Results - Discussion - References.
Campylobacter gracilis and C. rectus were, respectively, detected in 21.4 (6 of 28) and 30% (6 of 20) of the root canals associated with chronic asymptomatic periradicular lesions. Campylobacter gracilis was found in16.7% (2 of 12) of the cases diagnosed as acute apical periodontitis, whilst C. rectus was in 33.3% (two of six cases). In the abscessed cases, C. gracilis and C. rectus were detected in 23.5 (4 of17) and11.8% (2 of17) of the cases, respectively. Neither C. gracilis (P = 0.797), nor C. rectus (P = 0.539) was positively associated with clinical symptoms. In general, species-specific nPCR allowed the detection of C. gracilis in 21.1% (12 of 57) and C. rectus in 23.3% (10 of 43) of the samples taken from primary endodontic infections. The species were found together in only two asymptomatic teeth.
Reference DNA and clinical samples that were positive for either C. gracilis or C. rectus showed only one band of the predicted size. Specific primers generated no amplicons with genomic DNA from nontarget bacterial species. The detection limit of then PCR assay used in this study was approximately 10 cells as determined by amplification of serial dilutions of templates prepared from genomic DNA.
All clinical samples contained bacteria as demonstrated after the first round of amplification using universal primers for the 16S rDNA. A product of the appropriate size (1505 bp) was obtained from all samples, revealing that bacteria were present in all cases examined, demonstrating the suitability of the DNA for PCR analysis, and indicating the absence of inhibitors in the reaction mixture. Discussion.
The specificity of a microbiological diagnostic test is essential to avoid false positive results. In the present study, noevidence of cross-reactivity was observed when checking the C. gracilis- and C. rectus- specific primers against a panel of nontargeted oral species. Nonspecific amplification products were also absent. In addition, nPCR directed to16S rDNA is more sensitive and can still show improved specificity when compared with single PCR by allowing the second species-specific reaction to be performed with reduced background of necrotic tissue, pus debris, eukaryotic DNA and other regions of the bacterial DNA.
Campylobactergracilis have been found in infections of endodontic origin in prevalence values ranging from 1.5-55.6%of cases. Sundqvist et al. (1989) have recovered C. gracilis from 13.6% of canals containing black-pigmented rods. In another study, Sundqvist (1992) investigated the root canal microbiota of 65 teeth with intact pulp chambers and radiographic evidence of periradicular disease and found C. gracilis in only one case (1.5%). Gomes et al. (1996) isolated C. gracilis from 2.9% of 70 infected root canals. Le Goff et al. (1997) reported the highest prevalence value for this bacterial species when evaluating the microbiota of infected root canals in teeth without carious lesions and with intact crowns - 55.6% of cases. Sundqvist et al. (1998) have found this species in12.5% of the canals of teeth with failed endodontic treatment. Some studies evaluating the presence of microbial species in chronic periradicular lesions have also found C. gracilis. Wayman et al. (1992) revealed that C. gracilis was one of the five most commonly isolated bacteria in lesions with no detectable communication with the oral cavity. Recently, C. gracilis was also detected in periradicular lesions of asymptomatic teeth by DNA-DNA hybridization (Sunde et al. 2000).
Studies have revealed that C. rectus may also be present in endodontic infections, in prevalence values ranging from 7.1-27.3%. Ranta et al. (1988) investigated the microbiota of 62 cases of periradicular lesions and found C. rectus in 11.3% of the cases. Sundqvist et al. (1989) observed the occurrence of this bacterial species in 27.3% of 22 root canals. Further, Sundqvist (1992) has reported that C. rectus was isolated from 25% of 65 infected root canals. This species was positively associated with P. endodontalis, Peptostreptococcus micros, Selenomonas sputigena, F. nucleatum, Actinomyces sp. and Eubacterium sp., which may be partly dependent on the production of growth factors, such as formate, by most of these bacteria. Siqueira et al. (2000b) examined the microbiota of infected root canals using whole genomic DNA probes and the checkerboard DNA-DNA hybridization method and found C. rectus in 7.1% of the cases. Using the same method to investigate the microbiota associated with acute periradicular abscesses, Siqueira et al. (2001b) detected C. rectus in 7.4% of the abscessed cases. Other studies using the checkerboard DNA-DNA hybridization method to assess the microbiota present in periradicular lesions have reported the detection of C. rectus in a relatively high prevalence value (Gatti et al. 2000, Sunde et al. 2000).
