JOURNAL OF ENDODONTICS Copyright 2003 by The American Association of Endodontists

Printed in U.S.A. VOL. 29, NO. 4, APRIL 2003

Treponema socranskii in Primary Endodontic Infections as Detected by Nested PCR

Jose F. Siqueira, Jr., DDS, MSc, PhD, and Isabela N. Ro c as, DDS, MSc

Spirochetes have been frequently observed in root canal infections, but they were rarely identified. The purpose of this study was to investigate the prevalence of Treponema socranskii in primary endodontic infections using a species-specific nested polymerase chain reaction assay. Samples were collected from 60 teeth having carious lesions, necrotic pulps, and different forms of periradicular diseases. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first polymerase chain reaction products to detect a specific fragment of T. socranskii 16S rDNA. T. socranskii was detected in 11 of 28 asymptomatic cases (39.3%), five of 12 root canals associated with acute apical periodontitis (41.7%), and five of 20 cases diagnosed as acute periradicular abscesses (25%). There was no relationship between the presence of T. socranskii and the occurrence of symptoms. In general, this spirochete was detected in 21 of 60 samples of endodontic infections (35%). Findings suggest that T. socranskii can be involved in the pathogenesis of different forms of periradicular lesions.

Spirochetes were one of the first bacteria observed in humans by Antonie van Leeuwenhoek (16321723). In his pioneer study about root canal infections, Willoughby Dayton Miller (1) observed a high prevalence of spirochetes, which were in great number in some cases, and suggested that they could play an important role in purulent infections. The occurrence of spirochetes in infected root canals was further reported by innumerous studies (25), but species have been only recently identified (6 10). Dahle et al. (6) reported that four spirochete strains isolated from endodontic infections were found to have similarities to Treponema pectinovorum but probably represent two previously unidentified new species. We reported for the first time the occurrence of T. denticola, which has been implicated in the pathogenesis of severe marginal periodontitis, in cases of endodontic infec244

tions by means of polymerase chain reaction (PCR) methodology (8). This species was found in 34.5% of the canals associated with asymptomatic chronic periradicular lesions, 53.3% of the cases of acute apical periodontitis, and in 50% of the teeth with acute periradicular abscess. In general, T. denticola was found in 42.6% of the cases (8). These reports suggested that this spirochete can participate in the pathogenesis of periradicular diseases. Dot blot hybridization with species-specific oligonucleotide probes and amplified bacterial DNA has recently allowed the detection of T. maltophilum and T. socranskii in root canal infections (9). T. socranskii was also recently detected in pus samples from acute periradicular abscesses by checkerboard DNADNA hybridization (10). Many spirochetes are extremely fastidious in their nutrient requirement and have proven difficult or even impossible to culture. All spirochetes isolated from the oral cavity fall within the genus Treponema, and only nine species have been cultured and validly named: T. denticola, T. vincentii, T. pectinovorum, T. socranskii, T. maltophilum, T. medium, T. amylovorum, T. lecithinolyticum, and T. parvum. Many of these species have been implicated as etiological agents of a variety of oral diseases (714). T. socranskii is an anaerobic, helically coiled, motile bacterium. Cells are 0.16- to 0.18-m wide by 6- to 15-m long and have tapered ends with a slight bend or hook at one or both ends (11). This species has one periplasmic flagellum arising from each end, which creates a 1-2-1 axial filament arrangement (11). Fermentable carbohydrates are required as an energy source. Sugars such as glucose, fructose, maltose, mannose, sucrose, ribose, and galactose are fermented. Major metabolic products include ammonia, hydrogen sulfide, acetate, lactate, and succinate. The GC content of the DNA is 50 52 mol%. Three T. socranskii subspecies have been proposed: T. socranskii subspecies socranskii, T. socranskii subspecies buccale, and T. socranskii subspecies paredis (11). T. socranskii has been frequently detected in periodontitis patients and a pathogenetic role has been suggested for this species (1114). Using PCR, Takeuchi et al. (14) recently reported that detection frequencies of T. socranskii in plaque samples from aggressive periodontitis patients (71.1%) and chronic periodontitis patients (89.2%) were much higher than those from healthy subjects (30%). In aggressive and chronic periodontitis patients, this species was detected frequently at sites that showed deep periodontal pockets and severe attachment loss. T. socranskii was frequently detected together with T. denticola or P. gingivalis at the same sites, and coexistence of these microorganisms was

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frequently observed in deep periodontal pockets of aggressive periodontitis patients (14). The species T. socranskii had never been isolated from infected root canals by culture procedures. Nevertheless, recent studies using different molecular technologies have reported the occurrence of T. socranskii in infections of endodontic origin, albeit in a low percentage of the cases examined (9, 10). Therefore, the purpose of this study was to investigate the prevalence of T. socranskii in primary root canal infections associated with different forms of periradicular diseases by using a species-specific nested PCR method.

at 2500 g. Pellets were then resuspended in 100 l of bidistilled water, boiled for 10 min, and chilled on ice. After centrifugation to remove cell debris for 10 s at 9000 g at 4C, the supernatant was collected and used as the template for PCR amplification. Reference DNA from T. socranskii (S1 strain, Forsyth Dental Center) was also extracted to serve as a positive control for the primers used.

