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Introduction: Very little is known of the diversity and expression of virulence factors of serotypes of Aggregatibacter actinomycetemcomitans. Toxic activity on Chinese hamster ovary (CHO) cells and cdt and ltx genotyping were evaluated in A. actinomycetemcomitans serotypes., Methods: Forty-one A. actinomycetemcomitans isolates were analysed for CHO cell growth inhibition. Genotyping was performed by polymerase chain reactions specific to the ltx promoter region, serotype-specific and cdt region and by sequencing of cdtB., Results: cdtABC was detected in 40 strains. Analysis of the cdtA upstream region revealed 10 cdt genotypes. Toxicity to CHO cells was detected for 92.7% of the isolates; however, no correlation between the toxic activity and the cdt genotype was detected. Serotype c was more prevalent among Brazilian samples (68.0%). Four serotype b isolates from subjects with aggressive periodontitis were associated with high leukotoxin production and exhibited moderate to strong toxic activity in CHO cells, but were classified in different cdt genotypes. High levels of toxicity in CHO cells were not associated with a particular serotype; 57.1% of serotype a isolates presented low toxicity to CHO cells whereas the highly toxic strains belonged to serotypes b and c. Sequencing of cdtB revealed a single nucleotide polymorphism of amino acid 281 but this was not related to the toxic activity in CHO cells., Conclusion: Differences in prevalence of the low and highly cytotoxic strains among serotypes reinforce the hypothesis that serotype b and c isolates of A. actinomycetemcomitans are more virulent than serotype a strains.

Introduction: Cytolethal distending toxin (Cdt) is potentially one of several virulence factors of Aggregatibacter actinomycetemcomitans, the prime etiological agent of localized aggressive periodontitis (LAP). Little is known regarding the Cdt-specific antibody response in humans. The current study is a quantitative and qualitative evaluation of the toxin-specific antibody response in a cohort of LAP patients and age-, race- and sex-matched controls., Methods: Ninety-five subjects provided a total of 692 serum samples. Sera were analysed by enzyme-linked immunosorbent assays to determine the titers of antibody against the intact Cdt holotoxin as well as the individual subunit proteins (CdtA, CdtB, and CdtC). Neutralization of growth inhibition mediated by Cdt was evaluated in a modified colony-forming assay using Chinese hamster ovary cells., Results: Fourteen of the 95 subjects exhibited significant serum Cdt-binding activity. There were no differences in the percentages of seropositive individuals or in the mean antibody titers between the control and LAP groups. Binding activity was detected against each of the three Cdt subunit proteins in all of the positive samples. Neutralization of Cdt-mediated growth inhibition was detected in samples from all of the seropositive subjects (range 20-75%)., Conclusions: Cdt, a recently identified A. actinomycetemcomitans virulence factor, is capable of inducing a neutralizing antibody response indicating that the toxin is produced during natural infection of humans. The failure of a vast majority (20 of 23) of the LAP subjects to mount a significant anti-Cdt response may in part explain their relative susceptibility to the disease.

Introduction: Polymorphonuclear neutrophil (PMN) dysfunctions have been associated with severe forms of periodontitis. This study evaluated the correlation between PMN phagocytosis and oxidative burst with the subgingival microbiota of patients with generalized aggressive periodontitis (GAgP)., Methods: Heparinized peripheral blood samples were obtained from 18 GAgP patients and 11 periodontally healthy (PH) subjects, and PMNs were isolated on a Ficoll-Hypaque gradient. For phagocytosis analysis, PMNs were incubated with fluorescein-labeled Staphylococcus aureus. The oxidative burst was evaluated by incubation of PMNs with dihydroethidium and activation by S. aureus. The assays were examined using flow cytometry. Subgingival biofilm samples were obtained from periodontal sites with and without periodontitis and 24 species were detected by checkerboard., Results: A significantly lower phagocytosis rate was observed for patients with GAgP compared with PH subjects over time (P < 0.05). No differences between groups were found for superoxide production. GAgP patients presented significantly higher prevalence and levels of Porphyromonas gingivalis, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans serotype b than controls (P < 0.05). Significant negative correlations between T. forsythia and P. gingivalis and PMN functions were observed., Conclusions: GAgP subjects presented diminished phagocytic activity of peripheral PMNs and high prevalence and levels of classical periodontal pathogens.