Most studies using PCR methodology have either detected certain bacterial species never previously found in endodontic infections by culture (Conrads et al. 1997, Siqueira et al. 2000a, Jung et al. 2001, Rolph et al. 2001) or detected certain bacterial species usually at higher prevalence when compared with culture (Siqueira et al. 2001a, Hashimura et al. 2001). In the present study, C. gracilis was detected in 21.1% of the samples of all examined samples and C. rectus in 23.3%. These frequency rates as evaluated by highly sensitive nPCR assay were not significantly discrepant from culture studies. This suggests that the prevalence of these bacterial species has not probably been underestimated by culture and confirmed that they may be associated with endodontic infections in a reasonable number of cases. Because both bacterial species were found in asymptomatic as well as symptomatic infections inpractically similar frequencies, no association with symptoms could be detected by statistical analysis.
The mechanisms of pathogenicity of C. gracilis are poorly understood. Its virulence factors probably include lipopolysaccharide (LPS), hydrogen sulphide and succinate. Campylobactergracilis are usuallyless susceptible to antimicrobial agents than other oral Campylobacter species (Johnson et al.1986, Tanner et al.1992, Baron et al.1993, Lee et al.1993). Johnson et al. (1986) evaluated the in vitro activities of 17 antimicrobial agents against 46 clinical isolates of formate/fumarate-requiring anaerobic Gram-negative bacilli. Campylobacter gracilis showed some striking resistance, with penicillin being active against only 67%, the cephalosporins active against 67-89%, and clindamycin active against 67% of the strains tested. Because C. gracilis has been associated with serious deep-tissue infection, coupled with the relatively high frequency of antibiotic resistance, it has been considered as an important human pathogen (Johnson et al. 1985). Supportive evidence for this statement is still lacking.
Whereas the pathogenicity of C. gracilis has not been conclusively demonstrated, there is suggestive evidence that C. rectus is a pathogenic microorganism. Campylobacter rectus possesses some virulence factors that may be involved in the pathogenesis of periradicular diseases. They include an extracellular cytotoxin against polymorphonuclear neutrophils, LPS, a proteinaceous surface structure (S-layer), a native GroEL-like protein, tissue-damaging enzyme arylsulfatase and hydrogen sulphide (Gillespie et al. 1992, 1993, Okuda et al. 1997, Hinode et al. 1998, Zubery et al. 1998). The native GroEL-like protein is able to stimulate production of pro-inflammatory cytokines such as interleukin-6 and -8 (Hinode et al.1998). S-layer possessing C. rectus cells can be resistant to complement-mediated killing and phagocytic killing by leucocytes in the absence of speci- fic antibody (Okuda et al.1997). It has been demonstrated that C. rectus can cause soft tissue destruction following inoculation into subcutaneous tissue of mice (Kesavalu et al.1991). Live and heat-killed cells of C. rectus are also able to stimulate bone resorption in mice, possibly via LPS or other polysaccharide components (Zubery et al. 1998).
Taken together, the findings of this study indicated that C. gracilis and C. rectus participate in infections of endodontic origin. The possible involvement of these species with other human infections, including periodontal diseases and their potential virulence armamentarium might also implicate them in the pathogenesis of periradicular diseases. Nevertheless, whilst a pathogenetic role can be suspected for these species, clear evidence of causation is still lacking. Studies are necessary to elucidate the specific role played by C. gracilis and C. rectus in primary endodontic infections as well as their involvement in the pathogenesis of periradicular diseases.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. Journal of Molecular Biology 215, 403-10.
Ashimoto A, Chen C, Bakker I, Slots J (1996) Polymerase chain reaction detection of eight putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiology and Immunology 11, 266-73.
Baron EJ, Ropers G, Summanen P, Courcol RJ (1993) Bactericidal activity of selected antimicrobial agents against Bilophila wadsworthia and Bacteroides gracilis. Clinical Infectious Diseases 16 (Suppl. 4), S339-43.
Conrads G, Gharbia SE, Gulabivala K, Lampert F, Shah HN (1997) The use of a 16S rDNA PCR for the detection of endodontopathogenic bacteria. Journal of Endodontics 23, 433-8.
