Journal of Medical Microbiology (2007), 56, 110118

DOI 10.1099/jmm.0.46835-0

Phylogenetic analysis of bacterial and archaeal species in symptomatic and asymptomatic endodontic infections
M. M. Vickerman,1,2 K. A. Brossard,2 D. B. Funk,1 A. M. Jesionowski2 and S. R. Gill2
Correspondence M. M. Vickerman [email protected]
1

Department of Periodontics and Endodontics, University at Buffalo School of Dentistry, Buffalo, NY, USA Department of Oral Biology, University at Buffalo School of Dentistry, Buffalo, NY, USA

Received 14 July 2006 Accepted 24 August 2006

Phylogenetic analysis of bacterial and archaeal 16S rRNA was used to examine polymicrobial communities within infected root canals of 20 symptomatic and 14 asymptomatic patients. Nucleotide sequences from ~750 clones amplied from each patient group with universal bacterial primers were matched to the Ribosomal Database Project II database. Phylotypes from 37 genera representing Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria were identied. Results were compared to those obtained with species-specic primers designed to detect Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis, Peptostreptococcus micros, Enterococcus sp., Streptococcus sp., Fusobacterium nucleatum, Tannerella forsythensis and Treponema denticola. Since members of the domain Archaea have been implicated in the severity of periodontal disease, and a recent report conrms that archaea are present in endodontic infections, 16S archaeal primers were also used to detect which patients carried these prokaryotes, to determine if their presence correlated with severity of the clinical symptoms. A Methanobrevibacter oralis-like species was detected in one asymptomatic and one symptomatic patient. DNA from root canals of these two patients was further analysed using species-specic primers to determine bacterial cohabitants. Trep. denticola was detected in the asymptomatic but not the symptomatic patient. Conversely, Porph. endodontalis was found in the symptomatic but not the asymptomatic patient. All other species except enterococci were detected with the species-specic primers in both patients. These results conrm the presence of archaea in root canals and provide additional insights into the polymicrobial communities in endodontic infections associated with clinical symptoms.

INTRODUCTION
The use of molecular mechanisms for dening the taxonomy of the oral microbial ora has emphasized the complexity of these communities and has conrmed estimates that more than 500 bacterial species exist in the oral cavity (Paster et al., 2001). The composition of these complex microbial communities varies in different ecological niches, such as the gingival crevice, tongue, buccal mucosa and saliva. Less attention has been paid to the
Abbreviation: RDP-II, Ribosomal Database Project II. The GenBank/EMBL/DDBJ Trace Archive accession numbers for the bacterial 16S rDNA sequences are 13915683751391571307, and for the archaeal 16S rDNA sequences 13915680381391568374 and 13915572171391557263. A table showing the taxonomic distribution of bacterial phylotypes detected in the study is available as supplementary data with the online version of this paper.

microbial ora in infected root canals of teeth. Bacteria can gain access to the dental pulp through the crown or root surfaces in association with processes such as caries, periodontal disease or trauma. Any micro-organisms colonizing the normally sterile root-canal space can cause pulp necrosis and inammation in the surrounding bone. The microbial community within the root canal is thought to undergo ecological succession as nutritional, acidic and oxygenation changes occur in conjunction with bacterial growth (Sundqvist & Figdor, 2003). This chronic infectious process can progress asymptomatically, but dramatic clinical symptoms such as pain and swelling can occur. Although the patients immune system plays a key role in the response to endodontic infections, qualitative and quantitative changes in the bacterial composition within the root canal have been implicated in clinical presentation. Although bacterial cultivation studies have suggested that specic bacterial species such as strict anaerobes are associated with symptoms (Jacinto et al., 2003; Drucker &
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Bacterial and archaeal 16S rDNA in root canals numbers of male and female patients. Female subjects were excluded on the basis of pregnancy or lactation. Only one tooth was sampled from each patient.
Patient classication. Patients were classied as symptomatic if

