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J Dent Res 83(10):767-770, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Clinical

The Highly Leukotoxic JP2 Clone of Actinobacillus actinomycetemcomitans and Progression of Periodontal Attachment Loss

D. Haubek1,*, O.-K. Ennibi3, K. Poulsen2, N. Benzarti3, and V. Baelum1

1 Department of Community Oral Health and Pediatric Dentistry, Dental School, Faculty of Health Sciences, University of Aarhus, Vennelyst Boulevard, DK 8000 Aarhus C, Denmark; 2 Department of Medical Microbiology and Immunology, University of Aarhus, Denmark; and 3 Dental Faculty, University of Rabat, Morocco;

* corresponding author, dhaubek{at}odont.au.dk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The JP2 clone of Actinobacillus actinomycetemcomitans has been implicated in the etiology of periodontitis in adolescents. The aim of this two-year longitudinal study was to describe clinical attachment loss (CAL) progression and to assess its association with baseline occurrence of the JP2 and non-JP2 types of A. actinomycetemcomitans. Clinical re-examination of 121 adolescents in Morocco was performed. Progression of CAL ≥ 1 mm, ≥ 2 mm, ≥ 3 mm, and ≥ 4 mm on at least one site was found in 58%, 48%, 22%, and 6% of the subjects, respectively. Subjects who, at baseline, harbored the JP2 clone had a significantly higher progression of CAL than did subjects harboring non-JP2 types of A. actinomycetemcomitans. Subjects harboring non-JP2 types displayed a marginally higher CAL progression than did subjects who were culture-negative for A. actinomycetemcomitans.

KEY WORDS: Actinobacillus actinomycetemcomitans • leukotoxin • Morocco • progression • adolescent


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The JP2 clone of Actinobacillus actinomycetemcomitans—characterized by a 530-bp deletion in the promoter region of the leukotoxin gene operon, resulting in increased leukotoxin production (Brogan et al., 1994)—has most frequently been detected in individuals of African descent (Haubek et al., 1996, 1997; Contreras et al., 2000; Haraszthy et al., 2000). Cross-sectional studies have demonstrated a positive association between the presence of this clone and the occurrence of early-onset periodontitis (EOP) (Haubek et al., 1997, 2001; Haraszthy et al., 2000). Moreover, patients who harbor the JP2 clone have more advanced stages of the disease than patients without the clone (Haubek et al., 2002). We have previously suggested that A. actinomycetemcomitans may play a dual role in EOP: The JP2 clone acts as a traditional disseminating pathogen, whereas other types may be opportunistic pathogens that merely add to the pathogenic potential of the complex subgingival microflora (Haubek et al., 1996). However, a minimum requirement for a causal interpretation of these associations is the demonstration that the putative cause (the JP2 clone) precedes the effect (periodontitis initiation or progression). Hence, cross-sectional studies are intrinsically unable to disclose the temporal relationship between acquisition of the bacterium and initiation and progression of disease. Support for a causal role of the JP2 clone may be found in the family study conducted by Bueno and co-workers, demonstrating that LJP-susceptible individuals harboring A. actinomycetemcomitans with the 530-bp deletion were more likely to convert to a diseased status than were subjects who had variants containing the full-length leukotoxin promoter region (Bueno et al., 1998).

We have previously reported cross-sectional findings on a population of Moroccan adolescents, 9% of whom were found to be detectably infected by the JP2 clone of A. actinomycetemcomitans, and 15% of whom were found to have EOP, defined as the presence of one or more teeth with CAL ≥ 3 mm (Haubek et al., 2001). In this Moroccan population of adolescents, the present study describes the progression of CAL over a two-year period and its possible association with the baseline presence of the JP2 clone of A. actinomycetemcomitans.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The baseline study was carried out in 1999 and consisted of 301 Moroccan adolescents (mean age, 16.1 yrs; SD 1.4) (Haubek et al., 2001). Two years later, all 301 adolescents were invited to participate in a follow-up examination after a renewed ethics clearance of the study by the Unité de Coordination de L’Action Pedagogique, Royaume du Maroc. A total of 121 adolescents (40% of the baseline sample), 66 girls and 55 boys (mean age, 17.6 yrs; SD 1.5), gave their written informed consent and were re-examined at public schools (n = 106) or at the Dental Faculty in Rabat, Morocco (n = 15). At both baseline and follow-up, the clinical examination included the recording of CAL (in mm) in 4 sites (the buccal and oral aspects of the mesial and distal surfaces) of all fully erupted permanent teeth (except third molars), amounting to a potential maximum of 112 sites for examination. Procedures used in the baseline and follow-up examinations were identical. At both occasions, examinations were performed by the same examiner (O.-K.E.), who was unaware of the baseline findings at the time of the follow-up examination.

