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RESEARCH REPORT |
1 Department of Endodontics, Estácio de Sá University, Rio de Janeiro, Brazil; and
2 Institute of Microbiobiology Prof. Paulo de Góes, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil;
* corresponding author’s address, R. Herotides de Oliveira 61/601, Icaraí, Niterói, RJ, Brazil 24230-230; siqueira{at}estacio.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: endodontic microbiology • 16S rDNA • molecular biology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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It has been postulated that a comprehensive description of microbial communities inhabiting different habitats requires the use of techniques that sidestep cultivation (Hugenholtz et al., 1998). Genetic fingerprinting techniques represent a powerful tool for the investigation of the structure of microbial communities in diverse ecosystems and can be used for microbial identification. A commonly used strategy for the fingerprinting of complex bacterial communities consists of 16S rDNA-based PCR, followed by product analysis by denaturing gradient gel electrophoresis (DGGE) (Muyzer, 1999). The DGGE approach is based on electrophoresis of PCR products in polyacrylamide gels containing a linearly increasing gradient of DNA denaturants (Muyzer, 1999). In DGGE, DNA fragments of the same length, but with different base-pair sequences, can be separated. A single-base change in a sequence can be resolved (Fischer and Lerman, 1983), which provides PCR-DGGE with a great potential to identify closely related species based on 16S rDNA sequence divergence. In addition to being applied to the broad-range analysis of complex microbial communities (Muyzer and Smalla, 1998), the method can also be used to detect specific bacterial groups by altering the primer targets or by using a nested PCR (nPCR) approach (Ercolini, 2004).
We undertook the present study to investigate the occurrence of members of the phyla Spirochaetes and Deferribacteres in endodontic infections, and to examine their diversity through a culture-independent approach. Representatives of these phyla have been associated with periodontal diseases, and the possibility exists that these phyla also contain potential endodontic pathogens.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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nPCR-DGGE Assay
A 16S rDNA nPCR was used. Reverse-primer C90 (5'-GTTACGACTTCACCCTCCT-3') (Dewhirst et al., 2000), which is selective for spirochetes but also amplifies DNA from bacteria of the Deferribacteres phyla and Atopobium species (Paster et al., 2001), was used, along with a forward universal bacterial primer (5'-AGAGTTTGATCCTGGCTCAG-3') in the first round of PCR amplification, generating a PCR amplicon of about 1500 bp (base positions 8-1503 of the Escherichia coli 16S rDNA).
In the second round of amplification, a fragment located in the V6-to-V8 regions of the 16S rDNA (Tung et al., 2002) was amplified from the first PCR products with the following universal bacterial primers: 968f-GC (5'-CGCCCGCCGCGCGCGGCGGG CGGGGCGGGGGCACGGGGGGAACGCGAAGAACCTTAC-3') and 1401r (5'-CGGTGTGTACAAGACCC-3') (Nübel et al., 1996).
DNA extracted from clinical samples was used as target in the first PCR reaction, which was performed in a 50-µL mixture containing 40 pmol of each primer, 5 µL of 10X PCR buffer (Biotools, Madrid, Spain), 1.5 mM MgCl2, 1.25 U Tth DNA polymerase (Biotools), and 0.2 mM of each deoxyribonucleoside triphosphate (Biotools). Afterward, 1 µL of the PCR products generated in the first round of amplification was used as a template for the nested reaction in a PCR mixture comprised of 25 pmol of universal primers, 5 µL of 10X PCR buffer, 3.8 mM MgCl2, 2.5 U of Tth DNA polymerase, 0.2 mM concentration of each deoxyribonucleoside triphosphate, and sterile filtered milliQ water, to a final volume of 50 µL. Negative controls consisting of sterile milliQ water instead of sample were included with each batch of samples analyzed.
PCR amplification was performed in a DNA thermocycler (Primus 25/96, MWG-Biotech, Ebersberg, Germany). Cycling conditions were as follows: (first round) 30 cycles of denaturation at 94°C for 45 sec, annealing at 60°C for 45 sec, and extension at 72°C for 90 sec, and a final elongation step at 72°C for 15 min; and (second round) initial denaturation at 94°C for 2 min, followed by 28 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min, and a final step of 72°C for 10 min. Prior to DGGE analysis, the presence of PCR products was confirmed by electrophoresis in an agarose gel stained with ethidium bromide and viewed under UV transillumination.
DGGE was performed with use of the Dcode Universal Mutation Detection System (Bio-Rad Dcode, Richmond, VA, USA) at 75V and 60°C for 16 hrs in 0.5X TAE buffer [20 mM Tris-acetate (pH 7.4), 10 mM sodium acetate, 0.5 mM disodium EDTA]. The nPCR products (30 µL) were loaded onto 6% (w/v) polyacrylamide gels containing a linear gradient ranging from 20% to 70% denaturant [100% denaturant corresponded to 7 M urea and 40% (v/v) formamide]. Afterward, the gel was stained with SYBR green I nucleic acid gel stain (Molecular Probes, Leiden, The Netherlands) for 40 min, and then scanned in a Storm PhosphorImager (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK).
