Distribution of Actinobacillus actinomycetemcomitans serotypes in periodontal health and disease

A total of 177 Actinobacillus actinomycetemcomitans isolates from 136 periodontally healthy or diseased subjects were serotyped by indirect immunofluorescence and/or immunodiffusion assays. Serotype-specific rabbit antisera against A. actinomycetemcomitans serotypes a, b and c were used. All 3 serotypes were commonly found in the study subjects. Serotype b was dominant in subjects with periodontal disease and serotype c was the most common serotype in the healthy subjects. In the immunofluorescence assay, when 85 isolates were cultured anaerobically and fixed in acetone, or cultured aerobically in 10% CO2 and heat-fixed, 60 isolates revealed the same serotypes. The remaining 25 isolates reacted with 2 of the serotype-determining reagents. In the immunodiffusion assay, 22 of these 25 isolates reacted with one antiserum only. These results suggest differences in the distribution of A. actinomycetemcomitans serotypes between periodontal health and disease and point to possible variation in serotype determination due to bacterial growth and preparation procedures.     

Actinobacillus actinomycetemcomitans in human periodontal disease

Recent evidence implicates Actinobacillus actinomycetemcomitans in the etiology of localized juvenile periodontitis. This paper reviews the morphological, biochemical and serological charcteristics of A. actinomycetemcomitans, evidence incriminating it as a periodontopathogen, its importance in human nonoral infections, and virulence factors which may be involved in the pathogenesis of A. actinomycetemcomitans infections. A. actinomycetemcomitans is a non-motile, gram-negative, capnophilic, fermentative coccobacillus which closely resembles several Haemophilus species but which does not require X or V growth factors. The organism has been categorized into 10 biotypes based on the variable fermentation of dextrin, maltose, mannitol, and xylose and into 3 serotypes on the basis of heat stable, cell surface antigens. A. actinomycetemcomitans' primary human ecologic niche is the oral cavity. It is found in dental plaque, in periodontal pockets, and buccal mucosa in up to 36% of the normal population. The organism can apparently seed from these sites to cause severe infections throughout the human body such as brain abscesses and endocarditis. There is a large body of evidence which implicates A. actinomycetemcomitans as an important micro-organism in the etiology of localized juvenile periodontitis including: (1) an increased prevalence of the organism in almost all localized juvenile periodontitis patients and their families compared to other patient groups; (2) the observation that localized juvenile periodontitis patients exhibit elevated antibody levels to A. actinomycetemcomitans in serum, saliva and gingival crevicular fluid; (3) the finding that localized juvenile periodontitis can be successfully treated by eliminating A. actinomycetemcomitans from periodontal pockets; (4) histopathologic investigations showing that A. actinomycetemcomitans invades the gingival connective tissue in localized juvenile periodontitis lesions; (5) the demonstration of several pathogenic products from A. actinomycetemcomitans including factors which may: (a) facilitate its adherence to mucosal surfaces such as capsular polysaccharides; (b) inhibit host defense mechanisms including leukotoxin, a polymorphonuclear leukocyte chemotaxis inhibiting factor, and a lymphocyte suppressing factor (c) cause tissue destruction such as lipopolysaccharide endotoxin, a bone resorption-inducing toxin, acid and alkaline phosphatases, collagenase, a fibroblast inhibiting factor and an epitheliotoxin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Actinobacillus actinomycetemcomitans genotypes in relation to serotypes and periodontal status

Actinobacillus actinomycetemcomitans is prevalent in periodontitis but is found in some periodontally healthy individuals as well. The arbitrarily primed polymerase chain reaction (AP-PCR) was used to fingerprint clinical A. actinomycetemcomitans isolates of different serotypes to determine the association between individual clonal types and periodontal conditions. Fifteen different AP-PCR genotypes were distinguished among 93 A. actinomycetemcomitans isolates from 86 uncohabiting individuals with adult periodontitis, localized juvenile periodontitis or no periodontal destruction. The 3 most common AP-PCR genotypes accounted for 68% of the isolates. Seven of the remaining AP-PCR genotypes were found only in periodontitis. The isolates of a given AP-PCR genotype usually belonged to the same serotype. The distribution of the AP-PCR genotypes among serotype b isolates seemed to differ among the subject groups. The results revealed a major genetic dissimilarity between A. actinomycetemcomitans serotypes and suggested a relationship between some A. actinomycetemcomitans clones and periodontal disease.     

