Identification of putative pathogens in acute endodontic infections by PCR based on 16S rDNA

Jee-Hoon Kim; So Young Yoo; Sun-A Lim; Joong-Ki Kook; Sang-Soo Lim; Seul-Hee Park; Ho-Keel Hwang

doi:10.5395/JKACD.2003.28.2.178

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Original Article Identification of putative pathogens in acute endodontic infections by PCR based on 16S rDNA: Jee-Hoon Kim¹, So Young Yoo^2,3, Sun-A Lim², Joong-Ki Kook^2,3, Sang-Soo Lim¹, Seul-Hee Park¹, Ho-Keel Hwang^1,3; 2003;28(2):-183.
DOI: https://doi.org/10.5395/JKACD.2003.28.2.178
Published online: March 31, 2003

¹Department of Conservative Dentistry, College of Dentistry, Chosun University, Korea.

²Department of Oral Biochemistry, College of Dentistry, Chosun University, Korea.

³Oral Biology Research Institute, College of Dentistry, Chosun University, Korea.

Corresponding author (rootcanal@hanmail.net)

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Abstract
REFERENCES

Abstract

The purpose of this study was to investigate the frequency of 7 putative pathogens in endodontic infections. The specimens were collected from infected pulpal tissue of patients who were referred for root canal treatment to the department of conservative dentistry, Chosun University. Samples were collected aseptically using a barbed broach and a paper point. The cut barbed broaches and paper points were transferred to an eppendorf tube containing 500 ml of 1 X PBS. DNAs were extracted from the samples by direct DNA extraction method using lysis buffer (0.5% EDTA, 1% Triton X-100). Identification of 7 putative pathogens was performed by PCR based on 16S rDNA. The target species were as follows: Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Bacteroides forsythus, Actinobacillus actinomycetemcomitans, and Treponema denticola. Our data revealed that the prevalence of P. endodontalis was found in 88.6% (39/54), P. gingivalis 52.3% (23/44), P. nigrescens 18.2% (8/44), P. intermedia 15.9% (7/44), B. forsythus 18.2% (8/44), A. actinomycetemcomitans 2.3% (1/44), T. denticola 25% (11/44) of the samples. The high prevalence of P. endodontalis and P. gingivalis suggests that they may play an important role in the etiology of endodontic infections.
Keywords: 16S rDNA; Acute endodontic infections; PCR; Pathogens

Table 1

PCR primer pairs used for detection of putative oral pathogens in samples

Table 2

Prevalence of some bacterial species in infected root canals

Pe ; Porphylomonas endodontalis, Pg ; Porphylomonas ginginvalis, Pn ; Prevotella nigrescens, Pi ; Prevotella intermedia, Td ; Treponema denticola, Bf ; Bacteroide forsythus, Aa ; Actinobacillus actinomycetemcomitans ^*n ; Sample number

