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PubMed1990

[Analysis of antigens of Actinobacillus actinomycetemcomitans with monoclonal antibodies].

The purpose of this study was to analyze antigens of Actinobacillus actinomycetemcomitans. Fifteen hybridomas producing monoclonal antibodies (MAbs) against A. actinomycetemcomitans strain Y4 were obtained. These hybridomas were divided into three groups (Group 1, Group 2 and Group 3) on their MAbs' specificity. The MAbs (MAb S1-S8) produced by Group 1 hybridomas reacted with serotype b-specific antigen of A. actinomycetemcomitans. The MAbs (MAb L1-L3) produced by Group 2 hybridomas reacted with lipopolysaccharides (LPSs) of all serotypes of A. actinomycetemcomitans. The high-molecular-weight peak (peak A) and the low-molecular-weight peak (peak B) were separated by gel-filtration of the phenol-water extract (PWE) of strain Y4. MAb S5 reacted with peak A, and MAb L2 reacted with peak B. Peak B bound to a polymyxin affinity column, but peak A did not. These findings indicate that peak A was serotype-specific antigen and peak B was LPS. MAbs P1, P2, P3 and P4 produced by Group 3 hybridomas reacted with 81 kDa, 64 kDa, 64 kDa and 40 kDa protein antigens, respectively. Preincubation of strain Y4 whole cells with MAb P3 inhibited significantly the adherence of the cells to human buccal epithelium cells (HBECs). These findings suggest that the 64 kDa protein antigen might participate in the adherence of A. actinomycetemcomitans to HBECs.

Aggregatibacter actinomycetemcomitans↗
PubMed1991

Sensitivity of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus to oxidative killing.

We examined the killing of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by oxygen metabolites generated by the xanthine-xanthine oxidase (X-XO) system. This system generates a mixture of oxidants, including superoxide radical, hydrogen peroxide, hydroxyl radical, and possibly singlet oxygen. Differential sensitivity to the X-XO system was observed among strains of A. actinomycetemcomitans; notably, 2 catalase-deficient strains and 2 strains representative of serotypes b and c were the most susceptible. H. aphrophilus was not sensitive. The amount of oxidants produced by the X-XO system more closely correlated with killing than the ratio of oxidant production. Cytochrome c, superoxide dismutase, catalase, dimethyl sulfoxide, and desferrioxamine were used to determine the role of superoxide radical, hydrogen peroxide and hydroxyl radical in the bactericidal process. Hydrogen peroxide was the major bactericidal agent against A. actinomycetemcomitans. Superoxide anion participated in killing of A. actinomycetemcomitans to varying but lesser degrees. The intracellular generation of hydroxyl radical was implicated in the killing of several strains. We conclude that (i) strains of A. actinomycetemcomitans are differentially sensitive to the bactericidal effects of the X-XO system and (ii) of the oxidants produced by the X-XO system, hydrogen peroxide is the most bactericidal against A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1992

Actinobacillus actinomycetemcomitans, Capnocytophaga and Porphyromonas gingivalis in subgingival plaque of adolescents with Down's syndrome.

Levels of Actinobacillus actinomycetemcomitans, Capnocytophaga and Porphyromonas gingivalis were determined in subgingival plaque samples from 37 adolescents with Down's syndrome and 37 healthy controls matched with respect to age and sex. Gingival inflammation, supra- and subgingival calculus, periodontal pockets ( > 4 mm) and alveolar bone loss were registered. Alveolar bone loss was more frequent in Down's syndrome subjects (32%) than in the controls (3%). A. actinomycetemcomitans was detected in the subgingival plaque in 35% of the Down's syndrome adolescents and in 5% of the controls. On site level, A. actinomycetemcomitans and Capnocytophaga were more frequent in the subgingival plaque samples of Down's syndrome children than in those of controls. Comparing Down's syndrome subjects positive or negative for A. actinomycetemcomitans and Capnocytophaga, no significant differences were found in terms of gingival inflammation, periodontal pockets ( > 4 mm) or number of sites with alveolar bone loss. The results indicate an altered microbial composition of the subgingival plaque of Down's syndrome subjects compared with healthy controls, with higher frequency of A. actinomycetemcomitans.

Adolescent↗
PubMed1991

Transformation of Actinobacillus actinomycetemcomitans by electroporation, utilizing constructed shuttle plasmids.

