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Several bacteriophages associated with the oral bacterium Actinobacillus actinomycetemcomitans have been identified. Lysogeny might affect the virulence of this bacterium, which has been implicated in the etiology of juvenile and adult periodontitis. We have determined the presence of bacteriophage Aa phi 23-related DNA sequences among 185 A. actinomycetemcomitans strains belonging to 2 well-characterized collections and have related the findings to the population genetic structure of the collections. 2 cloned Aa phi 23-specific DNA probes were used in Southern blot hybridization experiments to detect homologous sequences in whole-cell DNA of the strains. DNA from 65 (35%) of the 185 strains hybridized to either of the DNA probes. The majority (74%) of the hybridizing strains showed an identical hybridization pattern, indicating presence of phage Aa phi 23. Whole-cell DNA from the remaining hybridizing strains hybridized to the probes with different patterns, indicating that DNA sequences related to but different from phage Aa phi 23 occur in these strains. The majority (81%) of the strains which harbored phage Aa phi 23 were of serotype a, whereas serotype d strains appeared to be resistant to infection with this phage. There was a clear correlation between hybridization patterns and genetic subdivisions based on our previous population genetic analyses of A. actinomycetemcomitans. However, there was no significant correlation between occurrence of Aa phi 23 among A. actinomycetemcomitans strains and the periodontal status of the patients from whom the isolates were obtained, suggesting that this bacteriophage does not significantly influence the virulence of A. actinomycetemcomitans.

Pulsed field gel electrophoresis was used to investigate nineteen strains of Actinobacillus actinomycetemcomitans. The genome was found to contain a single chromosome whose size we estimate to be 2300 kb from the sum of restriction fragments generated with rare cutting endonucleases. We detected the presence of large plasmids with sizes ranging from 35 to 300 kb. In some strains, extrachromosomal elements constitute over 20% of the total genome. Comparison of the profiles of ApaI digests of the 19 strains showed a high degree of polymorphism with 13 different profiles, providing a new tool for epidemiological studies.

The dnaKJ operon of Actinobacillus actinomycetemcomitans Y4 was cloned by the DNA-probing method using synthetic oligonucleotides designed on the basis of two conserved regions in DnaK/hsp70 proteins and sequenced by inverse PCR. The sequenced region was shown to contain two open reading frames coding for proteins analogous to DnaK and DnaJ.

The incidence of infection by Actinobacillus actinomycetemcomitans, one of the important pathogens in human periodontal diseases, has been reported to be associated with racial background and genetic factors. We attempted to determine the genetic regulation of immune responses to A. actinomycetemcomitans fimbriae, an attachment factor, using various inbred strains of mice. For this purpose, we synthesized an oligopeptide antigen using the amino acid sequence of the fimbriae and conjugated this antigen to branched lysine polymer resin beads. After immunization with the synthetic A. actinomycetemcomitans fimbrial antigen, serum antibody levels and the delayed-type hypersensitivity (DTH) reaction to the antigen were measured by enzyme-linked immunosorbent assay (ELISA) and footpad swelling responses, respectively. The strains of mice found to be high-IgG responders to the antigen were B10.HTT, B10.RIII, B10.A (5R) and B10.S (9R). These results indicate that mice with E(beta s):E(alpha k), E(beta r):E(alpha r) and E(beta b):E(alpha k) respond strongly to the synthetic peptide. All of the high-IgG responders showed a high DTH response. A cell transfer experiment confirmed that CD4 T cells mediated with DTH response to the synthetic peptide. Thus, the results of this study demonstrate that the immune responses to A. actinomycetemcomitans fimbriae are genetically controlled.

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.

In a search for clones from a lambda gt11 expression library of Actinobacillus actinomycetemcomitans (Aa) genomic DNA that expressed epitopes from a 70-kDa iron-repressible membrane protein, we inadvertently identified clones that encoded a member of the 90-kDa heat-shock protein (HSP 90) family. The gene appears to encode a homologue of HtpG, as the nucleotide sequence has approximately 70% identity with the Escherichia coli (Ec) and Vibrio fischeri htpG. Growth of an Aa htpG insertion mutant at 42 degrees C was reduced to 50% of the parent strain, similar to an Ec htpG deletion mutant. These data suggest that Aa HtpG performs a function similar to Ec HtpG.

Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are strongly associated with periodontitis. However, little is known about their distribution in periodontally healthy individuals, because culturing techniques are not sufficiently sensitive. A modified multiplex PCR was developed to address that question. Our method uses two species-specific forward primers in combination with a single reverse primer. These primers target variable and conserved regions of the 16S rRNA gene. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans and P. gingivalis cells. Primer specificity was tested against (i) six A. actinomycetemcomitans strains and four P. gingivalis strains, (ii) seven different species of oral bacteria, and (iii) supra- and subgingival plaque from 20 subjects. The multiplex PCR had a lower limit of detection of 2 A. actinomycetemcomitans and 30 P. gingivalis cells. Species-specific amplicons were obtained for all A. actinomycetemcomitans and P. gingivalis strains tested and did not occur with seven other bacterial species unless A. actinomycetemcomitans and P. gingivalis were added. Neither target species was detected in supragingival plaque; A. actinomycetemcomitans was detected in one subgingival specimen, and P. gingivalis was detected in another. When plaque samples were spiked with 10 A. actinomycetemcomitans cells and 100 P. gingivalis cells, species-specific amplicons were detected. These findings show our multiplex PCR to be highly sensitive and specific while allowing simultaneous detection of A. actinomycetemcomitans and P. gingivalis. This assay has potential applications in epidemiological studies, diagnosis, treatment planning, and monitoring of periodontal pathogens.

The purpose of the present investigation was to identify 2 putative periodontal pathogens: Eikenella corrodens and Actinobacillus actinomycetemcomitans by polymerase chain reaction (PCR) in vitro 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.

Periodontopathic Actinobacillus actinomyceremcomitans produces hemolysin and other leukotoxins. In the present study, two distinct clones which lysed horse erythrocytes were isolated by screening genomic DNA libraries of A. actinomycetemcomitans ATCC 43718 on blood agar plates. DNA hybridization analysis indicates that there were two distinct hemolytic genes present. Sonicated extracts from both Escherichia coli clones possessed hemolytic activities on horse, sheep and human erythrocytes, but not those of rabbit. Rabbit antiserum to A. actinomycetemcomitans ATCC 43718 whole cells inhibited the hemolytic activities of these clones.

The purpose of this study was to determine the distribution of the putative periodontal pathogens Prevotella intermedia, Prevotella nigrescens, the three oral Capnocytophaga species (C. ochracea, C. sputigena, C. gingivalis), as well as Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in plaque samples of periodontally healthy individuals. We chose a newly developed 16S rDNA directed polymerase chain reaction (PCR) and a previously described dot-blot hybridization assay to detect, differentiate, and quantify these bacteria directly in clinical samples. The subjects of these investigations were 66 sulcus fluid samples from 17 children (ages 3 to 5) attending a kindergarten, 48 sulcus fluid samples from 12 children (ages 9 and 10) from a primary school, and 25 subgingival plaque samples isolated from 6 different periodontally healthy dental students (ages 24 to 27). We were able to demonstrate the presence of P. nigrescens in 54 (kindergarten: 5; primary school: 33; students: 16) samples by PCR and quantified it by dot-blot hybridization. In addition, we found C. ochracea in 12 (kindergarten: 2; primary school: 10) samples by PCR reaction only. The other tested bacterial species were absent by the methods used. Furthermore we confirmed the specificity of our P. nigrescens-PCR in selected samples by enzyme electrophoresis.

