J. F. Siqueira Jr, I. N. Rocas, S. R. Moraes & K. R. N. Santos
Institute of Microbiology, Federal University of Rio de Janeiro, RJ, Brazil.
Department of Endodontics, Estacio de Sa University, RJ, Brazil.
Department of Microbiology, Immunology and Parasitology, School of Dentistry, Veiga de Almeida University, Rio de Janeiro, RJ, Brazil.

Introduction.
Several putative oral pathogens have been isolated from endodontic infections (Siqueira 1997). However, some pathogens, such as Bacteroides forsythus, Actinobacillus actinomycetemcomitans and Treponema denticola , have never or only rarely been found in root canal infections using cultural techniques. Recent studies using sensitive molecular diagnostic methods have allowed detection of microorganisms that are difficult or even impossible to culture in infections elsewhere in the human body, including within the root canal system (Relman et al . 1992, Greisen et al . 1994, Wang et al . 1996, Ashimoto et al . 1996, Conrads et al . 1997, Wade et al . 1997, Gonçalves & Mouton 1999, Jung et al . 2000, Siqueira et al . 2000a and b). In addition, some cultivable bacterial species have been detected in greater prevalence by molecular methods when compared with culture as not all strains within a species can be cultivable.
Conrads et al . (1997) and Gonçalves & Mouton (1999) have detected B. forsythus in infected root canals using the polymerase chain reaction method (PCR). Siqueira et al . (2000b), using a 16S rDNA-based PCR method, detected for the first time the occurrence of T. denticola in half of the infected teeth examined, regardless of whether symptoms were present. Xia et al . (2000) also reported for the first time the presence of Prevotella tannerae in infected root canals by means of PCR (60% of the cases). Siqueira et al . (2000a) examined the presence and levels of 42 bacterial species in 28 root canal samples using the checkerboard DNA-DNA hybridization method and found B. forsythus in 39.3% of cases, T. denticola in 17.9%, and A. actinomycetemcomitans in 3.6%. Sunde et al . (2000), also using the checkerboard DNA-DNA hybridization, reported the occurrence of A. actinomycetemcomitans and B. forsythus in more than 60% of the asymptomatic periradicular lesions examined.
The RNA components of the ribosome (rRNAs) are amongst the most evolutionary conserved macromolecules in all living systems (Woese 1987). The rRNA does not appear to undergo lateral gene transfer between species. Data from rRNA sequences can be used for accurate and rapid identification of known bacterial species, using techniques that do not require microbial cultivation (Tanner et al . 1994, Madigan et al . 2000). For diagnostic purposes, PCR has been used to amplify the DNA encoding species-specific signatures of the rRNA gene (Greisen et al . 1994, Ashimoto et al . 1996, Conrads et al . 1997, Siqueira et al . 2000b, Xia et al . 2000). This method has been reported (Mullis et al . 1994) to have high sensitivity (detection limit regarding the number of microbial cells in the sample) and specificity (detection of only the target microorganism).
The purpose of this study was to investigate the prevalence of selected oral pathogens in root canal samples, as well as the relationship with symptoms of endodontic infections, using a highly sensitive PCR method.

Materials and methods.

Specimen sampling.
Specimens were selected from patients that had been referred for root canal treatment to the department of Endodontics, Estácio de Sá University, Rio de Janeiro, Brazil. The characteristics of the cases examined are summarized in Table 1. Only single-rooted teeth from adult patients, all of them having carious lesions and necrotic pulps, were included. Selected teeth showed absence of periodontal pockets greater than 4 mm; the patient’s ages ranged from 18 to 60 years.
Samples were collected using strict asepsis as described previously (Siqueira et al . 2000a,b). Briefly, teeth were cleansed with pumice, isolated with rubber dam, and the surrounding field cleansed with 3% hydrogen peroxide and then decontaminated with a 2.5% sodium hypochlorite solution. 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 size 15 K-type file (Dentsply/Maillefer, Ballaigues, Switzerland) with the handle removed. 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. Two sequential paper points were then 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, USA) (TSB-DMSO). Samples were immediately frozen at –20 C.
After disinfection of the oral mucosa with 2% chlorhexidine gluconate, pus from the abscessed teeth was collected by aspiration using a sterile syringe, transferred to TSB-DMSO and frozen.

Table 1. Description of the clinical cases examined for the presence of each target microorganism.


a. Not including the abscessed teeth.
b. Number of teeth examined.
c. Number of teeth positive for the target microorganism.
d. Including S. anginosus, S. intermedius and S. constellatus species.