REFERENCES

1. Miller WD. Microorganisms of the human mouse. 1890;Philadelphia: S.S. White Dental Co.
2. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol. 1965;20: 340-349.Article PubMed
3. Sundqvist G. Bacteriological studies of necrotic dental pulps. 1976;Umea: Umea University.
4. Brown TA. Molecular biology labfax II. 1998;San Diego: Academic press.
5. Ashimoto A, Chen C, Bakker I, Slots J. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesion. Oral Microbiol Immunol. 1996;11: 266-273.PubMed
6. Brook I, Frazier EH, Gher ME. Aerobic and anaerobic microbiology of periapical abscess. Oral Microbiol Immunol. 1991;6: 123-125.Article PubMed
7. Socransky SS, Haffajee AD. Clinical periodontology and implant dentstry. 1997;3rd ed. Copenhagen: Munksgaard; 138-188.
8. Sundqvist GK, Eckerbom MI, Larsson AP, Sjogren UF. Capacity of anaerobic bacteria from necrotic dental pulps to induce purulent infections. Infect Immun. 1979;25: 685-693.Article PubMed PMC PDF
9. Van Winkelhoff AJ, Carlee AW, De Graaff J. Bacteriodes endodontalis and others black-pigmented Bacteriodes species in odontogenic abscesses. Infect Immun. 1985;49: 494-498.Article PubMed PMC PDF
10. Haapasalo M, Ranta H, Ranta K, Shah H. Black-pigmented bacteroides spp in human apical periodontitis. Infect Immun. 1986;53: 149-153.Article PubMed PMC PDF
11. Siqueira JF, Rôças IN, Oliveira JCM, Santos KRN. Molecular detection of black-pigmented bacteria in infections of endodontic origin. J Endod. 2001;27: 563-566.Article PubMed
12. Sundqvist G, Johansson E, Sjögen U. Prevalence of black-pigmented bacteria with endodontic infections. J Endod. 1989;15: 13-19.PubMed
13. Van Winkelhoff AJ. The role of black-pigmented bacteroides in human oral infections. J Clin Periodontol. 1988;15: 145-155.Article PubMed
14. Machado de Oliveira JC, Siqueira JF Jr, Alves GB, Hirata R Jr, Andrade AF. Detection of Porphyromonas endodontalis in infected root canals by 16S rRNA gene-directed Polymerase chain reaction. J Endod. 2000;26: 729-732.PubMed
15. Wahlfors J, Meurman JH. Simultaneous detection of Actinobacillus actinomycetemcomitans and Porphylomonas gingivalis by a rapid PCR method. J Dent Res. 1995;74: 1796-1801.Article PubMed PDF
16. Baumgartner JC, Watkins BJ, Bae KS, Xia T. Association of black-pigmented bacteria with endodontic infections. J Endod. 1999;25: 413-415.PubMed
17. Haapasalo M. Bacteroides spp in dental root canal infections. Endod Dent Traumatol. 1989;5: 1-10.Article PubMed
18. Siqueira JF, Rôças IN, Oliveira JCM, Santos KRN. Detection of putative oral pathogens in acute periradicular abscesses by 16S rDNA-directed Polymerase chain reaction. J Endod. 2001;27: 164-167.PubMed
19. Hashioka K, Yamasaki M, Nakane A, Horiba N, Nakamura H. The relationship between clinical symptoms and anaerobic bacteria from infected root canals. J Endod. 1971;18: 558-561.Article PubMed
20. Shah HN, Gharbia SE. Biochemical and chemical studies on strains designated Prevotella intermedia and proposal of a new pigmented species, Prevotella nigrescens sp. nov. Int J Syst Bacteriol. 1992;42: 542-546.Article PubMed
21. Gharbia SE, Haapasalo M, Shah HN. Characterization of Prevotella intermedia and Prevotella nigrescens isolates from periodontic and endodontic infection. J Periodontol. 1994;65: 56-61.PubMed
