Actinobacillus actinomycetemcomitans in Human Periodontal

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1 INFECTION AND IMMUNITY, Sept. 1980, p /80/ /08$02.00/0 Vol. 29, No. 3 Actinobacillus actinomycetemcomitans in Human Periodontal Disease: a Cross-Sectional Microbiological Investigation J0RGEN SLOTS,* HOMER S. REYNOLDS, AND ROBERT J. GENCO Department of Oral Biology and Periodontal Disease, Clinical Research Center, School of Dentistry, State University of New York at Buffalo, Buffalo, New York Actinobacillus actinomycetemcomitans is a facultative gram-negative bacterium which has been associated with severe oral and nonoral infections. This study examined its occurrence in the oral cavities of 10 normal juveniles, 11 normal adults, 10 juvenile periodontitis patients, and 12 adult periodontitis patients. Four deep periodontal pockets and two normal periodontal sites were sampled in the diseased patients, and six normal periodontal sites were sampled in the healthy individuals. In all subjects samples were obtained from the cheek, tongue, and saliva. Samples from a total of 172 normal periodontal sites, 83 deep periodontal pockets, 42 cheek mucosae, 42 tongue dorsa, and 42 salivas were examined. Isolation was performed by using a medium for selective isolation ofa. actinomycetemcomitans (Trypticase soy agar [BBL Microbiology Systems] supplemented with 10% serum and 75 pig of bacitracin per ml). The carrier rates were 20% for normal juveniles, 36% for normal adults, 50% for adult periodontitis patients, and 90% for juvenile periodontitis patients. A. actinomycetemcomitans was on average recovered in about fivefold-higher numbers from infected deep periodontal pockets than from infected normal subgingival areas. Samples of periodontal pockets generally contained 100-fold-more cells of A. actinomycetemcomitans than did samples of the cheek, tongue, and saliva. A. actinomycetemcomitans is commonly isolated from patients with juvenile periodontitis, often isolated from patients with adult periodontitis, and occasionally isolated from normal juveniles and adults. Its primary oral ecological niche appears to be dental plaque and periodontal pockets. Actinobacillus actinomycetemcomitans, a facultative gram-negative bacterium, appears to play an important role in certain types of periodontal disease. A. actinomycetemcomitans, or organisms likely to be A. actinomycetemcomitans, have been isolated in high numbers from young patients with rapidly progressing alveolar bone breakdown (16, 18, 21). Also, in monoinfected gnotobiotic rats, A. actinomycetemcomitans can cause alveolar bone destruction in 2 to 3 months (11). A. actinomycetemcomitans strains have been found to be cytotoxic for human polymorphonuclear leukocytes in vitro (2, 23; N. S. Taichman and J. M. A. Wilton, J. Dent. Res. Special Issue A, Abstr. Am. Assoc. Dent. Res. 1980, no. 224), the organism possesses a potent endotoxin (P. Kiley and S. C. Holt, J. Dent. Res. Special Issue A, Abstr. Am Assoc. Dent. Res. 1980, no. 974), and it can elaborate depolymerases which have the potential for destruction of the periodontal tissues (3, 8). Furthermore, recent serological studies demonstrated markedly higher serum antibody levels against cells of A. actinomycetemcomitans (R. J. Genco, N. A. Taichman, and C. A. Sadowski, J. Dent. Res. Special Issue A, Abstr. Am. Assoc Dent. Res. 1980, no. 246; J. L. Ebersole, D. E. Frey, M. A. Taubman, D. J. Smith, and R. J. Genco, J. Dent. Res. Special Issue A, Abstr. Am. Assoc. Dent. Res. 1980, no. 249) and against A. actinomycetemcomitans leukocidin (P. Baehni, C. C. Tsai, W. McArthur, N. S. Taichman, and R. J. Genco. J. Dent. Res. Special Issue A, Abstr. Int. Assoc. Dent. Res. 1979, no. 116; J. L. Ebersole, D. E. Frey, M. A. Taubman, D. J. Smith, R. J. Genco, and B. F. Hammond, J. Dent. Res. Special Issue A, Abstr. Am. Assoc. Dent. Res. 1980, no. 255) in juvenile periodontitis patients than in normal control subjects, indicating that antigenic components of A. actinomycetemcomitans may be involved in the pathogenesis of juvenile periodontitis. That A. actinomycetemcomitans has pathogenic potential is also suggested from studies of nonperiodontal infections. This organism is regularly isolated from actinomycosis lesions (3, 8, 10), and it has been recovered as the sole infecting agent from endocarditis lesions (17, 24), a brain abscess (14), a thyroid gland abscess (5), a urinary tract infection (22), and a case of vertebral osteomyelitis (15). It is not known whether A. actinomycetem-