The purpose of this study was to compare a commercially available DNA probe technique with conventional cultural techniques for the detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in subgingival plaque samples. Samples from 20 patients with moderate to severe periodontitis were evaluated at baseline and during a 15 months period of periodontal treatment. Paperpoints from 4 periodontal pockets per patient were forwarded to Omnigene for DNA probe analysis, and simultaneously inserted paperpoints from the same pockets were analyzed by standard culture techniques. In addition, mixed bacterial samples were constructed harbouring known proportions of 25 strains of A. actinomycetemcomitans, P. gingivalis and P. intermedia each. A relatively low concordance was found between both methods. At baseline a higher detection frequency was found for A. actinomycetemcomitans and P. gingivalis for the DNA probe technique; for P. intermedia the detection frequency by culture was higher. For A. actinomycetemcomitans, 21% of the culture positive samples was positive with the DNA probe. Testing the constructed bacterial samples with the DNA probe method resulted in about 16% false positive results for the 3 species tested. Furthermore, 40% of P. gingivalis strains were not detected by the DNA probe. The present data suggest that at least part of the discrepancies found between the DNA probe technique used and cultural methods are caused by false positive and false negative DNA probe results. Therefore, the value of this DNA probe method for the detection of periodontal pathogens is questionable.

We have found a new insertion sequence (IS), designated ISAa1, downstream of the S10 operon in Actinobacillus (Haemophilus) actinomycetemcomitans FDC Y4. ISAa1, the first IS element characterized in this organism, is 705 bp long and lacks terminal inverted repeats. This element displayed significant homology with IS200. Hybridization patterns of genomic DNA of seven A. actinomycetemcomitans strains with an internal ISAa1 probe varied depending on the serotypes, suggesting that ISAa1 might be a useful tool for epidemiological studies.

Actinobacillus actinomycetemcomitans, the etiologic agent of localized juvenile periodontitis, produces a potent leukotoxin that kills human neutrophils. The production of leukotoxin RNA can vary more than 50-fold among isolates of A. actinomycetemcomitans, and strains expressing high levels of leukotoxin RNA are most often found at sites of periodontal disease. To assess the relative contributions of transcription factors and promoter sequences in setting the disparate levels of leukotoxin RNA found, we have undertaken classical cis/trans analyses. First, the leukotoxin promoter regions from moderately leukotoxic (Y4) and minimally leukotoxic (ATCC 33384) strains of A. actinomycetemcomitans were cloned, sequenced, and compared with the previously sequences leukotoxin promoter region of the high-producer strain JP2. The Y4 and ATCC 33384 promoter regions each contain a 528-bp segment that is absent from JP2. Interestingly, the analysis of various deletion constructs in A. actinomycetemcomitans indicated that Y4, despite the large insertion, initiates leukotoxin RNA synthesis at the same promoter as JP2 does. To perform cis/trans analyses, these three leukotoxin promoter regions were cloned into a plasmid upstream of the reporter gene beta-galactosidase. Each plasmid was transformed into JP2, Y4, and ATCC 33384, and the beta-galactosidase levels were determined. The results indicated that the sequences responsible for down-regulating leukotoxin RNA levels in Y4 relative to JP2 are found within the transcribed region of the Y4 leukotoxin operon. Importantly, in ATCC 33384, strain-specific trans factors and promoter sequence differences are equally significant in determining the lower levels of leukotoxin RNA. We hypothesize that either strain ATCC 33384 has a negative regulatory protein (which is missing or mutated in JP2/Y4) or that JP2 and Y4 carry an activator that is missing or mutated in ATCC 33384.

Few vectors suitable for cloning in Actinobacillus actinomycetemcomitans, a periodontal pathogen, have been described. These plasmids were based either on the native A. actinomycetemcomitans replicon, pVT736-1, or derivatives of the IncP and IncQ family of plasmids. Their usefulness as cloning vectors is limited because of instability or size. Therefore, the ability of additional replicons to function in A. actinomycetemcomitans was evaluated. Derivatives of p15A, ColE1/f1, and pWV01 transformed A. actinomycetemcomitans efficiently and exhibited no structural instability in the new host. The stable maintenance of A. actinomycetemcomitans-specific DNA fragments inserted into the p15A derivative, pDMG4, demonstrated the ability of this plasmid to function as a cloning vector. In addition, pVT736-1 was used to clone selectable markers directly into A. actinomycetemcomitans. These constructs were structurally and segregationally stable.

