Microbiol. Immunol., 49(1), 9–16, 2005
Detection of Tannerella forsythia and/or Prevotella intermedia Might Be Useful for Microbial Predictive Markers for the Outcome of Initial Periodontal Treatment in Koreans Joong-Ki Kook1, Tomonori Sakamoto2, Kazuya Nishi2, Mi-Kwang Kim1, Jin-Hyo Seong3, Young Nam Son4, and Dong-Kie Kim*, 3 1
Department of Biochemistry, College of Dentistry, Chosun University, 375 Seo-Suk Dong, Dong-ku, Gwang-ju 501–759, Republic of Korea, 2Department of Oral Health, Okayama University Graduate School of Medicine and Dentistry, Okayama, Okayama 700–8525, Japan, 3Department of Preventive and Public Health Dentistry, College of Dentistry, Chosun University, 375 Seo-Suk Dong, Dong-ku, Gwang-ju 501–759, Republic of Korea, and 4Department of Computer Science and Statistics, College of Natural Sciences, Chosun University, 375 Seo-Suk Dong, Dong-ku, Gwang-ju 501–759, Republic of Korea Received May 13, 2004; in revised form, October 25, 2004. Accepted November 1, 2004
Abstract: A proportion of diseased sites in periodontal disease do not respond to the initial treatment, which might be due in part to the presence of specific microbial pathogens. The aim of this study was to clarify the value of microbial screening for predicting the outcome of periodontal treatment in Koreans using a polymerase chain reaction (PCR). This study enrolled 32 adults with periodontal disease. Microbial and clinical examinations were performed at the baseline and after the initial treatment (professional toothbrushing, scaling, and root planing). Subgingival plaque samples were taken from four sites in each subject (total 128 samples). PCR was used to detect the four putative pathogenic bacteria. There was an improvement in the average of each clinical measurement after the initial treatment. However, approximately half of the sites exhibiting bleeding upon probing (BOP) at the baseline still exhibited bleeding after treatment. There was a close association between the presence of BOP and the presence of Tannerella forsythia (formerly Bacteroides forsythus) and/or Prevotella intermedia. Furthermore, the sites harboring both T. forsythia and P. intermedia at the baseline had a poorer response to treatment than the sites where these two species were not detected. Therefore, microbial screening for T. forsythia and P. intermedia might be useful for predicting the treatment outcome in Koreans. Key words: Microbial marker, Periodontitis, Prevotella intermedia, Tannerella forsythia
Periodontitis refers to a collection of diseases that cause inflammation and a loss of the supporting structures of the teeth (32). Dental plaque is the major causative factor of periodontitis. The major putative pathogens known to be involved in severe periodontitis include Actinobacillus actinomycetemcomitans, Tannerrella forsythia (19, formerly Bacteroides forsythus), Porphyromonas gingivalis, Prevotella intermedia, Eikellena corrodens, Fusobacterium nucleatum, Micrococcus micros, Campylobacter rectus, and spirochetes (6, 10). Studies comparing the efficacy of different periodontal therapeutic regimens report that the sup-
pression or elimination of these bacterial species improves the clinical treatment response (11, 22, 24). Therefore, microbial monitoring for periodontal pathogenic bacteria might be useful for predicting the treatment outcome. Several putative periodontal pathogens have been detected in a number of studies using PCRs (1, 26, 32). The use of a PCR assay has resulted in large savings in time, costs, and experimental effort when compared with the other bacterial identification methods including cell culture, DNA probe methods, 16S rDNA sequencAbbreviations: Aa, Actinobacillus actinomycetemcomitans; BOP, bleeding upon probing; CAL, clinical attachment level; GI, gingival index; PCR, polymerase chain reaction; PD, probing depth; Pg, Porphyromonas gingivalis; Pi, Prevotella intermedia; PlI, plaque index; SRP, scaling and root planning; Tf, Tannerrella forsythia.
