Original Article

Comparison of culture and polymerase

chain reaction techniques in the
identification of Tannerella forsythia
in periodontal health and disease, an
in vitro study
Praveen Kumar Bankur, Aarati Nayak,1 Kishore Bhat,1 Rashmi Bankur,2
Reshma Naik,3 Nami Rajpoot1

Departments of Abstract:
Periodontology, Guru Background and Objectives: Various bacterial species from subgingival biofilm have demonstrated
Gobind Singh College aetiological relevance in the initiation and progression of periodontitis. The aim of this study was to detect
of Dental Sciences the presence of Tannerella forsythia (Tf) in subgingival plaque of periodontally healthy subjects and
and Research Centre, chronic periodontitis patients by using both culture and PCR technique and compare the two techniques.
Burhanpur, Madhya 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
Pradesh, 1Maratha
presence of T. forsythia using both techniques. Statistical analysis of the results was done using Chi‑square
Mandal’s NGH Institute test, sensitivity, and specificity tests. Results: Both techniques could detect T. forsythia in subgingival plaque
of Dental Sciences samples from healthy and periodontitis subjects. Periodontally healthy individuals and individuals with chronic
and Research Centre, periodontitis using the culture technique showed the presence of T. forsythia in 14 and 34%, respectively. PCR
Belgaum, 2Department technique showed the presence of T. forsythia in 20% healthy and 40% chronic periodontitis patients. T. forsythia
of Oral Pathology, detection in the periodontitis group was statistically significantly higher when compared to the healthy group by
M. R. Ambedkar Dental both culture and PCR technique (P = 0.019 and P = 0.029). PCR demonstrated high sensitivity and low specificity
College and Hospital, 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
3
Sathyadeep Dental
culture technique for detection of T. forsythia from the subgingival plaque samples.
Clinic, Bangalore,
Key words:
Karnataka, India
Culture technique, polymerase chain reaction, Tannerella forsythia
Access this article online
Website:
www.jisponline.com
INTRODUCTION data were available to associate T.  forsythia in
DOI: periodontal diseases in the Indian population.
10.4103/0972-124X.131312
Quick Response Code: T he oral ecosystem accommodates various
species of organisms from birth to death,
in health and disease. Many of these have been
This prompted us to undertake a study to detect
the presence of Tannerella forsythia in periodontal
health and disease using culture and the PCR
implicated in the pathogenesis of periodontal technique and compare the two techniques of
disease. The search is on to investigate the detection.
pathological potential of various bacteria
responsible for periodontal diseases. One such MATERIALS AND METHODS
organism has been Tannerella forsythia  (Tf),
a gram‑negative anaerobic rod with tapered A total of 100 subjects were grouped in two
Address for
correspondence: ends, described as fusiform bacteroides in one groups of 50 each based on the recordings of the
Dr. Praveen Kumar Bankur, of the culture report of progressing advanced following parameters. Plaque index,[2] Gingival
Department of periodontitis.[1] index,[2] Gingival Bleeding Index,[3,4] Probing
Periodontics, Guru Gobind Pocket Depth (PD) and Clinical Attachment Level
Singh College of Dental Past difficulties with identification and culture of (CAL). Study population by diagnosis, number,
Sciences and Research T. forsythia have probably been the main reasons and gender are shown in Table 1.
Centre, Burhanpur, why this organism has not been as extensively
Madhya Pradesh - 450 331,
studied as other putative periodontal pathogens. Periodontally healthy subjects showed no
India. E‑mail:
praveenbankur@gmail.com signs of gingival inflammation or bleeding
We researched to unearth data on the role of Tf on probing, probing depths being  ≤3  mm
Submission: 16-08-2013 in periodontal disease in Indian population. We and showed no clinical attachment loss. The
Accepted: 28‑10‑2013 were astonished to note that very little published periodontal disease group on the other hand
Journal of Indian Society of Periodontology - Vol 18, Issue 2, Mar-Apr 2014 155
 Bankur, et al.: Identification of Tannerella forsythia using culture and PCR technique

