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Campylobacter Gracilis and Campylobacter Rectus in Primary Endodontic Infections

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Campylobacter Gracilis and Campylobacter Rectus in Primary Endodontic Infections Campylobacter gracilis and Campylobacter rectus in primary endodontic infections J. F. Siqueira Jr1, 2 &I.N.Ro“ c¸as1, 2 1Department of Endodontics, Faculty of Dentistry, Esta¤ cio de Sa¤ University, and 2Laboratory of Oral Microbiology, Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Abstract 20) of the root canals associated with chronic asymptomatic periradicular lesions. Campylobacter Siqueira JF Jr, Ro“ c¸as IN. Campylobacter gracilis and Campylo- gracilis was found in 16.7% (2 of 12) of the cases bacter rectus in primary endodontic infections. International Endodon- diagnosed as acute apical periodontitis, whilst C. rectus tic Journal, 36,174^180,2003. was found in 33.3% (two of six cases). In the abscess- Aim A species-speci¢c nested polymerase chain reac- ed cases, C. gracilis and C. rectus were detected in tion (PCR) assay was used to investigate the occurrence 23.5 (4 of 17) and 11.8% (2 of 17) of the cases, res- of Campylobacter gracilis and C. rectus in primary root pectively. No association of these species with clinical canal infections. symptoms was observed (P > 0.01). In general, spe- Methodology Samples were collected from 57 sin- cies-speci¢c nPCR allowed the detection of C. gracilis gle-rooted teeth with carious lesions, necrotic pulps in 21.1% (12 of 57) and C. rectus in 23.3% (10 of 43) and radiographic evidence of periradicular disease. of the samples taken from primary endodontic infec- Twenty-eight cases were diagnosed as chronic asymp- tions. tomatic periradicular lesions, 12 cases as acute apical Conclusions Findings con¢rmed the assertion that periodontitis, and 17 cases as acute periradicular abs- both C. gracilis and C. rectus participate in infections cess. DNA was extracted from the samples and initially of endodontic origin and suggest a pathogenetic role ampli¢ed using universal 16S rDNA primers. A second with regard to periradicular diseases. round of ampli¢cation using the ¢rst PCR products was performed to speci¢cally detect C. gracilis or Keywords: Campylobacter gracilis, Campylobacter rec- C. rectus in the samples. tus, endodontic infections, nested PCR. Results Campylobacter gracilis and C. rectus were, respectively, detected in 21.4 (6 of 28) and 30% (6 of Received10June 2002; accepted16October 2002 Porphyromonas, Prevotella, Fusobacterium, Peptostrepto- Introduction coccus, Eubacterium and Campylobacter. Primary endodontic infection is caused by microorgan- Campylobacter gracilis is a nonmotile, nonspore form- isms colonizing the necrotic pulp tissue. In general, pri- ing, anaerobic Gram-negative rod with a formate- and mary infections are mixed and predominated by fumarate-requiring metabolism. Cells are small and anaerobic bacteria. Although more than 200 microbial straight,0.4 mmwide and 4^6 mm long, with round ends. species have been isolated/detected from infections of The G þ C content of the DNA is 43^47 mol% (Tanner endodontic origin, a more restricted group composed et al.1981,1992).This species was proposed and describ- of15^30 species has been implicated as candidate endo- ed by Tanner et al. (1981), a s Bacteroides gracilis. dontic pathogens (Siqueira 2002). Such putative patho- Vandamme et al. (1995) analysed the cellular fatty acids, gens belong to the genera Treponema, Bacteroides, respiratory quinones and proteins of B. gracilis,and compared the features with the corresponding chemo- taxonomic features of its closest relatives, the Campylo- Correspondence: Jose¤ F. Siqueira Jr, R. Herotides de Oliveira 61/601, bacters. Their results and previously published data for Icara|¤ , Nitero¤ i, RJ, 24230-230 Brazil (e-mail: [email protected]). genotypic and phenotypic characteristics were used to 174 International Endodontic Journal, 36, 174^180, 2003 ß 2003 Blackwell Publishing Ltd Siqueira & Ro“c¸as Campylobacter in endodontic infections reconsider the classi¢cation of this species, transferring associated with di¡erent forms of periradicular dis- it to the genus Campylobacter. eases usinga sensitive identi¢cation method^thenested Campylobacter rectus is a small, nonspore forming, PCR. asaccharolytic, microaerophilic Gram-negative rod cap- able of motility via a single polar £agellum. Cells are fre- Materials and methods quently straight, 0.5 mm wide and 4 mm long, but may Patients and specimen collection occasionally appear curved or helical. Regarding its metabolism, formate or hydrogen from several oral Adult patients ranging in age from18^60 years who had microorganisms serves as electrondonors, whilstnitrate been referred for either root canal treatment or emer- or fumarate from arpartate-producing microorganisms gency treatment to the department of Endodontics, serves as electron acceptors. The G þ C content of the Esta¤ cio de Sa¤ University, Rio de Janeiro, RJ, Brazil, were DNA is 42^46 mol% (Tanner et al. 1981, 1992). The spe- selected for this study. Fifty-seven