At baseline, subgingival plaque samples were collected on paper points from 2 periodontal pockets (pooled sample) and analyzed by cultivation for the presence of A. actinomycetemcomitans (Haubek et al., 2001). The leukotoxin gene promoter type (with the 530-bp deletion [JP2 type] and without the 530-bp deletion [non-JP2 type]) was determined by PCR with the primers ltx3 and ltx4 (Poulsen et al., 2003). One isolate from each individual positive for A. actinomycetemcomitans by cultivation was analyzed by PCR, except if colony morphology variants were observed. Different morphological types of A. actinomycetemcomitans were each tested by PCR. At baseline, A. actinomycetemcomitans could not be found in 37 individuals, 38 were positive for the non-JP2 type only, five were positive for the JP2 type only, and two individuals were positive for both non-JP2 and JP2 types of A. actinomycetemcomitans. Plaque samples from the remaining 39 (32.2%) individuals could not be analyzed, due either to overgrowth on the agar plates by other micro-organisms, e.g., yeasts, or to no growth at all because the transportation time of plaque samples was too long. To obtain the best possible estimate of the disease progression in the population studied, we invited all individuals to re-examination, regardless of the outcome of the cultivation of plaque samples at baseline.

At the follow-up examination, the participants were interviewed regarding smoking habits, diabetes, and periodontal treatment since baseline, and this information was provided by 118 of the 121 participants. None reported diabetes, and four subjects (3%) reported being smokers. Two persons had received periodontal treatment.

There was no indication of important differences with regard to the baseline characteristics of those who participated and those who did not participate in the follow-up examination, apart from non-participants being slightly older than participants (p < 0.05) (Table 1Go).


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  		Table 1. Baseline Characteristics of 121 Adolescents Who Participated in the Follow-up Examination and of the 180 Non-participants
 
Statistical Analysis
In the data analysis, two different periodontal outcomes were considered, one being the presence of CAL ≥ 3 mm or progression of CAL ≥ 3 mm in one or more teeth (Table 2Go), the other being the individual mean two-year CAL progression value, calculated as the mean change in CAL (in mm) over all sites examined (Fig.Go). The odds ratio for an association between the presence of JP2 and non-JP2 types of A. actinomycetemcomitans and the presence or progression of CAL was estimated by multivariable logistic regression analysis adjusted for the presence of the type of A. actinomycetemcomitans not addressed. The analyses were carried out with use of the program SPSS 11.0. Confidence intervals for the differences in mean two-year CAL between subgroups with different microbiological findings were calculated based on the unpaired samples option in the program Confidence Interval Analysis (Altman et al., 2002).


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  		Table 2. Adjusted Odds Ratios for an Association between the Presence of JP2 and Non-JP2 Types of A. actinomycetemcomitans and the Presence of CAL ≥ 3 mm and the Progression of CAL ≥ 3 mm
 


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  		Figure. The cumulative frequency distribution of persons according to their mean CAL in millimeters (mm) over 2 yrs. Results are given according to the presence of JP2 and non-JP2 types of A. actinomycetemcomitans (Aa) at baseline. Two individuals were positive for both JP2 and non-JP2 types of A. actinomycetemcomitans, and these were included in the group positive for the JP2 type only.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Presence and Progression of CAL
A total of 121 adolescents participated in the two-year follow-up examination. The baseline prevalence of CAL ≥ 1 mm, ≥ 2 mm, ≥ 3 mm, and ≥ 4 mm at one or more sites was 56.2%, 37.2%, 15.7%, and 6.6%, respectively (Table 1Go). At follow-up, the corresponding figures were 63.6%, 55.4%, 28.9%, and 11.6%. Progression of CAL ≥ 1 mm, ≥ 2 mm, ≥ 3 mm, and ≥ 4 mm in at least one site was found in 57.9%, 47.9%, 21.5%, and 5.8% of the subjects, respectively.

Progression of Disease According to Occurrence of the JP2 and Non-JP2 Types of A. actinomycetemcomitans
At baseline, the presence of the JP2 clone was associated with the presence of CAL ≥ 3 mm, whereas no association between carriage of non-JP2 types and CAL status was observed (Table 2Go). The presence at baseline of the JP2 clone was predictive for progression of CAL ≥ 3 mm over the following 2 yrs (OR = 14.5) (p < 0.01), whereas this was not the case for non-JP2 types of A. actinomycetemcomitans (OR = 1.0) (Table 2Go).