2 1014 SLOTS, REYNOLDS, AND GENCO comitans is associated with types of periodontal disease other than that of young individuals or in what incidence and proportions it inhabits normal periodontal sites. Also, besides the knowledge that it is a member of the periodontal microflora, little is known about the occurrence of A. actinomycetemcomitans on oral surfaces. To effectively control a potential pathogenic organism in the oral cavity, it seems essential to identify the organism's primary ecological habitat İn this paper, we report on the oral distribution of A. actinomycetemcomitans in 43 juveniles and adults. Normal periodontal sites, diseased periodontal pockets, cheek mucosal membrane, tongue dorsum, and saliva were sampled in each patient. Attempts were made to relate the oral A. actinomycetemcomitans infection to the clinical status of the periodontal tissues. MATERIALS AND METHODS Subjects. The following four groups of individuals were examined in the present study: 10 localized juvenile periodontitis patients, 13 to 23 years old, with rapidly advancing periodontal lesions (-6 mm of periodontal pocket depth as measured with a Michigan probe) on incisors and first molars; 10 juveniles, 14 to 21 years old, with no or only mild gingival inflammation (no bleeding from the periodontal tissues after applying 20 g of pressure with a Michigan probe) and no areas of a periodontal sulcus depth exceeding 3 mm; 12 adult periodontitis patients, 30 to 65 years old, with several teeth exhibiting a periodontal pocket depth greater than 6 mm; 11 adults (staff members of our department), 25 to 55 years old, with little gingival inflammation and no areas of periodontal sulcus depth exceeding 3 mm. Two juvenile periodontitis patients were siblings, and two normal juveniles were siblings; otherwise, the individuals examined were unrelated. None of the subjects had received dental or antibiotic treatment for at least 6 months before this study. Sample collection and processing. In each individual, samples were taken from at least six approximal subgingival areas, cheek mucosa, tongue dorsum, and saliva. Four deep periodontal lesions and two periodontal sites with no alveolar bone loss were routinely sampled in each periodontitis patient. Samples of periodontal pockets were collected as follows. Supragingival deposits were removed from the isolated teeth with sterile cotton balls. Three paper points (Johnson Fine Absorbent Points, Johnson & Johnson, East Windsor, N.J.) were subsequently inserted into the periodontal site until resistance was met or the paper points bent. Each was kept in place for 10 s and then transferred within 1 to 2 s to 9 ml of prereduced, anaerobically sterilized Ringer solution. Samples of cheek and tongue surfaces were taken with a Calgiswab, Type II (Inolex, Glenwood, Ill.), from an approximate sampling area of 1 cm2, and they were then transferred to 9 ml of the prereduced, anaerobically sterilized Ringer solution. Samples (1.0 ml) of whole, unstimulated saliva were collected by expectoration and transferred to 9 ml of the Ringer solution. INFECT. IMMUN. The bacterial deposits were dispersed by mixing in a Vortex mixer at the maximal setting for 60 s and serially diluted in 10-fold steps in the Ringer solution, and aliquots were transferred to a Petroff-Hausser bacteria counter (Hausser Scientific, Blue Bell, Pa.) to enumerate the total number of cells sampled. The bacterial cells were well dispersed, and only rarely were bacterial clumps present. Routinely, the bacterial counts of 200 to 400 small squares in the Petroff- Hausser bacteria counter were determined. The methodological error in determining the total cell counts was assessed by duplicate counting of 28 samples and was calculated by the equations Zd2/dn and d = [2(x,- x2)]/(xi + x2) (1), where xi and x2 represent the two values of repeated counting, and n represents the number of samples examined. The methodological error of total cell counting was 4.9%. Selective medium for A. actinomycetemcomitans Samples (0.1 ml) of suitable dilutions were plated on freshly prepared Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 75 jg of bacitracin per ml (Sigma Chemical Co., St. Louis, Mo.)