Warning: mkdir(): Permission denied in /home/virtual/lib/view_data.php on line 81

Warning: fopen(upload/ip_log/ip_log_2024-12.txt): failed to open stream: No such file or directory in /home/virtual/lib/view_data.php on line 83

Warning: fwrite() expects parameter 1 to be resource, boolean given in /home/virtual/lib/view_data.php on line 84
Detection of black-pigmented bacteria in infected root canals


Your browser does not support the NLM PubReader view.

Go to this page to see a list of supporting browsers.

Detection of black-pigmented bacteria in infected root canals

Article information

Restor Dent Endod. 2002;27(1):54-65
Publication date (electronic) : 2002 January 31
doi : https://doi.org/10.5395/JKACD.2002.27.1.054
Eun-Kyoung Kwon, Eun-Sook Kim, Ju-Seog Kwak, Hwang Lee, Su-Jong Lee, Mi-Kyung Im
Department of Conservative Dentistry, College of Dentistry, Wonkwang University, Korea.

Abstract

Black-pigmented bacteria have been implicated in the endodontic infections. This group of microorganisms includes Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens. The organisms display a wide variety of virulence factors that may be pertinent to acute endodontic infections.

The aim of this study was to identify P. endodontalis, P. gingivalis, P. intermedia, and P. nigrescens by using special potency disk test, filter paper spot test, 16S rRNA gene-directed PCR, and API 32A.

Microbial samples were collected from root canals of 33 intact teeth with necrotic pulp and/or apical periodontitis. Conventional laboratory methods were used for identification of the strains of black pigmented bacteria. Eighteen of 33 samples were positive for the growth of black-pigmented bacrteria. Five colonies were cultured from each pure cultured colonies from Brucella agar plate. Seventy seven colonies were positive for the growth of black-pigmented bacteria.

Thirty three of 77(42.6%) were identifed as P. nigrescens, 10 of 77(12.9%) were P. gingivalis, 6 of 77(7.8%) were P. endodontalis, 10 of 77(12.9%) were P. intermedia. On the contrary the reference strains of P. nigrescens, experimental strains of P. nigrescens was sensitive to kanamycin in special potency disk test.

16S rRNA gene PCR and API test after rapid presumptative identification methods, such as special potency disk test and filter paper spot test, would be accurate detection methods for black-pigemented bacteria.

References

1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg 1965. 20340–349.
2. Brook I, Frazier EH, Gher ME. Aerobic and anaerobic microbiology of periapical abscess. Oral Microbiol Immunol 1991. 6123–125.
3. Zavistoski J, Dzink J, Onderdonk A, Bartlett J. Quantitative bacteriology of endodontic infections. Oral Surg 1980. 49171–174.
4. Winkelhoff V, Steenbergen TJM, Graaff J. The role of black-pigmented Bacteroides in human oral infections. J Clin Periodontol 1998. 15145–155.
5. Winkelhoff AJ, Steenbergen TJM, Kippuw N, Graaff J. Further characterization of Bacteroides endodontalis, an asaccarolytic black-pigmented bacteroides species from the oral S Cavity. J Clin Microbiol 1985. 2275–79.
6. Oliveira JCM, Siqueira JF, Alves GB, Hirata R, Andrade AFB. Detection of Porphyromonas endodontalis in infected root canals by 16S rRNA gene-directed polymerase chain reaction. J Endod 2000. 26729–732.
7. Hofstad T. Pathogenicity of anaerobic gram-negative rods: possible mechanisms. Rev Infect Dis 1984. 6189–199.
8. Lamont RJ, Jenkinson HF. Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis. Microbiol Mol Biol Rev 1998. 621244–1257.
9. Shah HN, Collis MD. Proposal for reclassification of Bacteroides asaccarolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a new genus, Porphyromonas. Int J Syst Bacteriol 1988. 38128–131.
10. Shah HN, Collins MD. Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int J Syst Bacteriol 1990. 40205–208.
11. Winkelhoff AJ, Steenbergen TJM, Graaff J. Porphyromonas (Bacteroides) endodontalis:Its role in endodotal infections. J Endod 1992. 18431–433.
12. Steenbergen TJM, Winkelhoff AJ, Grenier D, Graff J. Bacteroides endodontalis sp. nov., an Asaccharolytic black-pigemented bacteroides species from infected dental root canals. Int J Syst Bacteriol 1984. 2118–120.
13. Slots J, Genco RJ. Direct hemagglutination technique for differentiating Bacteroides asaccarolyticus oral strains from nonoral strains. J Clin Microbiol 1979. 10371–374.
14. Herweijer JA, Loos BG, Neiders ME. Characterization of total membrane proteins of Porphyromonas endodontalis. J Endod 1992. 18620–624.
15. Stubbs S, Park SF, Bishop PA, Lewis MAO. Direct detection of Prevotella intermedia and P. nigrescens in suppurative oral infection by amplication of 16S rRNA gene. J Med Microbiol 1999. 181017–1022.
16. Gharbia SE, Haapasalo M, Shah HN, Kotiranta A, Lounatmaa K, Pearce MA, Devine DA. Characterization of Prevotella intermedia and Prevotella nigrescens isolates from periodontic and endodontic infections. J periodontol 1994. 6556–61.
17. 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. 42542–546.
18. Nisengard R, Newman MG. Oral microbiology and immunology 1991. second editionth ed. 213–216.
19. Baumgartner JC, Watkins BJ, Bae KS, Xia T. Association of black-pigmented bacteria with endodontic infections. J Endod 1999. 25413–417.
20. Bae KS, Baumgartner JC, Shearer TR, David LL. Occurence of Prevotella nigrescens and Prevotealls intermedia in infections of Endodontic Origin. J Endod 1997. 23620–623.
21. Sundqvist G, Johnasson E, Sjogren U. Prevalence of black-pigmented bacteroides species in root canal infections. J Endod 1989. 1513–19.
22. Siqueira JF, Rocas IN, Oliveira JCM, Santos KRN. Detection of putative oral pathogens in acute periradicular abscesses by 16S rDNA-directed polymerase chain reaction. J Endod 2001. 27164–167.
23. Siqueria JF, Rocas IN, Oliveira JCM, Santos KRN. Molecular detection of black-pigmented bacteria in infections of endodontic origin. J Endod 2001. 27563–566.
24. Kim YH, Kim SH. Diagnosis Clinical Microbiology 2000. 284–406.
25. Haapasalo M. Bacteroides spp. in dental root canal infections. Endod Dent Traumatol 1989. 51–10.
26. Bogen G, Slots J. Black-pigmented anaerobic rods in closed periapical lesions. Int Endod J 1999. 32204–210.
27. Slots J, Ashimoto A, Flynn MJ. Detection of putative periodontal pathogens in subgingival specimens by 16S ribosomal DNA amplication with the polymerase chain reaction. Clin Infect Dis 1995. 20S304–S307.
28. Steenbergen TJM, Menard C, Tijhof CJ, Mouton C, Graaff J. Comparison of three molecular typing methods in studies of transmission of Porphyronas gingivalis. J Med Microbiol 1993. 14416–421.
29. Summanen P, Baron EJ, Citron DM, Strong CA, Wexler HM, Finegold SM. Wadsworth Anaerobic bacteriology Manual 1993.
30. Moncla BJ, Braham P. Detection of sialidase (Neuraminidase) activity in actinomyces species by using 2'-(4-Methylumbelliferyl)α-D-N-Acetylneura-minic acid in a filter paper spot test. J Clin Microbiol 1989. 27182–184.
31. Moncla BJ, Braham P, Rabe LK, Hillier SL. Rapid presumptive identification of black-pigmented gram-negative anaerobic bacteria by using 4-methylumbelliferone derivatives. J Clin Microbiol 1991. 291955–1958.
32. Slots J. Enzymatic characterization of some oral and nonoral gram-negative bacteria with the API ZYM system. J Clin Microbiol 1981. 14288–294.
33. Wayman BE, Murata SM, Almeida RJ, Fowler CB. A bacteriological and histological evaluation of 58 periapical lesions. J Endod 1992. 18152–155.
34. Matto J, Asikainen S, Vaisanen ML, Rautio M, Saarela M, Summanen P, Finegold S, Jousimies-Somer H. Role of Porphyromonas gingivalis,Prevotella intermedia, and Prevotella nigrescens in extraoral and some odontogenic infections. Clin Infect Dis 1997. 25S194–S198.

