JICDRO is a UGC approved journal (Journal no. 63927)

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ORIGINAL ARTICLE
Year : 2010  |  Volume : 2  |  Issue : 2  |  Page : 64-69

Tracking of Actinobacillus actinomycetemcomitans in subgingival plaque of aggressive periodontitis patients


1 Department of Oral Pathology, Dr. D.Y. Patil Dental College and Hospital, Pimpri, Pune, India
2 Department of Oral Pathology, Government Dental College and Hospital, Nagpur, India
3 National Environmental Engineering Research Institute, Nagpur, India

Date of Web Publication20-Apr-2012

Correspondence Address:
Supriya Kheur
Department of Oral Pathology, Dr. D.Y. Patil Dental College and Hospital, Mahesh Nagar, Pimpri, Pune - 411 044, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0754.95257

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   Abstract 

Background: Actinobacillus actinomycetemcomitans is thought to be one of the etiological agents in aggressive periodontitis as well as indicated in various systemic diseases. Objective: To evaluate the prevalence of A. actinomycetemcomitans in the subgingival plaque of aggressive periodontitis patients. Study Design: Initially, under the selective growth conditions, the isolates were picked from the plaques and their identification was confirmed by polymerase chain reaction using primers specific for A. actinomycetemcomitans subgingival plaque of 15 patients diagnosed clinically and on radiographic criteria as aggressive periodontitis was inoculated on the Tryptic Soy agar with Bacitracin and Vancomycin culture media for 3-5 days under microaerophilic conditions. The positive colonies were selected based on biochemical tests for further analysis using reported primers for A. actinomycetemcomitans. Results: The results showed that 66.67% of aggressive periodontitis patients and 6.67% of control group of normal patients showed evidence of presence of A. actinomycetemcomitans in the subgingival microflora. Conclusion: This is the first study of its kind in an Indian population whereby almost all aggressive periodontitis patients showed evidence of A. actinomycetemcomitans.

Keywords: Actinobacillus actinomycetemcomitans , aggressive periodontitis, polymerase chain reaction, subgingival microflora


How to cite this article:
Kheur S, Hazarey VK, Kapley A, Purohit H. Tracking of Actinobacillus actinomycetemcomitans in subgingival plaque of aggressive periodontitis patients. J Int Clin Dent Res Organ 2010;2:64-9

How to cite this URL:
Kheur S, Hazarey VK, Kapley A, Purohit H. Tracking of Actinobacillus actinomycetemcomitans in subgingival plaque of aggressive periodontitis patients. J Int Clin Dent Res Organ [serial online] 2010 [cited 2019 Mar 21];2:64-9. Available from: http://www.jicdro.org/text.asp?2010/2/2/64/95257


   Introduction Top


Periodontal diseases are those affecting the supporting apparatus of the teeth including hard and soft tissues. It is an infectious disease characterized by alveolar bone destruction and tooth loss. Periodontal diseases are initiated by a complex network of molecular interactions between growing as a biofilm in the subgingival crevice and the host tissues that surround and support the teeth. The damage to host soft and hard tissues appears to be largely the result of multiple factors produced during the destructive chronic inflammatory response to bacteria and their products, as well as bacterial proteinases and cytotoxins released from colonizing biofilm on subgingival region. The pathogenesis profiles of these diseases depend on the complex genetic, microbial, immunological, and environmental factors that collectively determine the disease risk, progression, and course.

Aggressive periodontitis generally affects systemically healthy individuals less than 30 years old. [1] It encompasses distinct types of periodontitis that affect people who in most cases appear healthy. It tends to have a familial aggregation, and there is rapid rate of disease progression. The two main clinical features are that the amounts of microbial deposits are inconsistent with the severity of periodontal destruction and the progression of loss of attachment and bone may be self-arresting. The review of the literature shows an excellent contribution on association between Actinobacillus actinomycetemcomitans and aggressive periodontitis. A. actinomycetemcomitans has also been reported to be the causative agent or associated with various infectious diseases including bacterial endocarditis, pericarditis, meningitis, septicemia, and thyroid abscess. [2],[3],[4],[5],[6]

There is a relative paucity of studies in Indian population on association of aggressive periodontitis and A. actinomycetemcomitans The study was carried out to isolate A. actinomycetemcomitans from the subgingival plaque samples in aggressive periodontitis patients using culture and biochemical tests and to confirm the identity of this organism by polymerase chain reaction (PCR) procedure using specific primers.


