Comparison of culture and polymerase chain reaction techniques in the identification of Tannerella forsythia in periodontal health and disease, an in vitro study.

BACKGROUND AND OBJECTIVES: Various bacterial species from subgingival biofilm have demonstrated aetiological relevance in the initiation and progression of periodontitis. The aim of this study was to detect the presence of Tannerella forsythia (Tf) in subgingival plaque of periodontally healthy subjects and chronic periodontitis patients by using both culture and PCR technique and compare the two techniques.
MATERIALS AND METHODS: Pooled subgingival plaque samples were taken using sterile curettes from predetermined sites in 50 periodontally healthy subjects and from 50 periodontitis subjects. Samples were analyzed for the presence of T. forsythia using both techniques. Statistical analysis of the results was done using Chi-square test, sensitivity, and specificity tests.
RESULTS: Both techniques could detect T. forsythia in subgingival plaque samples from healthy and periodontitis subjects. Periodontally healthy individuals and individuals with chronic periodontitis using the culture technique showed the presence of T. forsythia in 14 and 34%, respectively. PCR technique showed the presence of T. forsythia in 20% healthy and 40% chronic periodontitis patients. T. forsythia detection in the periodontitis group was statistically significantly higher when compared to the healthy group by both culture and PCR technique (P = 0.019 and P = 0.029). PCR demonstrated high sensitivity and low specificity when compared to the culture technique.
CONCLUSION: The results indicated that T. forsythia was more prevalent in periodontitis patients when compared with healthy subjects. The PCR was found to be more sensitive than culture technique for detection of T. forsythia from the subgingival plaque samples.

INTRODUCTION

The oral ecosystem accommodates various species of organisms from birth to death, in health and disease. Many of these have been implicated in the pathogenesis of periodontal disease. The search is on to investigate the pathological potential of various bacteria responsible for periodontal diseases. One such organism has been Tannerella forsythia (Tf), a gram-negative anaerobic rod with tapered ends, described as fusiform bacteroides in one of the culture report of progressing advanced periodontitis.[1]

Past difficulties with identification and culture of T. forsythia have probably been the main reasons why this organism has not been as extensively studied as other putative periodontal pathogens.

We researched to unearth data on the role of Tf in periodontal disease in Indian population. We were astonished to note that very little published data were available to associate T. forsythia in periodontal diseases in the Indian population. This prompted us to undertake a study to detect the presence of Tannerella forsythia in periodontal health and disease using culture and the PCR technique and compare the two techniques of detection.

MATERIALS AND METHODS

A total of 100 subjects were grouped in two groups of 50 each based on the recordings of the following parameters. Plaque index,[2] Gingival index,[2] Gingival Bleeding Index,[34] Probing Pocket Depth (PD) and Clinical Attachment Level (CAL). Study population by diagnosis, number, and gender are shown in Table 1.

Table 1

Distribution of number and gender of study population

Periodontally healthy subjects showed no signs of gingival inflammation or bleeding on probing, probing depths being ≤3 mm and showed no clinical attachment loss. The periodontal disease group on the other hand included criteria like the presence of bleeding on probing, PD ≥ 5mm and CAL ≥ 3 mm. Patients on any antibiotics within 3 months prior to this study, periodontal treatment within 3 months prior to this study were excluded. We also excluded patients with any systemic diseases or conditions, smokers, pregnant, and lactating women. Written informed consent was obtained from each subject. The study was approved by the Ethical committee of Maratha Mandal's N.G.H. Institute of Dental Sciences and Research Centre, Belgaum. Pooled subgingival plaque samples were collected using sterile curettes and transported in 1 mL of reduced transport fluid (RTF). Culture and PCR procedures were carried out in the Laboratory of Molecular Biology and Immunology at Maratha Mandal's NGH Institute of Dental Sciences and Research Centre.