Objectives: The objectives of this study were to characterize the bacterial profile and to seek possible bacterial associations in the subgingival microbiota of early onset periodontitis/aggressive periodontitis patients by using two different techniques, culture and immunofluorescence., Material and Methods: The study group consisted of 66 systemically healthy individuals with evidence of early onset periodontitis - 41 females and 25 males aged 23-35 years (mean 31.1 +/- 3.1 years). Bacterial samples were collected from the deepest site in each quadrant, resulting in a total of 264 sites with a mean probing pocket depth of 6.6 +/- 1.5 mm. Samples were cultured anaerobically and in 10% CO(2) using selective and nonselective media, and isolates were characterized to species level. Indirect immunofluorescence using monoclonal antibodies was applied to detect Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia (Bacteroides forsythus, Tannerella forsythensis), Prevotella intermedia/Prevotella nigrescens, Campylobacter rectus, Peptostreptococcus micros and Actinomyces israelii., Results: 93.6% of sampled sites showed bleeding on probing and 23.5% were positive for suppuration. P. intermedia/P. nigrescens, P. gingivalis, and C. rectus were detected in 77.3-85.9% of samples using culture methods and in 85.6-91.3% using immunofluorescence. P. micros and A. actinomycetemcomitans were found, respectively, in 63.3% and 25.0% of all sites using culturing and in 58.7% and 27.7% sites using immunofluorescence. Significantly strong positive associations were observed between T. forsythia and C. rectus (odds ratio 109.46), and T. forsythia and P. gingivalis (odd ratio 90.26), whereas a negative association was seen between P. intermedia/P. nigrescens and A. actinomycetemcomitans (odds ratio 0.42). Coinfection by P. gingivalis, T. forsythia, P. intermedia/P. nigrescens and C. rectus was observed in 62.1% of the test sites, and in 89.4% of the studied subjects. The sensitivity of immunofluorescence for T. forsythia, C. rectus, P. intermedia/P. nigrescens and P. gingivalis was found to be very high (0.99-0.94) using culture as the reference detection method. The agreement between culture and immunofluorescence in detecting the presence or absence of the investigated species was 85.2-88.1% for P. gingivalis, P. intermedia/P. nigrescens, C. rectus, and T. forsythia, 75.9% for A. actinomycetemcomitans and 70.4% for P. micros., Conclusions: The microbial profile of the early onset/aggressive periodontitis population was complex. The agreement between the two detection methods was very high.

Cells of the periodontal pathogen Actinobacillus actinomycetemcomitans exhibit tight adherence to surfaces such as glass, plastic and hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize teeth and other surfaces. Tight adherence is mediated by long fibrils of bundled pili (fimbriae) that form on the surface of the cell. The flp-1 gene encodes the major pilin protein component of A. actinomycetemcomitans fimbriae. In this study we compared flp-1 DNA sequences from 43 strains of A. actinomycetemcomitans isolated in Europe, Japan and the United States and identified seven distinct flp-1 allelic classes. DNA and predicted protein sequences were almost completely conserved within each flp-1 class but were highly divergent between classes. Most amino acid substitutions occurred in the C-terminus of the pilin protein, a region that has been shown to be important for the bundling and adhesive properties of the pili. flp-1 classes correlated with serotypes and 16S rRNA genotypes in most strains. At least five strains showed evidence of horizontal transfer of flp-1 between strains of different serotypes and 16S rRNA genotypes. Four of the seven flp-1 classes were present in geographically diverse isolates. Strains representing all seven flp-1 classes, but not a strain carrying a transposon insertion in flp-1, bound avidly to polystyrene in an in vitro adherence assay. Strains representing six of the seven flp-1 classes were isolated from localized juvenile periodontitis patients, suggesting that phylogenetically diverse strains carry pathogenic potential. Our findings provide a framework for future biochemical, immunological and genetic studies of A. actinomycetemcomitans fimbriae.