Prevalence of Actinobacillus actinomycetemcomitans serotypes in Japanese patients with periodontitis

Oral Actinobacillus actinomycetemcomitans strains are serologically classified into 5 distinct groups, a to e. We examined the distribution of A. actinomycetemcomitans serotypes in Japanese patients with periodontitis. A total of 157 A. actinomycetemcomitans clinical isolates from diseased sites of 39 patients with periodontitis were serotyped by using serotype-specific rabbit antisera against. A. actinomycetemcomitans serotypes a, b, c, d and e strains. In the immunodiffusion assay, autoclaved extracts of 42, 6, 39, 9 and 41 A. actinomycetemcomitans clinical isolates reacted with serotypes a, b, c, d and e antisera, respectively. Although 37 patients were infected with a serotype strain, 2 patients harbored 2 different serotype strains, b/e and b/untypeable. To establish a correlation between serotype and genotype of A. actinomycetemcomitans clinical isolates from 2 patients who had different serotype strains, we used arbitrarily primed polymerase chain reaction (AP-PCR) to fingerprint clinical isolates of different serotypes. The AP-PCR genotype among 4 clinical isolates (b/e and b/untypeable) were identical to that of A. actinomycetecomitans Y4 (serotype b), indicating the presence of multiple A. actinomycetemcomitans serotypes which are genetically homogenous in the periodontally diseased sites of patients with periodontitis.     

A follow-up case report of Actinobacillus actinomycetemcomitans in human periodontal disease

The purpose of this investigation was to compare clinical and microbial parameters in a follow-up case report of adult subjects harboring Actinobacillus actinomycetemcomitans (Aa) with clinically matched subjects who did not have detectable Aa. 16 subjects with Aa and 16 subjects without Aa at the baseline examination were re-examined at an average of 46 months following collection of baseline data. Clinical measurements were recorded and subgingival plaque sampled and evaluated for microbial flora from each maxillary first molar. In 16 subjects with Aa at baseline, 4 sites in 3 subjects had detectable actinobacilli at the follow-up appointment. 26 sites in 13 individuals with Aa at baseline had a significantly increased gingival index at the follow-up visit (p less than or equal to 0.05), but there was no significant increase in probing depth or attachment loss. 32 sites in the 16 subjects without Aa at baseline still did not have detectable levels of this microorganism at the follow-up examination nor was there any significant difference between baseline and the follow-up appointment for the gingival index, probing depth and attachment level measurements. In subjects with Aa at baseline, 1 of 12 teeth without Aa and 5 of 20 teeth with Aa had been extracted prior to the follow-up visit. In this population group, having sites where Aa was detected, 6 of 9 teeth which had a probing depth greater than or equal to 5 mm were lost before the follow-up data collection appointment. In the control group, which did not have detectable Aa at baseline, 9 teeth with probing depths greater than or equal to 5 mm were not lost. These observations, although not proving, suggest in this population group, that deeper probing depths taken together with the presence of Aa may have placed an individual at greater risk of tooth loss.     

Intraoral distribution of Actinobacillus actinomycetemcomitans in young adults with minimal periodontal disease

The aim of the present study was to investigate the intraoral distribution of Actinobacillus actinomycetemcomitans in young adults with minor signs of periodontal disease but harboring the organisms in the oral cavity. 17 healthy volunteers, 20 to 27 years of age, participated. Samples from mucosal surfaces of the oro-pharyngeal cavity and saliva (n = 221) as well as subgingival plaque from every tooth (n =477) were selectively cultivated for A. actinomycetemcomitans. Species identity and presence of the leukotoxin encoding gene, ltxA, were checked by multiplex polymerase chain reaction. Moreover, the leukotoxin promoter region was analyzed. No isolate harbored a 530 bp deletion in the promoter region of the leukotoxin gene, signaling minimally toxic strains. 42.1 +/- 30.4% extracrevicular and 34.4 +/- 29.5% subgingival samples were culture-positive. In extracrevicular samples, the organism could easily be recovered from cheek mucosa (62%), saliva (59%) and the palatal tonsils (41%). Mean log-transformed numbers of A. actinomycetecomitans colony forming units (CFU/ml) in culture-positive material ranged between 1.8 from the hard palate and 2.3 from 10 microl saliva. The highest prevalence in subgingival plaque was observed at maxillary 3rd molars (55%) followed by maxillary lateral incisors (50%) and mandibular 3rd molars (41%). Mean log-transformed counts of CFU/ml ranged between 2.2 at maxillary 3rd molars and 3.4 at upper central incisors. When adjusted for jaw, site and tooth type, the odds of isolating higher numbers of the organism were increased with every mm probing depth by a factor of 1.35 (p <0.05). The odds ratio for bleeding on probing was 1.38. Thus, in this young adult population with minor periodontal disease, A. actinomyetemcomitans was mainly associated with some deviation from gingival health. Of concern might be a minority of subjects (29%) with an extremely wide distribution of the organism in the oral cavity.     

