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 827 of the 16S rRNA gene; Lane, 1991); 519r (5'-GWATTACCGCGGCKGCTG-3'; J01695; positions 536519; Lane, 1991
); 515f (synonym A19) (5'-GTGCCSGCMGCCGCGGTAA-3'; J01695; positions 515533; Kroes et al., 1999
); 1525r (synonym S17) (5'-AAGGAGGTGATCCAGCC-3'; J01695; positions 15411525; Kroes et al., 1999
); GV1r (5'-GATCATCGACTTGGGGGTC-3'; specific to clone 10B6; accession no. AJ428412; positions 258240, this study); GV2f (5'-ACCCCCAAGTCGATGATCG-3'; specific to clone 10B6; AJ428412; positions 241259; 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 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 (JukesCantor 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.
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RESULTS |
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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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DISCUSSION |
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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 DNADNA 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.
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ACKNOWLEDGEMENTS |
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Received 4 June 2002;
revised 28 September 2002;
accepted 4 October 2002.