Natsiou, 2000), these analyses have not been denitive (Sakamoto et al., 2006; Siqueira & Rocas, 2005). The development of 16S rRNA phylogenetic methodology has widened the scope of detectable micro-organisms to include uncultivable organisms that may play signicant, as yet undened, roles in pathogenesis (Eckburg et al., 2003; Kroes et al., 1999; Paster et al., 2001). This methodology has been applied to characterization of oral microbial populations of healthy patients (Aas et al., 2005), patients with periodontal disease (Kroes et al., 1999), and patients with endodontic infections (Munson et al., 2002; Saito et al., 2006; Sakamoto et al., 2006). The latter studies indicate that the root-canal ora contains novel phylotypes and is more complex than culturing studies have previously indicated. Furthermore, recent studies have suggested that members of the domain Archaea, prokaryotes that have been implicated as pathogens in human disease (Eckburg et al., 2003), are found in greater abundance in dental plaque from sites with periodontal disease than in plaque from non-diseased sites (Lepp et al., 2004). Although attempts to isolate archaea from root-canal samples by amplication of 16S rDNA were at rst unsuccessful (Siqueira et al., 2005), one recent study has conrmed that members of the methanogenic archaea, similar to Methanobrevibacter oralis, can be detected in rootcanal samples (Vianna et al., 2006). The purpose of this study was twofold: rst, to use PCR amplication of 16S rRNA sequences to identify bacterial phyla present in a pool of infected teeth of patients with and without clinical symptoms; and second, to conrm the presence of archaea in infected root canals and determine if their presence correlated with disease severity. In the present study, M. oralis-like 16S rDNA was amplied from one symptomatic and one asymptomatic patient. Root-canal DNA of these two patients was further analysed for bacterial content using species-specic primers.

they had an immediate history of spontaneous pain, pain to percussion, or pain upon palpation. The presence of swelling, lymphadenopathy or evidence of a sinus tract was considered symptomatic whether or not pain was present. Patients without the above criteria were considered asymptomatic. Of the 34 patients who met the criteria for inclusion in the study, 20 patients, including nine males and 11 females, were classied as symptomatic, and 14 patients, including eight males and six females, were classied as asymptomatic.
Endodontic sample collection. Samples from infected root canals

were collected using a modication of published methods (Fouad et al., 2002). The tooth was isolated with a dental dam and the surrounding eld was cleaned with 30 % hydrogen peroxide and decontaminated with 2.5 % sodium hypochlorite for 30 s. After removal of caries, involved dentin and existing restorations, the area was swabbed with 2.5 % sodium hypochlorite, then deactivated with sterile 5 % (w/v) sodium thiosulfate. Access to the pulp chamber and root canal was made with a new, sterile, #4 round bur without water spray. After minimal canal enlargement with sterile saline irrigant to allow access to the working length, dry, autoclaved paper points were placed within the canal space for 30 seconds. The paper points containing the absorbed root-canal contents were placed in sterile vials containing 1 ml 10 mM Tris-HCl, 1 mM EDTA, pH 8. Samples were then taken directly to the laboratory and stored at 280 uC until processing. Endodontic treatment proceeded routinely.
Preparation of microbial DNA from root-canal contents. Each

METHODS
Patient selection. Patients over the age of 21 years who presented

to the University of Buffalo Graduate Endodontics Clinic for treatment were assessed. Data collected for each patient consisted of gender, clinical examination results, diagnosis, duration of symptoms associated with the dental condition (e.g. pain, swelling), and any concomitant drug therapy and medical conditions. In accordance with University at Buffalo Human Institutional Review Board requirements, the patients in this review board-approved study were de-identied so that the collected history and specimen for each patient were correlated only by randomly assigned numbers.
Criteria for inclusion. Healthy patients with teeth that had no previous pulpal treatment and who were diagnosed with pulp necrosis, based on negative responses to thermal stimulation and electronic pulp-vitality testing when possible, and with chronic apical periodontitis, based on radiographic evidence of periapical radiolucency, were included in the study. Single and multirooted teeth were included. Patients with a history of antibiotic treatment within the previous 3-month period were excluded. Subjects were enrolled regardless of race, and attempts were made to include equivalent