Actinobacillus actinomycetemcomitans serotype e – biotypes,genetic diversity and distribution in relation to periodontal status

Actinobacillus actinomycetemcomitans isolates from 356 individuals were screened for identification of serotype e in order to investigate its distribution in relation to periodontal status. From subjects with serotype e, 1–6 isolates per subject (n=61) were genotyped using arbitrarily primed–polymerase chain reaction (AP-PCR) and apaH gene polymerase chain reaction–restriction fragment-length polymorphism (PCR-RFLP) analysis to determine the genetic heterogeneity within the serotype. Furthermore, one serotype e strain per subject was tested for fermentation of 8 carbohydrates for biotyping. Among patients with adult periodontitis (n=219), localized juvenile periodontitis (n=55) and other forms of early-onset periodontitis (n=18) serotypes b, a and c, respectively, were the most frequently detected serotypes. Non-periodontitis subjects (n=64) were predominantly colonized with serotype c. Serotype e was found in 30 (14%) adult periodontitis patients, 2 (11%) early-onset periodontitis patients and in 5 (8%) non-periodontitis individuals, but in none of the 55 localized juvenile periodontitis patients. AP-PCR distinguished 3 and apaH gene PCR-RFLP analysis 2 genotypes among the 61 A. actinomycetemcomitans serotype e isolates, one genotype per subject. The AP-PCR genotypes 1 and 3 represented the apaH genotype 1 and the AP-PCR genotype 2 the apaH genotype 2. On the basis of variable fermentation of galactose and xylose, 3 biotypes among A. actinomycetemcomitans serotype e were established. Contrary to the absence of A. actinomycetemcomitans serotype e in localized juvenile periodontitis patients, its detection frequency was comparable among other forms of periodontitis and periodontal health. Clinical serotype e isolates form at least 2 genetic types and 3 biotypes.     

Actinobacillus actinomycetemcomitans in destructive periodontal disease. Three-year follow-up results

BACKGROUND: Convincing data exist that A. actinomycetemcomitans is an etiologic agent of periodontal disease. The purpose of this longitudinal study was to evaluate A. actinomycetemcomitans as a diagnostic indicator for periodontal disease in treated and periodontally maintained patients. METHODS: Following comprehensive mechanical/surgical and supportive amoxicillin plus metronidazole therapy in 13 subjects with A. actinomycetemcomitans-associated destructive periodontal disease, we monitored subgingival A. actinomycetemcomitans at 4 individual sites in each patient up to 3 years post-therapy. The periodontal status was determined, and A. actinomycetemcomitans levels were quantitatively enumerated on TSBV agar in CFU/ml. Six patients with a persistence of subgingival A. actinomycetemcomitans at each reexamination within 3 years post-therapy were selected to be at risk for minor periodontal treatment outcomes and further recurrence of periodontal disease (test group). Seven subjects with a complete suppression of A. actinomycetemcomitans at each post-therapy visit served as controls. RESULTS: The periodontal parameters decreased from overall values of 6.39 mm (probing depth, PD) and 7.64 mm (clinical attachment level, CAL) at the outset to 3.81 mm (PD) and 5.62 mm (CAL) 2 years post-therapy (Friedman, P< or =0.05). At the 3-year reexamination, the PD/CAL scores increased to 4.03/5.78 mm. Among the 6 individuals (46%) with persistence of subgingival A. actinomycetemcomitans at the final 3-year visit (test group), periodontal status yielded increased levels of 4.45 mm (PD) and 6.60 mm (CAL). The control subjects (n = 7) revealed lower values of 3.67 mm (PD) and 5.09 mm (CAL). However, on a patient level, during the 3-year observational trial, the periodontal status of the 13 individuals was not statistically affected by subgingival infection with A. actinomycetemcomitans. CONCLUSIONS: Although in advanced periodontal disease, comprehensive mechanical and antimicrobial treatment is an appropriate regimen for sustained improvement of periodontal health, long-term control of subgingival infection with A. actinomycetemcomitans could not be achieved. In the maintenance care of destructive periodontitis, the persistence of A. actinomycetemcomitans is not a diagnostic parameter for periodontal disease.     