Article information Continued

Copyright © 2002 Korean Academy of Conservative Dentistry
(open-access) :

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Fig. 1

Fig. 1

Primary culture sample

Fig. 2

Fig. 2

Specific potency disk test of P. gingivalis(ATCC 33277)

P. gingivalis was resistant to kanamycin and colistin, and sensitive to vancomycin.

Fig. 3

Fig. 3

Specific disk potency test of P. intermedia(ATCC 25611)

P. intermedia was resistant to kanamycin and vancomycin, and sensitive to colistin.

Fig. 4

Fig. 4

Specific potency disk test of P. nigrescens(ATCC 33563)

P. nigrescens was resistant to kanamycin and vancomycin, and sensitive to colistin.

Fig. 5

Fig. 5

Special potency disk test of patient sample was identified P. nigrescens.

P. nigrescens was resistant to vancomycin, and sensitive to kanamycin and colistin.

Fig. 6

Fig. 6

Filter paper spot test result of patient sample was identified P. nigrescens.

from left to right: trypsin like enzyme, α-glucosidase, β-glutosidase, α-fucosidase P. nigrescens showed positive response to α-glucosidase and α-fucosidase.

Fig. 7

Fig. 7

PCR profiles of P. endodontalis, P. gingivalis, P. nigrescens, P. intermedia amplified with species specific primers for the 16S rRNA

SM: size marker(1kb DNA ladder)

lane 1: P. endodontalis(ATCC 35406)

lane 2: P. gingivalis(ATCC 33277)

lane 3: P. nigrescens(ATCC 33563)

lane 4: P. intermedia(ATCC 25611)

Fig. 8

Fig. 8

Agarose gel electrophoresis patterns of PCR products of samples

SM: size marker(1kb DNA ladder)

lane 1: sample No. 201(P. nigrescens)

lane 2: sample No. 2803(P. intermedia)

lane 3: sample No. 2806(P. intermedia)

lane 4: sample No. 2401(P. gingivalis)

lane 5: sample No. 2403(P. gingivalis)

lane 6: sample No. 3201(P. nigrescens)

lane 7: sample No. 3205(P. nigrescens)

Fig. 9

Fig. 9

RapidID 32A test result of clinical isolates

Table 1

Special potency disk test on ATCC type strains

Table 1

*R indicates resistant; S indicates sensitive

Table 2

Special potency disk test on clinical isolates

Table 2

*R indicates resistant; S indicates sensitive

Table 3

Filter paper spot test on ATCC type strains

Table 3

+, 90 to 100% of strains positive; -, 90 to 100% of strains negative

Table 4

Filter paper spot test on clinical isolates

Table 4

+, positive reaction observable after 15 min; -, No reaction detected after 15 min

Table 5

Number of strains clinical isolates identified using PCR

Table 5

*: total number of strains identified by PCR

Table 6

Number of strains clinical isolates identified using RapidID 32A

Table 6

*: total number of strains identified by PCR