“DETECTION OF SEROTYPE b OF AGGREGATIBACTER ACTINOMYCETEMCOMITANS AND EXPRESSION OF HIGHLY LEUKOTOXIC JP2 CLONE – AN IN VIVO STUDY.”
BRIEF RESUME OF THE INTENDED WORK
Need for study
Aggressive periodontitis is a major clinical problem that eventually results in loss of tissues supporting teeth at an early age. It is an infectious disease, but the complexity of the microflora that constitutes the biofilm in the affected periodontal pockets makes it difficult to determine the exact etiology. Early studies demonstrated that there is an association between disease and the presence of bacteria belonging to the species Actinobacillus actinomycetemcomitans, which was recently re-classified in the new genus Aggregatibacter. The primary habitat of Aggregatibacter actinomycetemcomitans is dental plaque, and this organism may also be isolated from part of healthy population.1
Aggregatibacter actinomycetemcomitans (A.actinomycetemcomitans)is a gram-negative, capnophilic coccobacillus that colonizes the human oral cavity. This gram negative anaerobe has been implicated in the etiology of localized juvenile periodontitis (LJP), a severe and rapid form of periodontal disease.2A.actinomycetemcomitans produces several potential virulence factors like leukotoxin, which is a 116-kDa secreted lipoprotein that specifically kills human polymorphonuclear leukocytes and macrophages; cytolethal distending toxin, an immunosuppressive factor which is homologous to a family of toxins expressed by several other gram–negative bacteria.3
An intra-species division of this microorganism is termed as serotype. The species is represented by 6 serotypes (from a – f).4Studies have shown that A. actinomycetemcomitans serotype b is more prevalent in periodontally diseased patients when compared to healthy subjects in who serotype cpredominates.2
A.actinomycetemcomitans shows a high degree of genetic diversity. A.actinomycetemcomitans isolates, demonstrating high leukotoxic activity, belong to the JP2 clone of serotype b. Notably, this clone demonstrates racial tropism and can be isolated from subjects originating from Africa but not necessarily residing in Africa.4 The JP2 clone of A.actinomycetemcomitans is characterized by a 530-bp (base-pair) deletion in the promoter region of the leukotoxin gene operon.5 Studies on the leukotoxin gene showed that strains carrying a 530-bp deletion result in 10 to 20 fold higher levels of increased leukotoxin production.3
Hence, the purpose of taking up this study is to detect the serotype b of Aggregatibacter actinomycetemcomitans and expression of the highly leukotoxic JP2 clone amongst aggressive periodontitis and non-periodontitis patients.
REVIEW OF LITERATURE
Poulsen K et al (1994)2 conducted a study to evaluate the genetic relationships among A.actinomycetemcomitans strains and possible associations of serotype with genetic polymorphisms within the leukotoxin gene operon. The results showed that non-serotypeable strains are serotype antigen-deficient variants originating from strains of the known serotypes. Serotype b and c strains may contain transmittable DNA sequences not found in strains of the other serotypes.
Haubek D et al (2004)5 described the clinical attachment loss progression and assessed its association with baseline occurrence of the JP2 and non-JP2 types of A. actinomycetemcomitans. They concluded that the presence of the JP2 clone in dental plaque is associated with an increased risk of progression of periodontitis in the Moroccan population where this clone is endemically present.
Haubek D et al (2007)1 conducted a study using markers of genetic diversification in the JP2 clone of A.actinomycetemcomitans to obtain information about its natural history and patterns of inter-individual transmission and global dissemination. They concluded that the JP2 clone of A.actinomycetemcomitans originated in the northern Mediterranean part of Africa and spread to west Africa, from which it was transmitted to other continents during the transatlantic slave trade in 16th to 18th century.
A research was conducted by Haubek D et al (2009)6 to examine the stability of subgingival infection with the JP2 clone of A.actinomycetemcomitans and to analyze the association between two-year infection profiles of the JP2 clone and the development of aggressive periodontitis in Moroccan juveniles. This study concluded that the relative risk for the development of aggressive periodontitis adjusted for the concomitant presence of other genotypes of A.actinomycetemcomitans was highest for individuals continuously infected with JP2 clone indicating a relationship between infectious dose and disease, which further substantiates the evidence for the JP2 clone as a causal factor in aggressive periodontitis. This may suggest that the JP2 clone is not a persistent member of the oral microbiota of carriers.
Sakellari D et al (2011)4 conducted a study to investigate the prevalence and distribution of different A. actinomycetemcomitans serotypes and the JP2 clone in Greek subjects. One pooled subgingival plaque samples from mesio-buccal surfaces of four first molars were collected and the samples were analyzed using polymerase chain reaction (PCR) for five serotypes of A.actinomycetemcomitans and the JP2 clone, using primers. Thus the study concluded that A.actinomycetemcomitans was more prevalent in untreated periodontitis subjects, but no clear predominance of a specific A. actinomycetemcomitans serotype and absence of the JP2 clone were observed in Greek population.