sample was thawed on ice, vortexed for 2 min and centrifuged at 2000 g for 5 min. After removal of the supernatant and paper point, the pellet, containing microbial cells, was resuspended in a solution containing 100 ml sterile molecular biology grade water, 100 ml 0.1 % (w/v) blue dextran solution, 200 ml cell lysis buffer (100 mM TrisHCl, pH 7.4, 20 mM EDTA, 5 M guanidine thiocyanate, 2 % Triton X-100) and 100 mg proteinase K, and incubated at 65 uC for 30 min. This mixture was then added to a tube containing 350 mg of sterile glass beads (FastRNA Pro Blue kit, Qbiogene) and placed in a FastPrep cell disrupter (Qbiogene) at 4 m s21 for 30 s. A 400 ml volume of 100 % benzyl alcohol was added and the solution was vortexed for 15 s. Nucleic acids were separated from cell-lysis products by centrifugation at 7000 g for 5 min. The upper aqueous phase was transferred to a new tube and precipitated with 1/10 vol. 3 M sodium acetate, pH 5.3, and 2 vols 100 % ethanol at 220 uC. The solution was then centrifuged at 16 000 g for 30 min to pellet the nucleic acids, which were then RNase-treated, reprecipitated and resuspended in 30 ml 10 mM Tris-HCl, 1 mM EDTA, pH 8. Aliquots were stored at 280 uC until used as template in PCR. The mean amount of DNA recovered per tooth was 8.83.8 mg, as determined spectrophotometrically. The recovered root-canal contents contained both human host and microbial DNA, so microbial target template concentrations could not be readily quantied. To examine the overall composition of the microbial communities in the asymptomatic and symptomatic groups, equal volumes of DNA from each patient preparation were pooled according to the asymptomatic and symptomatic classication.
Universal bacterial primers and PCR conditions. Each PCR mix contained ~250 ng DNA, 100 ng each forward and reverse primer, 200 mM dNTPs (Invitrogen), 25 mM MgCl2 and 2.5 U Platinum Taq DNA polymerase (Invitrogen) in the manufacturers buffer. All reactions were done with a 3 min 95 uC hot start followed by 35 cycles of 95 uC for 1 min, the primer-specic annealing temperature for 1 min, and 72 uC for 2 min, followed by a 30 min 72 uC

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M. M. Vickerman and others extension. Primers designed to anneal to conserved regions were used to amplify bacterial 16S rDNA sequences that contained intervening species-specic regions. The forward primer, 8F (59-AGAGTTTGATCCTGGCTCAG-39), was used with reverse primers 1391R (59-GACGGGCGGTGTGTRCA-39) or 1492R (59-GGGTTACCTTGTTACGACTT-39) with an annealing temperature of 56 uC and template DNA from the root-canal contents to amplify 1.4 and 1.5 kbp regions, respectively, of bacterial 16S rDNA (Baker et al., 2003). The products of primers 8F and 1391R and the products of primers 8F and 1492R with pooled DNA from asymptomatic patients as template, and the products of the same primer pairs using DNA template pooled from symptomatic patients, were cloned for sequence analysis. An equivalent number of clones were analysed from both the symptomatic and asymptomatic patients, for a total of ~1500 clones.
Detection of archaeal and bacterial species with specic oligonucleotide primers. Amplicon products of bacterial universal

the pools carried these micro-organisms. DNA samples from archaea-positive patients were individually characterized with bacterial species-specic primers. All negative reactions were repeated at least twice with unamplied DNA template to conrm that the target sequences were below detectable threshold levels. To conrm the specicity of the species-specic primers, randomly chosen cloned amplicons from each positive reaction were sequenced and conrmed to have 99 % similarity to GenBank entries for each species.
Cloning and sequencing. The completed PCR mixtures were electrophoresed on agarose gels; amplicons were eluted from the gel with a Qiaex bead kit (Qiagen) and cloned into pCR-4-TOPO vectors (Invitrogen). One-Shot TOP10 Competent Escherichia coli cells (Invitrogen) were transformed using the TOPO-TA Cloning kit (Invitrogen) according to the manufacturers instructions. Plasmid DNA from each transformant was prepared by a modied alkalinelysis method. Nucleotide sequences of the clone inserts were determined by cycle sequencing using BigDye Terminator (Applied Biosystems) and 3.2 pmol M13F (59-GTAAAACGACGGCCAG-39) and M13R (59-CAGGAAACAGCTATGAC-39) sequencing primers. Sequences were analysed on ABI 3730xl sequencers (Applied Biosystems). After trimming to remove vector sequence and adjusting for quality values, the mean single-sequence read length was ~700 nt. Bidirectional sequence reads of clone inserts provided near full-length 16S bacterial and archaeal sequences, which were used for phylogenetic analysis. Phylogenetic analysis. To identify bacterial rDNA amplied with

