490

Distribution of Actinobacillus actinomycetemcomitans and

Porphyromonas gingivalis by Subject Age Eugene D. Savitt, * and Ralph L. Kenf The possible associations between Periodontitis subject age and the distribution of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalti were examined using an extensive data bank of subgingival plaque specimens analyzed using DNA probes. The results suggest that A. actinomycetemcomitans is strongly related to subjects in the youngest age group (10 to 19 years) with decreasing prevalence and concentration levels in older age groups. In contrast, P. gingivalti showed a reverse relationship to subject age with highest prevalence and concentration levels in older subjects (30 years and older) compared to subjects in younger age groups. Statistical testing of these relationships were highly significant. (P < 0.0001). / Periodontol 1991; 62:490^194.

KeyWords: Actinobacillus actinomycetemcomitans; periodontitis/etiology; Periodontitis/

microbiology; Porphyromonas gingivalis;

age factors.

The role oíActinobacillus actinomycetemcomitans and Porin various forms of Periodontitis is well established.1^1 These 2 periodontal pathogens, which have been shown to produce disease in animal models,5-8 contain a variety of metabolic by-products directly injurious to host tissues and cells,9 and have been found commonly in destructive periodontal lesions.10 Much of the research has focused on the role of A. actinomycetemcomitans in the pathogenesis of juvenile Periodontitis.2'11 This species has been described as a major etiologic agent in juvenile Periodontitis.12 In contrast, P. gingivalis has been most strongly associated with adult Periodontitis.13 Despite this categorization of periodontal pathogens into adult and juvenile forms of Periodontitis, a relatively extensive retrospective study on adult Periodontitis subjects in Scandinavia indicated that A. actinomycetemcomitans may also play a significant role in adult

phyromonas gingivalis

Periodontitis cases.14 DNA probes have been shown to be an effective method for enumerating specific species of bacteria in dental plaque including P. gingivalis, Prevotella intermedia, A. actinomycetemcomitans, Fusobacterium nucleatum, Bacteroides forsythus, and Wolinella recta.15'11 The purpose of this investigation was to evaluate whether probe-detected prevalence of either A. actinomycetemcomitans or P. gingivalis 'Currently, private practice, Wellesley, MA; previously, OmniGene, Inc., Cambridge, MA. Forsyth Dental Center, Boston MA.

in plaque samples from periodontal sites in a large population of dental patients exhibited any association with the ages of the subjects. METHODS AND MATERIALS

Subjects for Study A data base of clinical samples from 180 dental offices distributed nationally was sorted based on the following criteria. Patients chosen for the study exhibited at least 1 site with 2 mm attachment loss; those who had been treated with antibiotics or received periodontal therapy in the past 6 months were excluded. Sites were chosen at the discretion of the dentist submitting the specimens. The number of sites sampled ranged from 1 to 4 sites per patient. Most commonly, 2 sites were sampled for each subject, usually the molars. The subjects were divided into groups based on age with intervals of 10 years. Probing depth and bleeding on probing at sites sampled were recorded.

Specimen Collection Sites were chosen based on a minimum of 2 mm attachment loss. If present, supragingival plaque was removed with a cotton roll or gauze pad. After removing saliva from the site, specimens were collected using a single medium endodontic point* inserted into the site for 10 seconds. The endodontic point was placed into a microcentrifuge tube *Johnson &

Johnson,

New

Brunswick, NJ.

Volume 62 Number 8

SAVTTT,

without transport medium and sent to the central for processing.5

developed in an automatic film developer. The log concentration of cells in each test sample was determined by comparing each sample spot with known quantities of target species which acted as positive controls. A negative control was also included on each filter using a 105 concentration of Actinomyces viscosus. A more precise analysis of each specimen was performed using a video imaging system. The autoradiograms were placed on a light box and the video image captured using the BioScan Optimas Video Imaging System.1 The data were transformed using the Probit Approximation formula18 with 3 log concentration of control cells and converted into equivalent cell