The mean individual two-year CAL progression among the 121 subjects included in the study was 0.06 mm (95% CI = [0.04; 0.08]) and did not differ statistically from the mean individual two-year CAL progression among the 82 subjects for whom microbiological data at baseline were available (mean = 0.06 mm; 95% CI = [0.03; 0.08]).

The distribution of individuals according to mean CAL progression is shown in the FigGo. The seven individuals who were culture-positive for the JP2 clone of A. actinomycetemcomitans at baseline had a mean two-year CAL progression of 0.20 mm (SD 0.3), compared with 0.05 mm (SD 0.1) in the 38 individuals who were JP2-negative and non-JP2-positive, and compared with 0.03 mm (SD 0.1) in the 37 individuals who were culture-negative for A. actinomycetemcomitans. The mean two-year CAL progression was higher in the individuals who harbored the JP2 clone whether compared with those who did not harbor the JP2 clone and were non-JP2-positive (mean difference = 0.15 mm; 95% CI = [0.03; 0.27]; p < 0.05), or compared with those who were culture-negative for A. actinomycetemcomitans (mean difference = 0.17 mm; 95% CI = [0.07; 0.28]; p < 0.05). No difference in mean two-year CAL progression was found between individuals harboring the non-JP2 clone of A. actinomycetemcomitans and those being A. actinomycetemcomitans-negative (mean difference = 0.02 mm; 95% CI = [-0.06; 0.02]).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first population-based study to demonstrate that the presence of the JP2 clone of A. actinomycetemcomitans results in an increased risk of progression of CAL over a two-year follow-up period. While this observation lends support to a causal role of the JP2 clone for the progression of periodontitis, it remains to be elucidated whether the JP2 clone plays a role in the initiation of the disease. Since disease was already prevalent at baseline, this question cannot be addressed on the basis of the present study population (Haubek et al., 2001).

The present follow-up study included only 40% of the individuals who participated in the baseline examination. However, the main reasons for dropout were unrelated to the topic of the study, since students were lost mainly due to change of school for educational reasons. Moreover, a comparison of the baseline findings with regard to the periodontal and microbiological findings between participants and non-participants revealed only minor differences (Table 1Go). Besides, the baseline results on the association between different types of A. actinomycetemcomitans and the presence of CAL ≥ 3 mm were reproduced when only participants in the follow-up study were considered (Table 2Go). These observations clearly argue against the presence of a significant selection bias.

Previous studies have described the progression of periodontitis among adolescents (Timmerman et al., 2000; Griffiths et al., 2001; Jenkins and Papapanou, 2001; Heitz-Mayfield et al., 2003). However, differences in age of the study population, follow-up periods, choice of data collection methods, and choice of measurements in the data analyses make direct comparison with this study impossible.

While the determination of disease progression was based on all 121 subjects, only 82 were included in the analysis of the associations with microbiological findings. Cultivation of the remaining 39 plaque samples collected at baseline could not be evaluated because of overgrowth with other micro-organisms or no growth at all. These methodological problems may be overcome by the use of PCR-based techniques directly on plaque samples, since PCR does not rely on the viability of samples and is less sensitive to the presence of other potentially dominating micro-organisms. However, the PCR-based assay for the detection of A. actinomycetemcomitans strains with different leukotoxin promoter structures directly on plaque samples has only recently been developed (Poulsen et al., 2003), and it was not available to us at baseline.

For each individual who was cultivation-positive for A. actinomycetemcomitans, only a single colony was isolated and tested for leukotoxin gene promoter structure, except for a few cases where different morphological types of colonies were present. The type of A. actinomycetemcomitans not detected was considered not present. However, for 89 plaque samples collected in the same Moroccan population and analyzed by both PCR and cultivation, we found only one sample in which PCR revealed the occurrence of both the JP2 and non-JP2 types of A. actinomycetemcomitans, whereas only one of the types was detected by cultivation (Poulsen et al., 2003). In conclusion, the presence of different types of A. actinomycetemcomitans may have been overlooked in very few of the subjects, though this does not invalidate the conclusions drawn in this study, due to the strong association between the presence of the JP2 clone and the progression of disease.

In summary, 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. Further studies are required to evaluate causal relationships.