Actinobacillus actinomycetemcomitans, a periodontal pathogen, has been strongly implicated in human periodontal disease. Advances in the molecular analysis of A. actinomycetemcomitans virulence factors have been limited due to the unavailability of systems for genetic transfer, transposon mutagenesis, and gene complementation. Slow progress can be traced almost exclusively to the lack of gene vector systems and methods for the introduction of DNA into A. actinomycetemcomitans. An electrotransformation system that allowed at least five strains of A. actinomycetemcomitans to be transformed with stable shuttle plasmids which efficiently replicated in both Escherichia coli and A. actinomycetemcomitans was developed. One plasmid, a potential shuttle vector designated pDL282, is 5.7 kb in size, has several unique restriction enzyme sites, and codes for resistance to spectinomycin and ampicillin. E. coli and A. actinomycetemcomitans were transformed with equal efficiencies of approximately 10(5) transformants per micrograms of DNA. Similar transformation efficiencies were obtained whether the plasmid DNA was isolated from A. actinomycetemcomitans or E. coli. In addition, frozen competent cells of A. actinomycetemcomitans yielded comparable efficiencies of transformation. Restriction enzyme analysis of pDL282 isolated after transformation confirmed the presence of intact donor plasmids. A plasmid isolated from A. pleuropneumoniae was also capable of transforming some isolates of A. actinomycetemcomitans, although generally at a lower frequency. The availability of these shuttle plasmids and an efficient transformation procedure should significantly facilitate the molecular analysis of virulence factors of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1992

Occurrence of periopathogens in smoker and non-smoker patients.

The aim of the present study was to elucidate the interrelationship between cigarette smoking and the occurrence of periopathogens, i.e., Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedius. The study was based on 145 patients with clinically severe periodontitis, 52 men (age range 32-73 years) and 93 women (age range 32-74 years). 83 patients were smokers (> or = 15 cigarettes/day) and 62 non-smokers. Bacterial samples were collected from one site with a probing depth > or = 6 mm for each individual. There were no statistically significant differences in bacterial counts between smokers and non-smokers (p > 0.05). The relative frequencies of smokers and non-smokers positive for A. actinomycetemcomitans were 31% and 31%, for B. gingivalis 42% and 44%, and for B. intermedius 65% and 53%, respectively. The differences between smoking groups were not statistically significant (p > 0.05). The occurrence in different combinations of these bacteria was also determined. There were no statistically significant differences between smoking and combinations of periopathogens (p > 0.05). The results suggest that smoker and non-smoker patients do not differ with regard to occurrence, relative frequency or different combinations of A. actinomycetemcomitans, B. gingivalis and B. intermedius.

Adult↗
PubMed1992

In vitro susceptibilities of Actinobacillus actinomycetemcomitans to a number of antimicrobial combinations.

The in vitro susceptibilities of Actinobacillus actinomycetemcomitans to 14 antimicrobial combinations were studied by using the checkerboard titration technique. The results, expressed as the range of the fractional inhibitory concentration indices, were as follows: for metronidazole or its hydroxymetabolite combined with cefixime, 0.2 to 0.6; for moxalactam, 0.2 to 0.6; for penicillin G, 0.3 to 0.6; for tobramycin, 0.8 to 2.0; for erythromycin, 0.8 to 1.7; for ciprofloxacin, 0.2 to 0.6; for tetracycline, 0.8 to 1.2. Our observations indicated that the beta-lactam antibiotics as well as ciprofloxacin act synergistically with both metronidazole and its hydroxymetabolite against A. actinomycetemcomitans. Synergistic interactions were independent of the individual MICs of the antibiotics tested. Erythromycin, tobramycin, and tetracycline combined with either metronidazole or its hydroxymetabolite showed additive to indifferent effects against the five strains of A. actinomycetemcomitans, with the fractional inhibitory concentration indices ranging from 0.8 to 2.0. A. actinomycetemcomitans was found to be highly susceptible to ciprofloxacin (MIC of ciprofloxacin for 90% of strains tested, 0.010 micrograms/ml) and cefixime (MIC of cefixime for 90% of strains tested, 0.8 micrograms/ml). The results indicate that in patients who are allergic to penicillin, cefixime and ciprofloxacin may be useful alternative antibiotics in combination with metronidazole for the treatment of A. actinomycetemcomitans-associated periodontitis.