Sequence Analysis
The 15 most intense bands were cut from the DGGE gel with a fresh sterile scalpel blade, placed in 50 µL of milliQ water, and left at 4°C for 24 hrs. A 5-µL quantity of the resulting solution was re-amplified by PCR with 968f-GC/1401r primers. Products were then purified in a PCR purification system (Wizard PCR Preps, Promega, Madison, WI, USA), and sequenced directly with the 1401r primer on the ABI 377 automated DNA sequencer with dye terminator chemistry (Amersham Biosciences). Sequences were analyzed by means of the BLAST program in the GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al., 1990). Sequences with less than 99% similarity to GenBank database entries were screened for chimeras by means of the CHECK-CHIMERA program of the Ribosomal Database Project (http://rdp.cme.msu.edu/html). Nucleotide sequences of close evolutionary relatives of our sequences were retrieved from the GenBank database. Each sequence was aligned to the closest matched sequences with the ClustalW multiple sequence alignment tool (Thompson et al., 1997). Neighbor-joining phylogenetic trees were constructed from the alignments with the use of MEGA version 2.1 software (Kumar et al., 2001). Distance matrix analyses were performed with a Jukes-Cantor correction. Robustness of the phylogeny was tested by bootstrap analysis with 500 iterations. The sequences of 8 bands excised from the DGGE gels have been deposited in the GenBank database under accession numbers AY597648 to AY597655.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The DGGE profiles generated by nPCR amplification of endodontic samples revealed distinct banding patterns from different clinical samples (Fig. 1
). Profiles contained both intense and faint DNA bands. No single band occurred in all profiles. The mean number of bands detected in the clinical samples was 6.5, ranging from 2 to 10.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Our results indicate that Flexistipes species may be common members of the endodontic microbiota, since at least one-half of the samples surveyed harbored these bacteria. Three sequences showed high similarity (> 99%) to Flexistipes oral clone BA121 or Flexistipes sp. E3-33. The other 2 sequences showed 95% and 97% similarities to these 2 oral clones, respectively, suggesting that they are different phylotypes of the Flexistipes group. The fact that Flexistipes species have never been consistently found in endodontic infections by culture suggests either that most phylotypes can be uncultivable, that they occur in numbers below the detection limits of culture approaches, or that they can present ambiguous phenotypic behaviors which can lead culture procedures to misidentification. The latter speculation is based on the fact that, in the only study that isolated a Flexistipes species from an infected root canal, identification was based on sequencing of the 16S rDNA and not by phenotype-based methods (Munson et al., 2002). Because some Flexistipes species are cultivable (Munson et al., 2002), efforts should be made to cultivate the most prevalent Flexistipes phylotypes found in endodontic infections, in an attempt to determine their pathogenicity and susceptibility to endodontic medicaments and procedures.
The occurrence of spirochetes in endodontic infections has long been obscured by difficulties in culturing these micro-organisms. Recent studies using species-specific nPCR approaches revealed a high prevalence of oral treponemes in those infections (Baumgartner et al., 2003; Rôças et al., 2003; Siqueira and Rôças, 2003 Siqueira and Rôças, 2004). In the present study, 5 of the excised bands yielded sequences with similarities to members of the Spirochaetes phylum, but none of them clustered with Treponema species. This was rather unexpected, given the high prevalence of treponemes in endodontic infections, as previously reported by species-specific nPCR. Differences may have been due to the higher sensitivity of species-specific nPCR when compared with nPCR-DGGE (Rôças et al., 2003; Ercolini, 2004). Moreover, it is possible that the faint bands observed in DGGE profiles, which were not sequenced, might have been from treponemes, suggesting that they were in low numbers in the samples examined.
It has been reported that all oral spirochetes belong to the genus Treponema (Dewhirst et al., 2000). However, our findings suggest that other spirochetes may be present as part of the oral microbiota. Nevertheless, a disadvantage of the DGGE assay is that the sequences generated are too short to disclose phylogenetic positions if no close match is present in the databases. Further studies should confirm or refute these findings.