22. Conrads G, Mutters R, Fischer J, Brauner A, Lutticken R, Lampert F. PCR reaction and dot-blot hybridization to monitor the distribution of oral pathogens within plaque samples of periodontally healthy individuals. J Periodontol. 1996;67: 994-1003.PubMed
23. Dahlén G, Wikström M, Renvert S, Gmür R, Guggenheim B. Biochemical and serological characterization of Bacteroides intermedius strains isolated from the deep periodontal pocket. J Clin Microbiol. 1990;28: 2269-2274.Article PubMed PMC PDF
24. Mättö J, Asikainen S, Väisänen ML. Role of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens in extraoral and some odontogenic infections. Clin Infect Dis. 1997;25: Suppl. S194-S198.PubMed
25. Milsom SE, Sprague SV, Dymock D, Weighman AJ, Wade WG. Rapid differentiation of Prevotella intermedia and Prevotella nigrescens by 16S rDNA PCR-RFLP. J Med Microbiol. 1992;44: 41-43.
26. Dahle UR, Tronstad L, Olsen I. Observation of an unusually large spirochete in endodontic infection. Oral Microbiol and Immunol. 1993;8: 251-253.
27. Hampp EG. Isolation and identification of spirochetes obtained from unexposed canals of pulp-involved teeth. Oral Surg Oral Med and Oral Pathol. 1957;10: 1100-1104.Article PubMed
28. Thilo BE, Bachni P, Holz J. Darkfield observation of bacterial distribution in root canals following pulp necrosis. J Endod. 1986;12: 202-205.PubMed
29. Trope M, Tronstad L, Rosenberg ES, Litsgarten MA. Darkfield microscopy as a diagnostic aid in differentiating exudates from endodontic and periodontal abscesses. J Endod. 1984;14: 35-39.
30. Siqueira JF, Rôças IN, Moraes SR, Santos KRN. Direct amplification of rRNA gene sequences for identification of selected oral pathogens in root canal infections. Int Endod J. 2002;35: 345-351.PubMed
31. Rôças IN, Siqueira JF, Santos KRN, Coelho MA. "Red complex" (Bacteroides forsythus, Porphylomonas gingivalis, and Treponema denticola) in endodontic infections: A molecular approach. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2001;91: 468-471.PubMed
32. Fenno JC, McBride BC. Virulence factor of oral treponemes. Anaerobe. 1998;4: 1-17.PubMed
33. Gon alves RB, Mouton C. Molecular detection of bacteriodes forsythus in infected root canals. J Endod. 1999;25: 336-340.PubMed
34. Conrads G, Gharbia SF, Gulabivala K, Lampert F, Shah HN. The use of a 16S rDNA PCR for the detection of endodontopathogenic bacteria. J Endod. 1997;23: 433-438.PubMed
35. Siqueira JF, Rôças IN, Souto R, De Uzeda M, Colombo AP. Microbiological evaluation of acute periradicular abscesses by DNA-DNA hybridization. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2001;92: 451-457.Article PubMed
36. Takemoto T, Kurihara H, Dahlen G. Characterization of Bacteriodes forsythus isolates. J Clin Microbiol. 1997;35: 1378-1381.PubMed PMC
37. Meyer DH, Fives-Taylor PM. The role of Actinobacillus actinomycetemcomitans in the pathogenesis of periodontal disease. Trends Microbiol. 1997;5: 224-228.PubMed
38. Siqueira JF, Rôças IN, Souto R, Uzeda M, Colombo AP. Checkerboard DNA-DNA hybridization analysis of endodontic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000;89: 744-748.PubMed
39. Sunde PT, Tronstad L, Eribe ER, Lind PO, Olsen I. Assessment of periradicular microbiota by DNA-DNA hybridization. Endod Dent Traumatol. 2000;16: 191-196.Article PubMed