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]
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
Table 1: Distribution of number and gender of study
population
Groups Male % Female % Total
Healthy 22 44.00 28 56.00
Chronic periodontitis 27 54.00 23 46.00
Total 49 98.00 51 102.00
as a negative control. Preparations were amplified in a DNA
thermocycler.
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.
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.
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
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
Groups Absent % Present % Total
Healthy 43 86.00 7 14.00 50
Chronic periodontitis 33 66.00 17 34.00 50
50 Total 76 76.00 24 24.00 100
50 Chi‑square=5.4823, df=1, P=0.01921*
100

Table 4: Comparison of prevalence of T. forsythia in the

Table 2: Nucleotide sequences of PCR primers used for subgingival plaque samples from both periodontally
identification of T. forsythia healthy subjects and chronic periodontitis patients by
Species Sequence Base position PCR technique
(amplicon lengths, bp) Groups Absent % Present % Total
T.forsythia 5 GCG TAT GTA 641 bp Healthy 40 80.00 10 20.00 50
(B.forsythus) ACC TGC CCG CA 3 Chronic periodontitis 30 60.00 20 40.00 50
5 TGC TTC AGT GTC Total 70 70.00 30 30.00 100
AGT TAT ACCT 3
Chi‑square=4.7623, df=1, P=0.02910*. PCR – Polymerase chain reaction

156 Journal of Indian Society of Periodontology - Vol 18, Issue 2, Mar-Apr 2014
 Bankur, et al.: Identification of Tannerella forsythia using culture and PCR technique

 

  





  
3UHYDOHQFH

3UHYDOHQFH

 
 
 

 





+HDOWK\ &KURQLF 2YHUDOO +HDOWK\ &KURQLF 2YHUDOO
SHULRGRQWLWLV SHULRGRQWLWLV

Graph l: Comparison of prevalence of T. forsythia in the subgingival plaque samples Graph 2: Comparison of prevalence of T. forsythia in the subgingival plaque
from healthy periodontium and chronic periodontitis by culture technique samples from healthy periodontium and chronic periodontitis by PCP technique

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

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 7 describes sensitivity and specificity of PCR technique using

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)
positive and PCR negative. Of the total samples 65 were
negative for both techniques, 19 positive in both technique.
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.
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.

Journal of Indian Society of Periodontology - Vol 18, Issue 2, Mar-Apr 2014 157
 Bankur, et al.: Identification of Tannerella forsythia using culture and PCR technique