Dieffenbach CW, Dveksler GS (1995) PCR Primer. A Laboratory Manual. Plain View, NY: Cold Spring Harbor Laboratory Press, 1995.
Gatti JJ, Dobeck JM, Smith C, White RR, Socransky SS, Skobe Z (2000) Bacteria of asymptomatic periradicular endodontic lesions identified by DNA-DNA hybridization. Endodontics and Dental Traumatology 16, 197-204.
Gillespie J, De Nardin E, Radel S, Kuracina J, Smutko J, Zambon JJ (1992) Production of an extracellular toxin by the oral pathogen Campylobacter rectus. Microbial Pathogenesis 12, 69-77.
Gillespie MJ, Smutko J, Haraszthy GG, Zambon JJ (1993) Isolation and partial characterization of the Campylobacter rectus cytotoxin. Microbial Pathogenesis 14, 203-15.
Gomes BPFA, Lilley JD, Drucker DB (1996) Clinical significance of dental root canal microflora. Journal of Dentistry 24, 47-55.
Hashimura T, Sato M, Hoshino E (2001) Detection of Slackia exigua, Mogibacterium timidum and Eubacterium saphenum from pulpal and periradicular samples using the Polymerase Chain Reaction (PCR) method. International Endodontic Journal 34, 463-70.
Hinode D, Yoshioka M, Tanabe S, Miki O, Masuda K, Nakamura R (1998) The GroEL-like protein from Campylobacter rectus: immunological characterization and interleukin-6 and -8 induction in human gingival fibroblast. FEMS Microbiology Letters167, 1-6.
Johnson CC, Reinhardt JF, Edelstein MA, Mulligan ME, George WL, Finegold SM (1985) Bacteroides gracilis, an important anaerobic bacterial pathogen. Journal of Clinical Microbiology 22, 799-802.
Johnson CC, Reinhardt JF, Mulligan ME, George WL, Finegold SM (1986) In vitro activities of 17 antimicrobial agents against the formate/fumarate-requiring, anaerobic Gram-negative bacilli. Diagnostic Microbiology and Infectious Disease 5, 269-72.
Jordan RCK, Daniels TE, Greenspan JS, Regezi JA (2001) Advanced diagnostic methods in oral and maxillofacial pathology. Part 1. molecular methods. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics 92, 650-69.
Jung I-Y, Choi B-K, Kum K-Y et al. (2001) Identification of oral spirochetes at the species level and their association with other bacteria in endodontic infections. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics 92, 329-34.
Kamma JJ, Diamanti-Kipioti A, Nakou M, Mitsis FJ (2000) Profile of subgingival microbiota in children with primary dentition. Journal of Periodontal Research 35, 33-41.
Kesavalu L, Holt SC, Crawley R, Borinski R, Ebersole JL (1991) Virulence of Wolinella recta in a murine abscess model. Infection and Immunity 59, 2806-17.
Le Goff A, Bunetel L, Mouton C, Bonnaure-Mallet M (1997) Evaluation of root canal bacteria and their antimicrobial susceptibility in teeth with necrotic pulp. Oral Microbiology and Immunology 12, 318-22.
Lee D, Goldstein EJ, Citron DM, Ross S (1993) Empyema due to Bacteroides gracilis: case report and in vitro susceptibilities to eight antimicrobial agents. Clinical Infectious Diseases 16 (Suppl. 4), S263-5.
Macuch PJ, Tanner AC (2000) Campylobacter species in health, gingivitis and periodontitis. Journal of Dental Research 79, 785-92.
McPherson MJ, Moller SG. (2000) PCR. Oxford, UK: BIOS Scientific Publishers Ltd.
NgY-L, Spratt DA, Sriskantharajah S, Rahbaran S, Gulabivala K (2002) Development of contemporary decontamination protocols for study of root canal flora by cultivation and molecular techniques. International Endodontic Journal 35, 85- 6 (Abstract R16).
Okuda K, Kigure T, Yamada S et al. (1997) Role for the S-layer of Campylobacter rectus ATCC33238 in complement-mediated killing and phagocytic killing by leukocytes from guinea pig and human peripheral blood. Oral Diseases 3, 113-20.
Rams TE, Feik D, Slots J (1993) Campylobacter rectus in human periodontitis. Oral Microbiology and Immunology 8, 230-5.