primers were used as templates for detection of Prevotella intermedia, Porphyromonas endodontalis, Porphyromonas gingivalis, Peptostreptococcus micros, Streptococcus sp., Fusobacterium nucleatum and Tannerella forsythensis using species-specic primers and annealing temperatures as shown in Table 1. Representative strains of each bacterial species were used as positive controls to optimize PCR conditions for each primer pair; limits of detection were determined with serial dilutions of DNA prepared from pure cultures using standard alkaline-lysis techniques (Sambrook et al., 1989). Unamplied DNA pools from asymptomatic and symptomatic patients were used directly with primers to amplify the 16S rDNA of archaea and Treponema denticola, which would not be amplied well with the bacterial universal primers (Choi et al., 1994; Kroes et al., 1999), and to amplify the tuf gene, encoding the Tu elongation factor, of Enterococcus sp. (Table 1). Individual patient samples were screened for the presence of archaea to determine which patients in

the universal primers, sequences with 97 % similarity were assembled and the individual assemblies were analysed by comparison with known phylotypes via the Sequence Match program of the Ribosomal Database Project II (RDP-II; version 3 release 9.38) (Cole et al., 2005). The relationships of the cloned assemblies to known

Table 1. PCR primers and conditions for microbial detection

Bold type in the primer sequence or annealing temperature indicates a modication from the reference listed. Micro-organism Prev. intermedia Porph. endodontalis Porph. gingivalis Pept. micros Streptococcus sp. Fusob. nucleatum Tann. forsythensis Trep. denticola Archaea Enterococcus sp. Forward and reverse primers 59-CGTGGACCAAAGATTCATCGGTGGA-39 59-CCGCTTTACTCCCCAACAAA-39 59-GCTGCAGCTCAACTGTAGTC-39 59-CCGCTTCATGTCACCATGTC-39 59-AGGCAGCTTGCCATACTGCG-39 59-ACTGTTAGCAACTACCGATGT-39 59-AGAGTTTGATCCTGGCTCAG-39 59-ATATCATGCGATTCTGTGGTCTC-39 59-AGAGTTTGATCCTGGCTCAG-39 59-GTACCGTCACAGTATGAACTTTCC-39 59-AGAGTTTGATCCTGGCTCAG-39 59-GTCATCGTGCACACAGAATTGCTG-39 59-TACAGGGGAATAAAATGAGATACG-39 59-ACGTCATCCCAACCTTCCTC-39 59-TAATACCGAATGTGCTCATTTACAT-39 59-TCAAAGAAGCATTCCCTCTTCTTCTTA-39 59-TCCAGGCCCTACGGG-3959-YCCGGCGTTGAMTCCAATT-39 59-TACTGACAAACCATTCATGATG-39 59-AACTTCGTCACCAACGCGAAC-39 Annealing temperature (6C) 57 57 57 57 55 55 57 60 50 55 Amplicon size (bp) 259 665 404 207 500 407 745 316 607 112 Reference Baumgartner et al. (1999) Bogen & Slots (1999) Bogen & Slots (1999) Conrads et al. (1997b) Conrads et al. (1997a) Conrads et al. (1997a) Tran & Rudney (1999) Ashimoto et al. (1996) Lepp et al. (2004) Ke et al. (1999)

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Bacterial and archaeal 16S rDNA in root canals phylotypes were expressed with S_ab scores, dened as the number of unique oligomers shared between the query sequence and the sequence in the RDP-II database, divided by the lowest number of unique oligomers in either of the two sequences. The top 20 hierarchical matches for each assembly consensus sequence were examined, and the sequence was designated the phylotype in the RDP-II database with the highest S_ab score. In the two cases in which the assembly sequences matched more than one phylotype with the same S_ab score, data are presented as the type species. Phylogenetic identication was veried to the genus level using the RDP-II database Classier program, which uses a naive Bayesian rRNA classier (version 1.0) to assign a sequence to a taxonomical hierarchy according to Holt et al. (1994).