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.

The epithelial cell invasiveness of Actinobacillus actinomycetemcomitans strains of different restriction fragment-length polymorphism (RFLP) groups associated with disease conversion and asymptomatic carrier status in localized juvenile periodontitis was examined. Twenty clinical isolates were studied for their ability to invade KB monolayers, using the quantitative gentamicin killing assay. Five isolates were found to be invasive, five were not invasive; and the other 10 did not invade better than an invasion negative control Haemophilus aphrophilus strain ATCC 19415. Using probe-specific DNA fingerprinting. 11 strains were assigned to RFLP group II (disease-associated); 4 to RFLP type XIII (carrier status associated); and the other to groups III, IV, V and VII. Eight isolates, all RFLP group II, were leukotoxin producers as determined by PCR amplification of the lkt promoter region. No correlation was found between invasiveness and RFLP group. Leukotoxin production was more associated with noninvasive than invasive strains.

This study examined the variable serum immunoglobulin G (IgG) levels to genetically distinct autologous Eikenella corrodens strains by enzyme-linked immunosorbent assay (ELISA). Twenty subjects, including 10 adult periodontitis patients, 5 juvenile periodontitis patients and 5 periodontally healthy subjects were examined. Each subject was colonized by 2-8 genetically distinct E. corrodens strains. The serum IgG levels to autologous E. corrodens within individuals were significantly different in 7 adult periodontitis patients, 4 juvenile periodontitis patients and a periodontally healthy subject. Poor correlation was found in diseased subjects between serum IgG levels to autologous strains and to reference strains ATCC 23834 or FDC 373. Four adult periodontitis patients and two juvenile periodontitis patients exhibited significant serum IgG levels to autologous E. corrodens strains (two standard deviations above the mean for periodontally healthy subjects); two of these six diseased subjects exhibited low serum IgG levels to reference strains and would have been classified as low immune responders if only reference strains had been used in ELISA. This study showed the importance of using autologous E. corrodens strains in the assessment of serum IgG immune responses to this organism.

A method was developed to evaluate the presence of immunoglobulin G (IgG) subclass (1-4) antibody to Actinobacillus actinomycetemcomitans, serotype b (strain Y4) in patients with early-onset periodontitis on a single nitrocellulose membrane. Sera from 30 early-onset periodontitis patients and gingival crevicular fluid samples from 2 patients were collected and tested with four different preparations of A. actinomycetemcomitans (Y4). The principle steps of the assay are: a) binding of the bacterial antigen (Y4) and the anti-human IgG antibody (capture antibody) in parallel lanes on nitrocellulose membranes; b) incubation of known concentrations of the IgG subclasses 1, 2, 3 and 4, as well as a dilution of serum and/or gingival crevicular fluid from patients in lanes perpendicular to the antigen lanes; c) incubation of the membranes with the corresponding peroxidase conjugated anti-human IgG subclass secondary antibody; d) detection of positive signals by enhanced chemiluminescence. The blots were evaluated by visual comparison to a series of blots containing known concentrations of IgG subclasses. The method was used to rapidly screen a relatively large number of patient sera and gingival crevicular fluid samples for IgG subclasses in a cost-effective assay. The predominant IgG subclass found in early-onset periodontitis was IgG2.