   Materials and Methods Top


Patient recruitment

In all, 15 patients who were clinically and radiographically diagnosed as suffering from aggressive periodontitis were included in this study [7] [Figure 1], [Figure 2] and [Figure 3].
Figure 1: Generalized form of aggressive periodontitis in a 22-year-old female patient

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Figure 2: Localized form of aggressive periodontitis with pathological migration of incisors and mobility in fi rst molars

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Figure 3: Generalized form of aggressive periodontitis with severe attachment loss seen in all teeth

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All the subjects were informed of the experimental protocol. All of them gave informed consent. The patient included in the study were neither been treated previously for periodontitis nor had taken antibiotics or oral antiseptics within preceding 6 months. For comparison, samples were taken from randomly selected periodontally normal individuals with no evidence of any other disease process of the same age group.

Clinical examination

The examination included evaluation of age, sex, and dietary habits. A clinical examination was performed on the mesiofacial and distofacial surfaces of all teeth. The examination included the assessment of oral hygiene, [8] bleeding on probing using a periodontal probe-present/absent, pocket depth, and attachment loss by measuring the distance from the CEJ to the depth of the pocket using a periodontal probe [Table 1].
Table 1: Clinical and diagnostic parameters used in our study

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Sample collection

For study group: After complete clinical and radiographic evaluation, the sites in each subject showing the maximum pocket depth were selected for bacteriological examination. Two such sites per patient were selected having more pocket depth than the observed average depth for that subject [Figure 4] and [Figure 5]. Thorough supragingival cleaning of the selected sites was performed. The samples were taken by a single pass of 11/12 Hufriedy's Gracey curette. The samples were collected in the sterile 1.5 ml tube and stored at 4°C until used for further analysis.
Figure 4: Site showing maximum disease activity for sample collection (pocket depth >10 mm)

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Figure 5: OPG showing generalized vertical bone loss, arc-shaped pattern (arrows)

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For control group: In normal subjects, the plaque samples were obtained from incisors and first molars and the samples were collected as mentioned earlier.

Culture analysis

All the 30 subgingival plaque samples (15 of the control and 15 of the study group) were directly inoculated on the Tryptic Soy agar with Bacitracin and Vancomycin (TSBV) agar, a selective medium for A. actinomycetemcomitans The samples were incubated in the anaerobic chamber with 5-10% CO 2 for 3-5 days at 37°C. [9] The growth of A. actinomycetemcomitans was then studied using gram staining and the various biochemical tests for presumptive diagnosis [10] [Figure 6].
Figure 6: Actinobacillus actinomycetemcomitans colonies (arrows) Tryptic soy agar with Bacitracin and Vancomycin

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The following biochemical tests were carried out viz. catalase test, oxidase test, and nitrate reduction test. Subsequently, identification and confirmation of A. actinomycetemcomitans were done by PCR using specific primers.

PCR analysis

DNA preparation/extraction protocol


A loopful of pure colonies was picked up from the culture plate and they were suspended in 100 ml of sterile double distilled water. The cell pellet was collected by centrifugation at 2500 × g. The pellet was washed twice with 1 ml of double distilled water and DNA was prepared by alkaline lysis using 0.5 N NAOH, as reported earlier. [11] The DNA template was stored at -20°C until further use. The following primers were used in the study to amplify a 253-bp product. [2]

Forward primer 5′ CTT TGC ACA TCA GCG TCA GTA CAT CCC CAA GG 3′ and reverse primer 5′ CGT GCC AGC AGC CGC GGT AAT AGG 3′.

The PCR mix contained 1× buffer, 3 mM MgCl 2 200 μM each dXTP, 5 pmol of each primer, and 2.5 units Taq polymerase (Perking Elmer, USA). Primers were synthesized by Bangalore Genei, India. The thermocycling conditions used were as follows: 30 cycles of 94°C for 1 minute and 72°C for 30 seconds.

Agarose gel electrophoresis of PCR products

The presence of A. actinomycetemcomitans was indicated by the presence of a single band of a 253 bp as amplification product in the PCR when viewed in the gel documentation system, Vilber Lormat, on a 1.5% agarose gel.


   Results Top


Of the 15 samples A. actinomycetemcomitans was detected by PCR in 10 patients (66.67%) suffering from aggressive periodontitis (study group) and 1 (6.67%) in the normal (control) patients. Amplicons were always a single band of 253 bp, i.e., the expected size with no non-specific amplification product [Figure 7].
Figure 7: Agarose gel electrophoresis of selected PCR products obtained from subgingival plaque samples. Lanes 2-17: clinical samples. Amplifi cation product 253 bp corresponds to Lanes 3-5, 8, 9, 11, 12, 14-17: positive for A. actinomycetemcomitans. Lanes 18 and 19: negative controls. M: molecular weight marker, DNA ladder of 1 kb.