Actinobacillus Infections↗
PubMed1992

Evaluation of the E test for antimicrobial susceptibility testing of Actinobacillus actinomycetemcomitans.

The E test was compared with an agar dilution method for antimicrobial susceptibility testing of Actinobacillus actinomycetemcomitans on Mueller-Hinton Haemophilus test medium. Eighty recent isolates of A. actinomycetemcomitans from 40 subjects and 3 reference strains were studied. Excellent agreement was obtained between the E test and the agar dilution test for benzylpenicillin (98.8%), ampicillin (97.5%), cefaclor (100%), cefuroxime (100%), tetracycline hydrochloride (100%) and ciprofloxacin (98.7%); 76.0% agreement was obtained for metronidazole and only 5.2% for cotrimoxazole. This study indicates that the E test is a reliable method to determine minimal inhibitory concentrations of antimicrobials against A. actinomycetemcomitans, except for metronidazole and cotrimoxazole.

Aggregatibacter actinomycetemcomitans↗
PubMed1992

Microbiological and clinical results of metronidazole plus amoxicillin therapy in Actinobacillus actinomycetemcomitans-associated periodontitis.

We report on the microbiological and clinical effects of mechanical debridement in combination with metronidazole plus amoxicillin therapy in 118 patients with Actinobacillus actinomycetemcomitans-associated periodontitis. Patients were categorized into 3 groups: 28 had localized periodontitis; 50 had generalized periodontitis, and 40 had refractory periodontitis. After initial treatment and metronidazole plus amoxicillin therapy 114 of 118 (96.6%) patients had no detectable A. actinomycetemcomitans. Significant reduction in pocket probing depth and gain of clinical attachment were achieved in almost all patients. Four patients were still positive for A. actinomycetemcomitans after therapy. Metronidazole resistance (MIC greater than 25 micrograms/ml) was observed in 2 of 4 strains from these patients. Patients still positive for A. actinomycetemcomitans or Porphyromonas gingivalis showed a significant higher bleeding tendency after therapy. It was concluded that mechanical periodontal treatment in combination with the metronidazole plus amoxicillin therapy is effective for subgingival suppression of A. actinomycetemcomitans in patients with severe periodontitis.

Adult↗
PubMed1992

Comparative clinical and microbiological effects of topical subgingival application of metronidazole 25% dental gel and scaling in the treatment of adult periodontitis.

The aim of the study was to compare the clinical and microbiological effects of topical application of a metronidazole gel and a single session of subgingival scaling in the treatment of adult periodontitis. An open, randomized controlled clinical study design was employed. Each of 24 subjects received the 2 treatments simultaneously each in 2 randomly selected quadrants of the dentition. The metronidazole, 25% gel was applied subgingivally on days 0 and 7. Scaling was carried out in one quadrant on day 0 and in one quadrant on day 7. Clinical and microbiological examinations were carried out before treatment and on days 21, 49, 91, 133, and 175 of the experimental period. The microbiological analyses included determination of total anaerobically cultivable bacteria, and relative proportions of anaerobes, aerobes, black-pigmented anaerobic Gram negative rods, Actinobacillus actinomycetemcomitans, streptococci, and spirochetes. Both treatments were effective in reducing probing pocket depth and bleeding on probing. Metronidazole tended to be a little better than scaling during the study period and the clinical effects of both treatments persisted during the whole 6 months observation period. Local metronidazole treatment induced a significant and long-lasting shift in the subgingival flora towards a composition more compatible with health and comparable to that obtained by mechanical debridement. Proportions of black pigmented anaerobes including Prevotella intermedia, and the number of spirochetes were significantly reduced after both treatments with a concomitant increase in the proportions of streptococci. While scaling resulted in a statistically significant increase in the proportion of A. actinomycetemcomitans, this was avoided after metronidazole treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

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.

Actinobacillus Infections↗
PubMed1992

Pericarditis, pneumonia and brain abscess due to a combined Actinomyces--Actinobacillus actinomycetemcomitans infection.

A patient is described in whom there developed a pulmonary infiltrate and constrictive pericarditis caused by a combined Actinomyces and Actinobacillus actinomycetemcomitans infection, presumably originating from his poor dentition. The diagnosis was only made following repeated thoracotomy. After surgery, long-term treatment with antibiotics led to complete clinical recovery. None the less, some months later he was found to have a brain abscess which resolved during a further course of antibiotics. The variable clinical picture of actinomycosis is discussed, as well as the role of other bacteria frequently associated with actinomycotic infection, in particular Actinobacillus actinomycetemcomitans. The therapeutic implications are described.