Anaerobic culture

Samples were vortexed for 5 min and serially diluted 10-fold in RTF; 100 μl of each dilution was plated in media containing blood agar with N-acetyl muramic acid (NAM) disks (10 mg/l). The blood agar plates were studied after 5 to 7 days of anaerobic incubation (80% N2; 10% H2; 10% CO2 at 37°C) [Figure 1]. Plates were carefully examined for the identification of T. forsythia based on the morphology of the colony, grams reaction [Figure 2] and using different standard biochemical tests (Indole, Catalase and Nitrate reductase test) to confirm the initial identification.[5]

Figure 1

Growth on blood agar plate showing T. forsythia adjacent to NAM disks

Figure 2

Gram's stain showing pleomorphic Gram negative bacilli of T. forsythia

PCR detection

DNA extraction

All samples were diluted in 200 μl of fresh Tris EDTA buffer, and vortexed for 1 min. 100 μl of lysis buffer II, 10 μl of proteinase K was added. The samples were kept in a water bath at 65°C for 2 h then in boiling water bath for 10 min. The DNA was stored at −20°C (deep freeze) until required for PCR analysis.

PCR assay

A conventional PCR method was used. The PCR was processed using 3.0 μl of sample (DNA template) added to 22 μl of reaction mixture containing reaction buffer, dNTP (deoxy nucleotide triphosphate), Taq DNA polymerase enzyme, primer, and molecular grade water. The primer used in this study is listed in Table 2.[6] Purified genomic DNA from T. forsythia (B. forsythus ATCC 43037) was used as a positive control and distilled water as a negative control. Preparations were amplified in a DNA thermocycler.

Table 2

Nucleotide sequences of PCR primers used for identification of T. forsythia

PCR cycling conditions for amplification using primers specific for T. forsythia included an initial denaturation step at 95°C for 2 min, followed by 36 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 1 min, an extension at 72°C for 1 min, and a final elongation step at 72°C for 2 min.[7] The amplicons were separated by Gel electrophoresis and viewed under UV transillumination.

Statistical analysis

A statistical software program, SPSS 9 was used for data analysis. The results were expressed as being either positive or negative. The Chi-square test was used to compare the detection percentages of T. forsythia in both healthy and those with periodontitis subjects, with a P < 0.05 defining significance. The techniques (Culture and PCR) were compared using sensitivity and specificity tests.

RESULTS

The prevalence of T. forsythia in the subgingival plaque samples, from both periodontally healthy and chronic periodontitis subjects, by culture technique detected Tf in only 14% of healthy as against 34% of chronic periodontitis subjects [Graph 1]. By the culture technique, the Tf detection in the periodontitis group was statistically significantly higher when compared to the healthy group (P = 0.019) as shown in Table 3.

Graph 1

Comparison of prevalence of T. forsythia in the subgingival plaque samples from healthy periodontium and chronic periodontitis by culture technique

Table 3

Comparison of prevalence of T. forsythia in the subgingival plaque samples from both periodontally healthy subjects and chronic periodontitis patients by culture technique

The prevalence of Tf by PCR technique detected Tf in only 20% of healthy as against 40% of chronic periodontitis subjects [Graph 2]. By the PCR technique, the Tf detection in the periodontitis group was statistically significantly higher when compared to the healthy group (P = 0.029) as shown in Table 4. Figure 3 showing ethidium bromide stained PCR products after gel electrophoresis.

Graph 2

Comparison of prevalence of T. forsythia in the subgingival plaque samples from healthy periodontium and chronic periodontitis by PCP technique

Table 4

Comparison of prevalence of T. forsythia in the subgingival plaque samples from both periodontally healthy subjects and chronic periodontitis patients by PCR technique

Figure 3

T. forsythia detected using PCR technique at 641bp. No amplification is seen in lane 1. DNA bands in lane 2 and 3 showing successful amplification of the target sequence. The gel also contains positive control (lane 4) (purified genomic DNA from T. forsythia ATCC 43037), negative control (lane 6)(distilled water) and a DNA ladder containing DNA fragments of defined length (100bp) for sizing the bands in the experimental PCR (lane 5)

Table 5 shows the comparative results regarding the detection of T. forsythia with both culture and PCR technique. Overall, 11 were PCR positive and culture negative, while 5 were culture positive and PCR negative. Of the total samples 65 were negative for both techniques, 19 positive in both technique.