The in vitro minimal inhibitory concentrations (MIC) and minimal bactericidal concentration (MBC) of roxithromycin and erythromycin against Actinobacillus actinomycetemcomitans were evaluated. Sixty-seven different A. actinomycetemcomitans isolated from periodontal pockets of 101 subjects with different forms of early-onset and adult periodontitis and three reference strains of A. actinomycetemcomitans (ATCC 29522, ATCC 29523, and NCTC 9710) were included in this study. Erythromycin showed poor in vitro activity against A. actinomycetemcomitans; roxithromycin, on the contrary, exhibited good in vitro activity. Moreover, roxithromycin showed the best in vitro antimicrobial activity against 17 serotype a and 12 serotype c subpopulations of A. actinomycetemcomitans; against 38 serotype b subpopulation of A. actinomycetemcomitans, roxithromycin was consistently active. Roxithromycin exhibited MBC values usually equal to, or one-fold higher than MIC values. All the MBC values of erythromycin were three- to four-fold higher than the respective MIC result. Since roxithromycin is characterized by high concentrations in serum and good penetration and diffusion into gingival tissue, it could be expected to pass into the gingival crevicular fluid at levels sufficiently high to inhibit A. actinomycetemcomitans in vivo. These data indicate that roxithromycin might be a potential candidate for therapeutic trials in patients with A. actinomycetemcomitans-associated periodontitis.

Myeloperoxidase is an enzyme released by polymorphonuclear leukocytes which has been used, experimentally, as an indicator of periodontal disease activity when measured in gingival crevicular fluid. There are three myeloperoxidase isoforms: MPO I, MPO II and MPO III. We examined the activities of myeloperoxidase isoforms released by neutrophils in response to serum-opsonized Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Eikenella corrodens, Capnocytophaga sputigena and Streptococcus sanguis. Isoform activities were determined using intermediate-pressure liquid chromatography and microenzyme assay. A. actinomycetemcomitans stimulated higher levels of myeloperoxidase release than any other oral bacteria unless pre-opsonized with serum (or protein-A-purified immunoglobulin) from an individual with localized juvenile periodontitis. Most oral bacteria stimulated the release of all myeloperoxidase isoforms with a profile enriched in MPO I and diminished in MPO III. Exceptionally, serum-opsonized A. actinomycetemcomitans stimulated myeloperoxidase isoform release in proportion to the neutrophil granule constituency with or without localized juvenile periodontitis serum pre-opsonization. Because myeloperoxidase isoform profiles reflect how neutrophils were stimulated, isoform analysis may refine future diagnostic tests based upon myeloperoxidase.

Sera from young patients with periodontal diseases have been shown to often contain highly elevated antibody levels to Actinobacillus actinomycetemcomitans, in particular serotype b. Such responses were reportedly predominated by antibodies of the immunoglobulin G2 (IgG2) subclass. The aim of this study was to investigate an ethnically diverse group of 14 early-onset periodontitis and 15 rapidly progressive periodontitis patients for the occurrence of elevated antibody titers against the five known A. actinomycetemcomitans serotypes, and to compare the patient's IgG subclass response profiles. Enzyme-linked immunosorbent assays were used to measure both total IgG and subclass specific IgG titers. Twenty-four subjects had markedly elevate total IgG levels against at least one serotype. The frequencies of high responses against serotypes a, b, c, d and e were 7, 11, 6, 4, and 4, respectively. Elevated antibody responses were predominated by IgG2, regardless of the serotype to which the response was directed. The serotype specificity of the host responses was further investigated by competitive binding studies with serotype-specific monoclonal antibodies. Twelve sera were found to contain antibodies capable of strongly inhibit the binding of monoclonal antibodies against a single serotype; four other sera had antibodies against epitopes of two, and one serum against those of three serotypes. The findings document broad serotype diversity in an ethnically heterogeneous group of patients and indicate that strong antibody responses to A. actinomycetemcomitans are predominated by IgG2 regardless of the serotype of the infective agent.