Table 5: Comparison of detection of T. forsythia by PCR Manti as well, in there study showed that 24% of periodontally
and culture in both healthy and periodontitis subjects healthy patients had T. forsythia in their plaque.[15]
Culture +ve Culture −ve Total
Healthy If a bacterial species is considered as a periodontal pathogen,
PCR +ve 6 4 10 it is expected to exist in most individuals with periodontal
PCR −ve 1 39 40 disease and infrequently detected in subjects with a healthy
Total 7 43 50 periodontium. The results of the present study showed a
Periodontitis higher prevalence of T.  forsythia in chronic periodontitis
PCR +ve 13 7 20 group than in the periodontally healthy group detected by
PCR −ve 4 26 30
Total 17 33 50
culture method [Table  3] confirming the findings of other
Combined studies.[12,16] In our study, when the presence of T. forsythia in
PCR +ve 19 11 30 chronic periodontitis was evaluated by culture technique, it
PCR −ve 5 65 70 demonstrated a prevalence of 34.0% in periodontitis patients.
Total 24 76 100 This is comparable to the study by Verner et al. where 33.3%
PCR – Polymerase chain reaction T. forsythia was detected by them.[17] High prevalence of this
pathogen (51%) has been reported using bacterial culture in
Table 6: Sensitivity and specificity of culture technique other studies done by Jervoe Storm et al.[18] In a study conducted
using PCR as standard reference for the detection of by Laura Lau et al. only 25% of the periodontitis patients were
T. forsythia in subgingival plaque samples detected positive for T. forsythia by culture.[13]
Healthy (%) Periodontitis (%) Combined (%)
Sensitivity 60.0 65.0 63.3 Comparison of the microbiologic results of the present study
Specificity 97.5 86.6 92.8 with those from other studies is complicated by the fact that
PPV 85.7 76.4 79.16 different methods were applied in each study and that the age
NPV 90.6 78.7 85.5
range varied between studies.
PCR – Polymerase chain reaction; PPV – Positive predictive value;
NPV –  Negative predictive value
PCR detected T.  forsythia more frequently than culture
Table 7: Sensitivity and specificity of PCR technique technique. These findings confirm those from previous
using Culture as standard reference for the detection of studies.[18,19] In the present study 20% of the periodontally
T. forsythia in subgingival plaque samples healthy subjects showed the presence of T. forsythia by PCR
technique. This was comparable with the results of Narayanan
Healthy (%) Periodontitis (%) Combined (%)
et  al. (2005),[7] and Catalina et  al., (2012),[11] in which 25 and
Sensitivity 85.7 76.4 79.1
15% of periodontally healthy subjects showed the presence of
Specificity 90.6 78.7 85.5
PPV 60.0 65.0 63.3 T. forsythia by PCR technique. Tan et al. in their study reported
NPV 97.5 86.6 92.8 45% of the healthy sites were positive for T. forsythia.[20] This
PCR – Polymerase chain reaction; PPV – Positive predictive value; could be due to the use of multiplex PCR in their study. Choi
NPV –  Negative predictive value et  al. showed the presence of T.  forsythia in 55% of healthy
subjects.[21] The difference in their numbers in comparison to
Results from this study have shown that T.  forsythia was that of our results could be due to identification technique using
successfully detected both in periodontally healthy individuals nucleic acid base approach rather than PCR.
and in chronic periodontitis patients by both the detection
techniques (Culture and PCR). In the present study 40% of the chronic periodontitis patients
showed the presence of T. forsythia by PCR technique [Table 4].
Various researchers have reported difficulty in cultivation This was comparative with a recent studies conducted by Abiko
of T. forsythia in culture media.[8‑10] In one of the recent study et al. (2010),[22] and Boyanova et al. (2009),[23] in which T. forsythia
conducted by Catalina Suzana Stingu, T.  forsythia was not was detected by PCR in 43% and 44.4% of chronic periodontitis
detected in any of the samples in periodontally healthy subjects patients, respectively. The similarity between our study and
by culture method.[11] Yano Higuchi in their study showed that conducted by Boyanova et al. was the nucleotide sequences
only 1.7% of the patients with culture positive results from of PCR primers used for the identification of T. forsythia.
periodontally healthy subjects.[12] Another study conducted
by Laura Lau and Sanz, showed that only 3.3% of healthy When reviewing the scientific literature, the evaluation of
individuals showed the presence of T.  forsythia by culture T. forsythia prevalence results in periodontitis patients using
method.[13,14] The results of the above stated studies project PCR technology points to variable results. A high prevalence
a low prevalence of T.  forsythia in healthy subjects. In our has been reported in Singapore 91%,[20] in the USA between
study, 14% of the periodontally healthy patients showed the 84.5 and 100%,[24,25] and in Ireland 78%.[26] The wide variation
presence of T. forsythia by the culture technique. This could be in results may be attributed to patient and method selection,
attributed to the fact that N‑acetyl muramic acid, an essential different bacterial serotypes and true geographical differences.
nutrient for the growth of T. forsythia was incorporated in the Studies with a uniform design need to be conducted globally
culture plates by us as against the above mentioned authors in order to understand the true prevalence and geographical
who had not incorporated this nutrient in their cuture media. distribution of this bacterial species. Only limited data
Our results are in agreement with Wyss,[1] who in his study are available on the subgingival microbial composition of
showed the dependence of proliferation of Bacteroides forsythus periodontitis subjects in developing countries.[27] Till date,
on exogenous N‑acetylmuramic acid. Kamma, Nakou and we found very little published data available to associate

158 Journal of Indian Society of Periodontology - Vol 18, Issue 2, Mar-Apr 2014
 Bankur, et al.: Identification of Tannerella forsythia using culture and PCR technique
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,
Journal of Indian Society of Periodontology - Vol 18, Issue 2, Mar-Apr 2014 159
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.
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How to cite this article: Bankur PK, Nayak A, Bhat K, Bankur R,
Naik R, Rajpoot N. Comparison of culture and polymerase chain
reaction techniques in the identification of Tannerella forsythia
in periodontal health and disease, an in vitro study. J Indian Soc
Periodontol 2014;18:155-60.
Source of Support: Nil, Conflict of Interest: None declared.

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160 Journal of Indian Society of Periodontology - Vol 18, Issue 2, Mar-Apr 2014
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