   ACKNOWLEDGMENTS
 
We thank Dr. Lamia Abdellaoui for her kind help with the registration of patients and Professor Sven Poulsen for his advice during the organization of the study. Professor Mogens Kilian is acknowledged for taking part in discussions of the results and for his help in preparation of the manuscript. We thank the Dean of the Dental Faculty of Rabat, Morocco, for permission to use the facilities at the Dental Faculty. Financial support was obtained from FUT-Calcin, the Research Fund of the Danish Dental Association, and the Aarhus University Research Foundation (F-2001-SUN-1-72).

Received November 12, 2003; Last revision June 11, 2004; Accepted July 22, 2004


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Altman DG, Machin D, Bryant TN, Gardner MJ (2002). Statistics with confidence. Confidence interval analysis. Version 2.0.0. 2nd ed. Bristol: BMJ Books.

Brogan JM, Lally ET, Poulsen K, Kilian M, Demuth DR (1994). Regulation of Actinobacillus actinomycetemcomitans leukotoxin expression: analysis of the promoter regions of leukotoxic and minimally leukotoxic strains. Infect Immun 62:501–508.[Abstract/Free Full Text]

Bueno LC, Mayer MPA, DiRienzo JM (1998). Relationship between conversion of localized juvenile periodontitis-susceptible children from health to disease and Actinobacillus actinomycetemcomitans leukotoxin promoter structure. J Periodontol 69:998–1007.[ISI][Medline]

Contreras A, Rusitanonta T, Chen C, Wagner WG, Michalowicz BS, Slots J (2000). Frequency of 530-bp deletion in Actinobacillus actinomycetemcomitans leukotoxin promoter region. Oral Microbiol Immunol 15:338–340.[ISI][Medline]

Griffiths GS, Duffy S, Eaton KA, Gilthorpe MS, Johnson NW (2001). Prevalence and extent of lifetime cumulative attachment loss (LCAL) at different thresholds and associations with clinical variables: changes in a population of young male military recruits over 3 years. J Clin Periodontol 28:961–969.[ISI][Medline]

Haraszthy VI, Hariharan G, Tinoco EMB, Cortelli JR, Lally ET, Davis E, et al. (2000). Evidence for the role of highly leukotoxic Actinobacillus actinomycetemcomitans in the pathogenesis of localized juvenile and other forms of early-onset periodontitis. J Periodontol 71:912–922.[ISI][Medline]

Haubek D, Poulsen K, Westergaard J, Dahlén G, Kilian M (1996). Highly toxic clone of Actinobacillus actinomycetemcomitans in geographically widespread cases of juvenile periodontitis in adolescents of African origin. J Clin Microbiol 34:1576–1578.[Abstract]

Haubek D, DiRienzo JM, Tinoco EMB, Westergaard J, Lopéz NJ, Chung CP, et al. (1997). Racial tropism of a highly toxic clone of Actinobacillus actinomycetemcomitans associated with juvenile periodontitis. J Clin Microbiol 35:3037–3042.[Abstract]

Haubek D, Ennibi OK, Poulsen K, Poulsen S, Benzarti N, Kilian M (2001). Early-onset periodontitis in Morocco is associated with the highly leukotoxic clone of Actinobacillus actinomycetemcomitans. J Dent Res 80:1580–1583.[Abstract/Free Full Text]

Haubek D, Ennibi OK, Abdellaoui L, Benzarti N, Poulsen S (2002). Attachment loss in Moroccan early-onset periodontitis patients in relation to infection with the JP2-type of Actinobacillus actinomycetemcomitans. J Clin Periodontol 29:657–660.[ISI][Medline]

Heitz-Mayfield LJ, Schätzle M, Löe H, Bürgin W, Ånerud Å, Boysen H, et al. (2003). Clinical course of chronic periodontitis. II. Incidence, characteristics and time of occurrence of the initial periodontal lesion. J Clin Periodontol 30:902–908.[ISI][Medline]

Jenkins WM, Papapanou PN (2001). Epidemiology of periodontal disease in children and adolescents. Periodontology 2000 26:16–32.[ISI][Medline]

Poulsen K, Ennibi OK, Haubek D (2003). Improved PCR for detection of the highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans in subgingival plaque samples. J Clin Microbiol 41:4829–4832.[Abstract/Free Full Text]

Timmerman MF, Van der Weijden GA, Abbas F, Arief EM, Armand S, Winkel EG, et al. (2000). Untreated periodontal disease in Indonesian adolescents. Longitudinal clinical data and prospective clinical and microbiological risk assessment. J Clin Periodontol 27:932–942.[ISI][Medline]




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