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Yoon, Kim, Song, Kim, Lee, and Kim: Detection of Bacterial Species in Chronic Periodontitis Tissues at Different Stages of Disease Severity
Communication

J Bacteriol Virol 2015;45(4):364-371.

Published online: 9 February 2015

DOI: https://doi.org/10.4167/jbv.2015.45.4.364

Detection of Bacterial Species in Chronic Periodontitis Tissues at Different Stages of Disease Severity

Da-Le Yoon1, §, Shukho Kim2, §, Hyesoon Song2, Yong-Gun Kim1, Jae-Mok Lee1, ∗, Jungmin Kim2, ∗

1Department of Periodontology, Kyungpook National University School of Dentistry, Daegu, Korea

2Department of Microbiology, Kyungpook National University School of Medicine, Daegu, Korea

∗Corresponding author: Jungmin Kim. Department of Microbiology, Kyungpook National University School of Medicine, Daegu 41944, Korea. Phone: +82-53-420-4845, Fax: +82-53-427-5664, e-mail: minkim@knu.ac.kr

∗Corresponding author: Jae-Mok Lee. Department of Periodontology, Kyungpook National University School of Dentistry, Daegu 41940, Korea. Phone: +82-53-600-7522, Fax: +82-53-427-3263, e-mail: leejm@knu.ac.kr

§ These authors equally contributed to this work.

∗∗ This research was supported by Kyungpook National University Research Fund (2012) and MEDIM foundation (2011).

Received 16 October 201527 October 2015       Accepted 30 October 2015

Copyright © 2015 Journal of Bacteriology and Virology

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.

Abstract

The goal of this research was to determine the relationship between the stage of chronic periodontitis and the presence of six bacterial pathogens (Aggregatibacter actinomycetemcomitans: AA, Fusobacterium nucleatum: FN, Porphyromonas gingivalis: PG, Prevotella intermedia: PI, Enterococcus faecalis: EF, and Parvimonas micra: PM). Forty-six chronic periodontitis patients visiting a dental hospital were included in this investigation. They were classified into four chronic periodontitis stages based on the sulcus bleeding index value and the probing depth. The tissue samples from the periodontal surgery were used for a direct PCR detection assay. A total of 49 samples from 46 patients were collected and classified into four chronic periodontitis groups (N: 6, P1: 13, P2: 18, P3: 12). The PCR assay showed that FN, PI, and PM were involved from the beginning of chronic periodontitis (P1), while AA and PG existed regardless of the disease stages. EF was strongly linked to the P3 stage of the disease. In order to assess the effect of dental treatments on patients with chronic periodontitis, EF should be a critical marker for P3 patients, while FN, PI, and PM would be good indicators for chronic periodontitis.

Keywords: Chronic periodontitis, Oral bacteria, PCR detection

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Table 1.
Primers used in this study
Primer name Primer sequence Target pathogen1 Amplicon size (bp)
AA F3 5′-TGCGTAGAGATGTGGAGGAA-3′ AA 165
AA B3 5′-GGCGGTCGATTTATCACGT-3′ AA 165
FN F3 5′-AGGCGATGATGGGTAGCC-3′ FN 214
FN B3 5′-AGCCGTCACTTCTTCTGTTG-3′ FN 214
PG F3 5′-GGTAAGTCAGCGGTGAAACC-3′ PG 218
PG B3 5′-GCGTGGACTACCAGGGTAT-3′ PG 218
PI F3 5′-ACGGCCTAATACCCGATGTT-3′ PI 193
PI B3 5′-CTGCCTCCCGTAGGAGTT-3′    
EF16s-F 5′-CGCTTCTTTCCTCCCGAGT-3′ EF 142
EF16s-R 5′-GCCATGCGGCATAAACTG-3′    
PM16s-F 5′-TCGAACGTGATTTTTGTGGAAA-3′ PM 85
PM16s-R 5′-GGTAGGTTGCTCACGTGTTACTCA-3′ PM 85

1 AA: Aggregatibacter actinomycetemcomitans, FN: Fusobacterium nucleatum, PG: Porphyromonas gingivalis, PI: Prevotella intermedia, EF: Enterococcus faecalis, PM: Parvimonas micra