*Address correspondence to Dr. Dong-Kie Kim, Department of Preventive and Public Health Dentistry, College of Dentistry, Chosun University, 375 Seo-Suk Dong, Dong-ku, Gwang-ju, 501–759, Republic of Korea. Fax: 82–62–226–3604. E-mail:
[email protected]
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ing, ribotyping, etc. Due to the advantages of PCR, it has been widely used for both diagnosing and identifying bacterial species (37). 16S rDNA can be effectively used for PCR assays because 16S rDNA is found universally in all prokaryotic organisms and comparative analysis of 16S rDNA has shown that variable sequence regions are interspersed with highly conserved regions (32). It is generally accepted that an initial treatment including professional toothbrushing and scaling and root planning (SRP) leads to a reduction in inflammation (12, 27), which might be manifested by a reduction in BOP. The loss of attachment is more likely to occur at the periodontal sites exhibiting persistent bleeding upon probing (BOP) (14, 16). Because some sites fail to improve with this initial treatment (35), it is important to identify the appropriate risk markers in order to predict the treatment outcome. The aim of this study was to evaluate the utility of microbial detection using PCR for predicting the outcomes of non-surgical periodontal treatment using both professional toothbrushing (Toothpick method) and SRP. In this study, the sites were categorized into two groups; sites where the BOP was resolved (responding group) and sites with the BOP remaining (non-responding group) after the initial treatment. The proportions of four periodontopathogens, A. actinomycetemcomitans, T. forsythia, P. gingivalis, and P. intermedia, detected at the baseline in the two groups were subsequently compared. Materials and Methods Subject selection. Thirty-two participants (19 males and 13 females, ages ranging from 23 to 66 years (mean 42.710.2), were enrolled in this study. All the participants were new referrals to the Department of Preventive Dentistry, Chosun University Dental Hospital, Gwang-ju, Korea. The selection criteria were as follows: (a) the presence of a site of a probing depth (PD) 4 mm or more in at least two quadrants, (b) without known systemic diseases, (c) not pregnant, (d) no periodontal treatment within the last 6 months, (e) without familial aggregation, (f) with some amounts of plaque accumulation, and (g) without pronounced phagocyte abnormalities. All subject had untreated chronic adult periodontitis. None of the participants had received antibiotic therapy during the past 3 months, and did not receive antibiotic therapy during the experimental period. All the participants provided informed consent to participate in this study. Clinical measurements. The selected periodontal sites were those exhibiting the deepest PD in each
quadrant. Where more than two sites were present with the same PD in a quadrant, the site was selected randomly. The PDs were measured at six sites on each tooth (mesiobuccal, buccal, distobuccal, distolingual, lingual, and mesiodistal). One hundred and twenty eight sites from 32 participants were used as the study sites. The clinical parameters measured were as follows: the plaque index (PlI) (25), the gingival index (GI) (18), PD, clinical attachment level (CAL), and BOP. In the last case, the sites that bled within 30 sec after probing were classified as BOP. The clinical measurements were carried out both at the baseline and after the initial treatment (29). One of the authors (a well-trained periodontal clinician) carried out all the clinical examinations. PDs and clinical attachment level (CAL) were measured using a periodontal probe (Color code probe, Hu-Friedy, Ill., U.S.A.). Probing force was at 0.25 N and calibrated by an electric balance. Subgingival plaque sampling. After the clinical measurements had been performed, each site was isolated with cotton rolls and air-dried. The visible supragingival plaque was removed, and a sterile paper point was inserted into the site, which was kept there for 30 sec. The paper point was then placed immediately into a microcentrifuge tube, which contained 1 ml of 1 PBS. The tubes were well mixed using a Vortex mixer and stored at 20 C until needed. The Chosun University Institutional Research Board approved the protocols of this study. Clinical treatment. The initial treatment consisted of professional tooth brushing using the Toothpick method (20) with scaling and root planning being performed without local anesthesia. The initial treatment was completed within 1 month, and the mean number of treatments between each examination was 2.7 (SD; 1.1). All the participants received oral hygiene instructions at the baseline. Polymerase chain reaction (PCR). For DNA extraction, a 10 µl aliquot of each stored sample was added to 10 µl of a 2 lysis buffer (2 mM EDTA, 1% Triton X100). The mixture was boiled for 10 min and then placed on ice. PCR was performed using an AccuPower® PCR PreMix (Bioneer Corp., Daejeon, Korea) containing 5 nmol of each deoxynucleoside triphosphate, 0.8 µmol KCl, 0.2 µmol Tris-HCl (pH 9.0), 0.03 µmol MgCl2, and 1 unit of Taq DNA polymerase. The bacterial genomic DNA and 20 pmol of each primer were added to the PCR PreMix tube. PCR was performed in a final volume of 20 µl. Table 1 shows the species-specific primers used for the PCR amplification of the 16S rRNA genes (1, 28). The PCR temperature profiles are as follows: For T.