-10% heat-inactivated horse serum-0.1% yeast extract-0.2% glucose, and the agar plates were incubated at 370C for four days in an anaerobic chamber (Coy Manufacturing Co., Ann Arbor, Mich.) with 85% N2-10% H2-5% CO2. This medium for selective isolation of A. actinomycetemcomitans grew as many colonies as, or more colonies of A. actinomycetemcomitans than did enriched brain heart infusion blood agar (BBL Microbiology Systems; our standard medium for isolating oral organisms) in each of 15 subgingival plaque samples tested. Also, incubation in humid 5% CO2 plus air or in 85% N2-10% H2-5% CO2 yielded approximately the same viable counts of A. actinomycetemcomitans. The selective medium for A. actinomycetemcomitans suppressed the remaining oral microflora by 3 to 6 logs. A. actinomycetemcomitans was distinguishable from the few other organisms growing on the medium (haemophili, eikenella, gram-negative anaerobic motile rods, fusobacteria, neisseria, and streptococci) on the basis of colony morphology and catalase activity. Four days growth of A. actinomycetemcomitans resulted in round, convex colonies, 0.5 to 1.0 mm in diameter with slightly irregular edges. The colonies were translucent and glistening, adhered to the medium, and were difficult to break up. Often, the colonies exhibited a star-like inner structure. The ability of A. actinomycetemcomitans to decompose 3% H202 applied on the primary isolation plate was useful in helping to identify the organism. The recovery rate of A. actinomycetemcomitans was calculated by dividing the counts of A. actinomycetemcomitans obtained from the selective medium with the total cell counts obtained by the direct microscopic enumeration described above. Characteristics of A. actinomycetemcomitans A minimum of two isolates of A. actinomycetemcomitans were subcultured from each site of each individual after 4 days of incubation. Culture purity of the isolates was checked by repeated subcultivation, and each pure culture was identified by Gram stain characteristics, morphology, growth in various incubation atmospheres, and biochemical reactions carried out by established procedures (6, 9).

3 VOL. 29, 1980 The organisms were small, nonmotile, gram-negative coccobacilli. The ability of the colonies to adhere to agar surfaces disappeared for some strains on repeated subculture. The strains initially grew in broth with a granular sediment strongly adherent to the bottom of the test tube, whereas the nonadherent variant showed uniform turbidity. The organisms exhibited poor growth in air but grew abundantly in 5% C02 plus air or in anaerobic conditions. All strains utilized carbohydrates fermentatively, decomposed H202, reduced nitrate, and were benzidine positive. They produced less than 1 meq of formic acid, acetic acid, and succinic acid per 100 ml in acidified cultures of peptone-yeast extract-glucose broth (9). Pyruvate and oxalacetate were utilized as shown by disappearance of the pyruvic acid and oxalacetic acid peaks in peptone-yeast extract-pyruvate culture (9). Lactate was not utilized, and threonine was not converted to propionate. None of the strains required X (hemin) or V (nicotinamide adenine dinucleotide) growth factors nor grew on MacConkey agar. None hydrolyzed casein, deoxyribonucleic acid, esculin, gelatin, starch, or urea, nor produced acetylmethylcarbinol, decarboxylases, H2S lecithinase, lipase, oxidase, or indole. All strains produced acid (ph decrease of >0.5 U compared with inoculated peptone-yeast extract broth [ph 6.3 to 6.9]) from glucose, fructose, and mannose, but not from adonitol, amygdalin, arabinose, cellobiose, dulcitol, esculin, galactose, glycerol, inositol, inulin, lactose, melezitose, melibiose, rhamnose, ribose, salicin, sorbitol, or sucrose. Variable fermentation results were found for dextrin, maltose, mannitol, and xylose. RESULTS Table 1 summarizes the recovery counts of A. actinomycetemcomitans in normal periodontal sites, on cheek mucosa, on dorsum of the tongue, and in saliva of the 10 juveniles and 11 adults TABLE 1. A. ACTINOMYCETEMCOMITANS AND PERIODONTITIS 1015 studied with minimal or no periodontal disease. The organism was isolated from only two normal juveniles and four normal adults. Two normal adults each harbored the organism in four subgingival areas, in saliva, and on the tongue and cheek mucosae; one normal adult harbored the organism in two periodontal sites, on the tongue, and on the cheek, and one normal adult had only one periodontal site infected with the organism. The organism was recovered from two periodontal sites and the cheek of one normal juvenile and from the cheek only of another normal juvenile. In the normal juvenile and adult individuals examined, A. actinomycetemcomitans was only present in approximately 10% of the normal periodontal study sites, and it comprised less than 5% of the microflora of all infected periodontal sites (Table 1). Its proportion of the cheek, tongue, and salivary flora was less than 1% (Table 1). Table 2 shows differences in microbial content of normal individuals between