single-rooted teeth cies was described by Tanner et al. (1981), a s Wolinella with carious lesions, necrotic pulps and radiographic recta, and further transferredtothegenus Campylobacter evidence of periradicular disease were included. (Vandamme et al.1991). According to clinical diagnosis, cases were classi¢ed as Campylobacter gracilis and C. rectus have been recov- follows: 28 cases of chronic asymptomatic periradicular ered from di¡erent forms of periodontal diseases and lesions, 12 cases of acute apical periodontitis, and 17 claimed to have a potential pathogenic role in such dis- cases of acute periradicular abscess. Diagnoses were eases (Rams et al.1993,Tanneret al. 1997, 1998, Kamma based onTorabineJad & Walton (1994). None of the teeth et al. 2000, Macuch & Tanner 2000). Studies have also had signi¢cant periodontal disease (pockets > 4mm isolated/detected these Campylobacter species from deep). endodontic infections in variable prevalence values Samples were obtained under strict asepsis.The tooth (Ranta et al. 1988, Sundqvist et al. 1989,1998, Sundqvist was cleansed with pumice and isolated from the oral 1992, Gome s et al.1996, Le Go¡ et al.1997, Siqueira et al. cavity with a rubber dam. Afterwards, the tooth and 2000b, 2001b). the surrounding ¢eld were cleansed with 3% hydrogen Because these species are not always easily identi¢ed peroxideand decontaminatedwitha 2.5% sodiumhypo- by conventional phenotype-based identi¢cation proce- chlorite (NaOCl) solution. Complete access preparations dures, it is possible that their prevalence has been under- were made using sterile burs without water spray. The estimated in primary endodontic infections. Molecular operative ¢eld, including the pulp chamber, was then technologies, particularly the polymerase chain reac- swabbed with 2.5% NaOCl as this solution has been tion (PCR)method, overcome manyof theproblems asso- demonstrated recently to be signi¢cantly more e¡ective ciated with traditional phenotype-based identi¢cation in decontaminating the operative ¢eld than 10% iodine methods. PCR has beenwidely used to identify microbial prior to sampling for PCR bacterial detection (Ng et al. species that are di⁄cult or impossible to cultivate, and 2002). NaOCl solution was then inactivated by sterile strains di⁄cult to identify due to a phenotypically con- 5% sodium thiosulphate. If upon opening the root canal vergent or divergent behaviour (Relman 1993, 1999). was dry, a small amount of sterile saline solution was The PCR methodology has the highest detection rate introduced into the canal. Samples were initially col- between the microbiological identi¢cation methods, lected by means of a size15 K-type ¢le (Dentsply/Maille- and under optimized conditions also shows high speci¢- fer, Ballaigues, Switzerland) with the handle cut o¡. city (McPherson & Moller 2000).The nested PCR (nPCR) The ¢le was introduced to a level approximately 1 mm technique is a modi¢cation of the PCR technology that short of the tooth apex, based on diagnostic radiographs, involves a ¢rst ampli¢cation reaction of a DNA sequence and a discrete ¢ling motion was applied. Two sequential with one set of primers followed by reampli¢cation us- paper points were placed to the same level and used to ing a second set of primers complementary to smaller soak up the £uid in the canal. Each paper point was speci¢c sequences within the ¢rst PCR product. nPCR retained in position for1 min. The ¢le and the two paper can show increased sensitivity and even improved points were then transferred to cryotubes containing speci¢city whencomparedwith single PCR (Die¡enbach 1 mL of 5% dimethyl sulphoxide in trypticase-soy broth & Dveksler 1995, McPherson & Moller 2000, Jordan (TSB-DMSO; Difco, Detroit, MI, USA). Samples were et al. 2001). immediately frozen at À20 8C. This study aimed to investigate the prevalence of Sampling of abscessed cases was performed after dis- C. gracilisand C. rectus inprimary endodontic infections infection of the oral mucosa with 2% chlorhexidine. ß 2003 Blackwell Publishing Ltd International Endodontic Journal, 36, 174^180, 2003 17 5 Campylobacter in endodontic infections Siqueira & Ro“c¸as Purulent exudate was aspirated with a sterile syringe, GGA ATT TAA GAG AGC TT-30 (forward primer, located transferred toTSB-DMSO and frozen. at base position 65^84 of the C. gracilis16S rDNA, Gen- Bank accession no. LO4320) and 50-CTT TCC CGA TTT ATC TTA TG-30 (reverse primer, located at base position DNA extraction 192^211 o f t he C. gracilis16S rDNA, GenBank accession Samples in TSB-DMSO were thawed to 37 8Cfor10min no. LO4320), producing a PCR amplicon of147 bp. and vortexed for 30 s. Microbial suspension was washed Speci¢c primers for C. rectus were 50-TTT CGG AGC three times with100 mL of double distilled water by cen- GTA AAC TCC TTT TC-30 (forwardprimer,locatedatbase trifugation for 2 min at 2500 Â g. Pellets were then position 415^437 of the C. rectus 16 S r D NA) a nd 5 0-TTT resuspended in 100 mL of double distilled water, boiled CTG CAA GCA GAC ACT CTT-30 (reverse primer, located for 10 min and chilled on ice.
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