MICROBIAL PREDICTION OF PERIODONTAL TREATMENT OUTCOME
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Table 1. Species-specific primers used for PCR Primer pairs (5'–3') A. actinomycetemcomitans AAA CCC ATC TCT GAG TTC TTC TTC ATG CCA ACT TGA CGT TAA AT T. forsythia GCG TAT GTA ACC TGC CCG CA TGC TTC AGT GTC AGT TAT ACC T P. gingivalis AGG CAG CTT GCC ATA CTG CG ACT GTT AGC AAC TAC CGA TGT P. intermedia CAA AGA TTC ATC GGT GGA GCC GGT CCT TAT TCG AAG
forsythia and P. gingivalis, denaturing at 94 C for 1 min, annealing at 60 C for 30 sec and extension at 72 C for 1 min; and for A. actinomycetemcomitans and P. intermedia, denaturing at 94 C for 30 sec, annealing at 55 C for 1 min and extension at 72 C for 1 min. Each of these thermal cycles was performed 36 times. Prior to the first cycle, the DNA was denatured at 95 C for 2 min. Following the final cycle, the PCR product was fully extended at 72 C for 10 min. The PCR detection limits for A. actinomycetemcomitans, T. forsythia, and P. gingivalis were in the range of 25–100 cells (1) and for P. intermedia was 5 pg of genomic DNA (28). The amplified PCR products were electrophoresed on a 1.5% agarose gel in a Tris-acetate buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.0). The amplification products were stained with ethidium bromide and visualized by UV transillumination. Statistical analysis. All the clinical and microbial parameters at each site at both the baseline and after initial treatment were compared. The differences in the PlI and GI were analyzed using the Wilcoxon matchedpairs signed-ranks test. A paired t-test was used to analyze the mean values and standard deviations for the PD and CAL. The proportion of the sites with a BOP was compared using McNemar test. McNemar test was used to analyze the differences in the detection frequency of each bacterium. The selected sites were categorized into two groups according to the presence or absence of BOP: in the responding group, sites that exhibited BOP at the baseline but not after the initial treatment, while in the nonresponding group, sites that exhibited BOP both before and after treatment. The clinical measures at the baseline were averaged, and the differences between the two groups (responding and non-responding) were compared using a t-test. The number of sites harboring each specific bacterial species in the two groups was compared using a χ2 test.
Size of amplicon (bp)
Reference
557
(1)
641
(1)
404
(1)
307
(28)
All statistical analyses were performed using a statistical software package (SPSS for Windows 10.0.7J, SPSS, Inc., Tokyo). Results Comparison of Microbial and Clinical Parameters at Baseline and after Treatment There was a significant reduction in the percentage of sites testing positive for each bacterium from the baseline to after treatment (Fig. 1). The proportion of P. gingivalis-harboring sites was 91% at the baseline, which decreased to 64% after the initial treatment (P0.01). There were significant reductions in the proportion of positive sites in the case of T. forsythia (from 79 to 38%, P0.01), P. intermedia (from 52 to 32%, P0.01), and A. actinomycetemcomitans (from 23 to 13%, P0.05). The changes in the clinical parameters are shown in Table 2. The mean PlI was 1.8 at the baseline and 0.8 after the initial treatment (P0.01), the GI was reduced from 1.7 to 0.7 (P0.01), and both the mean PD and CAL were also reduced (PD, from 4.8 to 3.6 mm, P0.01; CAL, from 4.9 to 4.3 mm, P0.01). Table 3 shows the changes in the BOP at the baseline and after treatment. The number of sites exhibiting BOP was reduced from 89.8% (115/128) at the baseline to 46.9% (60/128) after the initial treatment. At the baseline, 115 sites demonstrated BOP, and after the initial treatment (responding group) only 61 out of the 115 sites (53%) showed improvement. Comparison of Responding Sites and Non-Responding Sites at Baseline Table 4 shows the differences in the clinical measures between the responding and the non-responding group. The mean PlI, PD, and CAL were similar at the baseline, and there were no statistical differences
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Fig. 1. Percentage of sites testing positive for each of the bacterial species tested at the baseline and after the treatment. at the baseline, after the initial treatment. Pg, Porphyromonas gingivalis; Tf, Tannerrella forsythia; Pi, Prevotella intermedia; Aa, Actinobacillus actinomycetemcomitans. **P0.05, **P0.01, McNemar test, n128 sites.
