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 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.     

Immunodominant antigens of Actinobacillus actinomycetemcomitans serotypes a and c in high-responder patients

This study was undertaken to examine the characteristics of the immunodominant antigens of Actinobacillus actinomycetemcomitans serotypes a and c. The top responders for A. actinomycetemcomitans serotypes a and c were selected (19 for serotype a and 21 for serotype c) from 150 clinically characterized patients. Competition assays revealed that 9 of 19 of these patients were reacting specifically to serotype a and 12 of 21 for serotype c. Limiting dilution analysis on Western blots revealed that most antigen bands apparent at low dilution disappeared as the patient's serum was diluted. The antigen band(s) remaining at the endpoint or the dilution corresponding to the antibody titer were defined as immunodominant. For serotype a there were several different immunodominant antigens but none was present in more than half of the subjects. For serotype c the immunodominant antigens included a number of discrete bands and a diffuse smeared polysaccharide band. Only 2 of these antigens were present in the majority of the high-responders: 92% had the smeared antigen and 67% had a 15 kDa antigen. The 15 kDa band was a protein common to all A. actinomycetemcomitans serotypes. The smeared antigen was unaffected by protease K treatment and gave a reaction of identity with the serotype c specific rabbit antiserum. This rabbit antiserum is specific for a mannan carbohydrate and does not react with LPS (23). Therefore, the smeared immunodominant antigen appears to be a polysaccharide containing mannan.     

Actinobacillus actinomycetemcomitans proportion of subgingival bacterial flora in relation to its clonal type

We investigated whether certain Actinobacillus actinomycetemcomitans clones occur in elevated proportions in subgingival flora, and if the proportions relate to other bacteria in the samples. A total of 121 A. actinomycetemcomitans strains from 121 patients with periodontitis were serotyped and 60 strains were also genotyped. The 121 strains were divided into three groups and the 60 strains into two groups according proportion of A. actinomycetemcomitans. The samples from the 60 patients with genotyped strains were cultured for five other species. Among the 121 strains, serotype b occurred significantly more frequently in the high- (n = 14, proportions > 5%, mean = 18.09, SD = 20.07%) than low- (n = 49, proportions < or = 0.1%), mean = 0.04, SD = 0.03%) or intermediate-proportion groups (n = 58, proportions > 0.5%, mean = 1.31, SD = 1.24%). Genotype 3 occurred significantly more frequently in samples with low A. actinomycetemcomitans proportions (n = 28, < or = 0.1%, mean = 0.04, SD = 0.03%) than in those with high proportions (n = 32, > 0.1%, mean = 5.70, SD = 14.60%). No differences were seen in the detection frequencies or proportions of the five bacterial species between the samples with low or high A. actinomycetemcomitans proportions. The results indicate that certain clonotypes of A. actinomycetemcomitans may preferentially occur as low proportions, suggesting their controlled growth. Conversely, some serotype b clones may have a competitive advantage in subgingival flora.     

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.     

Occurrence of Aggregatibacter actinomycetemcomitans serotypes in subgingival plaque from United States subjects

This study examined the distribution pattern of Aggregatibacter actinomycetemcomitans serotypes in the subgingival plaque of subjects residing in the United States. A. actinomycetemcomitans was identified in 256 subgingival plaque samples from 161 subjects. For 190 of the 256 samples, the total cultivable bacteria and selected periodontal pathogenic species were determined. A. actinomycetemcomitans isolates were confirmed by a16S rDNA‐based PCR analysis, genotyped by arbitrarily‐primed PCR, and serotyped by PCR analysis of serotype‐specific gene clusters. A total of 82 distinct A. actinomycetemcomitans strains were identified. The serotype distribution pattern of the strains was 21 (25.6%) serotype a, 12 (14.6%) b, 41 (50%) c, 6 (7.3%) e, 1 (1.2%) f, and 1 (1.2%) non‐typeable. For 14 subjects where multiple colonies of A. actinomycetemcomitans were identified, 11 subjects (78.6%) were each infected by a single serotype, while the remaining three subjects (21.3%) were each infected by two serotypes of A. actinomycetemcomitans. There was an inverse relationship between the level of cultivable A. actinomycetemcomitans and Porphyromonas gingivalis. Within subgingival plaque of study cohort A. actinomycetemcomitans serotype c was the dominant serotype and comprised 50% of all strains, followed by (in order of detection frequency) serotypes a and b. Serotypes d, e, and f strains were either not detected or less frequently found. Serotype distribution patterns of subgingival A. actinomycetemcomitans may vary among subjects of different race orethnicity.     