The expression of the leukotoxin (ltx) operon varies significantly among Actinobacillus actinomycetemcomitans strains. The dual promoters driving ltx expression in the highly toxic strain JP2 have been previously characterized (J. M. Brogan, E. T. Lally, K. Poulsen, M. Kilian, and D. R. Demuth, Infect. Immun. 62:501-508, 1994), and genetic analyses of A. actinomycetemcomitans suggest that highly toxic strains like JP2 arose from minimally toxic strains, presumably by deletion of a 530-bp domain within the ltx promoter region (K. Poulsen, E. Theilade, E.T. Lally, D. R. Demuth, and M. Kilian, Microbiology 140:2049-2060, 1994). However, the ltx promoter of minimally toxic A. actinomycetemcomitans strains has not been well characterized. In this study, deletion and primer extension analyses showed that the ltx promoter of A. actinomycetemcomitans 652 is situated approximately 150 bp upstream of the ltxC gene and initiates transcription 138 nucleotides upstream of ltxC. In contrast to strain JP2, only a single promoter appears to drive ltx expression in 652. The 652 promoter resides within the 530-bp region that is absent from the JP2 promoter sequence, suggesting that the specific sequences controlling ltx expression differ in highly toxic and minimally toxic A. actinomycetemcomitans strains. In addition, ltx expression in strain 652 was shown to be induced three- to fourfold when cells were grown under anaerobic conditions. The induction of whole cell leukotoxicity, was accompanied by increases in the levels of Ltx polypeptide and the steady-state levels of ltx mRNA, suggesting that regulation occurred at the level of transcription. In contrast, the levels of leukotoxicity, Ltx polypeptide, and fix mRNA in strain JP2 were unaffected by anaerobic growth. These results suggest that the ltx operon is differentially regulated in highly toxic and minimally toxic A. actinomycetemcomitans strains and that the sequences controlling the oxygen-dependent regulation of ltx expression may reside within the 530-bp domain that is not present in highly toxic A. actinomycetemcomitans.

To clarify the source of re-emerging periodontal pathogens after treatment, we compared the ribotypes of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia/Prevotella nigrescens group and Campylobacter rectus before and after treatment in 7 periodontitis patients and in 6 of the spouses. The patients harbored A. actinomycetemcomitans, P. gingivalis, P. intermedia/P. nigrescens group or C. rectus in their subgingival or salivary samples before treatment. The respective bacterial species were not detected 1 month after treatment, but reappeared by 6 months later. When available, 4 random colonies of each of the 4 species were isolated from both subgingival and salivary samples at each sampling occasion, the isolates were subcultured, identified and typed applying pheno- and genotypic intraspecies characterization methods. Altogether 90 strains of A. actinomycetemcomitans, P. gingivalis, P. intermedia/P. nigrescens group and C. rectus were available from 2, 3, 2 and 4 patients, respectively. The pre- and post-treatment ribotypes of A. actinomycetemcomitans-, P. gingivalis- and P. intermedia/P. nigrescens group-isolates were identical in all respective patients. The pre- and post-treatment ribotypes of C. rectus were identical in 1 of 4 patients, whereas 2 patients harbored a previously not detected post-treatment ribotype and 1 patient harbored the initial and a previously not detected post-treatment ribotype. To study the possibility that periodontitis patients may acquire strains from the spouse after treatment, isolates of A. actinomycetemcomitans, P. gingivalis, P. intermedia/P. nigrescens group and C. rectus (n = 95) from the patients' spouses were ribotyped and compared with those of the patients. The patient exhibited his own post-treatment ribotypes, different from those of the spouse, of A. actinomycetemcomitans and P. gingivalis in 1 couple and of P. intermedia/P. nigrescens group and C. rectus in 1 couple. In the 2 patients who harbored a previously not detected post-treatment ribotype of C. rectus, one patient shared the new ribotype with the spouse, whereas the other one did not. Although an exogenous source cannot be fully ruled out, the patient's own oral flora seems to be the main source of re-emerging periodontal pathogens after treatment.

The bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis as the etiologic agent on the basis of several lines of circumstantial evidence. A matter of extensive debate is whether A. actinomycetemcomitans is an exogenous contagious pathogen or an opportunistic pathogen that resides in the normal oral microflora. Here we show evidence of a single clone of A. actinomycetemcomitans isolated from multiple patients with juvenile periodontitis in members of families of African origin living in geographically widespread areas. The clone is characterized by a 530-bp deletion in the leukotoxin gene operon, resulting in a significantly increased production of leukotoxin.

We have identified a significant level of variation in antibody responses to Actinobacillus actinomycetemcomitans outer membrane antigens (OMA). This study was designed to verify A. actinomycetemcomitans antigenic diversity that could contribute to maintaining this chronic infection despite the host immune response. A. actinomycetemcomitans strains (5 from different patients and 3 the same patient) were cultured and OMA prepared for Western immunoblotting studies. Antigen bands in the OMA were identified using 7 sera obtained from 3 adult periodontitis (AP) and 4 localized juvenile periodontitis (LJP) patients that were documented with elevated A. actinomycetemcomitans antibody and infection. Differences/similarities in the antigen patterns among the A. actinomycetemcomitans strains were assessed using a kappa similarity coefficient. Antigen bands in the A. actinomycetemcomitans strains ranged from 11-150 kDa; however, variation in antigen patterns were noted among the strains. Utilizing the human sera as probes for antigenic diversity, the 5 heterologous strains showed average K=0.23 (p < 0.05), while homologous A. actinomycetemcomitans strains had a K=0.48 (p < 0.02). The A. actinomycetemcomitans OMA were used as probes to describe variability in host antibody and as such presumptive evidence of antigenic diversity in A. actinomycetemcomitans that is colonizing each of the patients. The results showed average K=0.26 (p < 0.05) for the patients when tested against each of the heterologous strains, and K=0.14 when tested against the homologous strains (p > 0.1). Finally, antigen bands of M r 80, 65, 58, 31 and 20 kDa were demonstrated as antigens contributing to the antigenic diversity in A. actinomycetemcomitans.

The aim of this study was to develop a PCR reaction specific to Actinobacillus actinomycetemcomitans, which targets a widely conserved gene of this bacterium. Two sets of primers were designed based on published sequences of the 16S rRNA of several microorganisms. The first set amplifies a major part of the 16S small subunit rRNA gene of several strains of bacteria commonly found in the periodontal pocket. This reaction produced a 1306 bp-long product and served as a positive control. The second set was specific to A. actinomycetemcomitans and produced a 449 bp-long product. H. aphrophilus and E. coli yielded positive results with the control primers and negative results with the A. actinomycetemcomitans-specific primers. DNA-DNA hybridization was used to validate the identify of the amplified sequences. B. cereus, which is a common contaminator in the laboratory, and human DNA did not generate PCR products in either reaction. The developed primers seen useful for the identification of A. actinomycetemcomitans strains.

Based upon DNA sequence data and positive immunochemical reactivity of expressed protein, novel homologues of the FNR family were identified in four representative oral facultative anaerobes: Capnocytophaga ochracea, Capnocytophaga sputigena, Haemophilus aphrophilus, and Actinobacillus actinomycetemcomitans. The similarity to E. coli FNR and to HlyX (itself 71% similar to E. coli FNR, while regulating expression of hemolysin operon in Actinobacillus pleuropneumoniae) was estimated from the deduced partial amino acid sequence to be, in the above order of tested species, 98, 98, 86, and 85%, and 75, 75, 88, and 88%, respectively. The phylogenetic relatedness indicates a rather closer link of HlyX to the FNR homologues from both pathogens, H. aphrophilus and A. actinomycetemcomitans. The possibility that the A. actinomycetemcomitans FNR homologue functions as a redox-sensing transcriptional factor to regulate, in addition to anaerobic respiration, microaerobic expression of the leukotoxin operon (ltx gene) is suggested.

