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ORIGINAL ARTICLE
Year : 2016  |  Volume : 20  |  Issue : 2  |  Page : 141-144  

Occurrence of Aggregatibacter actinomycetemcomitans in Indian chronic periodontitis patients and periodontally healthy adults


Department of Molecular Biology and Immunology, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgaum, Karnataka, India

Date of Submission16-Mar-2015
Date of Acceptance11-Oct-2015
Date of Web Publication11-Apr-2016

Correspondence Address:
Dr. Vinayak Mahableshwar Joshi
Department of Molecular Biology and Immunology, R. S. No. 47A/2, Bauxite Road, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgaum - 590 010, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-124X.175171

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   Abstract 

Background: Aggregatibacter actinomycetemcomitans (Aa), an important primary periodontal pathogen, is known for its strong virulence characteristics that cause periodontal disease. We investigated Aa occurrence in Indian individuals using culture and 16 s rDNA polymerase chain reaction (PCR). Materials and Methods: A cross-sectional study with 100 participants each in the healthy and chronic periodontitis (CP) groups was conducted. The subgingival plaque was collected and immediately plated on selective media for Aa. The remaining plaque samples were used for DNA extraction. PCR was performed using specific primers for Aa. Statistical Analysis Used: The detection of bacteria and the clinical parameters between the groups were compared using the Mann–Whitney U-test. For assessing the agreement between the results of anaerobic culture and PCR, Kappa analyses were performed. Results: Aa levels using culture and PCR was 51% and 69% in the CP group and 12% and 30% in the healthy group, respectively. The two groups showed significant differences (P < 0.00001). The detection accuracy of culture and PCR was assessed, and the coefficient of accuracy (k) was highly significant in the healthy (0.3103; P < 0.0001) and CP groups (0.1536; P < 0.0497). Conclusions: Aa was predominantly found in the CP group compared with the healthy group, which is consistent with previous findings. Our results showed that both techniques can be used for detecting Aa. An ideal technique for detecting subgingival microorganisms should be carefully selected depending on the scope of the intended future work.

Keywords: Aggregatibacter actinomycetemcomitans, chronic periodontitis, culture, polymerase chain reaction, subgingival plaque


How to cite this article:
Joshi VM, Bhat KG, Kugaji MS, Ingalgi PS. Occurrence of Aggregatibacter actinomycetemcomitans in Indian chronic periodontitis patients and periodontally healthy adults. J Indian Soc Periodontol 2016;20:141-4

How to cite this URL:
Joshi VM, Bhat KG, Kugaji MS, Ingalgi PS. Occurrence of Aggregatibacter actinomycetemcomitans in Indian chronic periodontitis patients and periodontally healthy adults. J Indian Soc Periodontol [serial online] 2016 [cited 2019 Dec 13];20:141-4. Available from: http://www.jisponline.com/text.asp?2016/20/2/141/175171


   Introduction Top


Periodontitis, a disease of the structure surrounding the teeth, is caused primarily because of the periodontal microbiome and the host response interplay. Periodontal pathogens that mainly reside in the subgingival plaque are largely unidentified.[1] The occurrence of these pathogens can vary in individuals based on their country, ethnicity, and social status.[2],[3]Aggregatibacter actinomycetemcomitans (Aa) is an important primary periodontal pathogen and is known for its strong virulence characteristics that cause periodontal disease. Aa detection facilitates the development of a better treatment plan for patients with periodontitis.

Culture has been long known as the gold standard for identifying bacteria. However, culturing all periodontal bacteria is not possible because of the complex living conditions of the bacteria and certain inherent limitations in the culturing technique. Conversely, polymerase chain reaction (PCR) can identify organisms even at low levels; however, it is a close-ended technique that can identify only known set of organisms. Various other molecular techniques have been used to detect some of these pathogens.[4],[5] Despite some studies on Aa, the occurrence of this pathogen in Indians is highly unclear.[6],[7],[8] In this study, we investigated Aa occurrence in Indian individuals using culture and 16 s rDNA PCR.


   Materials and Methods Top


This cross-sectional study included 200 participants (Healthy participants: Healthy group (n = 100) and patients with chronic periodontitis (CP): CP group (n = 100) from in and around Belgaum. The participants were screened for inclusion and exclusion criteria. The participants were excluded if they smoked; received orthodontic therapy; antibiotic therapy; or professional cleaning within the past 3 months; required antibiotic coverage before dental treatment; consumed immunosuppressant, bisphosphonates; or steroids; or were diabetic. The participants in the healthy group were periodontally healthy and did not have sites with a pocket depth (PD) of >3 mm. The patients in the CP group were selected according to the classification of the American Association of Periodontology, International Workshop for Classification of Periodontal Diseases.[9] Participants with minimum 20 natural non-carious teeth and three posterior teeth (premolars and molars) in two quadrants (right or left maxillary and mandibular sections) and without interproximal restorations (fillings that extend to the sides of a tooth), ≥5 mm probing PDs, and ≥3 mm clinical attachment loss (CAL) at the test sites (indicative of periodontal pockets) were considered for CP group. The selected participants were presented with the study details, and informed consent was obtained from them. This study was reviewed and approved by the Ethical Committee of the Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgaum.