DNA extraction.
Samples in TSB-DMSO were thawed to 37 C for 10 min and vortexed for 30 s. Microbial suspensions were washed three times with 100 L of bidistilled water by centrifugation for 2 min 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 4 C, the supernatant was collected for testing. Reference DNA from B. forsythus, A. actinomycetemcomitans, Fusobacterium nucleatum, Actinomyces israelii, Actinomyces naeslundii genospecies 1, A. naeslundii genospecies 2 (formerly Actinomyces viscosus ) , Actinomyces odontolyticus, Streptococcus anginosus, Streptococcus intermedius, Candida albicans, Candida glabrata, Candida guilliermondii, and Candida parapsilosis was also extracted to serve as a control.

PCR identification.
Species-specific oligonucleotide primers were used to detect the target microbial species. A pair of ubiquitous bacterial primers that match almost all bacterial 16S rRNA genes at the same position but not 18S rRNA gene from eukaryotic cells was used as a positive control for the PCR reaction. It served to indicate the presence of bacteria in clinical samples. Ubiquitous sequences, and specific primers for B. forsythus and A. actinomycetemcomitans were as described by Ashimoto et al . (1996). Primer sequences for F. nucleatum, A. israelii and S. anginosus group (including S. anginosus, S. constellatus and S. intermedius ) were as proposed by Conrads et al . (1997), using a universal forward primer in combination with a highly taxon-specific reversed one. The fungus-specific, universal primer pair ITS3 and ITS4 was also used as described by Fujita et al . (1995). Table 2 lists the primers (Oligos Etc. Inc., Wilsonville, OR, USA) and the predicted amplicon lengths for the target microbial species.
Aliquots of 5 L of the supernatant from clinical samples or 1 L of the reference strain nucleic acid (200 ng L –1 ) were amplified. PCR was performed in a 50- L of reaction mixture containing 1 L of each primer (40 pmol), 5 L of 10X PCR buffer, 1.25 unit Taq DNA polymerase (Gibco BRL, Gaithersburg, MD, USA) and 0.2 mmol L –1 of each deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) (Gibco BRL). Earlier experiments found optimal MgCl 2 concentration in the mixture to be 2.0 mmol L –1 .
The temperature profile for B. forsythus and ubiquitous primer included an initial denaturation step at 95 C for 2 min, followed by 36 cycles of a denaturation step at 95 C for 30 s, a primer annealing step at 60 C for 1 min, an extension step at 72 C for 1 min, and a final extension at 72 C for 2 min following the last cycle. For A. actinomycetemcomitans , the temperature profile included an initial denaturation step at 95 C for 2 min, followed by 36 cycles of a denaturation step at 94 C for 30 s, a primer annealing step at 55 C for 1 min, an extension step at 72 C for 2 min and a final extension step of 72 C for 10 min. The PCR temperature profile for F. nucleatum, A. israelii and S. anginosus included 30 cycles of a denaturation step at 94 C for 1 min, a primer annealing step at 55 C (for A. israelii and S. anginosus ) or 60 C (for F. nucleatum ) for 1 min, and an extension step at 72 C for 2.5 min. For fungus-specific, universal primer pair, the temperature profile included an initial denaturation step at 94 C for 5 min, followed by 30 cycles of a denaturation step at 95 C for 30 s, a primer annealing step at 58 C for 30 s, an extension step at 72 C for 1 min, and a final extension at 72 C for 5 min following the last cycle.

Table 2. PCR primers used for detection of selected oral pathogens in root canal samples.


a. Base positions of ubiquitous primers are from Escherichia coli.

Preparations were overlaid with two droplets of mineral oil and amplified in a DNA thermocycler (PTC-100, MJ Research, Inc., Watertown, MA, USA). Amplicons were analysed by 1.5% agarose gel electrophoresis performed at 4 V cm –1 in Tris-borate EDTA buffer. The gel was stained with 0.5 g mL –1 ethidium bromide and photographed under ultraviolet light. As molecular weight markers, it was used both 100 bp and 1 kb DNA ladder digest (Gibco BRL).

Data analysis.
Prevalence of the target species was recorded as the percentage of the cases examined. The chi-squared test and odds ratio calculation were used to analyse the association between the target species and symptomatic cases (including teeth that were tender to percussion and abscessed cases). Significance for chi-squared test was established at 5% ( P < 0.05). Odds ratios above 2.0 were considered to be indicative of positive associations and odds ratios below 0.5 of negative associations. Only the microorganisms found in symptomatic cases were submitted to such evaluation.