between the two groups. The mean GI was 1.7 for the responding group and 1.8 for the non-responding group (P0.01). The association between the presence of the four periodontopathogens at the baseline and the treatment outcome is shown in Fig 2. P. gingivalis was the most frequently detected bacterial species at the baseline in both the responding and non-responding group sites. T. forsythia was found in 74% of the responding and 89% of the non-responding group sites, P. intermedia was found in 32% of the responding and 62% of the nonresponding group sites, and A. actinomycetemcomitans was found in 21% and 22% of the responding and nonresponding group sites, respectively. Statistically significant differences in the detection rates between the responding and the non-responding groups were observed for T. forsythia (P0.05) and P. intermedia (P0.01). Influence of Coexistence of T. forsythia and P. intermedia Table 5 shows the effect of the coexistence of P.
Table 2. Clinical parameters at the baseline and after the initial treatment Clinical parameter Plaque index Gingival index Probing depth Clinical attachment level Bleeding on probing
Baseline 1.80.7 1.70.5 4.81.2 4.91.4 89.8
—**— —**— —**— —**— —**—
After treatment 0.80.7 0.70.5 3.61.2 4.31.5 46.9
Statistical analysis Wilcoxon test Wilcoxon test Paired t-test Paired t-test McNemar test
The plaque index, gingival index, probing depth, and clinical attachment levels are expressed as meanSD. Bleeding on probing is expressed as the percentage of positive sites. ** P0.01, n128. Table 3. Number of sites exhibiting BOP at the baseline and after the initial treatment
Base line
BOP () BOP ()
After initial treatment BOP () BOP () 54 61 6 7 60 68
115 13 128
At the base line, 115 sites were BOP (), and 61 out of the 115 sites showed improvement after the initial treatment. Table 4. Comparison of the clinical parameters at the baseline in the responding and non-responding groups Clinical parameter Plaque index Gingival index Probing depth Clinical attachment level a)
Respondinga) 1.60.6 1.70.5 4.81.4 5.01.5
—N.S.— —**— —N.S.— —N.S.—
Non-responding 1.60.7 1.80.8 4.81.1 5.01.2
Sites in the responding group were those not exhibiting BOP after the initial treatment. ** P0.01, N.S.: not statistically significant, t-test.
MICROBIAL PREDICTION OF PERIODONTAL TREATMENT OUTCOME
intermedia and T. forsythia at the baseline along with the treatment outcome. When both T. forsythia and P. intermedia were present at the baseline, 40.7% of the sites improved. However, 78.9% of the sites had
improved after the initial treatment when neither T. forsythia nor P. intermedia were present at the base line. There were statistically significant differences in the treatment outcome between the sites with different bac-
Fig. 2. The proportion of sites harboring the putative periodontal pathogens in the responding group (RG) and non-responding group (Non-RG). Pg, Porphyromonas gingivalis; Tf, Tannerrella forsythia; Pi, Prevotella intermedia; Aa, Actinobacillus actinomycetemcomitans. Bacteria were not detected at the baseline. Bacteria were detected at the baseline. * P0.05, ** P0.01, χ2 test, n115 sites. Table 5. Detection of T. forsythia and P. intermedia at the baseline in the responding and nonresponding groups
BOP () sites Responding groupa) (No.) Non-responding groupb) Ratio of responding site (%) a) b)
Species detected at the base line Tf () Tf () Tf () Pi () Pi () Pi () 15 22 23 4 32 16 78.9 40.7 59.0
Tf () Pi () 1 2 33.3
The number of sites in the responding group not exhibiting BOP after initial treatment. The number of sites in the non-responding group where BOP remained after initial treatment.