Oligonucleotide Primers The pair of universal 16S rDNA primers were 5'-AGA GTT TGA TCC TGG CTC AG-3' and 5'-ACG GCT ACC TTG TTA CGA CTT-3'. These primers anneal at conserved regions near the 5' and 3' ends of 16S rDNA, generating a practically full-length 16S product corresponding to base position 8-1513 of the 16S rDNA sequence of Escherichia coli. The PCR oligonucleotide species-specific primers, 16S rDNA directed, for T. socranskii were 5'-GAT CAC TGT ATA CGG AAG GTA GAC A-3' and 5'-TAC ACT TAT TCC TCG GAC AG-3'. The amplicon length is 288 bp (13). Sequences consisted of specific forward and reverse primers (12, 13). Primers were synthesized by Oligos Etc., Inc. (Wilsonville, OR).

MATERIALS AND METHODS Specimen Sampling Specimens were selected from patients that had been referred for root canal treatment to the department of Endodontics, Esta cio de Sa University, Rio de Janeiro, RJ, Brazil. Cases of acute periradicular abscesses were selected from adult patients that had been referred for emergency treatment to three hospitals in Rio de Janeiro. Only teeth from adult patients (ages ranging from 18 to 60 yr), all of them having carious lesions and necrotic pulps were included. On the whole, 60 samples of endodontic infections were obtained. According to the forms of periradicular disease teeth were divided as follows: 28 cases of chronic asymptomatic periradicular lesions; 12 cases diagnosed as acute apical periodontitis (7 cases showing both spontaneous symptoms and tenderness to percussion and 5 cases showing only tenderness to percussion); and 20 cases of acute periradicular abscesses. Selected teeth showed an absence of periodontal pockets greater than 4 mm. Samples were collected using strict asepsis. The tooth was cleansed with pumice and isolated with a rubber dam. The tooth and the surrounding field were then cleansed with 3% hydrogen peroxide and decontaminated with a 2.5% sodium hypochlorite solution. Complete access preparations were made using sterile burs without water spray. The operative field, including the pulp chamber, was then swabbed with 2.5% sodium hypochlorite. This solution was inactivated with sterile 5% sodium thiosulfate. If the root canal was dry, a small amount of sterile saline solution was introduced into the canal. Samples were initially collected by means of a #15 K-type file (Dentsply/Maillefer, Ballaigues, Switzerland) with the handle cut off. The file was introduced to a level approximately 1 mm short of the tooth apex, based on diagnostic radiographs, and a discrete filing motion was applied. Afterwards, two sequential paper points were placed to the same level and used to soak up the fluid in the canal. Each paper point was retained in position for 1 min. The cut file and the two paper points were then transferred to cryotubes containing 1 ml of 5% dimethyl sulfoxide in trypticase-soy broth (Difco, Detroit, MI) (TSB-DMSO). Samples were immediately frozen at 20C. After disinfection of the oral mucosa with 2% chlorhexidine, pus from the abscessed teeth was collected by aspiration using a sterile syringe, transferred to TSB-DMSO, and frozen.

PCR Amplification Five microliters of the supernatant from clinical samples were used as target in the PCR reaction using universal 16S rDNA primers. Two microliters of the universal reaction were then used as a template for the nested specific reaction. PCR amplification was performed in 25 l of reaction mixture containing 0.2 M concentration of forward and reverse primers, 2.5 l of 10 PCR buffer, 1.25 units of Taq DNA polymerase (Gibco BRL, Gaithersburg, MD), and 25 M concentrations of each deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP) (Gibco BRL). Earlier experiments found optimal MgCl2 concentration in the mixture to be 1.5 mM. Preparations were amplified in a DNA thermocycler (Primus 25/96; MWG-Biotech, Ebersberg, Germany). The PCR temperature profile included an initial denaturation step at 97C for 1 min, followed by 26 cycles of a denaturation step at 97C for 45 s, a primer annealing step at 55C (for the universal 16S rDNA primer) or 53C (for T. socranskii) for 45 s, an extension step at 72C for 1 min and a final step of 72C for 4 min. Eight microliters of each PCR product were electrophoresed on a 1.5% agarose gel at 4 V/cm in Tris-borate-EDTA buffer. The gel was stained for 15 min with 0.5 g/ml of ethidium bromide and photographed under ultraviolet light. Positive reactions were determined by the presence of bands of the appropriate sizes. A 100-bp DNA ladder digest (Gibco BRL) was used as size marker.

Data Analysis DNA Extraction The samples in TSB-DMSO were thawed to 37C for 10 min and vortexed for 30 s. Microbial suspension was washed three times with 100 l of bidistilled water by centrifugation for 2 min Prevalence of T. socranskii was recorded as the percentage of the cases examined. The Chi-square test was used to analyze the association between T. socranskii and symptomatic cases (either acute apical periodontitis or acute periradicular abscesses). Significance for the Chi-square test was established at 5% (p 0.05).