491

Age Distribution of Subject Population

laboratory

Microbial Speciation of Plaque Samples DNA probes specific for A. actinomycetemcomitans (FDC Strain Y4) and P. gingivalis (ATCC Strain 33277) were developed as previously described15'16 and used to enumerate these target species in specimens collected from the patients described above. In brief, the specimens were eluted from the endodontic points and the total sample DNA was denatured.15 Aliquots were then equilibrated with loading buffer and applied to a nitrocellulose filter using a Slot Blot11 apparatus. Specimens were adsorbed onto the filters by vacuum filtration, rinsed, air dried, and baked for 1 hour at 80°C. Filters were prehybridized as previously described.16 Hybridization conditions included 10% dextran sulfate and 2 107 cpms of 32P-labeled DNA probe in 4 ml hybridization buffer for 3.5 hours at 65°C. Filters were washed, air-dried, and exposed to Kodak X-OMAT AR film with 70°C for 12 to 48 hours and an intensifying screen at

KENT

800

o 3

CO O o

E 3



values. Results in Tables 1 and 2 were evaluated using standard chi-square and Mantel-Haenszel chi-square analyses. Tables 3 and 4 were evaluated by Mantel-Haenszel chi-square.

RESULTS Clinical Parameters A total of 3,142 sites from 1,492 subjects comprised the data set (Fig. 1). Of this population, 376 sites were from subjects under 30 years and 1,225 sites were from subjects under 40 years of age. The remaining 1,917 sites were from adult subjects ranging in age from 30 to 78 years old. The age distribution for the subject population is shown by number of sites and subjects by 10 year intervals on Figure 1 with a mean subject age of 44 and a median age of 43. The approximate 2 to 1 ratio of sites to subjects across all age levels reflects the fact that 2 plaque specimens were ob-

tained from most subjects, with remaining subjects having either 1 or 3 sites sampled. In the adult group (ages 30 §BioTechnica Diagnostics/OmniGene, Inc.. "Schleicher and Schuell, Keene, NH.

'BioScan, Edmonds, WA.

Cambridge,

MA.

Birthdate

Figure 1.

Distribution 10 year intervals.

(in decades)

of subjects and sites sampled by age of subjects in

years and older) affected sites had a mean and median probing depth of 5 mm with 54% of sites that bled on probing. In the juvenile group (29 years and younger), affected sites had a mean probing depth of 6 mm and a median probing depth of 5 mm with 43% of sites that bled on probing.

Microbiological Parameters

Overall prevalence (slO3) in plaque specimens was 78% for P. gingivalis and 54% for A. actinomycetemcomitans. However, these prevalence figures were not uniform over age groups. Frequency distributions of all specimens over 4 count levels (104) levels of each species (Tables 3 and 4). In this

10-19

20-29

30-39

50-59

40-49

60-69

70-79

Age of Subject Figure 2. Distribution in mean log levels of P. gingivalis and A. actinomycetemcomitans within groups ofsubjects divided by 10 year age intervals. Table 3. Comparison of Minimal and Elevated Levels of P. gingivalis by Subject Age

Age (years)

Level: Sites

103) and log counts

for A. actinomycetemcomitans and P. gingivalti both appear to be characterized by significant age associated trends in plaque specimens from affected sites. A. actinomycetemcomitans was found more frequently and at higher levels in younger subjects than in older subjects 30 years and beyond. Thus, the greater association of A. actinomycetemcomitans with a juvenile population is once again demonstrated. In contrast, the prevalence and levels of P. gingivalis were higher in the older age groups than in age groups below 30 years. In 10 to 19 year olds, P. gingivalis was detected in 65% of specimens, but most counts (50%) were at 103 and only 2% were at ^lO5. A. actinomycetemcomitans was detected in 64% of these specimens and 17% had counts at >105. Approximately 1 of 3 juvenile subjects did not harbor detectable levels of A. actinomycetemcomitans using DNA probe detection. The negative findings may reflect other pathogens involved in juvenile Periodontitis and that some subjects may have another form of periodontal disease. The age associations for both organisms were primarily due to differences in prevalence and mean levels of specimens from subjects in the 10 to 19 and 20 to 29 year age groups relative to those from subjects aged 30 or more. P. gingivalis in particular exhibited only minor fluctuations in mean levels beyond age 30. Sampled sites in older and younger subjects had similar average probing pocket depths. Bleeding on probing was somewhat more frequent (54%) in older subjects than in younger ones (43%). While the study cannot rule out cohort effects, the data

tributor to the flora found in periodontal lesions of younger subjects and may be a less important contributor to Periodontitis in subjects beyond 30 years of age. These findings are at variance with the data from European studies on adult Periodontitis, which suggest that this species is a significant pathogen within the oral flora from older subjects. The species was found in 52% of both progressing and non-