All samples examined showed the amplicon of the ubiquitous bacterial primers. Only one band of the predicted size was present for each clinical sample. Such results suggested that components of the infected teeth did not significantly inhibit the DNA amplification reaction and indicate that bacteria were present in all cases.
The S. anginosus group was detected in 16.7%, F. nucleatum in 14.3%, and B. forsythus in 7.1% of the abscess samples. No pus sample yielded A. israelii, A. actinomycetemcomitans or fungal species. In general, B. forsythus was found in 20% of the cases (16 of 80), S. anginosus in 12% (6 of 50), F. nucleatum in 10% (6 of 60), A. israelii in 5% (2 of 40), and fungi in 2% (1 of 50). No sample was positive for A. actinomycetemcomitans.
The primers used yielded a single amplicon only with strains of the target species. The primer pair specific for the S. anginosus group amplified reference DNA from both S. anginosus and S. intermedius. No cross-reactivity with non-target species was observed for the test primers. Reference DNA and clinical samples that were positive for the microorganisms tested showed only one band of the predicted size (Fig. 1). Reference DNA from C. albicans , C. glabrata , C. parapsilosis , and C. guilliermondii yielded amplicons of 330 bp, 410 bp, 310 bp, and 360 bp, respectively. The clinical sample positive for fungi showed an amplicon of approximately 380 bp.

Figure 1. Agarose gel electrophoresis of representative PCR results.


Lane 1, 100 bp ladder;
Lane 2, root canal sample positive for Bacteroides forsythus;
Lane 3, negative control (without DNA);
Lanes 4 and 5, root canal samples positive for Actinomyces israelii;
Lanes 6 and 7, root canal samples positive for Streptococcus anginosus group;
Lane 8, reference DNA from Actinobacillus actinomycetemcomitans;
Lane 9, reference DNA from Candida albicans;
Lane 10, reference DNA from Candida glabrata;
Lane 11, reference DNA from Candida parapsilosis.

The species B. forsythus, F. nucleatum and S. anginosus , which were detected in symptomatic cases, showed no correlation with symptoms as evaluated by the chisquared test ( P > 0.05) and odds ratio calculation (1.37, 0.58, and 0.6, respectively).