subgingival areas with A. actinomycetemcomitans and with no cultivable A. actinomycetemcomitans. Periodontal sites infected with A. actinomycetemcomitans contained significantly higher numbers of bacteria and were richer in spirochetes than periodontal sites free of A. actinomycetemcomitans (Table 2). The clinical examination revealed that most periodontal sites infected with A. actinomycetemcomitans were more inflamed than the noninfected periodontal sites. In the adult periodontitis group, six patients yielded no recoverable A. actinomycetemcomitans (Table 3). One patient harbored the organism only in one normal periodontal site. Five Distribution ofa. actinomycetemcomitans in 10 juveniles and 11 adults with healthy periodontium No. of infected Total no. No. of in- No. of infected samples at following count: samples at Sample site of sam- fected following ples samples ' ,000-20, , ,000- S ,000 99, , , Juvenile Normal periodontal areas Cheek mucosa Tongue Saliva Adultb Normal periodontal areas Cheek mucosa Tongue Saliva 'A. actinomycetemcomitans was recovered from two normal juveniles (20%). b A. actinomycetemcomitans was recovered from four normal adults M36%).

4 1016 SLOTS, REYNOLDS, AND GENCO patients had 67 to 100% of the deep periodontal pockets examined infected with A. actinomycetemcomitans. Three of these five patients also harbored the organism in normal periodontal sites, whereas the two remaining patients showed no infection of A. actinomycetemcomitans in normal subgingival areas. The organism was recovered from the cheek of four patients and from the salivas of three patients. No adult periodontitis patients yielded A. actinomycetemcomitans from the dorsum of the tongue. TABLE 2. Differences in microbial content of subgingival areas with and without cultivable A. actinomycetemcomitans in 10 normal juveniles and 11 normal adults Presence TtPeri Avgspi of A. ac- Total no. Avg no. of or- sites rochete tinomy- of perio- * popula- r cetem-dontal ganmsms sam- with cotem- dsjntal pled (±SD)b spiro- tion Comi- sites' ~chetes (±SD) tans (%) Y With x (±20) (±53 x 105) Without X (±8) (±26 x 105) aone sample from a periodontal site with A. actinomycetemcomitans and thirteen samples from periodontal sites with no cultivable A. actinomycetemcomitans contained less than 5 x 104 organisms per ml (no bacterial cells detected in the counting area of the Petroff-Hausser bacteria counter); these samples were not included in this table. bprobability, < P < 0.01 (Student's t test). SD, Standard deviation. 'Probability, P < (Student's t test). TABLE 3. INFECT. IMMUN. In the adult patient group, A. actinomycetemcomitans constituted less than 1% of the total microflora in about 75% of the infected, diseased sites and normal periodontal sites studied (Table 3). Only one deep periodontal pocket and one normal periodontal site harbored the organism in proportions of 10 to 40%. The absolute number of A. actinomycetemcomitans recovered from infected sites averaged 407,300 organisms for deep periodontal pockets, 67,200 organisms for normal subgingival areas, 8,900 organisms for the cheek, and 13,700 organisms for the saliva. In contrast to the three other subject groups studied, all juvenile periodontitis patients except one were extensively infected with A. actinomycetemcomitans (Table 3). Seven juvenile periodontitis patients harbored A. actinomycetemcomitans in all deep periodontal pockets examined, and two patients had 50 and 60% of their deep periodontal pockets infected with the organism. Four patients showed A. actinomycetemcomitans in all normal subgingival areas tested, two patients had 25 and 60% of the normal subgingival areas infected, and two patients with A. actinomycetemcomitans in deep periodontal pockets showed no infection of normal periodontal sites. Nine juvenile periodontitis patients harbored the organism on the cheek mucosa and in the saliva, and five patients had the organism on the tongue. From one juvenile periodontitis patient, A. actinomycetemcomitans was not recovered from any oral site even with repeated sampling. In infected deep juvenile periodontitis pock- Distribution ofa. actinomycetemcomitans in 12 patients with adult periodontitis and 10 patients with juvenile periodontitis dtotal No. of No. of infected samples at following count: No. of infected samples at fol- Total No. of ~~~~~~~~~~~~~lowing %: Sample site no. of infected samples sa e 2,000-,999 20, , , ,000,000 <0 19,999 99, , , O '4. Adult" Deep periodontal pockets Normal periodontal areas Cheek mucosa Tongue Saliva '40.0 Juvenile' Deep periodontal pockets Normal periodontal areas Cheek mucosa Tongue Saliva aa. actinomycetemcomitans was recovered from six adult periodontitis patients (50o). ba. actinomycetemcomitans was recovered from nine juvenile periodontitis patients (90%).