Table 5

Comparison of detection of T. forsythia by PCR and culture in both healthy and periodontitis subjects

Table 6 describes sensitivity and specificity of culture technique using PCR as standard reference for the detection of Tf in subgingival plaque samples. The sensitivity of culture in healthy subjects was 60%, while the specificity, positive predictive value, and negative predictive values were 97.5, 85.7, and 90.6%, respectively. For periodontitis patients the sensitivity for culture technique was 65.0%, while the specificity, positive predictive value, and negative predictive values were 86.6, 76.4, and 78.7%, respectively.

Table 6

Sensitivity and specificity of culture technique using PCR as standard reference for the detection of T. forsythia in subgingival plaque samples

Table 7 describes sensitivity and specificity of PCR technique using Culture as standard reference for the detection of Tf in subgingival plaque samples, the sensitivity of PCR in healthy subjects was 85.7%, while the specificity, positive predictive value and negative predictive values were 90.6, 60.0, and 97.5%, respectively. For periodontitis patients the sensitivity was 76.4%, while the specificity, positive predictive value, and negative predictive values were 78.7, 65.0, and 86.6%, respectively.

Table 7

Sensitivity and specificity of PCR technique using Culture as standard reference for the detection of T. forsythia in subgingival plaque samples

DISCUSSION

The composition of bacterial plaque has been studied for many decades using various microbiological and molecular techniques, ranging from morphologic detection, cultivation to molecular probe analysis.

Results from this study have shown that T. forsythia was successfully detected both in periodontally healthy individuals and in chronic periodontitis patients by both the detection techniques (Culture and PCR).

Various researchers have reported difficulty in cultivation of T. forsythia in culture media.[8910] In one of the recent study conducted by Catalina Suzana Stingu, T. forsythia was not detected in any of the samples in periodontally healthy subjects by culture method.[11] Yano Higuchi in their study showed only 1.7% of the patients with culture positive results from periodontally healthy subjects.[12] Another study conducted by Laura Lau and Sanz, showed that only 3.3% of healthy individuals showed the presence of T. forsythia by culture method.[1314] The results of the above stated studies project a low prevalence of T. forsythia in healthy subjects. In our study, 14% of the periodontally healthy patients showed the presence of T. forsythia by the culture technique. This could be attributed to the fact that N-acetyl muramic acid, an essential nutrient for the growth of T. forsythia was incorporated in the culture plates by us as against the above mentioned authors who had not incorporated this nutrient in their cuture media. Our results are in agreement with Wyss,[1] who in his study showed the dependence of proliferation of Bacteroides forsythus on exogenous N-acetylmuramic acid. Kamma, Nakou and Manti as well, in there study showed that 24% of periodontally healthy patients had T. forsythia in their plaque.[15]

If a bacterial species is considered as a periodontal pathogen, it is expected to exist in most individuals with periodontal disease and infrequently detected in subjects with a healthy periodontium. The results of the present study showed a higher prevalence of T. forsythia in chronic periodontitis group than in the periodontally healthy group detected by culture method [Table 3] confirming the findings of other studies.[1216] In our study, when the presence of T. forsythia in chronic periodontitis was evaluated by culture technique, it demonstrated a prevalence of 34.0% in periodontitis patients. This is comparable to the study by Verner et al. where 33.3% T. forsythia was detected by them.[17] High prevalence of this pathogen (51%) has been reported using bacterial culture in other studies done by Jervoe Storm et al.[18] In a study conducted by Laura Lau et al. only 25% of the periodontitis patients were detected positive for T. forsythia by culture.[13]

Comparison of the microbiologic results of the present study with those from other studies is complicated by the fact that different methods were applied in each study and that the age range varied between studies.

PCR detected T. forsythia more frequently than culture technique. These findings confirm those from previous studies.[1819] In the present study 20% of the periodontally healthy subjects showed the presence of T. forsythia by PCR technique. This was comparable with the results of Narayanan et al. (2005),[7] and Catalina et al., (2012),[11] in which 25 and 15% of periodontally healthy subjects showed the presence of T. forsythia by PCR technique. Tan et al. in their study reported 45% of the healthy sites were positive for T. forsythia.[20] This could be due to the use of multiplex PCR in their study. Choi et al. showed the presence of T. forsythia in 55% of healthy subjects.[21] The difference in their numbers in comparison to that of our results could be due to identification technique using nucleic acid base approach rather than PCR.