We found that 17 (68%) of 25 Actinobacillus actinomycetemcomitans isolated from periodontally diseased sites had MICs of erythromycin > or = 8 mg/L. The isolates were hybridized with five rRNA methylase genes and gene mobility was determined. Twenty-four (96%) of 25 isolates hybridized with one or more rRNA methylase genes. Representative isolates were able to transfer erythromycin resistance to Haemophilus influenzae and Enterococcus faecalis recipients. The transconjugants were shown to carry rRNA methylase genes by DNA probe hybridization and had MICs of erythromycin 32 to 256 mg/L.

Detection of putative pathogens is critical for delineating the etiology and progression of periodontitis. In the present study, we have used a polymerase chain reaction (PCR) assay utilizing primers specific for the lkt A gene of Actinobacillus actinomycetemcomitans, the fimbrial gene of Porphyromonas gingivalis, and tdp A gene of Treponema denticola in order to determine the presence of these pathogens in subgingival plaque samples from periodontitis sites. These gene specific primers were also used to assess the detection of different strains of bacteria in the PCR assays. Primers for P. gingivalis detected P. gingivalis strain 33277, but no product was detected when primers were used with extracts from 4 species of Capnocytophaga, 3 species of Prevotella, 2 species of Porphyromonas other than P. gingivalis, Bacteroides levii, Escherichia coli, 3 strains of A. actinomycetemcomitans and 3 strains of T. denticola. PCR analysis using primers for the lkt A gene of A. actinomycetemcomitans also did not result in a product with any of these bacteria with the exception of a positive result with 3 different strains of A. actinomycetemcomitans. Primers selected from the tdp A gene of T. denticola did not identify any of the bacteria strains tested except T. denticola serovars a, b, and c. Thus, these primers were shown to amplify gene segments that are specific to either P. gingivalis (33277), A. actinomycetemcomitans (33384, 43717 and 43718) or T. denticola (35405, 33521 and 35404). The PCR assay may be used to rapidly detect the presence of periodontal pathogens in the future.

The aim of the present study was to determine by standard cultivation procedures the detection frequencies of Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Capnocytophaga species as well as various enteric rods in subgingival plaque samples form Romanian adult periodontitis patients. DNA probe analysis (Affirm DP Microbial Identification Test) was also used, parallel to cultivation, to identify P. gingivalis, A. actinomycetemcomitans, and B. forsythus, in deep (> or = 6 mm) and intermediate (4-5 mm) pockets in some of the subjects investigated. Paper points were used to sample 86 deep pockets in 36 patients and 27 intermediate pockets in 9 of the 36 patients. The chi 2 test was used to test for significance of differences between results obtained by cultivation and DNA analysis in both intermediate and deep pockets. P. gingivalis was recovered in a high percentage of the patients (75.8%) and sites (63.6%) examined, followed by P. intermedia, F. nucleatum, and A. actinomycetemcomitans, respectively. Capnocytophaga species were present in almost all subjects. Enteric rods were recovered in 61.1% of the patients and 55.8% of the sites. Except for this high prevalence of enteric rods, the present group of patients had the periodontal species monitored in %s similar to those commonly perceived in the West. The Affirm DP Test and cultivation showed poor correlation in detecting P. gingivalis, A. actinomycetemcomitans, and B. forsythus. The cultivation prevalence of P. gingivalis and P. intermedia in deep pockets was similar to their prevalence in intermediate ones. Overall, the prevalence of the periodontal pathogens investigated in the present Romanian periodontitis patients is similar to what has been revealed in matching Norwegian and other Western periodontitis patient populations. The high prevalence of enteric rods in the Romanian patients may have been an artifact resulting from prolonged transport of the samples in VMGA III.

