Eradication of Actinobacillus actinomycetemcomitans from the oral cavity in adult periodontitis

Eradication of Actinobacillus actinomycetemcomitans from the oral cavity seems to be a prerequisite for successful therapeutic outcome in patients periodontally infected with the organism. In view of the limited number of subgingival samples obtained in recent studies one cannot conclude, however, whether eradication has actually been achieved. In the present study clinical and microbiological parameters were monitored in 10 adult patients with A. actinomycetemcomitans-associated periodontitis during successive non-surgical and adjunctive metronidazole plus amoxicillin (or ciprofloxacin) (AB) therapy. In every patient, 13 extracrevicular samples and subgingival samples from the deepest site of every tooth present were selectively cultivated for A. actinomycetemcomitans. The organism was isolated in 47 ± 29% subgingival and 64 ± 31% extracrevicular samples. Six weeks following subgingival scaling, A. actinomyceterncomitans was detected in 37 ± 30% subgingival and 55 ± 38% extracrevicular samples (n.s.). Three months after antibiotic therapy, the organism was recovered from only 1 patient. At baseline, 7.5 ± 4.2% sites had a probing pocket depth (PPD) ≥7 mm. This proportion dropped to 2.3 ± 2.4% after scaling (p<0.05) and to 0.3 ± 0.4% after AB (p<0.05). The proportion of sites with clinical attachment loss (CAL) ≥6 mm dropped from 23.3 ± 13.3% to 17.7 ± 13.4% (p<0.05) and to 16.8 ± 14.6%. Statistical analysis revealed that the organism was strongly related, at baseline, to PPD ≥7 mm (odds ratio 9.8, p<0.001). Six weeks after scaling, the organism was associated with CAL ≥6mm (odds ratio 1.8, p = 0.02). After scaling, high counts of A. actinomycetemcomitans in excess of 10⁴ CFU/ml significantly interfered with attachment gain of ≥2 mm (odds ratio 0.24, p = 0.001). Based on the present findings, eradication of A. actinomycetemcomitans seems to be possible with adjunctive antibiotic treatment. Elimination of the organism after scaling was only weakly associated with clinical improvement.     

Distribution of a newly described species, Kingella oralis, in the human oral cavity

The oral distribution of Kingella oralis was investigated in 10 periodontally healthy subjects, 11 untreated adult periodontitis patients and 6 untreated localized juvenile periodontitis patients. From each subject, 6–8 each of supra- and subgingival tooth samples, 4 mucosa samples and a saliva sample were examined by culture for the presence of K. oralis. K. oralis was found in at least one oral site in 26 of the 27 study subjects, and in at least one tooth site in each of these 26 positive subjects. Its prevalence in dental plaque ranged from 23% to 59% in different subject groups. The mean percentage of K. oralis in total microbiota in the dental plaque ranged from 0.40% in the periodontally healthy group to 4.60% in localized juvenile periodontitis subjects. The organism was a significant species in a few periodontitis sites, constituting >5% of the total microbiota.     

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.     

Characterization and expression of adjacent proline iminopeptidase and aspartase genes from Eikenella corrodens

Two adjacent genes involved in nitrogen metabolism from Eikenella corrodens, with a potential role in pathogenesis, were studied. Proline iminopeptidase (Pip) activity, which may be essential for energy production and protection against host immune mechanisms, is exhibited by E. corrodens. Analysis of Pip-expressing clones revealed an ORF of 939 bases with a predicted amino acid sequence identity of 67% to the Pip of Neisseria gonorrhoea. 200 bp downstream from pip, an ORF of 1395 bases, encoding a protein with 87% identity to a putative aspartase from the Neisseria meningitidis genome sequence, was identified. Enzymatic function was confirmed with a complemented Escherichia coli aspartase deficient mutant. The E. corrodens aspartase was found to be 77% identical to the Haemophilus influenzae aspartase sequence, which was originally identified on the basis of its ability to bind plasminogen. However, the E. corrodens aspartase had no such activity. Southern hybridization indicated both genes to be single copy and conserved within the genomes of a diverse panel of E. corrodens isolates from health and disease.     

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.     