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   Discussion Top


This study was carried out to correlate the microbial presence of A. actinomycetemcomitans in the proportion of aggressive periodontitis patients in this region using culture and PCR (with specific primers). The objective of this study was to evaluate the presence of these bacteria in the subgingival plaque of aggressive periodontitis patients. The utility of PCR using specific primer the detection of periodontopathogens such as A. actinomycetemcomitans was also studied.

This study revealed that all the patients of aggressive periodontitis were below 30 years of age, and a definite female predilection was seen. Few patients also gave history of tobacco consumption in one form or the other and further studies can be done to evaluate the effect of tobacco in this condition. The presence of this pathogen in normal individual in several studies shows similar results. [12] This could be explained on the basis of leukotoxin production, a factor considered important for virulence of A. actinomycetemcomitans varies significantly among strains of this species, which appears to be regulated by 2 upstream promoters. The toxin genes of A. actinomycetemcomitans have been designated Itx C, Itx A, Itx B, and Itx D in transcriptional order. Itx B and D code for proteins that are involved in transporting the toxin to the surface of the cell, while Itx C posttranslationally activates the toxin. The virulent strain has a 530-bp deletion in the promoter region that may be involved in 10-20 times more leukotoxin production. [13]

Among various detection methods, PCR displays the best detection limits identifying as few as three to five cells and shows no cross reactivity under optimized amplification conditions. [14],[15] In this study, PCR was used for rapid tracking of A. actinomycetemcomitans. We have also used extracted DNA of direct plaque samples and could amplify the expected 253-bp product (data not shown). Hence, adding in effective control of the disease could extend the method as the diagnostic tool even at early stage of infection.


   Conclusion Top


This study highlights and demonstrates for the first time using molecular tools the association of A. actinomycetemcomitans in Indian subpopulation, in aggressive periodontitis. More detailed studies are advocated to correlate the periodontopathogens in systemic diseases as well as to evaluate the pathogenic and nonpathogenic strains of A. actinomycetemcomitans.

 
   References Top

1.Tonetli MS, Mombelli A. Early onset periodontitis. Ann Periodontol 1999;4:39-52.  Back to cited text no. 1
    
2.Garcia L, Tercero JC, Legido B, Ramos JA, Alemany J, Sanz M. Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedius and Porphyromonas gingivalis by multiplex PCR, J Periodontol Res 1998;33:59-64.  Back to cited text no. 2
    
3.Zambon JJ. Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontol 1985;12:1-20.  Back to cited text no. 3
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4.Slots J. Subgingival microflora and periodontal disease. J Clin Periodontol 1979;6:351-82.  Back to cited text no. 4
[PUBMED]    
5.Socransky SS, Haffajee AD. The bacterial etiology of destructive periodontal disease: Current concepts. J Periodontol 1992;63:322-31.  Back to cited text no. 5
[PUBMED]    
6.Ebersole JL, Cappelli D, Sandoval MN. Subgingival distribution of Actinobacillus ctinomycetemcomiftans in periodontitis. J Clin Periodontol 1994;21:65-75.  Back to cited text no. 6
[PUBMED]    
7.Baer PN. A case of periodontitis: A clinical entity. J Periodontol 1971;42:516-20.   Back to cited text no. 7
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8.Silness J, Loe H. Periodontal disease in pregnancy. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1974;22:121-35.  Back to cited text no. 8
    
9.Slofts J. Selective medium for isolation of Actinobacillus Actinomycetemcomitans. J Clin Microbiol 1982;15:606-9.  Back to cited text no. 9
    
10.Slots J. Salient biochemical characteristics of Actinobacillus Actnomycetemcomitans. Arch Microbiol 1982;131:60-7.  Back to cited text no. 10
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11.Kapley A, Lampel K, Purohit HJ. Development of duplex PCR for the detection of Salmonella and Vibrio in drinking water. World J Microbiol Biotech 2000;16:457-8.  Back to cited text no. 11
    
12.Slots J, Reynold HS, Genco RJ. Actinobacillus actinomycetemcomitans in human periodontal disease: A cross sectional microbiological investigations. Infect Immun 1980;29:1013-20.  Back to cited text no. 12
    
13.Borgan JM, Cally ET, Poulsen K, Kilian M, Demuth DR. Regulation of Actinobacillus actinomycetemcomitans leukotin expression: Analysis of the Promoter region of leukotoxic and minimally leukotoxic Strains. Infect Immun 1994;62:501-8.  Back to cited text no. 13
    
14.Zambon JJ, Haraszthy VI. The laboratory diagnosis of periodontal Infections. Periodontology 2000 1995;7:69-82.  Back to cited text no. 14
    
15.Chen C, Slots J. Microbiological tests for Actinobacillus actinomycentemcomitans and Porphyromonas gingivalis. Periodontology 2000 1999;20:53-64.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
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