Natural distribution of oral Actinobacillus actinomycetemcomitans in young men with minimal periodontal disease

A total of 1005 subgingival and extracrevicular samples from 201 male recruits, 18–25 yr old, were selectively cultivated for Actinobacillus actinomycetemcomitans. The organism was isolated in 55 subjects (27%); 9.5% of pooled subgingival plaque samples from first molars, 14% cheek mucosa, 20% dorsum of tongue and 20% saliva samples were culture-positive. In order to divide the study population into distinct clinical categories, cluster analysis was performed, based on previous caries experience, probing pocket depth categories, bleeding scores, visible plaque and calculus. Two clusters (n=86 and n=92, respectively) were identified with no or minimal periodontal disease (mean±standard deviation % of periodontal probing depth 1–2 mm 78.7±10.4% and 57.4±12.6%, respectively; virtually no periodontal probing/depth in excess of 4 mm) and a relatively low DMF-S (22±13). A third cluster (n=22) had, in contrast, a high DMF-S (47.7±173) and a relatively high % of periodontal pockets of ≥5 mm (5.9 ±5.2%). Prevalence of A. actinomycetemcomitans in this cluster was 41%, while the organism was found in 23% and 27% in the minimally diseased populations (p<0.15). Whereas no heterogeneity of associations between subgingival and extracrevicular occurrence of the organism could be ascertained in different clusters, the organism was significantly more often identified in extracrevicular material, especially dorsum of tongue samples, compared with subgingival plaque (McNemar's X²=12.45, p<0.001). Multiple linear regression analysis revealed the number of A. actino-mycetemcomitans positive samples as well as the % of sites bleeding on probing being positively associated with the % of sites with a probing pocket depth of ≥5 mm (R2=0.345, p≤0.0001). The present large-scale investigation points to the wide distribution of this putative periodontopathogen in young individuals with minimal periodontal disease.     

Distribution of biotypes and antimicrobial susceptibility of Actinobacillus actinomycetemcomitans

Eighty isolates of Actinobacillus actinomycetemcomitans from 30 Brazilian periodontitis patients were examined to determine the distribution of biotypes and in vitro antimicrobial susceptibility. Seventy-seven percent of the isolates belonged to biotype X. All A. actinomycetemcomitans isolates were susceptible to cefoxitin, imipenem and tetracycline.     

Relationship of Actinobacillus actinomycetemcomitans serotypes to periodontal condition: prevalence and proportions in subgingival plaque

No study available has utilized the new classification scheme (the consensus report of the American Academy of Periodontology 1999) to determine the prevalence of Actinobacillus actinomycetemcomitans in different periodontal conditions. The purpose of this study was to investigate prevalence and proportions of A. actinomycetemcomitans serotypes in subgingival plaque samples from a young Taiwanese population with aggressive periodontitis, chronic periodontitis and no periodontal disease. A total of 221 subgingival plaque samples from 171 diseased subjects (70 had aggressive periodontitis, and 101 had chronic periodontitis) (mean age 25.0 +/- 8.2 yr) and 50 periodontally healthy subjects (mean age 18.4 +/- 9.5 yr) were screened for A. actinomycetemcomitans. Serotypes of A. actinomycetemcomitans were determined by an indirect immunofluorescence assay using serotype-specific polyclonal antisera to A. actinomycetemcomitans strains ATCC 29523 (serotype a), ATCC 43728 (serotype b) and ATCC 33384 (serotype c). Prevalence (% of positive samples) of A. actinomycetemcomitans was 84.3% in aggressive periodontitis, 60.4% in chronic periodontitis, and 64.0% in periodontally healthy subjects. Proportions of A. actinomycetemcomitans (mean percentage per total bacteria) in periodontally healthy subjects were significantly lower than in aggressive periodontitis subjects. The proportion of serotype b in subjects with aggressive periodontitis and subjects with chronic periodontitis were significantly greater than that in periodontally healthy subjects. The proportion of serotype c in periodontally healthy subjects was much higher than that in chronic periodontitis subjects. The results of this study suggest that prevalence and proportions of A. actinomycetemcomitans are significantly greater in patients with aggressive periodontitis than in those with chronic periodontitis. Serotype b is the predominant serotype of A. actinomycetemcomitans in patients with diseased periodontal conditions. Serotype c is a more common serotype detected in periodontally healthy subjects.     