Sensitivity of Actinobacillus actinomycetemcomitans and Capnocytophaga spp. to the bactericidal action of LL-37: a cathelicidin found in human leukocytes and epithelium

The bactericidal activity of synthetic LL-37, a cathelicidin, was assessed against Actinobacillus actinomycetemcomitans (three strains) and Capnocytophaga spp. (three strains). All strains were sensitive to LL-37, and exhibited 99% effective dose of 7.5-to-11.6 micrograms/ml. An amidated form of LL-37, pentamide-37, killed with about the same efficacy as LL-37. Partial inhibition of killing was noted at physiologic concentrations of NaCl, and complete inhibition was observed at 400 mM NaCl. At approximately the 99% effective dose--i.e., 10 micrograms/ml--LL-37 also lost activity against A. actinomycetemcomitans in the presence of native or heat-inactivated 10-15% normal human AB serum. Pentamide-37 was less sensitive to serum inhibition than LL-37. In conclusion, certain oral, gram-negative bacteria are sensitive to the bactericidal activity of LL-37 at low concentrations of serum and salt, a condition likely to be found within the membrane-delimited phagolysosome. Modified forms of LL-37, such as pentamide-37, may be more suitable for future therapeutic application in the presence of serum.     

Significance of serum antibody against surface antigens of Actinobacillus actinomycetemcomitans in patients with adult periodontitis

This study was undertaken to examine the prevalence of Actinobacillus actinomycetemcomitans , its serotype distribution and the serum immune responses against its surface antigens in 41 Japanese patients with adult periodontitis. The dominant A. actinomycetemcomitans serotype isolated was serotype c. Immunoblot analysis of 3 serotypes of A. actinomycetemcomitans -sonicated antigens and the patient sera revealed that the reactivities with serotype c were the most frequent and that heat-stable surface serotype-specific antigen appeared to be immunodominanl. Elevated serum immunoglobulin G titers to extracted lipopolysaccharide and fimbriae antigen of A. actinomycetemcomitans were noted for the patient sera by enzyme-linked immunosorbent assay. High serum immunoglobulin G tilers to the fimbriae antigen detected in patients without cultivable A. actinomycetemcomitans suggested Ihe possibilily that the clicked antibody to the antigen played a role in eliminating A. actinomycetemcomitans from the periodontal lesions.     

DNA probe detection of Eikenella corrodens, Wolinella recta and Fusobacterium nucleatum in subgingival plaque

This cross-sectional study used species-specific DNA probes to examine subgingival plaque specimens for the presence of Eikenella corrodens, Wolinella recta, and Fusobacterium nucleatum in adults with untreated periodontitis or gingivitis and in healthy controls. W. recta and F. nucleatum were more prevalent in diseased sites from the periodontitis group when compared with the controls (81% vs 22% and 83% vs 20% respectively). E. corrodens was detected in 62% of the control sites and 81% of the periodontitis sites. Because the control sites commonly contained this organism, E. corrodens may not be useful in differentiating between health and disease. In addition, the relationship between the prevalence of W. recta and F. nucleatum and the prevalence of the established periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides intermedius and Bacteroides gingivalis, was examined. Positive detection of W. recta and F. nucleatum correlated closely with the presence of A. actinomycetemcomitans, B. intermedius and B. gingivalis. Therefore, W. recta and F. nucleatum do not appear to be unique indicators of periodontal disease.     

Detection of Eikenella corrodens and Actinobacillus actinomycetemcomitans by use of the polymerase chain reaction (PCR) in vitro and in subgingival plaque

Abstract The purpose of the present investigation was to identify 2 putative penodontal pathogens: Eikenella corrodens and Actinobacillus actinoinycetemcoiniiuns by polymerase chain reaction (PCR) in vilro and in subgingival plaque. On the basis of published sequences coding for 16S rRNA two primer pairs were designed which amplify a 410 bp sequence from E. corrodens DNA and a 547 bp fragment from A. actinomycetemcomitans DNA. respectively. As few as 50 cells could be detected from pure bacterial cultures. Each of the two primer pairs was found to be specific in that it did not give any amplification product neither with cell lysates from the respective alternative bacterium nor with lysates obtained from other putative periodontal pathogens and other bacteria. The PCR method developed turned out to be a simple, rapid and reliable diagnostic tool for the detection of the target microorganisms in clinical samples.     

