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. After centrifugation to at base position 992^1012 of the C. rectus 16 S r D NA). remove cell debris for10 s at 9000 Â g at 4 8C, the super- Predicted amplicon size is 598 bp. Campylobacter rectus natant was collected and used as the template for PCR primers were as described by Ashimoto et al.(1996). ampli¢cation. Reference DNA from C. gracilis ATCC Primers weresynthesized byOligos Etc. Inc. (Wilsonville, 33236 and from C. rectus ATCC 33238 was also extracted OR, USA). to serve as positive control for the primers used.Negative controls included the PCR mixture without DNA templ- nPCR assay ate orcontainingextracted DNA from Actinobacillusacti- nomycetemcomitans (ATCC 43718), Bacteroides forsythus A 16S rDNA-based nPCR detection method was used to (ATCC 43037), C. gracilis (for C. rectus primers), C. rectus detect C. gracilis and C. rectus in clinical samples. Ali- (for C. gracilis primers), C. showae (ATCC 51146), Cap- quots of 5 mL of the supernatant from clinical samples nocytophaga ochracea (ATCC 27872), Fusobacterium were used as target in the ¢rst PCR reaction using uni- nucleatum (ATCC 25586 and 10953), Porphyromonas versal 16S rDNA primers. PCR ampli¢cation was endodontalis (ATCC 35406), Po. gingivalis (ATCC 33277), performed in 25 mL of reaction mixture containing Prevotella intermedia (ATCC 25611), Pr. nigrescens (ATCC 0.2 mm concentration of forward and reverse universal

33563), Streptococcus intermedius (ATCC 27335) and Tre- primers,2.5 mLof10Â PCR bu¡er,2 mm MgCl2,1.25units ponema denticola (B1strain, Forsyth Dental Institute). of Tth DNA polymerase (Biotools, Madrid, Spain) and 25 mm concentration of each deoxyribonucleoside tri- phosphate (dATP, dCTP, dGTP and dTTP) (Gibco BRL, Oligonucleotide primers Gaithersburg, MD, USA). In the ¢rst PCR reaction, a practically full-length 16S Afterwards,1 mL of the universal reaction was used as rDNAwas ampli¢ed using a pair of universal 16S rDNA template for the nested speci¢c reaction. The second primers, which consisted of a forward universal primer PCR reaction, used to assess the occurrence of C. gracilis (50-AGA GTT TGA TCC TGG CTC AG-30, base position or C. rectus, was performed in 50 mL of reaction mixture 8^27 relative to Escherichia coli16S rDNA) and a reverse containing 1 mm of each speci¢c primer, 5 mLof10Â 0 universal primer (5 -ACG GCT ACC TTG TTA CGA CTT- PCR bu¡er (Gibco BRL),2 mm MgCl2,1.25unitTth DNA 30, base position1,493^1513 relative to E. coli 16S rDNA). polymerase (Biotools) and 0.2 mm of each deoxyribonu- PCR oligonucleotide primers speci¢c for C. gracilis cleoside triphosphate (Gibco BRL). PCR reactions were were designed using the published DNA data from the performed in 25-well microtitre plates. GenBank. Brie£y, 16S rDNA sequences from the Gen- Preparations were ampli¢ed in a DNA thermocycler Bank database were aligned, and variable areas between (Primus 25/96, MWG-Biotech, Ebersberg, Germany). species were identi¢ed. Upstream and downstream The PCR temperature pro¢le for the universal reaction C. gracilis primers were designed from these areas and included an initial denaturation step at 97 8Cfor1min, blast (Altschul et al.1990) was used toverify their speci- followed by 26 cycles of a denaturation step at 97 8Cfor ¢city by comparing primer sequences with all available 45 s, a primer annealing step at 55 8Cfor45s,anexten- sequences in the GenBank database. BLAST search sion step at 72 8C for 1 min and a ¢nal step of 72 8Cfor revealed no likely cross-reactivity with other related or 4 min.Temperature pro¢le for the second round of amp- unrelated oral species. Primer speci¢city was further li¢cation speci¢c for C. gracilis included an initial dena- tested against DNA from the reference strains used as turation step at 95 8C for 2 min, followed by 26 cycles controls. The PCR oligonucleotide species-speci¢c pri- of a denaturation step at 94 8Cfor30s,aprimeranneal- mers, 16S rDNA-directed, for C. gracilis were 50-AAC ing step at 53 8C for 1 min, an extension step at 72 8C