A novel selective medium for isolation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

Background and Objective:  Conventional selective media have been used for the selection of Aggregatibacter ( Actinobacillus ) actinomycetemcomitans in clinical samples. The proportion of A. actinomycetemcomitans grown on the selective media in vitro may not reflect the true counts in vivo because of the low selectivity. A novel selective medium, designated AASM, was developed for the isolation of A. actinomycetemcomitans .
Material and Methods:  AASM was prepared by adding of 200 μg/mL of vancomycin and 10 U/mL of bacitracin to AAGM, which contains dextrose, sodium bicarbonate, trypticase soy, yeast extract and agar. Clinical efficacy was evaluated by the recovery, on AASM, of A. actinomycetemcomitans from subgingival samples of 44 periodontally healthy subjects and 76 patients with chronic periodontitis.
Results:  All serotypes (a–f) of A. actinomycetemcomitans strains grew well, and the average growth recovery of A. actinomycetemcomitans on AASM medium was 94.4% (80.0–109.7%) of that on AAGM. The exclusive rate of other bacteria was 99.9% in clinical samples cultured on AASM. A. actinomycetemcomitans was not detected in periodontally healthy persons but was detected in 25 (32.9%) patients with chronic periodontitis. The predominant serotype was c, detected in 11 subjects.
Conclusion:  The new selective medium, AASM, was highly selective for A. actinomycetemcomitans , eliminated possible false-positive results and was useful for the isolation of A. actinomycetemcomitans from clinical samples.     

Distribution of Actinobacillus actinomycetemcomitans serotypes and Porphyromonas gingivalis in Japanese adults

Strains of the bacterium Actinobacillus actinomycetemcomitans found in the human oral cavity are divided into five serotypes, a, b, c, d, and e. In this study, A. actinomycetemcomitans serotypes and Porphyromonas gingivalis were isolated from 656 subgingival sites in systemically healthy Japanese adults. A. actinomycetemcomitans was detected in 19.5% of 328 Japanese subjects, while 27.1% of subjects were positive for P. gingivalis. Of 75 A. actinomycetemcomitans-positive sites, only one serotype was detected in 39 sites (52.0%). The numbers of sites in which two different serotypes and three different serotypes were detected were 18 (25.0%) and 7 (9.3%), respectively. A. actinomycetemcomitans serotype c was detected more frequently in sites that were positive for both P. gingivalis and A. actinomycetemcomitans (76.9%) than in sites that were P. gingivalis-negative and A. actinomycetemcomitans-positive (33.9%). In addition, serotype c was detected much more frequently than the other serotypes (<16%) in sites that were positive for both P. gingivalis and A. actinomycetemcomitans. These findings suggest that the characteristics of serotype c may differ from those of the other serotypes. This report is the first to use PCR to describe the distribution of A. actinomycetemcomitans serotypes in humans and to examine the association between the distribution of A. actinomycetemcomitans serotypes and the presence of P. gingivalis.     

Inhibition of Actinobacillus actinomycetemcomitans leukotoxicity by bacteria from the subgingival flora

Actinobacillus actinomycetemcomitans produces a pore-forming leukotoxin that lyses human polymorphonuclear leukocytes and monocytes. Certain proteolytic bacteria may coexist with A. actinomycetemcomitans in periodontal pockets. We aimed therefore to examine whether oral bacteria can modify the leukotoxicity of A. actinomycetemcomitans. A total of 55 strains representing 45 bacterial species of the subgingival flora were tested. Each strain was incubated with the highly toxic strain of A. actinomycetemcomitans HK 1519 and the leukotoxic activity of the suspension against human polymorphonuclear leukocytes was determined from the activity of the lactate dehydrogenase released upon lysis of the leukocytes. Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica and Prevotella loeschii inhibited the leukotoxicity of A. actinomycetemcomitans cells as well as the activity of leukotoxin purified from the same strain. The bacterial strains without the ability to block leukotoxic activity also failed to destroy pure leukotoxin even after 5 h of incubation. The proteolytic degradation of leukotoxin by P. gingivalis was mainly dependent on the activity of the enzymes R- and K-gingipains. P. intermedia and P. nigrescens also degraded the leukotoxin by enzymes. The results imply a role of the periodontal microflora in modifying the virulence of A. actinomycetemcomitans by destroying its leukotoxin.     