Localized juvenile periodontitis and Actinobacillus actinomycetemcomitans in a Brazilian population

Localized juvenile periodontitis (LJP) has been used as a model for studying periodontal disease, and its prevalence is considered to be higher in third-world countries (0.3–8%) than in industrialized countries (0.1%). Mostly, the disease has been associated with Actinobacillus actinomycetemcomitans ( A.a. ) but lack of association has also been reported. The aim of this study was to identify LJP patients in geographically different Brazilian populations and assess the presence of A.a. in their periodontal lesions. 7843 children, 12–19–years of age, from the cities of Rio de Janeiro, Votorantim and Belo Horizonte were screened, and LJP patients were identified by strict clinical and radiographical criteria. A final LJP prevalence of 0.3%, with a 99% confidence interval between 0.16%) to 0.47%, was found. The prevalence in the subpopulations varied between 0.1–1.1% in the different areas. Subgingival bacterial samples were obtained from the oral cavity of 25 patients and their family members. 80% of these patients, 39.5% of their family members, 35.3% of their parents, and 43.9% of all siblings were culture positive for A.a. All but one of the families had at least one member in addition to the patient who was culture positive for A.a. In 3 families, >1 member showed radiographic and clinical signs of LJP. 30% of non-LJP subjects coming from one of the areas with higher LJP prevalence harbored A.a. We conclude that LJP is highly associated with A.a. in this Brazilian population.     

Characterization of serum antibody to Actinobacillus actinomycetemcomitans GroEL-like protein in periodontitis patients and healthy subjects

Recent evidence suggests that molecular mimicry between bacterial and human heat shock protein 60 (hsp60) is involved in various conditions of autoimmune and infectious diseases. Many periodontopathic bacteria have been reported to express GroEL-like protein that is homologous to human hsp60. In this study, the presence of antibodies to the hsp60 of Actinobacillus actinomycetemcomitans in the sera of periodontitis patients and periodontally healthy control subjects was evaluated by enzyme-linked immunosorbent assay using a recombinant A. actinomycetemcomitans GroEL as an antigen. Furthermore, their cross-reactivity with Escherichia coli GroEL and Mycobacterium bovis BCG hsp65 was examined. The mean values of antibody were 0.624 (range 0.088-1.113) and 0.728 (range 0.217-1.296) in control subjects and periodontitis patients, respectively. The antibody levels to A. actinomycetemcomitans after absorption with E. coli GroEL and M. bovis BCG clearly decreased in both control subjects and periodontitis patients. The remaining antibody levels to A. actinomycetemcomitans GroEL after absorption with M. bovis BCG hsp65 were higher than those with E. coli GroEL, indicating higher cross-reactivity with E. coli GroEL. These results suggest that not only periodontitis patients but also periodontally healthy subjects may be infected with A. actinomycetemcomitans but that the part of the antibody could be derived from the cross-reactivity with E. coli GroEL. Any relationship of the antibody to the disease, however, remains to be determined.     

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.     

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.     

Characterization of serum antibody to Actinobacillus actinomycetemcomitans GroEL‐like protein in periodontitis patients and healthy subjects

Recent evidence suggests that molecular mimicry between bacterial and human heat shock protein 60 (hsp60) is involved in various conditions of autoimmune and infectious diseases. Many periodontopathic bacteria have been reported to express GroEL‐like protein that is homologous to human hsp60. In this study, the presence of antibodies to the hsp60 of Actinobacillus actinomycetemcomitans in the sera of periodontitis patients and periodontally healthy control subjects was evaluated by enzyme‐linked immunosorbent assay using a recombinant A. actinomycetemcomitans GroEL as an antigen. Furthermore, their cross‐reactivity with Escherichia coli GroEL and Mycobacterium bovis BCG hsp65 was examined. The mean values of antibody were 0.624 (range 0.088–1.113) and 0.728 (range 0.217–1.296) in control subjects and periodontitis patients, respectively. The antibody levels to A. actinomycetemcomitans after absorption with E. coli GroEL and M. bovis BCG clearly decreased in both control subjects and periodontitis patients. The remaining antibody levels to A. actinomycetemcomitans GroEL after absorption with M. bovis BCG hsp65 were higher than those with E. coli GroEL, indicating higher cross‐reactivity with E. coli GroEL. These results suggest that not only periodontitis patients but also periodontally healthy subjects may be infected with A. actinomycetemcomitans but that the part of the antibody could be derived from the cross‐reactivity with E. coli GroEL. Any relationship of the antibody to the disease, however, remains to be determined.     

