J Korean Acad Conserv Dent.  2002 Jan;27(1):54-65. 10.5395/JKACD.2002.27.1.054.

Detection of black-pigmented bacteria in infected root canals

• Kwon EK
• Kim ES
• Kwak JS
• Lee H
• Lee SJ
• Im MK

• KMID: 2175548
• DOI: http://doi.org/10.5395/JKACD.2002.27.1.054

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.

MeSH Terms

Agar
Bacteria
Brucella
Dental Pulp Cavity
Genes, rRNA
Kanamycin
Periapical Periodontitis
Polymerase Chain Reaction
Porphyromonas endodontalis
Porphyromonas gingivalis
Prevotella intermedia
Prevotella nigrescens
Tooth
Virulence Factors
Agar
Kanamycin
Virulence Factors

Figure

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  Fig. 1 Primary culture sample

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  Fig. 2 Specific potency disk test of P. gingivalis(ATCC 33277) P. gingivalis was resistant to kanamycin and colistin, and sensitive to vancomycin.

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  Fig. 3 Specific disk potency test of P. intermedia(ATCC 25611) P. intermedia was resistant to kanamycin and vancomycin, and sensitive to colistin.

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  Fig. 4 Specific potency disk test of P. nigrescens(ATCC 33563) P. nigrescens was resistant to kanamycin and vancomycin, and sensitive to colistin.

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  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.

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  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.

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  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)

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  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)

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  Fig. 9 RapidID 32A test result of clinical isolates

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