Comparison of amplicons from universal and species-specic primers Although the universal primers detected a wide range of bacterial phylotypes, PCR with species-specic primers conrmed the limitations of universal primers, which cannot anneal equally well to all microbial 16S rDNA (Baker et al., 2003; de Lillo et al., 2006) and did not amplify DNA from all the bacteria present in the template. Similarly, the number of PCR cycles could bias amplication in favour of more abundant species by allowing self-annealing in later amplication cycles (Acinas et al., 2005), resulting in a less representative sample of the community. Although Prev. intermedia was not detected with universal primers in either asymptomatic or symptomatic patients (Table 2), speciesspecic primers were able to successfully amplify Prev. intermedia 16S rDNA with 99 % similarity to the expected sequence from a pool of DNA from symptomatic patients (Table 3). However, species-specic primers could not detect Prev. intermedia in the DNA of asymptomatic patients, suggesting that the relative numbers of this species may be lower in these patients. Conversely, universal primers detected peptostreptococci only in the asymptomatic patients (Table 2), whereas the species-specic primers were able to detect Pept. micros in both patient groups (Table 3). Similarly, Porph. endodontalis and Tann. forsythensis, which were not detected with universal primers (Table 2), were successfully amplied from both asymptomatic and symptomatic patients when species-specic primers were used (Table 3). Streptococcus sp. and Fusob. nucleatum were detected with both universal and speciesspecic primers (Tables 2 and 3) from asymptomatic and symptomatic patients. Although Porph. gingivalis was detected with universal primers only in asymptomatic patients (Table 2), species-specic primers detected this species in both patient groups (Table 3). As expected, Trep. denticola was detected only with species-specic primers. However, this species was detected only in the asymptomatic patients (Table 3), suggesting that it is absent or less abundant in the symptomatic patients, in contrast to reports elsewhere (Siqueira & Rocas, 2005). Enterococcal speciesspecic primers did not amplify the tuf gene from patient DNA, even when the amount of template in the reaction mix was increased by up to threefold. The absence of an enterococcal tuf amplicon was supported by the lack of 16S rDNA universal primer amplicons that were 60 % similar to any enterococcal species in the RDP-II database (data not shown). Nevertheless, other studies have also failed to detect this species in root canals (Cheung & Ho, 2001), and the abundance of Enterococcus faecalis in root canals has been reported to be variable (Sedgley et al., 2006). Bacterial communities in asymptomatic and symptomatic patients The bacterial phyla detected with universal primers included Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria (Supplementary Table S1 in JMM online). Although others have also detected low levels of
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RESULTS AND DISCUSSION