Clinical procedure and sample collection

Clinical measurements included PD, CAL using a University of North Carolina-15 probe, and plaque and gingival indices (plaque index [PI] and gingival index [GI], respectively). CP diagnosis was established by full mouth examination.

The subgingival plaque was collected using a universal curette after air drying the area and carefully removing the supragingival plaque with sterile cotton rolls. The subgingival dental plaque was collected from the most apical portion of the accessible probing depth and transferred into a vial containing reduced transport fluid medium. For each patient, six sites were harvested and pooled into one plaque sample. Samples were collected from the posterior and anterior teeth. If the first molar tooth was missing, then the next most distal tooth was accessed. If all the molar teeth were missing, then the most distal tooth in that quadrant was sampled.

Culture

The plaque samples were immediately plated on selective media for Aa after a 10-fold dilution (10−3). Aliquots (100 µl) of the diluted sample were spread on the (trypticase soy, serum, bacitracin, and vancomycin) agar and Dentaid plates.[10],[11] The plates were stored at 37°C in a microaerophilic environment (5% CO2 and 95% N2). After 7 days, the plates were examined for the presence of translucent, circular, and slightly convex colonies with a star like inner structure, which indicated the presence of Aa. From each plate, data were recorded as colony forming units/mL.

The remaining plaque samples were then used for DNA extraction. The plaque sample was vortexed and washed thrice in Tris-ethylenediaminetetraacetic acid (EDTA) buffer, pH 7.5, containing 1M Tris (Himedia, Mumbai, Maharashtra, India), and 0.5 M EDTA (Himedia). Lysis buffer I (50 µl) containing 1 M Tris, 0.5M EDTA, Triton X-100 (SD Fine Chemicals Ltd., Mumbai, Maharashtra, India), and lysis buffer II (50 µl) containing 50 mM potassium chloride (Himedia, Mumbai, Maharashtra, India), 50 mM magnesium chloride (Himedia, Mumbai, Maharashtra, India), 0.45% Tween-20 (Himedia, Mumbai, Maharashtra, India), and 0.45% Nodient P-40 (Himedia, Mumbai, Maharashtra, India), followed by 5 µl of proteinase K (10 mg/mL) (Chromous Biotech, Bangalore, Karnataka, India), were added and incubated at 60°C for 2 h and then kept in a boiling water bath for 10 min to inactivate the enzyme. The sample was then centrifuged, and the supernatant containing the DNA was aliquoted in a separate tube and stored at −20°C until further processing.[12]

Polymerase chain reaction

PCR was performed using specific primers for the 16 S rDNA gene of Aa (forward: 5'-GCTAATACCGCGTAGAGTCGG-3'nd reverse: 5'-ATTTCACACCTCACTTAAAGGT-3'). PCR amplification was performed in the Veriti thermal cycler (Applied Biosystems, Grand Island, NY, USA), using a 20 µl reaction mixture (Chromous Biotech, Bangalore, Karnataka, India), containing dNTP mixture (10 mM each), 10 × PCR buffer containing 15 mM of MgCl2, Taq DNA polymerase (1.5 units/reaction), and DNA templates (≤1 µg/reaction). Primer concentration of 25 pM was used. The following thermal cycler conditions were applied: Initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 72°C for 1 min, and a final extension of 72°C for 5 min.[13]

PCR products of the amplified sample were detected on 2% agarose by agarose gel electrophoresis. A 100-bp DNA ladder was loaded onto the gel simultaneously with the samples. The gel was stained with 0.5 µg/mL ethidium bromide for 30 min. The gel was visualized, and the results were recorded using the Gel doc system (Major Science, Saratoga, CA, USA). The molecular size of 443 bp was detected by comparing the band position with the 100-bp DNA ladder.

Statistical analysis

The results were compared by considering the group wise presence or absence of Aa. The mean clinical measurements, such as GI, PI, PD, and CAL, were computed for each participant, and the scores were averaged across the participants in each group. A P < 0.05 was considered significant. Aa detection and the clinical parameters between the healthy and CP groups were compared using the Mann–Whitney U-test. For assessing the agreement between the results of anaerobic culture and PCR for detecting Aa, Kappa analyses were performed. The Spearman correlation coefficient was calculated (SPSS Inc. Released 2001. SPSS for Windows, Version 11.0. Chicago) to describe the agreement of the results of both methods of Aa detection.