The objective of this study was to determine whether a relationship exists between antibody reactive with the Actinobacillus actinomycetemcomitans leukotoxin and the severity of periodontal disease. Serum concentrations of antibody reactive with the leukotoxin were determined for 119 early-onset periodontitis patients and 59 non-periodontitis subjects using limiting dilution analysis on Western blots. Immunoglobulin G (IgG) antibody reactive with the A. actinomycetemcomitans leukotoxin ranged from undetectable to 29 micrograms/ml (mean = 3.13 +/- 0.97 micrograms/ml for the generalized early-onset periodontitis and 2.17 +/- 0.86 micrograms/ml for the localized juvenile periodontitis patients vs 0.32 +/- 0.24 ng/ml for 59 non-periodontitis controls), and the dominant subclass was IgG1. Analysis of the relationship between antibody reactive with A. actinomycetemcomitans sonicate, A. actinomycetemcomitans leukotoxin and attachment loss patterns indicates that seropositive generalized early-onset periodontitis patients had decreased attachment loss compared with patients lacking this antibody. The statistical relationship appeared to be stronger for the sonicate than the purified leukotoxin. These data suggest that antibody reactive with A. actinomycetemcomitans leukotoxin may be protective in early-onset periodontitis, but given that the sonicate appeared better than the leukotoxin alone, it is not likely that leukotoxin is the only antigen of importance to host defense.

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.

Immunoglobulin A1 (IgA1) proteases secreted by oral Prevotella and Capnocytophaga species specifically cleave IgA1 at the same peptide bond in the hinge region, leaving intact monomeric Fab and Fc fragments. Assuming that Prevotella- and Capnocytophaga-induced Fab fragments of IgA1 expose a specific immunogenic neoepitope at the cleavage site, we established an enzyme-linked immunosorbent assay to measure human serum antibodies to this neoepitope as indirect evidence of in vivo activity of Prevotella and Capnocytophaga IgA1 proteases. The assay used a monoclonal antibody with specificity for the neoepitope, and the ability to block binding of the monoclonal antibody to the neoepitope was investigated. Absorption of sera with Prevotella melaninogenica-induced Fab fragments of IgA1 resulted in removal of antibodies blocking binding of the monoclonal antibody, whereas absorption with Fab fragments induced by bacterial IgA1 proteases of other cleavage specificities did not remove blocking antibodies. Consequently, we assume that the antibodies detected had been induced by a neoepitope an the Fab fragment of IgA1 exposed exclusively after cleavage with IgA1 proteases from Prevotella and Capnocytophaga, indicating in vivo activity of these IgA1 proteases. Evidence, though indirect, of in vivo activity of Prevotella and Capnocytophaga IgA1 proteases was present in 42 of 92 sera examined and in a significantly higher proportion of sera from adults with periodontal disease compared with control individuals. No correlation with disease was observed for the juvenile periodontitis groups.

Periodontal diseases comprise a heterogeneous group of infections that are difficult to distinguish on a clinical basis alone. The purpose of the present investigation was to group periodontitis subjects according to their elevated serum antibody levels to specific subgingival species. A total of 119 subjects (19-70 years) with evidence of prior periodontal destruction were monitored at 2-month intervals (maximum 8 visits), prior to therapy, using clinical parameters measured at 6 sites per tooth. The probing attachment level was measured twice at each visit, and an increase of > 2.5mm at a site was used to define subjects with progressing disease. Serum samples were obtained from each subject at each visit and the level of antibody determined by enzyme-linked immunosorbent assay to 12 subgingival species. Subgingival plaque samples were taken from the mesial aspect of all teeth in each subject at each visit, and the levels of 14 different subgingival species were determined using a colony-lift method and DNA probes. Subjects were grouped by cluster analysis of their elevated antibody levels using a simple matching coefficient. Ninety-two subjects fell into 9 clusters with 100% similarity; 29 subjects in one cluster group exhibited elevated antibody to none of the test species. Seven subjects in a second cluster group showed elevated antibody to Bacteroides forsythus. Subjects in the other 7 clusters showed elevated antibody to Actinobacillus actinomycetemcomitans serotype a only or in combination with B. forsythus, A. actinomycetemcomitans serotype b, Prevotella intermedia or Porphyromonas gingivalis.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