Table 2.
PCR detection results of 49 samples including their clinical information
Sample number Sampling data (yyyy.mm.dd) Sex1/Age Chronic periodontitis2 AA3 FN PG PI EF PM
1 2014.05.30 M/54 P2 ＋4 ＋ ＋ ＋ - ＋
2 2014.04.02 F/45 P2 ＋ ＋ ＋ ＋ - ＋
3 2014.03.18 M/52 P1 ＋ ＋ ＋ ＋ - ＋
4 2014.04.21 F/53 N ＋ - ＋ - - -
5 2014.03.25 M/48 P2 ＋ ＋ ＋ ＋ - ＋
6 2014.04.09 F/18 N ＋ ＋ ＋ - - -
7 2014.06.13 M/60 P2 ＋ ＋ ＋ - - ＋
8 2014.06.13 F/57 P2 ＋ ＋ ＋ ＋ - ＋
9 2014.05.16 M/58 P1 ＋ ＋ ＋ ＋ - -
10 2014.01.28 M/52 P2 ＋ ＋ ＋ ＋ - ＋
11 2014.06.27 M/42 P2 ＋ ＋ ＋ - - -
12 2014.04.09 F/68 N ＋ ＋ ＋ - - ＋
13 2014.04.03 M/35 P2 ＋ ＋ ＋ - - ＋
14 2014.04.25 M/51 P2 ＋ ＋ ＋ ＋ ＋ ＋
15 2014.04.02 F/59 P2 ＋ ＋ ＋ ＋ - ＋
16 2014.03.07 M/39 N ＋ ＋ ＋ ＋ - ＋
17 2014.06.16 M/54 P2 ＋ ＋ ＋ ＋ - ＋
18 2014.02.24 F/42 P2 ＋ ＋ ＋ ＋ - ＋
19 2014.04.16 M/53 N - - - - - -
20 2014.05.09 F/48 N ＋ ＋ ＋ - - -
21 2014.02.07 M/60 P2 ＋ - ＋ - - -
22 2014.04.11 F/64 P2 ＋ ＋ ＋ ＋ - ＋
23 2014.04.07 M/49 P1 ＋ ＋ ＋ - - -
24 2014.03.19 F/43 P2 ＋ - - - - -
25 2014.03.28 M/46 P3 ＋ ＋ ＋ ＋ ＋ ＋
26 2014.06.16 F/53 P3 ＋ ＋ ＋ ＋ - ＋
27 2014.04.11 F/51 P3 ＋ ＋ ＋ ＋ - ＋
28 2014.04.11 F/35 P3 ＋ ＋ ＋ - - ＋
29 2014.06.17 F/53 P3 ＋ ＋ ＋ ＋ ＋ ＋
30 2014.04.07 F/59 P3 - - - - ＋ ＋
31 2014.04.08 M/53 P3 ＋ ＋ ＋ ＋ ＋ ＋
32 2014.06.20 M/45 P3 ＋ ＋ ＋ ＋ ＋ ＋
33 2014.03.14 F/52 P3 + ＋ ＋ ＋ ＋ ＋
34 2014.04.07 F/42 P3 ＋ ＋ ＋ ＋ ＋ ＋
35 2014.04.02 F/55 P3 ＋ ＋ ＋ ＋ ＋ ＋
36 2014.04.21 F/47 P3 ＋ ＋ ＋ ＋ ＋ ＋
37 2014.05.09 M/49 P1 ＋ ＋ ＋ ＋ - ＋
38 2014.03.28 M/61 P1 ＋ ＋ ＋ ＋ - ＋
39 2014.04.03 M/47 P1 ＋ ＋ ＋ ＋ - ＋
40 2014.06.10 F/55 P1 ＋ ＋ ＋ - - ＋
41 2014.04.30 F/46 P1 - ＋ ＋ ＋ - ＋
42 2014.04.11 F/51 P1 ＋ ＋ ＋ ＋ - ＋
43 2014.02.28 M/38 P1 ＋ ＋ ＋ ＋ - ＋
44 2014.03.26 M/48 P2 ＋ ＋ ＋ - - ＋
45 2014.06.09 M/54 P1 ＋ ＋ ＋ ＋ - ＋
46 2014.04.28 F/48 P1 ＋ ＋ ＋ ＋ - ＋
47 2014.04.02 M/37 P2 ＋ ＋ ＋ ＋ - ＋
48 2014.04.08 M/48 P2 - ＋ ＋ ＋ - ＋
49 2014.04.11 M/52 P1 ＋ ＋ ＋ ＋ - ＋

1 M: male, F: female

2 Refer Materials & Methods for the abbreviation

3 AA: Aggregatibacter actinomycetemcomitans, FN: Fusobacterium nucleatum, PG: Porphyromonas gingivalis, PI: Prevotella intermedia, EF: Enterococcus faecalis, PM: Parvimonas micra

4 ＋: PCR positive, -: PCR negative.

Table 3.
Detection of six pathogenic bacteria from dental tissue samples by PCR
Bacteria1 No. of PCR positive samples/No. of samples used for PCR (%)
Sample groups classified by clinical measurement
N, Normal gingiva P1, Mild chronic periodontitis (PAL2 ≤ 2 mm) P2, Moderate chronic periodontitis (PAL ≤ 4 mm) P3, Severe chronic periodontitis (PAL > 4 mm)
AA 5/6 (83.3) 12/13 (92.3) 17/18 (94.4) 11/12 (91.6)
FN 4/6 (66.7) 13/13 (100) 15/18 (83.3) 11/12 (91.6)
PG 5/6 (83.3) 13/13 (100) 16/18 (88.8) 11/12 (91.6)
PI 1/6 (16.7) 11/13 (84.6) 12/18 (66.7) 10/12 (83.3)
EF 0/6 (0) 0/13 (0) 1/18 (5.5) 9/12 (75.0)
PM 2/6 (33.3) 12/13 (92.3) 15/18 (83.3) 12/12 (100)

1 AA: Aggregatibacter actinomycetemcomitans, FN: Fusobacterium nucleatum, PG: Porphyromonas gingivalis, PI: Prevotella intermedia, EF: Enterococcus faecalis, PM: Parvimonas micra

2 PAL: probe attachment level.

Table 4.
Result of statistical analysis to determine the association of six bacteria with the states of chronic periodontitis
Bacteria1 P value
N versus P1 P1 versus P2 P2 versus P3
AA 0.554 0.811 0.765
FN 0.028∗ 0.121 0.511
PG 0.130 0.214 0.803
PI 0.004∗ 0.259 0.311
EF - 0.387 0.0000772∗
PM 0.007∗ 0.462 0.136

1 AA: Aggregatibacter actinomycetemcomitans, FN: Fusobacterium nucleatum, PG: Porphyromonas gingivalis, PI: Prevotella intermedia, EF: Enterococcus faecalis, PM: Parvimonas micra

∗ Statistical significance (p < 0.05).

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