Sequence analysis of cloned 16S rDNA amplicons from universal primers Cloned amplicons generated from PCR with DNA from the two patient pools and universal primers were sequenced, assembled and compared with sequences of known species in the RDP-II database for phylotype identication. In some cases, multiple different assemblies matched most closely to the same sequence in the RDP-II database, but with a different similarity, as indicated by the Sequence Match (S_ab) score. Sequences were conrmed to the genus level with a greater than 95 % condence threshold for symptomatic and asymptomatic samples, using the RDP-II database Classier program. Taxonomic classications and distribution of most prevalent phylotypes are shown in Supplementary Table S1 in JMM online. Asymptomatic and symptomatic patients had only seven phylotypes in common: Dialister invisus (GenBank accession no. AF287787); Fusob. nucleatum [most similar to phylotype ChDC OS50 (AF543300)]; Prevotella sp. [most similar to oral clone F045 (AY005056)]; Veillonella parvula (AY995769), Lactobacillus gasseri (AY730721), Solobacterium moorei (AY044915) and Atopobium parvulum (X67150). The remaining clones were exclusive to either the asymptomatic or the symptomatic patients, although multiple assemblies matched most species (Table 2). The results of this study support recent studies using culture-independent methods that indicate that the polymicrobial communities in infected root canals may be more complex than previously appreciated (Sundqvist & Figdor, 2003; Siqueira & Rocas, 2005). A recent review (Siqueira & Rocas, 2005) of studies that use molecular methodologies has indicated that independently of the presence of symptoms, the most prevalent non-treponemal endodontic bacteria are Porph. endodontalis, Tann. forsythensis, Pseudoramibacter alactolyticus, Dialister pneumosintes, Filifator alocis, Porph. gingivalis and Propionibacterium propionicum. In the present study, universal primers did not detect Porph. endodontalis, Tann. forsythensis or D. pneumosintes (Table 2), suggesting that they were not present in high abundance in our patient pools or that the universal primers did not anneal well to the 16S rDNA of these species.
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Table 2. Bacterial species and phylotypes detected in each group of patients by amplication of 16S rDNA with universal primers
Asymptomatic* Anaeroglobus geminatus Bacteroidales oral clone MCE7_20 Brachymonas denitricans Campylobacter gracilis Catonella morbi Cryptobacterium curtum Desulfovibrio sp. D4 Desulfovibrio sp. LNB1 Dialister (genus), uncultured bacterium Dialister sp. E2_20 Eikenella corrodens Eikenella sp. MDA2346-4 Eubacterium (genus), Eubacteriaceae oral clone P2PB_46 P3 Eubacterium inrmum Eubacterium nodatum Eubacterium sp. oral clone JS001 Eubacterium sp. oral strain A35MT Eubacterium sulci Filif. alocis Firmicutes oral clone CK057 Firmicutes oral clone MCE3_120 Firmicutes sp. oral strain FTB41 Fusobacterium genomosp. C2 Fusob. nucleatum subsp. animalis Haemophilus aphrophilus Haemophilus paraphrophilus Lactobacillus catenaformis Lactobacillus plantarum Megasphaera sp. oral clone MCE3_141 Mogibacterium timidum Olsenella profusa O. uli Parascardovia denticolens Pept. micros Peptostreptococcus sp. oral clone BS044 Peptostreptococcus stomatis Peptostreptococcus sp. oral clone FG014 Peptostreptococcus sp. oral clone FL008 Porph. gingivalis Porphyromonas sp. oral clone P2PB_52 P1 Prevotella baroniae Prevotella buccae Prevotella denticola Prevotella genomosp. C2 Prevotella genomosp. P4 Prevotella oralis Prevotella oulorum Prevotella sp. E7_34 Prevotella sp. oral clone DA058 Prevotella tannerae Propionibacterium avidum Prop. propionicum Pseud. alactolyticus S_ab rangeD 0.6440.757 0.7080.986 0.835 0.7440.985 0.7540.980 0.909 0.993 0.721 0.6560.951 0.8530.975 0.917 0.958 0.8080.957 0.7760.927 0.961 0.764 0.6090.979 0.936 0.7510.959 0.821 0.6470.901 0.740 0.933 0.959 0.9350.975 0.979 0.957 0.835 0.970 0.7100.953 0.9530.981 0.6600.975 0.984 0.7421.000 0.7410.969 0.743 0.7210.830 0.7210.975 0.998 0.7170.968 0.7640.980 0.7730.961 0.961 0.9100.948 0.8220.978 0.964 0.958 0.6750.989 0.967 0.8770.960 0.978 0.837 0.7340.994 Symptomatic Eubacterium sp. oral clone BU061 Fusob. nucleatum Fusobacterium sp. oral clone CY024 Fusobacterium sp. oral clone CZ006 Granulicatella adiacens Lactobacillus johnsonii Lactobacillus paracasei Lactobacillus salivarius Mogibacterium neglectum Neisseria bacilliformis Neisseria ava Prevotella melaninogenica Prevotella nigrescens Prevotella sp. oral clone FU048 Rothia dentocariosa Sphingomonas sp. P2 Streptococcus gordonii Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus parasanguinis Streptococcus salivarius Streptococcus sanguinis Veillonella sp. oral clone AA050 Veillonella sp. oral clone X042 S_ab range 0.985 0.7140.990 0.786 0.975 0.986 0.770 0.845 0.9291.000 0.983 0.9500.959 0.9720.981 0.958 0.992 0.9280.980 0.994 0.990 0.9320.985 0.6950.988 0.917 0.7440.988 0.7970.991 1.000 0.9790.984 0.811 0.9480.965

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Bacterial and archaeal 16S rDNA in root canals

Table 2. cont.
Asymptomatic* Selenomonas genomosp. C2 Selenomonas sp. oral clone CS002 Selenomonas sp. oral clone FT050 Selenomonas sp. oral clone P2PA_80 P4 Selenomonas sputigena Shuttleworthia satelles Slackia exigua Streptococcus intermedius Streptococcus sp. NJ9704 Synergistes genomosp. C1 Synergistes sp. P4G_18 P1 Veillonella parvula S_ab rangeD 0.6690.942 0.7410.981 0.746 0.7400.927 0.6370.974 0.9280.962 0.956 1.000 0.7470.808 0.744 0.968 0.977 Symptomatic S_ab range

*The PCR template was a pool of equal volumes of DNA prepared from each of the asymptomatic patients (n=14) or symptomatic patients (n=20). Each species listed is the reference strain in the RDP-II database that had the highest similarity to the queried assembly sequences using the Sequence Match program. Entries in bold type indicate that members of these genera were unique to the symptomatic patient pool. DS_ab scores indicate the degree of match of assembly consensus sequences to each named phylotype in the RDP-II database. The top 20 hits were examined. In cases where the assemblies matched more than one phylotype in the database with the same S_ab score, the ATCC type strain matched is shown. When multiple different assemblies matched the same phylotype, the range of S_ab scores is shown.