Likelihood of transmitting Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in families with periodontitis

This study examined the frequency of spouse-to-spouse and parent-child transmission of the periodontal pathogens Actinobacillus actinomycetemcomitans (124 subjects in 47 families) and Porphyromonas gingivalis (78 subjects in 31 families). The two test organisms were recovered from subgingival and tongue surface specimens using established microbiological techniques. Arbitrarily primed polymerase chain reaction (AP-PCR) was used to genetically characterize isolates of the test species. The probability of isolating identical AP-PCR types of A. actinomycetemcomitans and P. gingivalis in family members by chance was estimated from the AP-PCR genotype distribution of the two species among unrelated individuals. A probability of 5% or less for occurrence by chance alone suggests intra-familial transmission. With a bacterium-positive spouse, A. actinomycetemcomitans revealed inter-spousal transmission in 4/11 (36%) married couples and P. gingivalis in 2/10 (20%) married couples. Parent-child transmission of A. actinomycetemcomitans took place in 6/19 (32%) families. P. gingivalis was not transmitted from parent to child in any of the study families. The intra-familial transmission of A. actinomycetemcomitans and P. gingivalis may in part explain a familial pattern of periodontitis and may have important prophylactic and treatment implications.     

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.     

The immunoreactivity of systemic antibodies to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in adult periodontitis

Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis secrete several potent virulence factors and are known to be two of the major periodontal pathogens. In the present case-control study, the systemic immunoreactivity to A. actinomycetemcomitans exotoxins, cytolethal distending toxin (Cdt) and leukotoxin was analyzed in adult subjects with periodontitis and in periodontally healthy controls. Furthermore, systemic immunoreactivity to P. gingivalis was analyzed in these subjects. Reactivity to the A. actinomycetemcomitans toxins was determined in bioassays that quantified neutralizing antibodies, and P. gingivalis antibodies were detected by enzyme-linked immunosorbent assay (ELISA). The results showed a significantly enhanced immunoreactivity to P. gingivalis in the subjects with periodontitis, while the reactivity to A. actinomycetemcomitans leukotoxin showed no significant difference between patients and controls. However, combined immunoreactivity to leukotoxin and Cdt was more prevalent in the subjects with periodontitis than in the controls. In addition, immunoreactivity to leukotoxin correlated to periodontitis in men but not in women. In conclusion, data from the present study indicate that immunoreactivity to P. gingivalis is frequent in adult periodontitis, while the role of A. actinomycetemcomitans seems to be more complex and depends on gender of the infected subject as well as the virulence of the bacteria.     

Sensitivity of Actinobacillus actinomycetemcomitans and Capnocytophaga spp. to the bactericidal action of LL‐37: a cathelicidin found in human leukocytes and epithelium

The bactericidal activity of synthetic LL‐37, a cathelicidin, was assessed against Actinobacillus actinomycetemcomitans (three strains) and Capnocytophaga spp. (three strains). All strains were sensitive to LL‐37, and exhibited 99% effective dose of 7.5‐to‐11.6 μg/ml. An amidated form of LL‐37, pentamide‐37, killed with about the same efficacy as LL‐37. Partial inhibition of killing was noted at physiologic concentrations of NaCl, and complete inhibition was observed at 400 mM NaCl. At approximately the 99% effective dose – i.e., 10 μg/ml – LL‐37 also lost activity against A. actinomycetemcomitans in the presence of native or heat‐inactivated 10–15% normal human AB serum. Pentamide‐37 was less sensitive to serum inhibition than LL‐37. In conclusion, certain oral, gram‐negative bacteria are sensitive to the bactericidal activity of LL‐37 at low concentrations of serum and salt, a condition likely to be found within the membrane‐delimited phagolysosome. Modified forms of LL‐37, such as pentamide‐37, may be more suitable for future therapeutic application in the presence of serum.