Actinobacillus Infections↗
PubMed1992

Construction of mutants of Actinobacillus actinomycetemcomitans defective in serotype b-specific polysaccharide antigen by insertion of transposon Tn916.

Mutants of Actinobacillus actinomycetemcomitans strain Y4 defective in the capsular-like serotype b-specific polysaccharide antigen (SPA) were constructed by inserting the transposon Tn916. Southern blot analysis suggested that the transposon was inserted into a variety of different sites on the chromosome. Whole cells from two mutants (strains ST1 and ST2) lacked reactivity with a monoclonal antibody to SPA of A. actinomycetemcomitans Y4 (mAb S5) in enzyme-linked immunosorbent assay, but those from another nine mutants (e.g. strains ST3 and ST5) reacted very weakly with mAb S5. Immunodiffusion tests showed that mAb S5 or rabbit antiserum against whole cells of strain Y4 produced a fused precipitin band with purified SPA and autoclaved extract from strain Y4, but no precipitin band with autoclaved extracts from these four mutants. The hydrolysate of autoclaved extract from strain Y4 contained equal amounts of rhamnose and fucose, component sugars of SPA. The hydrolysates of autoclaved extracts from strains ST1 and ST2 contained a trace amount of rhamnose, but not fucose. Those of autoclaved extracts from strains ST3 and ST5 contained a trace amount of fucose, but not rhamnose. All of these SPA-defective mutants reacted with a mAb to lipopolysaccharide of strain Y4. The cell hydrophobicity of SPA-defective mutants was higher than that of the parent strain. These mutant clones will be useful for analysing the gene complex responsible for the synthesis of SPA of A. actinomycetemcomitans and the regulation of expression of the polysaccharide.

Aggregatibacter actinomycetemcomitans↗
PubMed1992

Longitudinal monitoring of humoral antibody in subjects with destructive periodontal diseases.

Fifty-one subjects (16-61 years old) with evidence of prior destructive periodontal disease were monitored clinically and immunologically at bi-monthly intervals for up to 5 yr. Periodontal disease activity, determined as new attachment loss, was detected in 33 of these subjects. Only 4 of 51 subjects failed to show an elevated serum antibody level to any of the 18 subgingival species tested. The antibody level threshold established for periodontally healthy subjects was exceeded most often in diseased subjects with serum antibody to Actinobacillus sp., P. gingivalis, E. corrodens, C. concisus, F. nucleatum and P. intermedia in that order. In general, most serum antibody levels to subgingival species remained relatively consistent for periods as long as 5 yr. However, major increases and decreases in antibody could be detected to at most one or two species in individual subjects. In addition, prolonged, steady increases and decreases in antibody to specific species could be detected in certain subjects. These findings suggest that major changes occurring in serum antibody may reflect fluctuations in the nature of the infection. Differences were observed in the antibody level to specific species when subjects were divided into subsets on the basis of clinical criteria. These included high levels of antibody to A. actinomycetemcomitans Y4 in LJP and RPP subjects and to A. actinomycetemcomitans 29523 in LJP and GJP subjects.

Adolescent↗
PubMed1991

Analysis of in vitro lymphoproliferative responses and antibody formation following subcutaneous injection of Actinobacillus actinomycetemcomitans and Wolinella recta in a murine model.

The potential of Wolinella recta and Actinobacillus actinomycetemcomitans to cause abscesses and induce an immune response was tested in BALB/c mice. Mice were injected subcutaneously with W. recta, A. actinomycetemcomitans or a mixture of these 2 microorganisms. Mice injected with A. actinomycetemcomitans alone, or with both organisms, demonstrated abscesses at the injection site 2 days later, from which pure cultures of A. actinomycetemcomitans were isolated. Mice injected with W. recta had small, flat abscesses at the injection site from which no bacteria could be cultured. W. recta was cultured from injection sites only when associated with A. actinomycetemcomitans. Mice developed positive serum IgG antibody responses to W. recta by 20 days post-injection but not to A. actinomycetemcomitans whether injected in pure culture or mixed infection. In vitro lymphoproliferative responses following injection of W. recta and/or A. actinomycetemcomitans resulted in increased lymphocyte reactivity in unstimulated cultures and decreased in vitro responses to phytohemagglutinin. In vitro lymphoproliferative responses to Escherichia coli LPS or Salmonella typhimurium LPS were depressed in mice injected with A. actinomycetemcomitans, but not in mice injected with W. recta.