Tables & Figures

Table 1

PCR primer pairs used for detection of putative oral pathogens in samples

Table 2

Prevalence of some bacterial species in infected root canals

REFERENCES

1. Miller WD. Microorganisms of the human mouse. 1890;Philadelphia: S.S. White Dental Co.
2. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol. 1965;20: 340-349.Article PubMed
3. Sundqvist G. Bacteriological studies of necrotic dental pulps. 1976;Umea: Umea University.
4. Brown TA. Molecular biology labfax II. 1998;San Diego: Academic press.
5. Ashimoto A, Chen C, Bakker I, Slots J. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesion. Oral Microbiol Immunol. 1996;11: 266-273.PubMed
6. Brook I, Frazier EH, Gher ME. Aerobic and anaerobic microbiology of periapical abscess. Oral Microbiol Immunol. 1991;6: 123-125.Article PubMed
7. Socransky SS, Haffajee AD. Clinical periodontology and implant dentstry. 1997;3rd ed. Copenhagen: Munksgaard; 138-188.
8. Sundqvist GK, Eckerbom MI, Larsson AP, Sjogren UF. Capacity of anaerobic bacteria from necrotic dental pulps to induce purulent infections. Infect Immun. 1979;25: 685-693.Article PubMed PMC PDF
9. Van Winkelhoff AJ, Carlee AW, De Graaff J. Bacteriodes endodontalis and others black-pigmented Bacteriodes species in odontogenic abscesses. Infect Immun. 1985;49: 494-498.Article PubMed PMC PDF
10. Haapasalo M, Ranta H, Ranta K, Shah H. Black-pigmented bacteroides spp in human apical periodontitis. Infect Immun. 1986;53: 149-153.Article PubMed PMC PDF
11. Siqueira JF, Rôças IN, Oliveira JCM, Santos KRN. Molecular detection of black-pigmented bacteria in infections of endodontic origin. J Endod. 2001;27: 563-566.Article PubMed
12. Sundqvist G, Johansson E, Sjögen U. Prevalence of black-pigmented bacteria with endodontic infections. J Endod. 1989;15: 13-19.PubMed
13. Van Winkelhoff AJ. The role of black-pigmented bacteroides in human oral infections. J Clin Periodontol. 1988;15: 145-155.Article PubMed
14. Machado de Oliveira JC, Siqueira JF Jr, Alves GB, Hirata R Jr, Andrade AF. Detection of Porphyromonas endodontalis in infected root canals by 16S rRNA gene-directed Polymerase chain reaction. J Endod. 2000;26: 729-732.PubMed
15. Wahlfors J, Meurman JH. Simultaneous detection of Actinobacillus actinomycetemcomitans and Porphylomonas gingivalis by a rapid PCR method. J Dent Res. 1995;74: 1796-1801.Article PubMed PDF
16. Baumgartner JC, Watkins BJ, Bae KS, Xia T. Association of black-pigmented bacteria with endodontic infections. J Endod. 1999;25: 413-415.PubMed
17. Haapasalo M. Bacteroides spp in dental root canal infections. Endod Dent Traumatol. 1989;5: 1-10.Article PubMed
18. Siqueira JF, Rôças IN, Oliveira JCM, Santos KRN. Detection of putative oral pathogens in acute periradicular abscesses by 16S rDNA-directed Polymerase chain reaction. J Endod. 2001;27: 164-167.PubMed
19. Hashioka K, Yamasaki M, Nakane A, Horiba N, Nakamura H. The relationship between clinical symptoms and anaerobic bacteria from infected root canals. J Endod. 1971;18: 558-561.Article PubMed
20. Shah HN, Gharbia SE. Biochemical and chemical studies on strains designated Prevotella intermedia and proposal of a new pigmented species, Prevotella nigrescens sp. nov. Int J Syst Bacteriol. 1992;42: 542-546.Article PubMed
21. Gharbia SE, Haapasalo M, Shah HN. Characterization of Prevotella intermedia and Prevotella nigrescens isolates from periodontic and endodontic infection. J Periodontol. 1994;65: 56-61.PubMed
22. Conrads G, Mutters R, Fischer J, Brauner A, Lutticken R, Lampert F. PCR reaction and dot-blot hybridization to monitor the distribution of oral pathogens within plaque samples of periodontally healthy individuals. J Periodontol. 1996;67: 994-1003.PubMed
23. Dahlén G, Wikström M, Renvert S, Gmür R, Guggenheim B. Biochemical and serological characterization of Bacteroides intermedius strains isolated from the deep periodontal pocket. J Clin Microbiol. 1990;28: 2269-2274.Article PubMed PMC PDF