Most oral microorganisms may be considered opportunistic pathogens that cause infections after a change in local environmental conditions. After necrosis, the pulp tissue loses its defence ability and may theoretically be colonized by any oral microorganism. However, the environmental conditions within the root canal system will select the species that will survive and establish an infectious disease. The mere presence of a microorganism in infected root canals does not necessarily assure that it is involved in the pathogenesis of periradicular diseases (Siqueira et al. 1998). Nevertheless, since the species detected in the present study have been associated with infections in diverse oral sites, it is reasonable to suppose that they may participate in the pathogenesis of periradicular diseases.
Four groups of oral streptococci are recognized based on 16S rRNA gene sequence comparisons (Whiley & Beighton 1998). The S. anginosus group includes the species S. anginosus, S. constellatus, and S. intermedius. The primer pair used in this study does not differentiate between these three species (Conrads et al. 1997). The S. anginosus group has been reported to be the most common streptococcal species found in infected root canals (Sundqvist 1994). In the present study, only B. forsythus, F. nucleatum and members of the S. anginosus group were detected in symptomatic cases, including the abscessed teeth. Nonetheless, no correlation could be established since they were also found in a significant number of asymptomatic cases. It is entirely possible that these species were present in higher numbers in symptomatic cases. However, due to the qualitative nature of the PCR assay employed in the present study, any statement in this regard may be considered speculative.
The species A. actinomycetemcomitans has been implicated in the etiology of some forms of periodontal diseases (Meyer & Fives-Taylor 1997). Although this species has been detected by DNA-DNA hybridization in extraradicular infections (Sunde et al. 2000) and occasionally in cases of intraradicular infections (Siqueira et al. 2000a), it was not found in any root canal sample examined by PCR in the present study. This finding corroborates most of the studies regarding the root canal microbiota and suggests that A. actinomycetemcomitans is not a relevant endodontic pathogen. It is possible that some environmental differences between the root canal system and the periodontal pocket may influence colonization by this microorganism. On the other hand, the molecular method used in this study confirmed previous findings that another important periodontal pathogen, B. forsythus, may be also involved in the pathogenesis of periradicular infections (Conrads et al. 1997, Gonçalves & Mouton 1999, Siqueira et al. 2000a).
The fungus-specific primer pair used in the present investigation (ITS3 and ITS4) amplifies a large portion of the 5.8S ribosomal DNA region, the adjacent internal transcribed spacer (ITS) region, and a small portion of the 28S rDNA region, generating PCR products of different lengths depending on the fungal species (Fujita et al. 1995, Shin et al. 1997). It is able to amplify DNA from most, if not all, fungi. PCR assay using this primer pair has been reported to be rapid, reliable and effective in identifying more than one species of yeast in mixed cultures with no cross-reactivity with bacteria or human cells (Fujita et al. 1995, Shin et al. 1997). In the present study, only one clinical case yielded fungi. Precise identification after amplification with ITS3 and ITS4 primers requires use of additional methods, such as DNA sequencing, which was not performed in the present study. The amplicon length of the DNA amplified was greater than that found and reported for most Candida species (Fujita et al. 1995, Shin et al. 1997). Our findings confirmed that fungi are rarely found in primary root canal infections (Möller 1966, Sundqvist 1976). Fungi are considered opportunistic pathogens that cause secondary infections after changes in the ecosystem that permit their overgrowth. Because of this, fungi have been found associated with persistent and/or secondary root canal infections, including teeth refractory to endodontic therapy (Nair et al. 1990, Waltimo et al. 1997).
The rRNA gene directed-PCR assay as used in this study has been reported to possess detection sensitivity of 25–100 colony-forming units (Fujita et al. 1995, Ashimoto et al. 1996, Conrads et al. 1997). Theoretically, an assay with such sensitivity can still fail to detect a few positive cases. This is particularly true when one considers that such a small aliquot of the root canal sample was used to detect each target microorganism. Nonetheless, all the cases having a meaningful number of microorganisms will register a positive result. Moreover, the sensitivity level is still considerably greater when compared with that of culture, which is reported to range from 103 to 104 CFUs (Zambon & Haraszthy 1995).
A question arises when one is using a highly sensitive method such as PCR to assess clinical samples. Since the PCR protocols commonly used for direct microbial detection in a sample are qualitative, what is the number of cells present in a sample? It is well known that microbial virulence associated with a critical number of cells is required for the induction of an infectious disease. It is possible that some microbial species detected in this study were in such low numbers that they were insufficient to cause disease. This could explain why certain cultivable species are found in a higher number of cases when PCR is used, although the fact that some strains within a species are uncultivable should also be taken into account. Quantitative PCR assays are warranted to evaluate the levels of specific microorganisms in endodontic infections and thereby elucidate this question.
Culturing requires at least an 8-h incubation of the sample in culture medium and then biochemical and other tests to identify the microorganism. The time required for identification can be even longer for slowly growing microorganisms or in samples with low microbial counts. The former is especially true for identification of fastidious anaerobic bacteria, which are commonly found in endodontic infections. When traditional culture methods are used, the laboratory can require 7–14 days to identify anaerobic bacteria, whilst some molecular methods (such as PCR) can provide information in only a few hours. As it now stands, the method used in this study can be performed in approximately 5 h and can thus be very useful for rapid clinical diagnosis. This is especially important in cases of life-threatening infections.
The rRNA gene based-PCR is superior to culture in clinical situations such as infections caused by microorganisms with unusual growth requirements that are difficult or even impossible to culture, and specimens taken during antimicrobial treatment (Rantakokko-Jalava et al. 2000, Jordan & Durso 2000). In addition, it has the potential for excellent sensitivity and a shorter turnaround time than those of culture-based protocols. The PCR assay used in this study does not require prior sample preparation and time-consuming laboratory procedures, and is sensitive, specific, and reproducible (Fujita et al. 1995, Ashimoto et al. 1996, Conrads et al. 1997). Therefore, direct molecular approach appears to be a valuable tool for the rapid and reliable diagnosis of infectious diseases, as well as for research purposes.
Although the impact of molecular genetic methods on the knowledge of the root canal microbiota has not been dramatic, such approaches have allowed the recognition of new putative endodontic pathogens. The well-directed use of these methods will provide additional valuable information regarding the identification and understanding of the causative factors associated with endodontic diseases, helping to elaborate more successful treatment strategies.

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