Detection of black-pigmented bacteria in infected root canals

Eun-Kyoung Kwon; Eun-Sook Kim; Ju-Seog Kwak; Hwang Lee; Su-Jong Lee; Mi-Kyung Im

doi:10.5395/JKACD.2002.27.1.054

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Original Article Detection of black-pigmented bacteria in infected root canals: Eun-Kyoung Kwon, Eun-Sook Kim, Ju-Seog Kwak, Hwang Lee, Su-Jong Lee, Mi-Kyung Im; Journal of Korean Academy of Conservative Dentistry 2002;27(1):54-65.
DOI: https://doi.org/10.5395/JKACD.2002.27.1.054
Published online: January 31, 2002

Department of Conservative Dentistry, College of Dentistry, Wonkwang University, Korea.

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Abstract
REFERENCES

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.

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Fig. 1

Primary culture sample

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

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

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

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

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

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

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

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

RapidID 32A test result of clinical isolates

Table 1

Special potency disk test on ATCC type strains

^*R indicates resistant; S indicates sensitive

Table 2

Special potency disk test on clinical isolates

^*R indicates resistant; S indicates sensitive

Table 3

Filter paper spot test on ATCC type strains

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

Table 4

Filter paper spot test on clinical isolates

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

Table 5

Number of strains clinical isolates identified using PCR

^*: total number of strains identified by PCR

Table 6

Number of strains clinical isolates identified using RapidID 32A

^*: total number of strains identified by PCR

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Detection of black-pigmented bacteria in infected root canals

J Korean Acad Conserv Dent. 2002;27(1):54-65. Published online January 31, 2002

DOI: https://doi.org/10.5395/JKACD.2002.27.1.054

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Figure

Detection of black-pigmented bacteria in infected root canals

Fig. 1 Primary culture sample

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

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 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 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 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 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 RapidID 32A test result of clinical isolates

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Detection of black-pigmented bacteria in infected root canals

Table 1

Special potency disk test on ATCC type strains

^*R indicates resistant; S indicates sensitive

Table 2

Special potency disk test on clinical isolates

^*R indicates resistant; S indicates sensitive

Table 3

Filter paper spot test on ATCC type strains

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

Table 4

Filter paper spot test on clinical isolates

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

Table 5

Number of strains clinical isolates identified using PCR

^*: total number of strains identified by PCR

Table 6

Number of strains clinical isolates identified using RapidID 32A

^*: total number of strains identified by PCR

Table 1 Special potency disk test on ATCC type strains

^*R indicates resistant; S indicates sensitive

Table 2 Special potency disk test on clinical isolates

^*R indicates resistant; S indicates sensitive

Table 3 Filter paper spot test on ATCC type strains

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

Table 4 Filter paper spot test on clinical isolates

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