5 VOL. 29, 1980 TABLE 4. A. ACTINOMYCETEMCOMITANS AND PERIODONTITIS 1017 ets, counts of A. actinomycetemcomitans averaged 515,402 organisms, and 21 of 27 of the infected lesions contained more than 100,000 cells of the organism (Table 3). In infected normal subgingival areas, the total counts of A. actinomycetemcomitans averaged 130,350 organisms. Whether the three normal sites which harbored more than 100,000 A. actinomycetemcomitans cells (Table 3) actually were undergoing initial alveolar bone breakdown could not be established because the patients studied were subjected to intensive periodontal treatment within a few months following initial examination. The total number ofa. actinomycetemcomitans recovered from infected cheeks, tongues, and salivas of juvenile periodontitis patients averaged 4,755, 10,071, and 110,387 organisms, respectively. The consistency of the A. actinomycetemcomitans infection in juvenile periodontitis was examined in seven patients by duplicate sampling during the 1- to 4-month pretreatment period. Of a total of 44 deep and normal periodontal sites tested, 36 sites gave similar results with repeated sampling in terms of presence or absence of A. actinomycetemcomitans. Eight periodontal sites varied between culture-positive and culture-negative results; however, when A. actinomycetemcomitans was isolated from these subgingival areas, an average of only 55,000 cells of the organism was recovered. Statistical analysis revealed that A. actinomycetemcomitans was found in significantly higher proportions and higher numbers in infected periodontal lesions of juvenile periodontitis patients than in infected periodontal lesions of adults (at the 5% significance level analyzed by means of the Mann-Whitney test for ordinal data [25]). In infected juvenile periodontitis patients, the salivary counts of A. actinomycetemcomitans tended to be higher than those of adult periodontitis patients, but this was not considered statistically significant (0.1 < P < 0.2). By utilizing a correlation analysis for dichotomous nominal scale data (25), significant positive associations (at the 5% significance level) were found in the incidence ofa. actinomycetemcomitans in infected individuals for deep periodontal pockets and normal periodontal sites, periodontal pockets and cheek, tongue and saliva, and cheek and saliva. The correlations between periodontal pockets and saliva, periodontal pockets and tongue, and tongue and cheek were not statistically significant. As previously described, the present isolates of A. actinomycetemcomitans had many physiological properties in common. On the basis of variable fermentation of dextrin, maltose, mannitol, and xylose, however, ten "biotypes" of A. actinomycetemcomitans could be established (Table 4). It was of interest that all oral study sites of each individual were inhabited by one biotype only. An exception was one juvenile periodontitis patient who was infected with two biotypes. Also, the same biotype was found in the repeat samplings from seven juvenile periodontitis patients. Two siblings with juvenile periodontitis furthermore harbored the same biotype. No obvious differences in the distribution of biotypes between the four subject groups examined could be established (Table 4). DISCUSSION Organisms of this study designated A. actinomycetemcomitans conformed to the description of the species in Cowan and Steel's Manual for the Identification of Medical Bacteria (6) and in Bergey's Manual of Determinative Bac- Biotypes ofa. actinomycetemcomitans and their distribution in periodontally healthy and diseased individuals Fermentation by:a N1 Carbohy- Y4, NJ (1), NA (2),b drate ATCC AP (5), NJ (1), JP (2), JP (1), NA (1), 29522, P (5), J (1) AP (1) NA (1) AP (2) A (1) AP (1) JP (1) AP (1) ATCC ATCCNA) A() AP) Dextrin Maltose Mannitol Xylose a +, Fermentation of carbohydrate (ph decrease of >0.5 U compared with inoculated basal medium); -, no fermentation of carbohydrate; NJ, isolate from normal juvenile; JP, isolate from juvenile periodontitis patient; NA, isolate from normal adult; AP, isolate from adult periodontitis patient; Y4, strain isolated from a juvenile periodontitis patient by S. S. Socransky, Boston, Mass.; ATCC 29522, ATCC 29523, and ATCC 29524, strains obtained from the American Type Culture Collection, Rockville, Md. b Number of individuals is shown within parentheses.