Elevated serum IgG titer and avidity to Actinobacillus actinomycetemcomitans serotype c in Japanese periodontitis patients

AIM: The purpose of this study was to characterize serum antibody responses to different serotypes of Actinobacillus actinomycetemcomitans strains in various forms of periodontitis and to determine whether any specific type of A. actinomycetemcomitans was associated with any specific form of periodontitis in a Japanese population. METHODS: Sonicated whole cell and autoclaved serotype antigens of A. actinomycetemcomitans were used. Serum IgG titer and avidity to A. actinomycetemcomitans were measured by enzyme-linked immunoabsorbant assay (ELISA) and ammonium thiocyanate-dissociation ELISA, respectively, in 46 aggressive periodontitis patients (8 localized, 38 generalized), 28 chronic periodontitis patients, and 18 periodontally healthy subjects. The presence of A. actinomycetemcomitans in plaque and saliva samples was determined using polymerase chain reaction. RESULTS: Generalized aggressive and chronic periodontitis patients exhibited significantly higher IgG titers than healthy subjects to both sonicated and autoclaved antigens of serotype c strains, while IgG titer to serotype b (Y4) was significantly higher in localized aggressive periodontitis patients compared to healthy subjects. No A. actinomycetemcomitans was detected in localized aggressive periodontitis patients. A. actinomycetemcomitans-positive patients exhibited significantly higher IgG titer and avidity to serotype c than A. actinomycetemcomitans-negative patients. In A. actinomycetemcomitans-positive patients, a significantly positive correlation was observed between antibody titer and avidity to serotype c. A. actinomycetemcomitans-positive patients with generalized aggressive periodontitis showed lower IgG avidities to serotype c than those with chronic periodontitis, though no statistically significant difference was found. CONCLUSION: A. actinomycetemcomitans serotype c may play a significant role in chronic and generalized aggressive periodontitis, while A. actinomycetemcomitans serotype b may be associated with localized aggressive periodontitis in a Japanese population.     

伴放线放线杆菌与牙周病相关细胞凋亡关系的研究

牙周病是口腔的常见病和多发病,但其具体机制至今尚未完全明了。大量研究证实,细胞凋亡在牙周病的发生和发展过程中起重要作用。伴放线放线杆菌是牙周炎主要致病菌之一,可产生多种毒力因子。本文就伴放线放线杆菌的各种毒力因子与细胞凋亡及伴放线放线杆菌与牙周组织内多种细胞凋亡的关系进行综述。     

Quantitative aspects of the subgingival distribution of Actinobacillus actinomycetemcomitans in a patient with localized juvenile periodontitis

Abstract The subgingival microflora in a patient with localized juvenile periodontitis was studied. Of the 97 sites investigated, 28 (29%) showed attachment loss. A correlation was found between the number of Actinobacillus actinomycetemcomitans cells and the clinical attachment level and probing pocket depth. Of the 97 test sites, 70 (73%) were positive for A. actinomycetemcomitans. Of the total number of A. actinomycetemcomitans cells isolated from this patient, more than 99% were found at sites with attachment loss, <1 % being present at sites without attachment loss. The mean percentage of A. actinomycetemcomitans was 21.2% at sites with attachment loss and 0.45% at sites without attachment loss. The distribution of Porphyromonas gingilis showed a symmetrical pattern, being present at the 1st molar and 2nd premolar sites in all quadrants and at the lower incisor sites. This species was absent at multiple sites showing overt attachment loss.     

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.     

Frequency and stability of mono-or poly-infection by Actinobacillus actinomycetemcomitans serotypes a, b, c, d or e

The aims of the study were to determine the prevalence of simultaneously multiple Actinobacillus actinomycetemcomitans serotypes in one individual, stability of infection by the same serotype and the occurrence of previously not described serotypes of A. actinomycetemcomitans. The serotypes of 515 clinical isolates of A. actinomycetemcomitans from 91 Finnish, Caucasian subjects, including 321 follow-up samples from 51 subjects, were determined with immunodiffusion assay. Most subjects (n = 86, 95%) were infected with one serotype only; 466 (91%) isolates from 80 subjects belonged to serotype a (25% of isolates/25 subjects), b (25% of isolates/27 subjects) or c (41% of isolates/30 subjects). Fifteen isolates from 4 subjects reacted with the antiserum raised against previously untypable clinical strain IDH 781 (serotype d) and 18 isolates from 5 subjects with the antiserum raised against strain IDH 1705 or IDH 3096 (serotype e). Sixteen (3%) isolates from 5 subjects remained untypable. The same infecting A. actinomycetemcomitans serotype(s) persisted for the 1-6 years of follow-up. In conclusion, the study indicates a rare simultaneous occurrence of multiple oral A. actinomycetemcomitans serotypes, the stability of infection by the same serotype(s) and the existence of serotypes of A. actinomycetemcomitans not previously described.     