Deferribacteres (Saito et al., 2006), none was detected in the present study. Similarly, the TM7 phylotype, which has been reported in dental plaque (Brinig et al., 2003) and endodontic samples (de Lillo et al., 2006), was not detected. Bacterial species previously reported as potential pathogens in endodontic infections were detected. D. invisus, Olsenella uli and Synergistes sp., which have been recovered from

symptomatic infections in earlier studies (Rocas & Siqueira, 2005), were detected in our asymptomatic, but not symptomatic patients (Table 2). Granulicatella adiacens, which has been reported in symptomatic patients (Rocas & Siqueira, 2005), was found exclusively in symptomatic patients (Table 2), supporting the possibility that this species has traits associated with clinical symptoms, or

Table 3. Microbial amplicons detected in root-canal samples with universal archaeal and species-specic primers
Micro-organism detected Archaea Prev. intermedia Porph. endodontalis Porph. gingivalis Pept. micros Streptococcus sp. Fusob. nucleatum Tann. forsythensis Trep. denticola Enterococcus sp. Asymptomatic pooled* +D 2d + + + + + + + 2 Symptomatic pooled + + + + + + + + 2|| 2 Asymptomatic patient #10 + 2 2 + + + + + + 2 Symptomatic patient #37 + + + + + + + + 2 2

*Template DNA was a pool of equal volumes either from each of the asymptomatic patients (n=14) or from each of the symptomatic patients (n=20). DNA preparations from root canals of patients #10 and #37, the only patients with detectable archaea, were further examined individually. D+, a detectable PCR product that was conrmed by nucleotide sequence analysis to be 99 % similar to the expected species sequence; 2, no amplicon detected when entire reaction was electrophoresed on 0.7 % agarose gel and stained with ethidium bromide. d > 0.1 ng Prev. intermedia ATCC 25611 chromosomal DNA was necessary for detection of this amplicon under the reaction conditions in this study. > 0.05 ng Porph. endodontalis ATCC 35406 chromosomal DNA was necessary for detection of this amplicon. || > 0.04 ng Trep. denticola ATCC 35405 chromosomal template was necessary for detection of this amplicon. > 0.05 ng Ent. faecalis strain JH2-2 chromosomal DNA was necessary for detection of this amplicon. http://jmm.sgmjournals.org 115