6 1018 SLOTS, REYNOLDS, AND GENCO teriology (4). The present isolates produced catalase and failed to ferment lactose or sucrose, characteristics which are important in differentiating between A. actinomycetemcomitans and the closely associated species Haemophilus aphrophilus (13). However, data from our laboratory revealed that the present species A. actinomycetemcomitans includes groups of organisms with different biochemical and antigenic properties, indicating that the taxonomic position of these organisms is still not sufficiently clarified. The present results indicated that the occurrence of A. actinomycetemcomitans in the oral cavity is related to the presence of periodontal inflammation. The organism was recovered from 6 of 12 adult periodontitis patients and from 9 of 10 juvenile periodontitis patients. In contrast, only 6 of the 21 individuals with generally healthy periodontal tissues harbored the organism. Also, when present in normal individuals, A. actinomycetemcomitans preferentially inhabited periodontal sites which exhibited some degree of inflammation and were rich in spirochetes. A high subgingival proportion of spirochetes is suggestive of gingival inflammation (20). The positive correlation between A. actinomycetemcomitans and periodontal pocket depth and gingival inflammation as demonstrated in this study has also been reported by Tanner et al. (21). Juvenile periodontitis patients were more extensively infected with A. actinomycetemcomitans than the adult periodontitis patients. A. actinomycetemcomitans constituted more than 5% of the total microflora in 24% of all juvenile periodontal lesions and in 16% of all normal periodontal sites in juvenile periodontitis patients. Only 2% of deep periodontal pockets and 5% of normal periodontal sites in adult periodontitis patients harbored the organism in proportions of more than 5%. By utilizing a reproducible sampling technique (20), 78% of infected juvenile periodontitis lesions were shown to harbor more than 100,000 A. actinomycetemcomitans cells, whereas only 47% of infected adult periodontitis lesions exhibited more than 100,000 cells of the organism. In patients with localized juvenile periodontitis, Slots (18) previously demonstrated that gram-negative facultative rods, many of which likely belonged to A. actinomycetemcomitans, made up 5% (range, 0 to 17%) of the deep periodontal pocket isolates and 8% (range, 0 to 30%) of the normal periodontal site isolates. Newman et al. (16) also recovered A. actinomycetemcomitans (groups III and IV [21]) in high proportions from several periodontosis (localized juvenile periodontitis) lesions. A recent study of Tanner INFECT. IMMUN. et al. (21) found that A. actinomycetemcomitans constituted 2 to 26% of the cultivable microflora in five deep periodontal pockets of three patients 20 to 29 years old. When comparing the present results with those of previous studies, it should be remembered that our A. actinomycetemcomitans data are expressed as a percentage of the total cell counts as determined by direct microscopy and not as a percentage of the total number of cultivable microorganisms. Since advanced periodontal lesions contain 30 to 50% spirochetes, selenomonas, and other organisms which are difficult to grow (20) and since healthy or slightly inflamed periodontal sites harbor fewer motile organisms and have a microflora which more readily can be cultivated (19), the percentage of A. actinomycetemcomitans in deep periodontal pockets will appear higher, and the difference between diseased and normal periodontal sites will be increased if the recovery counts are related to numbers of cultivable organisms. Expression of A. actinomycetemcomitans in relation to the total cell counts was chosen because such results are independent of variations in recovery efficiency of the total microflora and because they also take into account organisms which cannot be grown by available techniques, but which may possess periodontopathic potential. Our microbiological data, along with the recognized pathogenic potential of A. actinomycetemcomitans and recent immunological