progressing sites.13

Tabulations retrieved from the laboratory's data base essentially the site based values given in Tables 1 and 2. Obtaining subject data would have entailed considerable further effort which did not seem warranted given the small generally balanced number of sites per subject and the very high levels of significance obtained in the site based analysis. These findings may have implications in the selection of antibiotics for the reduction and eradication of these species in clinical practice. Antibiotics such as metronidazole might be an appropriate drug for treating adults in the United States since the susceptibility pattern for this drug includes most known periodontal pathogens with the important exception of A. actinomycetemcomitans. In younger patients, where A. actinomycetemcomitans is often found in elevated levels, tetracyclines may be more effective. were

REFERENCES 1. Zambón JJ, Reynolds HS, Slots J. Black-pigmented Bacteroides spp. in the human oral cavity. Infect Immun 1981; 32:198-203. 2. Zambón JJ. Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontal 1985; 12:1-20. 3. Kornman KS, Robertson PB. Clinical and microbiological evaluation of therapy for juvenile Periodontitis. / Periodontal 1985; 56:443446. 4. Slots J. Bacterial specificity in adult Periodontitis. / Clin Periodontal 1986; 13:912-917. 5. Irving JT, Newman MG, Socransky SS, Heeley JD. Histological changes in experimental periodontal disease in rats mono-infected with a gram-negative organism. Archs Oral Biol 1975; 20:219-220. 6. Socransky SS, Gibbons RJ. Required role of Bacteroides melaninogenicus in mixed anaerobic infections. J Infect Dis 1965; 115:247253. 7. Slots J, Gibbons RJ. Attachment of Bacteroides melaninogenicus subsp. asaccharolyticus to oral surfaces and its possible role in colonization of the mouth and of periodontal pockets. Infect Immun 1978; 19:254264. 8. Holt, S, Ebersole E, Felton J. Brunsvold M, Kornman K. Implantation of Bacteroides gingivalis in nonhuman primates initiates progression of Periodontitis. Science 1988; 239:55-57. 9. Slots J, Genco RJ. Black-pigmented Bacteroides species, Capnocytophaga species, and Actinobacillus actinomycetemcomitans in human periodontal disease: Virulence factors in colonization, survival, and tissue destruction. J Dent Res 1984; 63:412-421. 10. Slots J, Listgarten MA. Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal diseases. / Clin Periodontal 1988; 15:85-93. 11. Genco RJ, Zambón JJ, Christersson LA. Use and interpretation of microbiological assays in periodontal diseases. Oral Microbiol Immunol 1986; 1:73-79. 12. Holt SC, Ebersole J, Felton J, Brunsvold M, Kornman KS. Implan-

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DISTRIBUTION OF A. ACTINOMYCETEMCOMITANS AND P. GINGIVALIS BY AGE

tation of Bacteroides gingivalis in nonhuman primates initiates progression of Periodontitis. Science 1988; 259:55-57. 13. Slots J. Importance of black-pigmented Bacteroides in human periodontal disease. In: Genco RJ, Mergenhagen, SE, eds. Host-Parasite Interactions in Periodontal Diseases. Washington: American Society for Microbiology, 1982:27-45 14. Bragd L, Dahlén M, Wikström M. The capability of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedia to indicate progressive Periodontitis. A retrospective study. / Clin Periodontol 1987; 14:95-99. 15. Lippke J, Savitt ED. Diagnostic methods for Periodontitis. Diag Clin Testing 1989; 27:35-39.

16. French

J Periodontol August 1991

CK, Savitt ED, Simon SL,

et al. DNA probe detection of Oral Microbiol Immunol 1986; 1:58-62. 17. Savitt ED, Keville M, Peros W. DNA probes in the diagnosis of periodontal microorganisms. Arch Oral Biol 1990; 35:153S-160S. 18. Abramowitz M, Stegun IA, eds. Handbook of Mathematical Functions with Formulas, Graphs, and Mathematical Tables. New York: Dover Publications, Inc. 1972:62.

periodontal pathogens.

Send reprint requests to Dr. Eugene Savitt, 333 Washington St., Wellesley, MA 02181. Accepted for publication February 25, 1991.

Distribution of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by subject age.

The possible associations between periodontitis subject age and the distribution of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis ...
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