The occurrence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in subgingival plaque in 24 juvenile periodontitis patients was determined using DNA probe. 36 samples of subgingival plaque from 36 pockets having > or = 6 mm depth, > or = 3 mm of loss of attachment, and bleeding on probing and/or suppuration were taken from 18 patients with localized juvenile periodontitis (LJP, age range 12-24 years); and 12 samples from 6 patients with generalized juvenile periodontitis (GJP, age range 23-26 years). As control, an equal numbers of samples from healthy sites in the same patients were studied. P. gingivalis was found in 17 of 18 LJP patients, and in 31 of 36 diseased sites in those patients. P. intermedia was found in 15 out of the 18 LJP patients and in 28 of the 36 diseased sites. A. actinomycetemcomitans was present in 7 of the 18 LJP patients, and in 9 of the 36 diseased sites, and was not found in any GJP patients. All GJP patients had P. gingivalis (11 out of 12 diseased sites) and P. intermedia (all of the diseased sites). None of the three bacterial species was detected in healthy sites of GJP patients, and were found in healthy sites in only 2 of 18 LJP patients. The high prevalence and high levels of P. gingivalis and P. intermedia found in the LJP and GJP patients studied, suggest that there are populations affected by juvenile periodontitis in which this type of periodontitis is more associated with these species than with A. actinomycetemcomitans.

This review focuses on two topics that in the past decade have significantly enhanced our understanding of the mechanisms by which bacteria produce disease in humans: population structures of bacterial pathogens and tactics used by these bacteria to gain entry to mammalian cells. Studies in these areas have identified a number of virulence factors of some medically important pathogens. Very recent applications of this knowledge in efforts to understand mechanisms by which the most extensively studied "putative" periodontal pathogens, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, may contribute to the pathogenesis of periodontitis are presented. Knowledge of the population structures of these periodontopathic bacterial species and the mechanisms used by these bacteria to subvert host cell communication systems and functions will shape future approaches to diagnosis, prevention, and treatment of periodontal diseases.

The purpose of this study was to assess the sensitivity and specificity of the PCR in detecting Actinobacillus actinomycetemcomitans. The PCR's detection capability was compared with those of three other methods: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conventional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was amplified by PCR with primers TT-15 and TT-16. For CH, the PCR product was labeled with digoxigenin and used as a hybridization probe. Nucleotide sequence analysis of the PCR product of A. actinomycetemcomitans 1D4 and 1664 and three clinical isolates revealed complete homology among the tested strains, with only one base substitution (at position 1344) in comparison with the published sequence. With artificially infected subgingival plaque, the detection limit of PCR for A. actinomycetemcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR (CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of subgingival plaque samples from 35 patients with periodontal disease and 10 periodontally healthy subjects revealed that CE-PCR and CH had the highest overall rate of A. actinomycetemcomitans detection (both 58%), followed by PCR and culture (both 42%). With CH as the "gold standard", the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respectively. The CE-PCR provided acceptable positive and negative predictive values (> or = 70%) when the prevalence of A. actinomycetemcomitans varied between 30 and 70%. PCR alone provided comparable predictive values over a narrower range of prevalence rates (30 to 50%), while culture did not afford acceptable predictive values at any prevalence rate. PCR and CE-PCR were found to be superior to culture with presumptive biochemical identification and should be the preferred methods for the detection of A. actinomycetemcomitans in subgingival plaque.

We developed a colorimetric microtiter plate-based assay for the detection and quantification of polymerase chain reaction-amplified DNA fragment specific for Actinobacillus actinomycetemcomitans. We amplified the 396-bp leukotoxin-specific DNA fragment by using two oligonucleotide primers, one carrying a biotin group at the 5' end and another one with a digoxigenin at the 5' end. Following amplification, the biotinylated polymerase chain reaction products were applied to a microtiter well precoated with avidin. The colorimetric detection and quantification were achieved by an enzyme-linked immunosorbent assay using alkaline phosphatase-conjugated anti-digoxigenin antibody. The detection limit of the colorimetric assay was found to be as little as 500 fg of purified A. actinomycetemcomitans DNA and as few as 50 A. actinomycetemcomitans. Therefore, this colorimetric assay was able to estimate the amount of A. actinomycetemcomitans in subgingival plaque samples. We concluded that the colorimetric assay of the PCR product is a very useful method not only to detect the presence of A. actinomycetemcomitans but also to quantify the amount of A. actinomycetemcomitans in large numbers of subgingival plaque samples.