AIMS & OBJECTIVES
To investigate distribution of serotype b of Aggregatibacter actinomycetemcomitans in subgingival plaque among aggressive periodontitis and non-periodontitis subjects.
To detect the presence of highly leukotoxic JP2 clone and correlate its severity among aggressive periodontitis and non-periodontitis subjects.
7 MATERIALS AND METHODS
7.1 Source of data:
60 subjects, aged 15-35 years, of which 30 diagnosed as aggressive periodontitis cases and 30 non-periodontitis cases will be selected from the Department of Periodontics, PMNM Dental college, Bagalkot.
All potential participants will be clearly explained regarding the need and design of the study. A written informed consent will be obtained from all recruits.
7.2 Methods of collection of data
The study will be carried out on selected 30 subjects with aggressive periodontitis and 30 with non-periodontitis. The participants will be categorized in to the following two groups:
Group I: 30 Subgingival plaque samples from subjects with aggressive periodontitis.
Group II: 30 Subgingival plaque samples from patients with non-periodontitis.
CLINICAL EXAMINATION TO ASSESS THE PERIODONTAL CONDITION
Plaque index (Silness and Loe)
Probing Pocket Depth
Clinical loss of attachment.
Clinical assessments using the above mentioned parameters will be performed. Samples will be collected on the subsequent day. One pooled subgingival plaque sample (collected plaque sample will be placed in one test tube for each subject) from the mesio-buccal surfaces of four first molars of the selected subjects will be taken using a sterile Gracey curette after isolation with cotton roles, air drying and removal of supragingival plaque. The samples obtained will be placed in thioglycolate broth and will be sent to laboratory for identification of Aggregatibacteractinomycetemcomitans serotype b and JP2 clone by culture technique and polymerase chain reaction (PCR).
Subjects will be considered as non-periodontitis cases when they display no probing depth or probing attachment level >3mm and no radiographic signs of bone loss.4
Patients will be considered as aggressive periodontitis according to the analytical criteria of the American Academy of Periodontology (AAP-1999).7
Subjects should be systemically healthy and not received periodontal treatment for atleast 6 months prior to sampling and recording.
Systemic diseases like diabetes mellitus, hepatitis, and HIV infection etc known to influence the periodontal disease.
Diseases of oral hard and soft tissue except caries and periodontitis.
Pregnant women and lactating mothers.
Use of antibiotics and analgesics within three months prior to study.
LABORATORY INVESTIGATIONS The subgingival plaque samples from the selected subjects will be adequately and individually transferred in thioglycolate broth to the laboratory for identification of Aggregatibacter actinomycetemcomitans serotype b and JP2 clone using standard culture methods for A.actinomycetemcomitans and PCR for serotyping and JP2 clone.
Statistical analysis: The data so gathered from the clinical examinations and laboratory investigations will be subjected to statistical analysis.
7.3 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS TO BE CONDUCTED IN THE PATIENTS?
Yes, subgingival plaque samples have to be obtained from the patients participating in the study.
7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM THE INSTITUTION?
1. Haubek D, Poulsen K, Kilian M. Microevolution and Patterns of Dissemination of the JP2 Clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans. Infect Immun 2007 June;3080–3088.
2. Poulsen K, Theilade E, Lally ET, Demuth DR, Kilian M. Population structure of Actinobacillus actinomycetemcomitans: A framework for studies of disease associated properties. Microbiology 1994;140:2049-2060.
3. Kaplan JB, Schreiner HC, Furgang D, Fine DH. Population structure and genetic diversity of Actinobacillus a. strains isolated from localized juvenile periodontitis patients. J Clin Microbiol 2002;40:1181-1187.
4. Sakellari D, Katsikari A, Slini T, Ioannidis I, Konstantinidis A, Arsenakis M. Prevalence and distribution of Aggregatibacter Actinomycetemcomitans serotypes and the JP2 clone in a Greek population. J Clin Periodontol 2011;38:108–114.
5. Haubek D, Ennibi OK, Poulsen K, Benzarti N, Baelum V. The highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans and progression of periodontal attachment Loss. J Den Res 2004;83:767-770.
6. Haubek D, Ennibi OK, Vaeth M, Poulsen S, Poulsen K. Stability of the JP2 clone of Aggregatibacter actinomycetemcomitans. J Den Res 2009;88(9):856-860.
7. Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol 1999;4:1-6.