24. Mättö J, Asikainen S, Väisänen ML. Role of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens in extraoral and some odontogenic infections. Clin Infect Dis. 1997;25: Suppl. S194-S198.PubMed
25. Milsom SE, Sprague SV, Dymock D, Weighman AJ, Wade WG. Rapid differentiation of Prevotella intermedia and Prevotella nigrescens by 16S rDNA PCR-RFLP. J Med Microbiol. 1992;44: 41-43.
26. Dahle UR, Tronstad L, Olsen I. Observation of an unusually large spirochete in endodontic infection. Oral Microbiol and Immunol. 1993;8: 251-253.
27. Hampp EG. Isolation and identification of spirochetes obtained from unexposed canals of pulp-involved teeth. Oral Surg Oral Med and Oral Pathol. 1957;10: 1100-1104.Article PubMed
28. Thilo BE, Bachni P, Holz J. Darkfield observation of bacterial distribution in root canals following pulp necrosis. J Endod. 1986;12: 202-205.PubMed
29. Trope M, Tronstad L, Rosenberg ES, Litsgarten MA. Darkfield microscopy as a diagnostic aid in differentiating exudates from endodontic and periodontal abscesses. J Endod. 1984;14: 35-39.
30. Siqueira JF, Rôças IN, Moraes SR, Santos KRN. Direct amplification of rRNA gene sequences for identification of selected oral pathogens in root canal infections. Int Endod J. 2002;35: 345-351.PubMed
31. Rôças IN, Siqueira JF, Santos KRN, Coelho MA. "Red complex" (Bacteroides forsythus, Porphylomonas gingivalis, and Treponema denticola) in endodontic infections: A molecular approach. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2001;91: 468-471.PubMed
32. Fenno JC, McBride BC. Virulence factor of oral treponemes. Anaerobe. 1998;4: 1-17.PubMed
33. Gon alves RB, Mouton C. Molecular detection of bacteriodes forsythus in infected root canals. J Endod. 1999;25: 336-340.PubMed
34. Conrads G, Gharbia SF, Gulabivala K, Lampert F, Shah HN. The use of a 16S rDNA PCR for the detection of endodontopathogenic bacteria. J Endod. 1997;23: 433-438.PubMed
35. Siqueira JF, Rôças IN, Souto R, De Uzeda M, Colombo AP. Microbiological evaluation of acute periradicular abscesses by DNA-DNA hybridization. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2001;92: 451-457.Article PubMed
36. Takemoto T, Kurihara H, Dahlen G. Characterization of Bacteriodes forsythus isolates. J Clin Microbiol. 1997;35: 1378-1381.PubMed PMC
37. Meyer DH, Fives-Taylor PM. The role of Actinobacillus actinomycetemcomitans in the pathogenesis of periodontal disease. Trends Microbiol. 1997;5: 224-228.PubMed
38. Siqueira JF, Rôças IN, Souto R, Uzeda M, Colombo AP. Checkerboard DNA-DNA hybridization analysis of endodontic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000;89: 744-748.PubMed
39. Sunde PT, Tronstad L, Eribe ER, Lind PO, Olsen I. Assessment of periradicular microbiota by DNA-DNA hybridization. Endod Dent Traumatol. 2000;16: 191-196.Article PubMed

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Identification of putative pathogens in acute endodontic infections by PCR based on 16S rDNA

J Korean Acad Conserv Dent. 2003;28(2):178-183. Published online March 31, 2003

DOI: https://doi.org/10.5395/JKACD.2003.28.2.178

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Identification of putative pathogens in acute endodontic infections by PCR based on 16S rDNA

Table 1

PCR primer pairs used for detection of putative oral pathogens in samples

Table 2

Prevalence of some bacterial species in infected root canals

Table 1 PCR primer pairs used for detection of putative oral pathogens in samples

Table 2 Prevalence of some bacterial species in infected root canals

Pe ; Porphylomonas endodontalis, Pg ; Porphylomonas ginginvalis, Pn ; Prevotella nigrescens, Pi ; Prevotella intermedia, Td ; Treponema denticola, Bf ; Bacteroide forsythus, Aa ; Actinobacillus actinomycetemcomitans ^*n ; Sample number