findings that most juvenile periodontitis patients exhibit markedly higher levels of serum and gingival crevice fluid immunoglobulin G (IgG) antibodies (R. J. Genco, N. A. Taichman, and C. A. Sadowski, J. Dent, Res. Special Issue A, Abstr. Am. Assoc. Dent. Res. 1980, no. 246; P. A. Murray and R. J. Genco, J. Dent. Res. Special Issue A, Abstr. Am. Assoc. Dent. Res. 1980, no. 245) to A. actinomycetemcomitans than normal control subjects, point to the importance of this organism in certain types of human periodontal disease. However, whether A. actinomycetemcomitans is an etiological agent in periodontal disease or whether the organism is a secondary invader of the established periodontal lesion can only be determined by examining its occurrence throughout disease development and by monitoring the effect on the periodontal tissues of its removal from the periodontal pocket area. The ecology study revealed that 47% of the subgingival samples, 17% of the tongue samples, 40% of the cheek mucosa samples, and 29% of the saliva samples contained A. actinomycetemcomitans. In addition, periodontal pockets often contained 100-fold or more cells of A. actinomycetemcomitans than the other oral sites studied. These data suggest that dental plaque and

7 VOL. 29, 1980 the periodontal pocket area are a main oral reservoir of A. actinomycetemcomitans. This hypothesis is consistent with the finding that A. actinomycetemcomitans in vitro can attach to tooth surfaces and form dental plaque (12). It was of interest that A. actinomycetemcomitans often was recovered from the periodontal pocket samples without being isolated from the corresponding saliva samples. Statistically significant correlations, on the other hand, were found between the presence of A. actinomycetemcomitans in saliva and on tongue dorsum and cheek mucosa. These data seem to indicate that most salivary A. actinomycetemcomitans, in agreement with findings for other oral bacterial species (7), originate from the tongue and other oral mucosal surfaces. The finding that only 21 of the 43 individuals examined had detectable A. actinomycetemcomitans in the oral cavity and that many infected subjects only harbored the organism at a few oral sites is of interest. Bacteroides melaninogenicus and Bacteroides asaccharolyticus also vary considerably in incidence and porportions from person to person and from site to site in the oral cavity (J. J. Zambon, H. S. Reynolds, and J. Slots, J. Dent. Res. Special Issue A, Abstr. Am. Assoc. Dent. Res. 1980, no. 967), whereas major dental plaque organisms including Streptococcus sanguis, Streptococcus mitior, and Actinomyces naeslundii can be isolated from numerous oral sites of virtually all individuals. When evaluating the possibility of eliminating an organism from the oral cavity, it may be important to distinguish between universal and occasional members of the indigenous oral microflora. A universal indigenous organism presumably cannot be eliminated from the oral microflora, at least not for a prolonged period of time, whereas an oral organism which only sporadically appears in the human population may be removable from the infected individuals. We have recently shown that A. actinomycetemcomitans is highly susceptible to tetracycline in vitro (J. Slots and P. M. Lobbins, Abstr. Annu. Meet. Am. Soc. Microbiol. 1980, C129, p. 296) and that most juvenile periodontitis patients respond favorably to tetracycline therapy (unpublished data). Studies are in progress to determine to what extent periodontal infections of A. actinomycetemcomitans can be resolved by the use of tetracycline and other periodontal treatments. In summary, our studies (i) point to a positive relationship between A. actinomycetemcomitans and periodontal pocket depth and gingival inflammation, (ii) reveal that localized juvenile periodontitis patients, in particular, are extensively infected with this organism, and (iii) suggest that dental plaque and periodontal pockets A. ACTINOMYCETEMCOMITANS AND PERIODONTITIS 1019 are its primary oral ecological niche. 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