Journal List > J Korean Acad Conserv Dent > v.27(1) > 1056030

Kwon, Kim, Kwak, Lee, Lee, and Im: Detection of black-pigmented bacteria in infected root canals

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.

Figures and Tables

Fig. 1
Primary culture sample
jkacd-27-54-g001
Fig. 2
Specific potency disk test of P. gingivalis(ATCC 33277)
P. gingivalis was resistant to kanamycin and colistin, and sensitive to vancomycin.
jkacd-27-54-g002
Fig. 3
Specific disk potency test of P. intermedia(ATCC 25611)
P. intermedia was resistant to kanamycin and vancomycin, and sensitive to colistin.
jkacd-27-54-g003
Fig. 4
Specific potency disk test of P. nigrescens(ATCC 33563)
P. nigrescens was resistant to kanamycin and vancomycin, and sensitive to colistin.
jkacd-27-54-g004
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.
jkacd-27-54-g005
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.
jkacd-27-54-g006
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)
jkacd-27-54-g007
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)
jkacd-27-54-g008
Fig. 9
RapidID 32A test result of clinical isolates
jkacd-27-54-g009
Table 1
Special potency disk test on ATCC type strains
jkacd-27-54-i001

*R indicates resistant; S indicates sensitive

Table 2
Special potency disk test on clinical isolates
jkacd-27-54-i002

*R indicates resistant; S indicates sensitive

Table 3
Filter paper spot test on ATCC type strains
jkacd-27-54-i003

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

Table 4
Filter paper spot test on clinical isolates
jkacd-27-54-i004

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

Table 5
Number of strains clinical isolates identified using PCR
jkacd-27-54-i005

*: total number of strains identified by PCR

Table 6
Number of strains clinical isolates identified using RapidID 32A
jkacd-27-54-i006

*: total number of strains identified by PCR

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. 20:340–349.
crossref
2. Brook I, Frazier EH, Gher ME. Aerobic and anaerobic microbiology of periapical abscess. Oral Microbiol Immunol. 1991. 6:123–125.
crossref
3. Zavistoski J, Dzink J, Onderdonk A, Bartlett J. Quantitative bacteriology of endodontic infections. Oral Surg. 1980. 49:171–174.
crossref
4. Winkelhoff V, Steenbergen TJM, Graaff J. The role of black-pigmented Bacteroides in human oral infections. J Clin Periodontol. 1998. 15:145–155.
crossref
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. 22:75–79.
crossref
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. 26:729–732.
crossref
7. Hofstad T. Pathogenicity of anaerobic gram-negative rods: possible mechanisms. Rev Infect Dis. 1984. 6:189–199.
crossref
8. Lamont RJ, Jenkinson HF. Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis. Microbiol Mol Biol Rev. 1998. 62:1244–1257.
crossref
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. 38:128–131.
crossref
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. 40:205–208.
crossref
11. Winkelhoff AJ, Steenbergen TJM, Graaff J. Porphyromonas (Bacteroides) endodontalis:Its role in endodotal infections. J Endod. 1992. 18:431–433.
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. 2:118–120.
13. Slots J, Genco RJ. Direct hemagglutination technique for differentiating Bacteroides asaccarolyticus oral strains from nonoral strains. J Clin Microbiol. 1979. 10:371–374.
crossref
14. Herweijer JA, Loos BG, Neiders ME. Characterization of total membrane proteins of Porphyromonas endodontalis. J Endod. 1992. 18:620–624.
crossref
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. 18:1017–1022.
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. 65:56–61.
crossref
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. 42:542–546.
crossref
18. Nisengard R, Newman MG. Oral microbiology and immunology. 1991. second edition. 213–216.
19. Baumgartner JC, Watkins BJ, Bae KS, Xia T. Association of black-pigmented bacteria with endodontic infections. J Endod. 1999. 25:413–417.
crossref
20. Bae KS, Baumgartner JC, Shearer TR, David LL. Occurence of Prevotella nigrescens and Prevotealls intermedia in infections of Endodontic Origin. J Endod. 1997. 23:620–623.
crossref
21. Sundqvist G, Johnasson E, Sjogren U. Prevalence of black-pigmented bacteroides species in root canal infections. J Endod. 1989. 15:13–19.
crossref
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. 27:164–167.
crossref
23. Siqueria JF, Rocas IN, Oliveira JCM, Santos KRN. Molecular detection of black-pigmented bacteria in infections of endodontic origin. J Endod. 2001. 27:563–566.
crossref
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. 5:1–10.
crossref
26. Bogen G, Slots J. Black-pigmented anaerobic rods in closed periapical lesions. Int Endod J. 1999. 32:204–210.
crossref
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. 20:S304–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. 14:416–421.
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. 27:182–184.
crossref
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. 29:1955–1958.
crossref
32. Slots J. Enzymatic characterization of some oral and nonoral gram-negative bacteria with the API ZYM system. J Clin Microbiol. 1981. 14:288–294.
crossref
33. Wayman BE, Murata SM, Almeida RJ, Fowler CB. A bacteriological and histological evaluation of 58 periapical lesions. J Endod. 1992. 18:152–155.
crossref
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. 25:S194–S198.
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