A gingival explant culture system was utilized to evaluate the reactivity of local immunoglobulins produced by juvenile periodontitis tissue. Gingival explant culture supernatant fluids were screened, via a standardized dot-immunobinding assay, for antibodies reactive to: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, Eikenella corrodens, Peptostreptococcus micros, Peptostreptococcus anaerobius, Capnocytophaga ochracea, Eubacterium nodatum and Fusobacterium nucleatum and one nonoral microorganism, Porphyromonas asaccharolytica. Of the 75 juvenile periodontitis supernatant fluids tested, the organisms that reacted with the highest numbers of supernatant fluids were E. nodatum (72%) and A. actinomycetemcomitans (49%). More juvenile periodontitis than healthy tissue samples showed supernatant fluid reactivity to P. intermedia, C. ochracea, E. nodatum and P. micros. No significant difference was observed between the juvenile periodontitis group supernatant fluids reactivity and the supernatant fluids of the other periodontal disease groups tested. Cluster analysis revealed the association, as determined by supernatant fluid reactivity, of P. micros and C. ochracea in the juvenile periodontitis group. The data from this investigation are consistent with a hypothesis of multiple possible etiologies of periodontal destruction in juvenile periodontitis and other forms of periodontal diseases.

Associations between recovery of Actinobacillus actinomycetemcomitans from samples of subgingival plaque, and samples of buccal mucosa, tongue and unstimulated saliva were studied in 107 subjects. Ten subjects had gingivitis, 18 localized juvenile periodontitis, 45 rapidly progressive periodontitis and 32 adult periodontitis. Two children suffered from prepubertal periodontitis. Heterogeneity tests for associations in different study populations yielded nonsignificant results. Mantel-Haenszel's common odds ratios were 52.9, 37.2 and 19.8 for respective associations between pooled subgingival samples, and cheek, saliva and tongue samples. Significant McNemar's chi-square of 5.88, 11.25 and 16.96 for respective associations pointed to secondary occurrence of A. actinomycetemcomitans in extracrevicular samples. Multiple linear regression yielded a significant influence of the number of deep periodontal pockets of 7 mm or more and a negative influence of the diagnosis "adult periodontitis" on the log-transformed number of colony-forming units of A. actinomycetemcomitans in samples from cheek mucosa in patients infected with the organism. Extracrevicular occurrence of A. actinomycetemcomitans seems to reflect total subgingival numbers of the organism. Especially sampling cheek mucosa appears to be a promising tool in the diagnosis of a periodontal infection with A. actinomycetemcomitans.

Actinobacillus actinomycetemcomitans has been strongly implicated in the etiology of localized juvenile periodontitis. Techniques used in the identification of this periodontal pathogen include cultural, biochemical, immunological and DNA hybridization analysis. In this study, we report the use of polymerase chain reaction (PCR) to amplify unique sequences of A. actinomycetemcomitans. Specific oligonucleotide primers LKT2 and LKT3 were designed to hybridize to the A. actinomycetemcomitans lktA gene, which encodes leukotoxin, a putative A. actinomycetemcomitans virulence factor. The LKT2 and LKT3 primers amplified lktA-specific sequences from all 12 A. actinomycetemcomitans strains tested. In another set of experiments, 13 other bacterial species, most of which are normal residents of the oral cavity, were tested with these primers. These PCR amplifications also contained 2 additional primers, RRN4 and RRN5, which served as positive controls; RRN4 and RRN5 were designed to amplify specific sequences of eubacterial 16S ribosomal DNA (rDNA). PCR amplifications of all bacterial species tested, including A. actinomycetemcomitans, yielded 16S rDNA-specific DNA fragments. Furthermore, each bacterial species tested, with the exception of A. actinomycetemcomitans, failed to amplify lktA sequences. The LKT and RRN primers were used in further PCR experiments to detect A. actinomycetemcomitans directly from gingival fluid samples. The results clearly demonstrate the simplicity, rapidity, specificity and accuracy of the LKT primers in the identification of A. actinomycetemcomitans.