Ranta H, Haapasalo M, Ranta K et al.(1988) Bacteriology of odontogenic apical periodontitis and effect of penicillin treatment. Scandinavian Journal of Infectious Diseases 20, 187-92.
Relman DA (1993) The identification of uncultured microbial pathogens. Journal of Infectious Diseases 168, 1-8.
Relman DA (1999) The search for unrecognized pathogens. Science 284, 1308-10.
Rolph HJ, Lennon A, Riggio MP et al. (2001) Molecular identification of microorganisms from endodontic infections. Journal of Clinical Microbiology 39, 3282-9.
Siqueira JF Jr (2002) Endodontic infections: concepts, paradigms and perspectives. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 94, 281-93.
Siqueira JF Jr, Rocas IN, Favieri A, Santos KRN (2000a) Detection of Treponema denticola in endodontic infections by 16S rRNA gene directed polymerase chain reaction. Oral Microbiology and Immunology15, 335-7.
Siqueira JF Jr, Rocas IN, Oliveira JCM, Santos KRN (2001a) Molecular detection of black-pigmented bacteria in infections of endodontic origin. Journal of Endodontics 27, 563-6.
Siqueira JF Jr, Rocas IN, Souto R, Uzeda M, Colombo AP (2000b) Checkerboard DNA-DNA hybridization analysis of endodontic infections. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics 89, 744-8.
Siqueira JF Jr, Rocas IN, Souto R, Uzeda M, Colombo AP (2001b) Microbiological evaluation of acute periradicular abscesses by DNA-DNA hybridization. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics 92, 451-7.
Sunde PT, Tronstad L, Eribe ER, Lind PO, Olsen I( 2000) Assessment of periradicular microbiota by DNA-DNA hybridization. Endodontics and Dental Traumatology 16, 191-6.
Sundqvist G (1992) Associations between microbial species in dental root canal infections. Oral Microbiology and Immunology 7, 257-62.
Sundqvist G, Figdor D, Persson S, Sjogren U (1998) Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics 85, 86-93.
Sundqvist G, Johansson E, Sjogren U (1989) Prevalence of black pigmented Bacteroides species in root canal infections. Journal of Endodontics15, 13-9.
Tanner ACR, Badger S, Lai C-H, Listgarten MA, Visconti RA, Socransky SS (1981) Wolinella gen. nov., Wolinella succinogenes (Vibrio succinogenes Wolin et al.) comb. nov., and description of Bacteroides gracilis sp. nov., Wolinella recta sp. nov., Campylobacter concisus sp. nov. & Eikenella corrodens from humans with periodontal disease. International Journal of Systematic Bacteriology 31, 432-45.
Tanner A, Lai C-H, Maiden M (1992) Characteristics of oral Gram-negative species. In: Slots J, Taubman MA, eds. Contemporary Oral Microbiology and Immunology. St Louis: Mosby, 299-341.
Tanner A, Maiden MF, Lee K, Shulman LB, Weber HP (1997) Dental implant infections. Clinical Infectious Diseases 25 (Suppl. 2), S213-7.
Tanner A, Maiden MF, Macuch PJ, Murray LL, Kent RL Jr (1998) Microbiota of health, gingivitis, and initial periodontitis. Journal of Clinical Periodontology 25, 85-98.
Torabinejad M, Walton RE (1994) Periradicular lesions. In: Ingle JI, Bakland LK, eds. Endodontics, 4th edn. Baltimore: Williams &Wilkins, 439-64.
Vandamme P, Daneshvar MI, Dewhirst FE et al. (1995) Chemotaxonomic analyses of Bacteroides gracilis and Bacteroides ureolyticus and reclassification of B. gracilis as Campylobacter gracilis comb. nov. International Journal of Systematic Bacteriology 45, 145-52.
Vandamme P, Falsen E, Rossau R et al. (1991) Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter General nov. International Journal of Systematic Bacteriology 41, 88-103.
Wayman BE, Murata SM, Almeida RJ, Fowler CB (1992) A bacteriological and histological evaluation of 58 periapical lesions. Journal of Endodontics18, 152-5.
Zubery Y, Dunstan CR, Story BM et al. (1998) Bone resorption caused by three periodontal pathogens in vivo in mice is mediated in part by prostaglandin. Infection and Immunity 66, 4158-62.