17 6 International Endodontic Journal, 36, 174^180, 2003 ß 2003 Blackwell Publishing Ltd Siqueira & Ro“c¸as Campylobacter in endodontic infections for1 minanda ¢nal step of 72 8Cfor2min.Forthedetec- versal primers for the 16S rDNA. A product of the tion of C. rectus, the second round of ampli¢cation con- appropriate size (1505 bp) was obtained fromall samples, sisted of an initial denaturation step at 95 8Cfor2min, revealing that bacteria were present in all cases exam- followed by 26 cycles of a denaturation step at 95 8Cfor ined, demonstrating the suitability of the DNA for PCR 30 s, a primer annealing step at 60 8Cfor1min,an analysis, and indicating the absence of inhibitors in extension step at 72 8C for 1 min, and a ¢nal extension the reaction mixture. at 72 8C for 2 min following the last cycle. PCR amplicons were analysed by electrophoresis in a Discussion 1.5% agarosegelat 4 VcmÀ1inTris^borate^EDTAbu¡er. The gel was stained for 15 min with 0.5 mgmLÀ1 ethi- The speci¢city of a microbiological diagnostic test is dium bromide and visualized under ultraviolet light. essential to avoid false positive results. In the present Positive reactions were determined by the presence of study,noevidence ofcross-reactivity was observedwhen bands of the appropriate sizes. A 100-bp DNA ladder checking the C. gracilis-andC. rectus- speci¢c primers digest (Gibco BRL) served as the molecular size marker. against a panel of nontargeted oral species. Nonspeci¢c ampli¢cation products were also absent. In addition, nPCRdirectedto16SrDNA is moresensitiveand can still Data analysis show improved speci¢city when compared with single Prevalence values of C. gracilis and C. rectus were recor- PCR by allowing the second species-speci¢c reaction ded as the percentage of the cases examined. The chi- to be performed with reduced background of necrotic square test was used to analyse the association between tissue, pus debris, eukaryotic DNA and other regions of these bacterial species and the occurrence of symptoms. the bacterial DNA. Signi¢cance for chi-square test was established at 1% Campylobactergracilis have been found in infections of (P < 0.01). endodontic origin in prevalence values ranging from 1.5^55.6% of cases. Sundqvist et al.(1989)haverecovered C. gracilis from 13.6% of canals containing black-pig- Results mented rods. In another study, Sundqvist (1992) investi- Campylobacter gracilis and C. rectus were, respectively, gated the root canal microbiota of 65 teeth with intact detected in 21.4 (6 of 28) and 30% (6 of 20) of the root pulp chambers and radiographic evidence of periradicu- canals associated with chronic asymptomatic periradi- lar disease and found C. gracilis in only one case (1.5%). cular lesions. Campylobacter gracilis was found in 16.7% Gomes et al. (1996) isolated C. gracilis from 2.9% of 70 (2 of 12) of the cases diagnosed as acute apical period- infected root canals. Le Go¡ et al. (1997) reported the ontitis, whilst C. rectus was in 33.3% (two of six cases). highest prevalence value for this bacterial species when In the abscessed cases, C. gracilis and C. rectus were evaluating the microbiota of infected root canals in detected in 23.5 (4 of17) and11.8% (2 of17) of the cases, teeth without carious lesions and with intact crowns ^ respectively. Neither C. gracilis (P ¼ 0.797), nor C. rectus 55.6% of cases. Sundqvist et al. (1998) have found this (P ¼ 0.539) was positivelyassociated with clinical symp- species in12.5% of the canals of teethwith failed endo- toms. In general, species-speci¢c nPCR allowed the dontic treatment. Some studies evaluating the presence detection of C. gracilis in 21.1% (12 of 57) and C. rectus of microbial species inchronic periradicularlesionshave in 23.3% (10 of 43) of the samples taken from primary also found C. gracilis.Wayman et al. (1992) revealed that endodontic infections. The species were found together C. gracilis was one of the ¢ve most commonly isolated in only two asymptomatic teeth. bacteria in lesions with no detectable communication Reference DNAand clinical samples that were positive with the oral cavity.Recently, C. gracilis was also detect- for either C. gracilis or C. rectus showed only one band ed in periradicular lesions of asymptomatic teeth by of the predicted size. Speci¢c primers generated no DNA^DNA hybridization (Sunde et al. 2000). amplicons with genomic DNA from nontarget bacterial Studies have revealed that C. rectus may also be pre- species.ThedetectionlimitofthenPCRassayusedinthis sent in endodontic infections, in prevalence values ran- study was approximately 10 cells as determined by ging from 7.1^27.3%. Ranta et al. (1988) investigated ampli¢cation of serial dilutions of templates prepared the microbiota of 62 cases of periradicular lesions and from genomic DNA. found C. rectus in 11.3% of the cases. Sundqvist et al. All clinical samples contained bacteria as demon- (1989) observed the occurrence of this bacterial species strated after the ¢rst round of ampli¢cation using uni- in 27.3% of 22 root canals. Further, Sundqvist (1992)