Enterococci are potential pathogens in many human body sites. This study determined the subgingival occurrence and the in vitro antimicrobial susceptibility of enterococci in 100 persons with early-onset periodontitis and 545 persons with advanced adult periodontitis. Subgingival microbial samples were collected with paper points, transported in VMGA III and plated onto anaerobic enriched brucella blood agar or selective Enterococcosel agar (BBL Microbiology Systems). Enterococcal speciation was performed using commercial micromethod kit systems. In vitro sensitivity was determined using a commercial kit system and an agar dilution assay. Subgingival enterococci occurred in 1% of early-onset periodontitis patients and in approximately 5% of adult periodontitis patients. Enterococcus faecalis was the only enterococcal species recovered, and all but one isolate belonged to the same biotype. In vitro antimicrobial sensitivity testing revealed subgingival enterococci resistant to therapeutic levels of penicillin G, tetracycline, clindamycin and metronidazole, but relatively sensitive to ciprofloxacin and amoxicillin/potassium clavulanate (Augmentin). Enterococci may populate periodontal pockets as superinfecting organisms and, in heavily infected patients, may contribute to periodontal breakdown.

Peptostreptococcus micros is a recognized pathogen in medical infections, and its association with progressive periodontitis was examined in this study. P. micros was isolated from paper-point subgingival samples on anaerobic enriched blood agar plates and identified on the basis of cellular and colonial morphology and selected biochemical tests. In a cross-sectional study involving 907 people with advanced adult periodontitis, 127 with early-onset periodontitis, and 12 with localized juvenile periodontitis, P. micros in these patient groups occurred with a prevalence of 58-63%. In culture-positive patients, P. micros averaged 12-15% of total viable counts. P. micros demonstrated similar occurrence and proportional recovery in all age groups. In a longitudinal study of 91 adult periodontitis patients on maintenance therapy, P. micros demonstrated a significantly higher prevalence in disease-active than in disease-inactive patients (47% vs 14%). Mechanical subgingival debridement and 0.12% chlorhexidine pocket irrigation was unable to eradicate subgingival P. micros from 18 of 22 adult periodontitis patients. In vitro antimicrobial susceptibility testing showed P. micros to be sensitive to therapeutic levels of penicillin, clindamycin and metronidazole. Our findings indicate that P. micros is a potential pathogen in adult periodontitis. The methods for its eradication from subgingival sites remain to be determined.

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.

This investigation characterized and compared outer membrane proteins (OMP) of the closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by means of SDS-PAGE patterns and reactions on immunoblots with rabbit antiserum against A. actinomycetemcomitans FDC Y4. Reactions with serum from a patient with Papillon Lefévre syndrome (PLS), from whom periodontal wild strains of A. actinomycetemcomitans had been isolated, were also studied. OMP were purified with selective solubilization from lyophilized cells of 10 wild and 4 reference strains of A. actinomycetemcomitans and 4 reference strains of H. aphrophilus. OMP profiles from wild and reference strains of A. actinomycetemcomitans were similar while those from A. actinomycetemcomitans and H. aphrophilus differed. The most prominent difference was absence of a heat modifiable protein in H. aphrophilus strains. Immunoblotting revealed strong common antigens in most strains, including a heat modifiable protein with mol wt 34 kDa, as well as a 29 kDa and a 16.5 kDa protein. Treatment with pronase and sodium periodate confirmed the protein nature of the major OMP antigens.