The distribution of Actinobacillus actinomycetemcomitans in human plaque

The association between Actinobacillus actinomycetemcomitans and periodontal disease in juveniles has been well documented. The purpose of this investigation was to determine the prevalence and proportions of A. actinomycetemcomitans in supragingival and subgingival plaque samples from the maxillary first molars of a large number of young adults. The study population included 284 adults, aged 20–40, ranging in periodontal disease status from healthy to moderate periodontitis but with the majority exhibiting early periodontal disease. The clinical characteristics of probing depth, attachment level, plaque index, and gingival index were measured. Supragingival and subgingival plaque samples were evaluated microscopically for microbial forms. They were also cultured on supplemented blood agar and various selective agar media including selective media for A. actinomycetemcomitans. The prevalence of A. actinomycetemcomitans in subgingival and supragingival plaque for individuals in the population was 13.0% (37/284) and 4.9% (14/284), respectively. Proportions of actinobacilli, based on total anaerobic counts, were found at or below 1% in 87% of 47 subgingival sites from 37 subjects. Supragingival and subgingival sites with actinobacilli were compared to sites without actinobacilli. Subgingival sites with A. actinomycetemcomitans had a significantly higher mean plaque index, with 79% of these sites having a plaque index greater than 1.0 compared to 30% of sites without actinobacilli. The mean gingival index, probing depth, and attachment level of sites with actinobacilli were also higher, but not significantly, than those without. Of the microbial forms enumerated, only spirochetes had a significantly higher mean proportion at subgingival sites when compared to sites without actinobacilli. Mean proportions of the cultivable microorganisms, Veillonella spp. and Streptococcus spp., were significantly lower at sites with A. actinomycetemcomitans. Differences in the mean proportions of certain microorganisms were compared between the 47 subgingival sites with actinobacilli divided into three groups by probing depth. Mean proportions of A. actinomycetemcomitans were significantly higher at intermediate probing depths between 3.0 and 5.0 mm compared to deeper sites with probing depths above 5.0 mm. On the other hand, dark-pigmented Bacteroides spp. mean proportions were significantly higher at deeper probing depths than at either intermediate or shallow, less than or equal to 3.0 mm, probing depths. There were no significant differences in the mean proportions of spirochetes between shallow, intermediate, or deeper probing depths of the 47 subgingival sites with actinobacilli.     

Cytolethal distending toxin of Actinobacillus actinomycetemcomitans. Occurrence and association with periodontal disease

The cytolethal distending toxin (Cdt) of Actinobacillus actinomycetemcomitans, a periodontal pathogen, is a newly described cytotoxin with immunosuppressive properties, capable of causing cell cycle arrest of lymphocytes. The objectives of this study were to investigate the occurrence of A. actinomycetemcomitans with the cdt genotype in the subgingival plaque of periodontitis patients and to determine the association of this bacterial genotype with periodontal disease. A total of 146 subgingival plaque samples from periodontitis patients were assayed by the PCR method using oligonucleotide primers targeting the cdt operon of A. actinomycetemcomitans. Primers targeting the leukotoxin gene A (ltxA) of A. actinomycetemcomitans was used to determine the occurrence of the bacteria in the plaque samples at baseline. At baseline, A. actinomycetemcomitans was detected in 106 out of 146 (73%) diseased sites studied. Among the 106 diseased sites found to harbor A. actinomycetemcomitans, 13 sites were positive for the bacteria with the cdt genotype (12%). Out of the 13 positive sites, 10 sites were obtained from patients diagnosed with aggressive periodontitis (77%). Thus, A. actinomycetemcomitans with the cdt genetic subtype has low occurrence in the subgingival plaque of periodontitis patients. However, a strong association was observed between the presence of the bacteria and aggressive forms of periodontitis. Thus, the cytotoxic and immunosuppressive properties of A. actinomycetemcomitans Cdt may function to cripple the host immunity and contribute to the pathogenesis of aggressive periodontitis.     

Electron microscopy of phages in serotypes of Actinobacillus actinomycetemcomitans

Actinobacillus actinomyetemcomitans, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzas, Haemophilus parainfluenzae, Pasteurella haemolytica and Pasteurella multocida strains were examined by transmission electron microscopy for the presence of bacteriophages. Phages were detected in serotype a (SUNY 75) and e (UOH 1705) and in the fresh clinical isolates UOH Q1243 and UOH Q1247 of A. actinomycetemcomitans. Phages were not found in serotype b, c and d strains of A. actinomycetemcomitans , in the fresh clinical isolate UOH Q1244 of this species or in old strains (including reference strains) of related species from the Actinobacillus-Haemophilus-Pasteurella group.