ß 2003 Blackwell Publishing Ltd International Endodontic Journal, 36, 174^180, 2003 17 7 Campylobacter in endodontic infections Siqueira & Ro“c¸as

has reported that C. rectus was isolated from 25% of 65 associated with serious deep-tissue infection, coupled infected root canals. This species was positively asso- with the relatively high frequency of antibiotic resis- ciated with P. endodontalis, Peptostreptococcus micros, tance, it has been considered as an important human Selenomonas sputigena, F. nucleatum, Actinomyces sp. pathogen (Johnson et al.1985).Supportiveevidencefor and Eubacterium sp., which may be partly dependent this statement is still lacking. on the production of growth factors, such as formate, Whereas the pathogenicity of C. gracilis has not been by most of these bacteria. Siqueira et al. (2000b) exam- conclusively demonstrated, there is suggestive evidence ined the microbiota of infected root canals using whole that C. rectus is a pathogenic microorganism. Campy- genomic DNA probes and the checkerboard DNA^DNA lobacter rectus possesses some virulence factors that hybridization method and found C. rectus in 7.1% of may be involved in the pathogenesis of periradicular dis- the cases. Using the same method to investigate the eases. They include an extracellular cytotoxin against microbiota associated with acute periradicular absces- polymorphonuclear neutrophils, LPS, a proteinaceous ses, Siqueira et al.(2001b)detectedC. rectus in 7.4% of surface structure (S-layer), a native GroEL-like protein, the abscessed cases. Other studies using the checker- tissue-damaging enzyme arylsulfatase and hydrogen board DNA^DNA hybridization method to assess the sulphide (Gillespie et al. 1992, 1993, Okuda et al.1997, microbiota present in periradicular lesions have report- Hinode et al. 1998, Zubery et al. 1998). The native ed the detection of C. rectus in a relatively high preva- GroEL-like protein is able to stimulate production of lence value (Gatti et al. 2000, Sunde et al. 2000). pro-in£ammatory cytokines such as interleukin-6 and Most studies using PCR methodology have either -8 (Hinode et al. 1998). S-layer possessing C. rectus cells detected certain bacterial species never previously can be resistant to complement-mediated killing and found in endodontic infections by culture (Conrads phagocytic killing by leucocytes in the absence of speci- et al. 1997, Siqueira et al. 2000a, Jung et al. 2001, Rolph ¢c antibody (Okuda et al.1997). It has been demonstrated et al. 2001) or detected certain bacterial species usually that C. rectus can cause soft tissue destruction following at higher prevalence when compared with culture inoculation into subcutaneous tissue of mice (Kesavalu (Siqueira et al.2001a,Hashimuraet al. 2001). In the pre- et al.1991). Live and heat-killed cells of C. rectus are also sent study, C. gracilis was detected in 21.1% of the sam- able to stimulate bone resorption in mice, possibly via ples of all examined samples and C. rectus in 23.3%. LPS or other polysaccharide components (Zubery et al. These frequency rates as evaluated by highly sensitive 1998). nPCR assay were not signi¢cantly discrepant from cul- Taken together, the ¢ndings of this study indicated ture studies. This suggests that the prevalence of these that C. gracilis and C. rectus participate in infections of bacterial species has not probably been underestimated endodontic origin.Thepossible involvementof these spe- by culture and con¢rmed that they may be associated cies with other human infections, including periodontal with endodontic infections in a reasonable number of diseases and their potential virulence armamentarium cases. Because both bacterial species were found in might also implicate them in the pathogenesis of perira- asymptomatic as wellas symptomatic infections in prac- diculardiseases. Nevertheless, whilstapathogeneticrole tically similar frequencies, no association with symp- can be suspected for these species, clearevidence of cau- toms could be detected by statistical analysis. sation is still lacking. Studies are necessary to elucidate The mechanisms of pathogenicity of C. gracilis are the speci¢c role played by C. gracilis and C. rectus in pri- poorly understood. Its virulence factors probably in- mary endodontic infections as well as their involvement clude lipopolysaccharide (LPS), hydrogen sulphide and in the pathogenesis of periradicular diseases. succinate. Campylobactergracilis are usuallyless suscep- tible toantimicrobialagents thanotheroral Campylobac- References ter species (Johnson et al. 1986, Tanner et al.1992, Baron et al.1993,Leeet al.1993). Johnson et al. 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ß 2003 Blackwell Publishing Ltd International Endodontic Journal, 36, 174^180, 2003 17 9 Campylobacter in endodontic infections Siqueira & Ro“c¸as

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18 0 International Endodontic Journal, 36, 174^180, 2003 ß 2003 Blackwell Publishing Ltd