Hybridization patterns of Actinobacillus actinomycetemcomitans serotypes a-e detected with an rRNA gene probe

This study reports a genetic characterization of Actinobacillus actinomycetemcomitans strains in relation to serotypes by using rRNA gene restriction patterns. Eighty-eight clinical strains were isolated from 20 unrelated subjects at one or several occasions. The strains were serotyped by using serotype-specific rabbit antisera against serotypes a, b, c, d or e. Three subjects harbored 2 A. actinomycetemcomitans serotypes, 15 subjects I serotype and 2 subjects untypable strains. Chromosomal DNA was digested with restriction endonuclease Cla I, Bam HI, Bgl I or Hind III and hybridized to the rrn B ribosomal RNA operon of the Escherichia coli chromosome. Isolates belonging to the same serotype were genetically identical in the same individual but nonidentical if they belonged to different serotypes. Isolates of the same or different serotypes were genetically nonidentical in different individuals. The banding patterns of A. actinomycetemcomitans isolates recovered from the same individuals during several years always remained identical. The hybridization method using pKK3535 as a probe seemed suitable as an epidemiological tool for comparing the clonal identity of A. actinomycetemcomitans strains.     

Black-pigmented Bacteroides species and Actinobacillus actinomycetemcomitans in subgingival plaque of adult Kenyans

A microbiological study was performed of the subgingival plaque on 2 sites in each of 20 adults originating from a rural area 40 km outside Nairobi, Kenya. The recovery rate of B. gingivalis was 70%, of B. intermedius 100% and of A. actinomycetemcomitans 40% of the subjects, and 50%, 90% and 28%, respectively, of the sites. The isolated strains exhibited similar biochemical characteristics and antibiotic susceptibility pattern as type strains of these species. The high recovery rate of these 3 bacterial species in adult Kenyans was a rather surprising finding, since pathological pocketing was found only sporadically. Furthermore, the results of 2 methodological approaches tested demonstrated that such microbiological studies can be carried out in countries with limited laboratory facilities.     

Temperate bacteriophages are common among Actinobacillus actinomycetemcomitans isolates from periodontal pockets

Actinobacillus actinomycetemcomitans is a suspected etiologic agent in destructive periodontal diseases. The detection of bacteriophages in A. actinomycetemcomitans in the subgingival plaque of patients with rapidly destructive forms of periodontitis led to the hypothesis that bacteriophage infection might increase the virulence of this bacterium (19). A. actinomycetemcomitans was isolated from 68 subjects from the Netherlands and Switzerland with localized juvenile periodontitis, rapidly progressing periodontitis, or adult periodontitis, and was tested for the presence of temperate bacteriophage with the overlay plate technique. More than half of the A. actinomycetemcomitans strains were found to release bacteriophage which formed individual plaques on indicator strains. Electron microscopy of preparations from 7 strains revealed virions with an icosahedral head and a contractile tail typical for double-stranded DNA bacteriophages. The presence of A. actinomycetemcomitans carrying temperate bacteriophage was not correlated with the composition of the subgingival microflora nor with the clinical form of periodontal disease. Destructive periodontal disease of subjects with phage-carrying A. actinomycetemcomitans was not more severe than of subjects with phage-free A. actinomycetemcomitans as determined by several clinical parameters. In contrast, the pocket depth and the attachment loss were significantly lower for adult periodontitis subjects with phage-carrying A. actinomycetemcomitans . It seems unlikely that the frequently occurring temperate bacteriophages increase significantly the virulence of A. actinomycetemcomitans .     

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)     

Plasmids in Actinobacillus actinomycetemcomitans strains isolated from periodontal lesions of patients with rapidly destructive periodontitis

Several strains of Actinobacillus actinomycetemcomitans newly isolated from periodontal lesions of patients with rapidly destructive periodontitis were all shown to possess identical plasmid profiles consisting of 4 plasmids. The largest plasmid, 20 MegaDalton (MDa), was also found in reference strains. Two different methods were used for isolation of the plasmids; the large 20 MDa plasmid (pHRP1) was found using the Kado and Liu method only. The 3 small plasmids of 7.0, 5.2 and 4.0 MDa (pHRP2, pHRP3, pHRP4), respectively, were seen using the Birnboim and Doly method. These plasmids are so far to be regarded as cryptic; no phenotypical characters have been linked to their presence. The large 20 MDa plasmid was found in all strains examined, and may be a genotypical marker for the A. actinomycetemcomitans species.