   Results Top


[Table 1] shows the clinical parameters of the healthy and CP groups. A statistically significant difference was observed between the two groups for all the clinical parameters (P < 0.00001). The healthy group had lower values of GI, PI, PD, and CAL than the CP group.
Table 1: Clinical parameters of the healthy and the chronic periodontitis group

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Aa levels using culture was 51% and 12% in the CP and healthy groups, respectively. The two groups showed significant differences (P < 0.00001) [Table 2]. Similarly, Aa detection using PCR was more statistically significant in the CP group (69%) than in the healthy group (30%) (P < 0.00001) [Table 3]. In the healthy group, three samples were PCR-negative but culture positive, and 21 samples were PCR positive but culture negative. The detection accuracy of culture and PCR was assessed (Kappa analyses), and the coefficient of accuracy (k) was highly significant in the healthy group 0.3103 (P < 0.0001) [Table 4].
Table 2: Comparison of healthy and CP groups with culture outcome in Aa micro-organism

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Table 3: Comparison of healthy and CP groups with PCR outcome in Aa micro-organism

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Table 4: Comparison of culture and PCR methods in healthy group

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In the CP group, 12 samples were PCR-negative but culture positive, and 30 samples were PCR positive but culture negative. The coefficient of accuracy (k) was 0.1536, which was statistically significant (P < 0.0497) [Table 5].
Table 5: Comparison of culture and PCR methods in CP group

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   Discussion Top


Aa, a putative periodontal pathogen, is universally found in periodontally healthy as well as patients with CP, particularly more in the aggressive form of the periodontal disease.[4],[14],[15],[16]

In this study, we detected Aa using specific culture media and a molecular method of PCR for highly specific conserved regions of 16 s rDNA from the subgingival plaque samples of periodontally healthy individuals and patients with CP.

Only a few studies have attempted to identify the oral bacteria by culture or molecular methods in Indians.[4],[5],[6] In addition, scant data exist on Aa occurrence in healthy Indians. However, various studies on Aa occurrence in healthy individuals have reported findings similar to those of our study.[17],[18],[19],[20],[21] A couple of studies have reported very low levels of Aa in healthy individuals, which is in contrast to our findings.[13],[22]

In this study, the two detection methods showed higher Aa occurrence in the healthy group compared to the results reported by Mahalakshmi et al. (2012).[6]

Aa, a putative periodontal pathogen, is universally found in healthy individuals and patients with periodontitis.[3],[13],[14],[18],[20],[21],[22],[23] Because of its virulence characteristics and tissue invasive ability, Aa occurs at higher levels in patients with periodontitis than in healthy individuals,[13],[17],[18],[20],[21],[22],[23] which is supported by the findings of our study.

Aa occurrence varies in percentage with different geographical locations and ethnicity, as reported by previous studies conducted in the Asian and South and North American individuals. Our results are similar to the findings of various previous studies conducted on different population subgroups and ethnicities, which showed that Aa occurrence was higher in patients with CP.[13],[17],[18],[20],[21],[23],[24] Furthermore, this was observed with both culture and PCR. A comparison of the two groups revealed a significantly higher level of Aa occurrence in patients with CP than that in healthy participants.

Culture is still considered as the gold standard for identifying bacteria. PCR is a more sensitive and specific technique of the two. Therefore, we used both the techniques for identifying Aa in our study. On comparison, PCR was found to be more efficient in identifying Aa, which was consistent with previous study results.[25],[26],[27] Although we used specific culture media and specific primers for PCR to identify Aa, the results varied. The varying results (culture negative and PCR-positive) were obtained possibly because of the inherent inability of the culture technique to identify bacteria from samples with low levels of organisms and its requirement of viable organisms. The PCR-negative and culture positive result may be related to technical inadequacies, such as inhibitory substances in plaque samples or the presence of genetic variation in the sequence of the organism. These inhibitory substances can inhibit PCR. The PCR-negative samples can be reassessed after adding substances that remove the inhibitory substances, but that would add to the cost and labour involved. Future studies can be planned to determine the serotypes present in the healthy Indians and Indian patients with periodontitis because no related data are available.

Our results indicate that both techniques can be used for identifying Aa. Culture can facilitate the detection of a wide variety of organisms, identification of unexpected species, and determination of antibiotic sensitivity, and, therefore, is the most objective technique and the gold standard. Given the complex nature of the biofilm and the variability of the oral flora, PCR can overcome many restrictions of anaerobic culture, such as identification of the taxa present at lower levels and provide quicker results than culture.

In conclusion, the presence of Aa in this subset of the Indian population was consistent with the previous findings, with the organisms dominating in the disease than in healthy. The ideal technique for detecting subgingival microorganisms should be carefully selected depending on the scope of the intended future work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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