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 »  Home  »  Endodontic Articles 4  »  Direct amplification of rRNA gene sequences for identification of selected oral pathogens in root canal infections
Direct amplification of rRNA gene sequences for identification of selected oral pathogens in root canal infections
Introduction - Materials and methods.



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.

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.

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.