Although Actinobacillus actinomycetemcomitans has been recognized as a primary etiological agent in localized juvenile periodontitis, questions remain concerning the source of infection, mode of transmission, and relative virulence of strains. DNA fingerprinting analysis, using a randomly cloned chromosomal DNA fragment as a probe, revealed that previously characterized strains of A. actinomycetemcomitans displayed significant restriction site heterogeneity which could be applied to the typing of clinical isolates of this bacterium such that individual strains or variants could be traced within subjects from localized juvenile periodontitis families. Hybridization data derived from an analysis of bacterial isolates obtained from families participating in an ongoing longitudinal study of the disease showed that a single individual could be infected with more than one strain or variant of A. actinomycetemcomitans and that various members of the same family could harbor different strains or variants of the bacterium. In several cases the clinical isolates were matched to characterized laboratory strains by comparing hybridization patterns generated by digestion of the DNA with several restriction enzymes in independent reactions. Thus, probe-specific DNA fingerprinting of A. actinomycetemcomitans will permit us to determine if particular strains or variants are frequently associated with sites of periodontal destruction. Attention could then be focused on determining the virulence properties of those strains or variants that have in vivo significance.

Localized prepubertal periodontitis has been described as a host-defect mediated form of bacterially induced periodontitis, with an early onset and rapid progression around a few teeth in children prior to puberty. To further our understanding of the etiology of this disease, we have examined the microbiological components of subgingival dental plaque in 9 children with localized prepubertal periodontitis to determine if patterns of putative pathogens existed, and have compared these results with those obtained from 4 children with no periodontitis. Subgingival plaque samples were plated onto a selective medium for Actinobacillus actinomycetemcomitans and onto a non-selective medium for anaerobes, and the predominant cultivable microbiota of 2 sites per child was determined. The subgingival microbiota of children with localized prepubertal periodontitis clearly differs from non-diseased children in the detection of high levels of several suspected pathogens, including A. actinomycetemcomitans, Bacteroides intermedius, Eikenella corrodens, and Capnocytophaga sputigena. These putative pathogens were found in various combinations. These findings suggest that localized prepubertal periodontitis is associated with specific subgingival bacteria which are generally not found in children without periodontitis.

Loss of crestal alveolar bone at primary teeth was ascertained radiographically in a dental school clinical population of 2264 children. 19 patients (0.84%) demonstrated distinct periodontal bone destruction around one or more primary teeth; in only 2 of these patients had periodontal disease been identified in previous clinical examinations. A microbiological study of 35 subgingival samples from 9 available patients revealed a high prevalence of black-pigmented Bacteroides spp., mainly Bacteroides intermedius. Actinobacillus actinomycetemcomitans and Capnocytophaga spp. were predominant organisms in some samples. The present data indicate that localized prepubertal periodontitis is more common than previously realized and is associated with bacteria generally regarded as major periodontal pathogens.

Samples of subgingival plaque from 67 children, 5-7 years of age, were examined for the presence of certain suspected periodontal pathogenic species using the conventional technique of anaerobic sonification, dilution and spiral plating. When this technique was compared with a direct plating procedure which involved no preliminary dispersion and dilution of plaque specimens, it was found that the direct method resulted in double the frequency of children in whom black-pigmented Bacteroides (BPB) were detected and a 10-times increase in the number of subjects harbouring Actinobacillus actinomycetemcomitans. Samples from the tongue, tonsils and saliva were also plated using the direct technique. BPB were detected less commonly in the plaque specimens (61.3% of children) than in saliva (89.5%), or on the tongue (86.6%) and tonsils (97.1%). Expressed as percentages of a pooled sample of the total BPB population, the most frequently detected species in plaque were Bacteroides intermedius (44.4%) and Bacteroides melaninogenicus (48.0%). The most prevalent isolate in all other oral sites was B. melaninogenicus. Expressed as percentages of children in whom BPB were detected, the most frequently isolated species from plaque using the conventional dilution technique was B. intermedius (21.3%), whereas other BPB species were present in fewer than 5% of children. Fusobacterium nucleatum and Capnocytophaga species were isolated most frequently from plaque but were also commonly detected in the various other oral sites.