DGGE is a reliable molecular tool that permits the construction of a ‘picture’ of the structure of the microbiota (Muyzer, 1999). An additional advantage is that it allows for the identification of community members by the sequencing of excised bands (Muyzer and Smalla, 1998). Our findings pointed to the applicability of the PCR-DGGE method for identification of members of the endodontic microbiota, including previously uncharacterized bacteria. The fingerprint generated also allowed for the analysis of the diversity of these bacterial groups in endodontic samples, with a mean number of 6.5 intense and faint bands per profile. Intensity of bands in the DGGE gel is supposedly related to the density of corresponding bacterial phylotypes within the sample (Murray et al., 1996). In this study, dominant (intense) bands excised from the gel were sequenced, and comparison with database sequences revealed phylotypes of all target bacterial groups. Overall, the most intense bands belonged to Flexistipes phylotypes, which indicates their dominance among the bacteria targeted herein.
Findings from the present study indicate that uncultivated species of the phyla Spirochaetes and Deferribacteres, as well as from the Atopobium genus, can be part of the endodontic microbiota, and therefore may participate in the etiology of periradicular diseases. Analysis of these data also expands the list of phyla represented in endodontic infections to include Deferribacteres. Ongoing studies from our laboratory, using specific probes for Deferribacteres phylotypes, are under way to investigate the prevalence of these bacteria in different types of endodontic infections.
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ACKNOWLEDGMENTS |
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Received April 15, 2004; Last revision January 14, 2005; Accepted February 24, 2005
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REFERENCES |
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Baumgartner JC, Khemaleelakul SV, Xia T (2003). Identification of spirochetes (treponemes) in endodontic infections. J Endod 29:794–797.[ISI][Medline]
Dewhirst FE, Tamer MA, Ericson RE, Lau CN, Levanos VA, Boches SK, et al. (2000). The diversity of periodontal spirochetes by 16S rRNA analysis. Oral Microbiol Immunol 15:196–202.[ISI][Medline]
Ercolini D (2004). PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. J Microbiol Methods 56:297–314.[ISI][Medline]
Fischer SG, Lerman LS (1983). DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc Natl Acad Sci USA 80:1579–1583.
Hugenholtz P, Pitulle C, Hershberger KL, Pace NR (1998). Novel division level bacterial diversity in a Yellowstone hot spring. J Bacteriol 180:366–376.
Hutter G, Schlagenhauf U, Valenza G, Horn M, Burgemeister S, Claus H, et al. (2003). Molecular analysis of bacteria in periodontitis: evaluation of clone libraries, novel phylotypes and putative pathogens. Microbiology 149:67–75.
Kumar PS, Griffen AL, Barton JA, Paster BJ, Moeschberger ML, Leys EJ (2003). New bacterial species associated with chronic periodontitis. J Dent Res 82:338–344.
Kumar S, Tamura K, Jakobsen IB, Nei M (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245.
Munson MA, Pitt-Ford T, Chong B, Weightman A, Wade WG (2002). Molecular and cultural analysis of the microflora associated with endodontic infections. J Dent Res 81:761–766. Erratum in J Dent Res 82:69, 247, 2003.
Murray AE, Hollibaugh JT, Orrego C (1996). Phylogenetic compositions of bacterioplankton from two California estuaries compared by denaturing gradient gel electrophoresis of 16S rDNA fragments. Appl Environ Microbiol 62:2676–2680.[Abstract]
Muyzer G (1999). DGGE/TGGE a method for identifying genes from natural ecosystems. Curr Opin Microbiol 2:317–322.[ISI][Medline]
Muyzer G, Smalla K (1998). Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek 73:127–141.[ISI][Medline]
Nübel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann RI, et al. (1996). Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178:5636–5643.
Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, et al. (2001). Bacterial diversity in human subgingival plaque. J Bacteriol 183:3770–3783.
Paster BJ, Falkler WA Jr, Enwonwu CO, Idigbe EO, Savage KO, Levanos VA, et al. (2002). Prevalent bacterial species and novel phylotypes in advanced NOMA lesions. J Clin Microbiol 40:2187–2191.
Rôças IN, Siqueira JF Jr, Andrade AFB, Uzeda M (2003). Oral treponemes in primary root canal infections as detected by nested PCR. Int Endod J 36:20–26.[ISI][Medline]
Siqueira JF Jr (2003). Taxonomic changes of bacteria associated with endodontic infections. J Endod 29:619–623.[ISI][Medline]
Siqueira JF Jr, Rôças IN (2003). PCR-based identification of Treponema maltophilum, T. amylovorum, T. medium, and T. lecithinolyticum in primary root canal infections. Arch Oral Biol 48:495–502.[ISI][Medline]
Siqueira JF Jr, Rôças IN (2004). Treponema species associated with abscesses of endodontic origin. Oral Microbiol Immunol 19:336–339.[ISI][Medline]
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997). The CLUST-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882.
Tung C-S, Joseph S, Sanbonmatsu KY (2002). All-atom homology model of the Escherichia coli 30S ribosomal subunit. Nat Struct Biol 9:750–755.[ISI][Medline]
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