Abscess↗
PubMed1992

Immunosuppressive effect induced by Actinobacillus actinomycetemcomitans: effect on immunoglobulin production and lymphokine synthesis.

The soluble sonicated extract (SE) from Actinobacillus actinomycetemcomitans inhibited primary T cell-dependent antibody responses in vivo. The production of IgG and IgM to sheep red blood cells (SRBC) was depressed when mice were treated with high concentrations of SE plus SRBC. Preinjection of SE 3 days prior to SRBC completely inhibited IgG production. SE plus SRBC-primed mice showed markedly depressed CD4/CD8 ratios relative to phosphate-buffered saline plus SRBC- or SRBC-immunized mice. SE-sensitized mice showed low blastogenic activity to concanavalin A (Con A) depending on sensitized periods induced by SE. This inhibitory mechanism was, in part, clarified by a suppression of IL-2 synthesis, IL-2 receptor expression and IL-6 secretion by the splenic T cells stimulated with Con A. These results support the hypothesis that the severe infection of A. actinomycetemcomitans suppresses the immune response by affecting CD4/CD8 ratios, followed by lymphokine production and finally antibody responses.

Aggregatibacter actinomycetemcomitans↗
PubMed1992

Immunoglobulin G subclass response of localized juvenile periodontitis patients to Actinobacillus actinomycetemcomitans Y4 lipopolysaccharide.

Sera from patients with localized juvenile periodontitis (LJP) often contain markedly elevated immunoglobulin G (IgG) antibody titers to serospecific determinants of the lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. The objective of the present study was to define the subclass distribution of the IgG antibody response of LJP patients to this key cell envelope antigen. IgG subclass antibody responses to A. actinomycetemcomitans LPS were quantified in an enzyme-linked immunosorbent assay with human IgG subclass-restricted monoclonal antibodies. Serum antibody concentrations were calculated by heterologous interpolation of a dose-response curve constructed by using human-mouse chimeric antibodies. Sixteen of 17 LJP serum samples tested contained significantly greater concentrations of IgG2 than IgG1 antibodies reactive toward A. actinomycetemcomitans LPS. Geometric mean antibody concentrations of IgG1 and IgG2 were 7.8 and 136.5 micrograms/ml, respectively, among LJP patients with elevated IgG titers to LPS (94% of whom were black). However, both IgG1 and IgG2 antibody concentrations were significantly greater than the corresponding values obtained from sera from LJP patients with low IgG titers to LPS. Among LJP patients with elevated IgG titers to A. actinomycetemcomitans LPS, serum IgG2 concentration and total IgG concentration were also significantly elevated compared with both low-titered LJP sera and sera from periodontally healthy race-matched controls. The results of this study indicate that the humoral response of a predominantly black population of LJP patients to A. actinomycetemcomitans includes the production of LPS-reactive IgG antibodies which are primarily of the IgG2 subclass.

Adolescent↗
PubMed1991

Identification of genomic clonal types of Actinobacillus actinomycetemcomitans by restriction endonuclease analysis.

To evaluate its utility in discriminating different strains, restriction endonuclease analysis was applied to 12 strains of Actinobacillus actinomycetemcomitans (3 serotype a, 5 serotype b, and 4 serotype c strains). DNA isolated from each strain was digested by 12 different restriction endonucleases, and the electrophoretic banding patterns of the resulting DNA fragments were compared. The DNA fragment patterns produced by SalI, XhoI, and XbaI for the 12 A. actinomycetemcomitans strains were simple (less than 30 bands) and allowed us to recognize easily 10 distinct genomic clonal types. The three serotype a strains exhibited distinctly different clonal types from one another, the five serotype b strains exhibited an additional four distinct clonal types, and the four serotype c strains showed another three different clonal types. The other endonucleases tested were less useful in typing A. actinomycetemcomitans. We conclude that restriction endonuclease analysis is a powerful tool for typing and discerning genetic heterogeneity and homogeneity among A. actinomycetemcomitans strains. It should, therefore, be very useful for epidemiologic studies.

Aggregatibacter actinomycetemcomitans↗