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RESULTS In general, species-specific nested PCR for T. socranskii detected this bacterial species in 21 of 60 samples of endodontic infections (35%). T. socranskii was detected in 11 of 28 asymptomatic cases (39.3%), and in five of 20 pus samples aspired from cases diagnosed as acute periradicular abscesses (25%). Of the cases diagnosed as acute apical periodontitis, this species was found in four of seven cases showing both spontaneous symptoms and tenderness to percussion (57.1%) and in one of five cases showing only tenderness to percussion (20%). Thus, five of the cases diagnosed as acute apical periodontitis yielded positive results for T. socranskii (41.7%). There was no relationship between T. socranskii and the occurrence of symptoms (p 0.05). Reference DNA and clinical samples that were positive for T. socranskii showed only one band of the predicted size (285 bp). All 60 samples contained bacteria as demonstrated by amplification using the universal 16S-rDNA primers. Only one band of the predicted size (1505 bp) was present for each root canal sample. Such results suggested that components of the necrotic pulp tissue or the purulent exudate did not significantly inhibit the DNA amplification reaction and indicated that bacteria were present in all cases.

DISCUSSION The species T. socranskii has been only recently detected in infected root canals through molecular methods. However, prevalence values were very low. Using PCR amplification with eubacteria universal primers and subsequent dot-blot hybridization with species-specific oligonucleotide probes, Jung et al. (9) detected T. socranskii in only 2.7% of the root canal samples examined. A similarly low prevalence value for T. socranskii was observed by us using whole genomic probes in the checkerboard DNADNA hybridization method to investigate the microbiota associated with acute periradicular abscesses (10). Curiously, the prevalence data regarding T. socranskii reported in the present study are somewhat divergent from those previous molecular studies. Differences between results may probably have been because of different reasons. For instance, the nested PCR used in this study to detect T. socranskii is more sensitive than the dot blot hybridization used by Jung et al. (9). Another factor might help to explain the discrepancies observed between the studies. In addition, to detect T. socranskii in a few cases, Jung et al. (9) did not find T. denticola in any root canal sample examined. Conversely, we previously detected T. denticola in a relatively high prevalence in our samples (8). The possibility exists that differences may have also been due to a geographical influence on the composition of oral and consequently root canal microbiota. With regard to our own results with the checkerboard DNADNA hybridization when examining abscessed cases (10), the apparent reason for differences in prevalence values may have been because of the lower detection rate of the checkerboard method compared with the nested PCR assay used in the present study. Whereas checkerboard DNADNA hybridization detected T. socranskii in 3.7% of the abscessed cases (10), the nested PCR used in this study detected this spirochete in 25% of the cases diagnosed as acute periradicular abscesses. Evidence suggests that T. socranskii is a pathogenic spirochete. After subcutaneous injection in mice, Kesavalu et al. (15) observed that T. socranskii induced well-demarcated, dose-dependent, and

raised subcutaneous abscesses, which were similar in time of onset, lesion progression, and duration of healing compared with other treponemes, including T. denticola. Even though T. socranskii has been demonstrated to be pathogenic, very little is known about its virulence factors. T. socranskii has the ability to locomote in highly viscous environments that render other prokaryotes immobile (16). The locomotory ability can enable the bacterium to invade tissues, escape from phagocytes, and may also position the treponeme in the microbial consortium in such a way that it can be strategically located near an active source of nutrient. Alkaline and acid phosphatases were reported to be the major enzymatic activities present in T. socranskii and both enzymes may play a role in pathogenicity (15). In addition, this species produce an enzyme, located at the cell surface, that hydrolyzes hyaluronic acid and chondroitin sulphate (17). This enzyme may play a role in the invasiveness of the bacterium and in the induction of tissue destruction by degradation of connective tissue ground substance. Outer envelope proteins of T. socranskii possess the ability to bind to laminin, type I, III, IV, or V collagen (18). Such a property may be important to the adhesion of the bacterium to host tissues. Other probable virulence factors of T. socranskii include major surface protein (msp)-like proteins, which may have porin activity (19), heat shock protein (20), peptidoglycan, and metabolites such as ammonia, hydrogen sulfide, acetate, lactate, and succinate. Because of the relatively high prevalence of T. socranskii in infections of endodontic origin, as detected in this study by nested PCR, its involvement with severe periodontal tissue destruction and the apparent pathogenicity of the microorganism, our findings suggest that this spirochete may participate in the pathogenesis of different forms of periradicular diseases and should be included in the set of suspected endodontic pathogens.
Dr. Siqueira is chairman and professor, and Dr. Ro c as is assistant professor, Department of Endodontics, Faculty of Dentistry, Esta cio de Sa University, Rio de Janeiro. Dr. Siqueira is also associate researcher, Laboratory of Oral Microbiology, Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Address requests for reprints to Jose F. Siqueira Jr., R. Herotides de Oliveira 61/601, Icara , Nitero i, RJ, Brazil 24230-230.

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