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that it thrives in multispecies communities associated with symptoms. The prevalence of amplicons that were most similar to Fusob. nucleatum in the symptomatic patients was noteworthy (Supplementary Table S1), supporting previous studies with broad-range PCR primers that suggest that levels of this species may be greatly underestimated in endodontic abscesses (Wade et al., 1997). The symptomatic patients also had a greater number of facultative streptococci than the asymptomatic patients, suggesting that the former infections may have a less reduced microbial community, consistent with earlier infections with a higher pO2 (Sundqvist & Figdor, 2003). Overall, the diversity of the microora of asymptomatic patients was more complex, with a larger number of phylotypes represented (Table 2). The approach of pooling DNA from patients in the asymptomatic and symptomatic groups and sequencing a large number of clones was aimed at minimizing the bias of patient variability. Nevertheless, the point at which an infection progresses to cause clinical symptoms may be affected by host variables in addition to the bacterial composition of the infection. Indeed, relative abundance rather than the presence of specic bacteria may also affect symptoms (Drucker & Natsiou, 2000). Quantication was not a goal of the present study, but future analyses using methodologies such as quantitative real-time PCR may clarify this issue. Clinical presentations of patients carrying archaea in root canals Universal primers for archaeal 16S rDNA amplied a ~600 bp product from both pools of DNA from asymptomatic and symptomatic patients. In order to identify which patients had archaea in their root-canal infections, the DNA from each individual patient in the pools was used as template in PCR. Amplicons were recovered from two patients. The rst, designated patient #10, was female and classied as asymptomatic. Her upper-left second premolar had recurrent decay around a large amalgam restoration and a 2 mm apical radiolucency. The patient reported no previous or current symptoms. The tooth tested non-vital with no response to electrical pulp testing or thermal stimuli. There was no sensitivity to palpation or percussion. The associated medical history indicated that the patient was taking birth-control pills, iron supplements and albuterol for asthma. The second patient, designated patient #37, was male and classied as symptomatic. He presented with a 1-month history of spontaneous, intermittent, throbbing pain that was elicited by pressure and mastication, and that was relieved with non-steroidal anti-inammatory agents. Clinical examination showed a large composite restoration with recurrent marginal decay on his upper-left rst premolar. The tooth gave no response to stimulation with cold or electric pulp testing, indicating pulp necrosis, and was sensitive to palpation and percussion. Although there was no intraoral or extraoral swelling, there was a sinus tract exiting to the buccal mucosa that was traced to the 465 mm
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apical radiolucency associated with the infected premolar. The patient had a history of hypertension satisfactorily controlled with beta-blockers. The patient reported no other medical conditions, and had taken no other medications except for ibuprofen for toothache pain during the previous 3-month period. In both patient #10 and patient #37, the overall surrounding periodontal conditions were sound. Adjacent teeth were healthy, responding normally to vitality tests, with no pocketing 3 mm. Detection of M. oralis-like clones Archaeal amplicons from the pools and from each patient were cloned. Up to six randomly selected clones of each of the pooled or individual patient templates were sequenced. Although these amplicons theoretically contained 16S rDNA sequences from all the archaeal phylotypes present in the root canals, all of the cloned sequences agreed with each other, suggesting that the amplied sequence represented either the only, or the predominant, member of this species. In all instances, the sequences of the archaeal amplicons were 100 % identical to that of an uncultured M. oralis-like species rst isolated from the oral cavity (GenBank accession no. AY374553; Lepp et al., 2004). The sequences were also 100 % identical to one of the two Methanobrevibacter species identied in root-canal contents by 16S rDNA amplication (GenBank accession no. DQ251044; Vianna et al., 2006). As with our results comparing species-specic and universal primers, the detection of archaea in root-canal samples emphasizes the limits of detection and sensitivity of PCR methodologies. Stringencies of PCR conditions (e.g. Mg2+ concentrations, temperatures and times) and primer sequences can result in over- or underdetection of target DNA that may, in turn, lead to incorrect conclusions about the presence or absence of a microbial species. Previous studies using different oligonucleotide primers have been unsuccessful at amplifying archaeal DNA (Siqueira et al., 2005). However, with the primers and conditions used in our study, archaeal 16S rDNA amplicons were readily detected. Our PCR conditions differed from those used to identify the same species of archaea in asymptomatic patients with root-canal infections (Vianna et al., 2006). Although the presence of archaea in subgingival plaque has been correlated with the severity of periodontal disease (Lepp et al., 2004), the present study detected archaea in both asymptomatic and symptomatic patients. Patient #37 had a draining sinus tract that may have allowed ingress of oral bacteria. No such obvious route of entry was clinically obvious in the asymptomatic patient #10. Furthermore, both patients had healthy surrounding periodontium. The medical and dental histories may provide future insights into the role that archaea and other microbial species play in the disease process in individual patients. Archaealbacterial communities The archaeon genus detected in these studies, Methanobrevibacter, consists of strict anaerobes that
Journal of Medical Microbiology 56

Bacterial and archaeal 16S rDNA in root canals

metabolize hydrogen to produce methane. Although only one sequence of M. oralis-like amplicon was detected from the two individual patients, the sequence was identical to that of a clone previously identied from dental plaque and from root-canal contents. This low diversity of Methanobrevibacter 16S rDNA sequences isolated from different patients has been previously noted in oral and nonoral strains (Kulik et al., 2001). Root-canal DNA from patients #10 and #37 was examined with species-specic bacterial 16S rDNA primers (Table 3) to gain further insights into polymicrobial communities that contain Methanobrevibacter species. It has been suggested that metabolic competition for hydrogen with sulfate-reducing bacteria (Vianna et al., 2006), such as the Desulfovibrio species detected in the pooled DNA of asymptomatic patients in the present study, or treponemal species (Siqueira et al., 2005), might inhibit the coexistence of these bacteria with methanogenic archaea. Although the two patients with Methanobrevibacter were not examined for Desulfovibrio, they were examined for Trep. denticola. Interestingly, although patient #37 supported this hypothesis and did not carry detectable levels of Trep. denticola, patient #10 carried both the M. oralis-like strain and Trep. denticola. Future quantitative and qualitative studies of metabolic competitors in individual infected root canals colonized with methanogenic archaea may provide more insights into the nutritional interdependence of these microbes.

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ACKNOWLEDGEMENTS
This work was supported by United States Public Health Service (USPHS) grants T32DE7034 and DE014868 and the University at Buffalo Division of Endodontics Funds. We thank Dr J. Cole of the RDP-II for his helpful advice. We also thank T. D. Felmet and A. R. Munsterman for excellent technical assistance, and members of the A. Sharma and J. Zambon laboratories (University at Buffalo Department of Oral Biology) who provided Prevotella, Porphyromonas, Fusobacterium and Tannerella strains used as positive controls in these studies.

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