In the present study 40% of the chronic periodontitis patients showed the presence of T. forsythia by PCR technique [Table 4]. This was comparative with a recent studies conducted by Abiko et al. (2010),[22] and Boyanova et al. (2009),[23] in which T. forsythia was detected by PCR in 43% and 44.4% of chronic periodontitis patients, respectively. The similarity between our study and that conducted by Boyanova et al. was the nucleotide sequences of PCR primers used for the identification of T. forsythia.

When reviewing the scientific literature, the evaluation of T. forsythia prevalence results in periodontitis patients using PCR technology points to variable results. A high prevalence has been reported in Singapore 91%,[20] in the USA between 84.5 and 100%,[2425] and in Ireland 78%.[26] The wide variation in results may be attributed to patient and method selection, different bacterial serotypes and true geographical differences. Studies with a uniform design need to be conducted globally in order to understand the true prevalence and geographical distribution of this bacterial species. Only limited data are available on the subgingival microbial composition of periodontitis subjects in developing countries.[27] Till date, we found very little published data available to associate T. forsythia in health and periodontal diseases in the Indian population. To the best of our knowledge, this could well be one of the first studies that specifically investigated, the prevalence of T. forsythia in the subgingival plaque samples isolated from healthy subjects and periodontitis patients in India.

The overall results of our study have shown that the conventional PCR detection method identified T. forsythia in health and disease, with a high degree of sensitivity when compared with traditional culture technique. Comparing both procedures, PCR yielded a significantly higher prevalence than culture in both periodontally healthy individuals and periodontitis patients. This could be explained by the lower detection threshold of PCR which is a highly sensitive diagnostic tool. The comparative low detection of T. forsythia by the culture technique could be attributed to the fastidious growth requirements of this bacterial species.

The significant difference between PCR and culture was also demonstrated when comparing counts of the total T. forsythia positive samples [Table 5]. Overall, in five patients a positive culture result was associated with a negative PCR. This could be justifiable, considering the inhibition of PCR reaction may occur due to the presence of inhibitory substances in plaque samples.[28] It could also be attributed to contamination of samples and PCR reagents with irrelevant DNA from various sources. Error may also occur during sampling, DNA extraction/isolation, template amplification and visualization of the results. On the other hand, in 11 patients a positive PCR result was associated with a negative culture analysis. This can be explained by the fact that culture has lower detection capability than PCR.[29] It has been stated before and we ascertain the fact that T. forsythia has stringent growing conditions and hence a difficult microorganism to culture.

When comparing, the diagnostic techniques used in our study, the PCR demonstrated high sensitivity (79.1%) against culture technique (sensitivity of 63.3%). The PCR showed low specificity (85.5%) compared to culture with specificity of 92.8%. This supports the observations done by Laura Lau et al. (2004),[13] Jervoe et al. (2005),[18] who compared culture with PCR for detection of T. forsythia in subgingival plaque samples. Their studies also demonstrated high sensitivity and low specificity for PCR technique.

In summary of the results discussed, Tannerella forsythia was successfully detected in subgingival plaque samples of periodontally healthy individuals and individuals with chronic periodontitis using both culture and the PCR technique. T. forsythia detection in the periodontitis group was statistically significantly higher when compared to the healthy group by both culture and PCR technique (P = 0.019 and P = 0.029). When the comparisons were made between PCR and culture techniques, PCR showed that it is a highly sensitive (79.1%) technique for detection of T. forsythia than the culture technique with 63.3% sensitivity. The high sensitivity of PCR technology justifies its use in the studies of periodontal microflora. However, it is deemed important to state, that PCR and other molecular diagnostic assays cannot replace the traditional culture technique. The major advantages of the culture technique, to name a few, are its capability to detect multiple bacterial species coincidentally, to detect unexpected bacteria, characterize microbiota, as well as allow the determination of antibiotic resistance. This study has been a small effort on our part to investigate the role of two diagnostic methods in the identification of T.forsythia in a small section of the Indian population.