Table 6. Statistical differences between the sites based on the presence/absence of T. forsythia and P. intermedia at the baseline in the responding and non-responding groups in Table 5 Species detected at the base line Tf () Tf () Tf () Tf ()
Pi () Pi () Pi () Pi ()
Ratios of responding sites (%) 78.9 40.7 59.0 33.3
P valuea)
P valueb)
P valuec)
0.004d) 0.130 0.099
— — 0.080 0.799
— — — 0.830
P value based on the χ2 test, significant differences compared to the sites without both species. P value based on the χ2 test, significant differences compared to the sites with both species. c) P value based on the χ2 test, significant differences compared to the sites with T. forsythia. d) It indicates that P values of less than 0.0083 were obtained, which mean statistically significant differences between two groups. Bonferroni’s modification was used for this multiple comparison. Differences were considered as statistically significant at P0.0083. a)
b)
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terial harboring patterns (Table 6). Discussion The result showed that there is a close association between the positivity of BOP and the detection of T. forsythia and/or P. intermedia in Koreans. Furthermore, sites harboring both T. forsythia and P. intermedia at the baseline had a poorer response to treatment than those sites where these two species were not detected. Surprisingly, the presence of P. gingivalis and A. actinomycetemcomitans was not associated with the outcome of the initial periodontal treatment, which is in contrast to that observed with T. forsythia or P. intermedia. It has been reported that after periodontal therapy, the sites with undetectable levels of P. gingivalis, P. intermedia and/or A. actinomycetemcomitans show greater clinical improvement and stability than the sites that remain infected with these species (4, 33, 36). T. forsythia is a gram-negative, anaerobic, fusiform bacterium (34). The presence of T. forsythia in the subgingival flora was reported to be associated with severe periodontal disease (8, 9, 38). The virulence factors that have been identified thus far are the trypsin-like protease (7), a sialidase (13), and N-benzoyl-Val-Gly-Argp-nitroanilide-specific protease (23). In addition, the high hemolytic activity of P. intermedia might be involved in the pathogenicity of P. intermedia in the progression of periodontal disease (21). Hemoglobin or hemin resulting from the hemolytic degradation of red blood cells might be a major source of the exogenous iron for the growth of black pigmented bacteria including P. intermedia itself (17). This indicates that the detection of T. forsythia and P. intermedia might be useful for predicting the outcome of the initial periodontal treatment. BOP is generally accepted as a site-specific periodontal parameter for predicting the progression of periodontal disease. BOP can result in a loss of attachment if it is observed continually (12, 16). However, without BOP, the periodontal condition can be maintained (15). In periodontal lesions, the pocket epithelium is thin and partially destroyed (30), and these may manifest as BOP. However, BOP only reflects the current condition at the site. A more reliable predictor of future disease progression would be valuable, and the bacterial parameters may provide an important advancement in this regard. It has been reported that a non-surgical treatment decreases the levels of gingival inflammation (3, 13, 39), subgingival plaque (34), and putative pathogenic bacteria (5). The results also confirm the clinical effects of the initial treatment including professional
toothbrushing and SRP. The mean PD, CAL, GI, and PlI were reduced after the initial treatment. Approximately 90% of the examined sites exhibited BOP at the baseline, and the initial treatment reduced the proportion of sites with BOP to approximately half of that at the baseline. There was a significant reduction in the number of sites harboring P. gingivalis, P. intermedia, T. forsythia, and A. actinomycetemcomitans after the initial treatment. The clinical parameters improved and the number of the sites harboring putative periodontal pathogens was reduced. The proportion of periodontal pathogens detected in this study are in accordance with other reports, i.e. P. gingivalis, T. forsythia, and P. intermedia were frequently detected at the baseline. The detection frequency of T. forsythia at the baseline was 79%. T. forsythia has previously been detected at a high frequency in the BOP positive sites (31), which was confirmed in the present study. The proportion of sites harboring A. actinomycetemcomitans was approximately 23% at the baseline, while Choi et al. (2) reported that 74% of the sites harbored A. actinomycetemcomitans in Korean adults with periodontitis. One explanation for the discrepancy in the A. actinomycetemcomitans detection rate between the studies may be the differences in the detection methods employed. In conclusion, this study confirmed the effect of the initial periodontal treatment on the clinical and microbial parameters. In addition, monitoring both T. forsythia and P. intermedia using PCR might be useful for predicting the outcome of the initial periodontal treatment using professional toothbrushing and SRP in Koreans. This study was supported by research funds from Chosun University, 2003.
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