The identification of periodontal pathogens by conventional methods is time-consuming and difficult. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.) and Porphyromonas gingivalis (P.g.) was developed for rapid and easy determination of these risk-indicator bacteria in human periodontal disease. The PCR primers were designed to hybridize to various regions of 16S rRNA genes, and a hot-start technique was used to obtain maximum sensitivity and specificity. This method can detect both of these bacteria in subgingival plaque samples at concentrations as low as 5 to 50 cells per sample. The sensitivity, however, was even 10 times better when the bacteria were analyzed in a water suspension. Since the only step between sample collection and the actual analysis is a brief centrifugation of the patient sample, the detection can be readily carried out in four hours. The performance of the method was studied with 36 patient samples. The results showed that the PCR method detected A.a. (44% vs. 25%, respectively) and P.g. (56% vs. 42%, respectively) more often than the conventional culture in plaque samples. Thus, our multiplex PCR method is rapid and more effective than conventional protocols in detecting these periodontal pathogens.

Membrane exposure with bacterial contamination is often considered one of the main reasons for the lack of predictability in achieving complete regeneration of periodontal defects. Ten cellulose membranes, retrieved from 7 patients treated for Class II furcation lesions in lower molars with at least 4 mm of exposure at time of retrieval were studied. Contamination of exposed membranes was studied using SEM analysis of four surfaces of the membrane, upper external, lower external, upper internal, and lower internal surfaces. DNA probe analysis of three periodontopathic bacteria, Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinmycetemcomitans, was carried out for specimens collected from the external and internal sulci. The results suggest that bacterial contamination of the membranes could be controlled if proper pre- and postoperative care is followed, since significant amounts of any of the three periodontopathogenic bacteria studied were not found. The SEM analysis corroborated the DNA probe analysis since the predominant morphotypes detected were not suggestive of periodontopathogenic bacteria. The importance of membrane contamination and of root concavities in the lack of predictability of the GTR procedure is discussed.

A series of cloning vectors has been constructed from pACYC177 and pACYC184. These vectors contain a MCS, the lacZ alpha reporter gene, and P15A ori. Plasmids pMBL18, pMBL19 and pMBLcos express the bla gene encoding ampicillin resistance, while pMCL200 and pMCL210 contain the cat gene encoding chloramphenicol resistance. In addition, the cosmid pMBLcos was constructed with the P15A ori. These vectors are useful for cloning or subcloning genes which are unstable and/or toxic in high-copy-number vectors.

The objective of this study was to assess the effectiveness of surgical debridement versus various guided tissue regeneration techniques for the treatment of localized juvenile periodontitis (LJP). Seven patients, 14 to 18 years old, were selected on the basis of having; 1) vertical bone loss on at least 3 first molars; 2) probing attachment loss > or = 5 mm; 3) presence of Actinobacillus actinomycetecomitans (Aa) as determined by the DNA probe test; 4) no systemic diseases; and 5) not taken antibiotics for 6 months prior to treatment. Each patient was randomly assigned by first molar involvement to one of four treatment modalities: 1) surgical debridement alone (DEBR); 2) ePTFE membrane alone (G); 3) ePTFE membrane plus root conditioning (G + D); or 4) ePTFE membrane plus root conditioning plus composite graft (G + D + B). Standardized clinical attachment level measurements (CAL) were taken at baseline, 6, 12, and 18 months. Hard tissue measurements were evaluated radiographically (RAD) at baseline, 6, 12, and 18 months, and volumetrically at baseline and 12 months post-surgery. At 12 months re-entry, all residual defects were re-treated with a composite graft consisting of calcium sulfate + DFDBA + doxycycline. Results were evaluated 6 months post-retreatment using CAL and RAD measurements. Statistical analysis of the data showed: 1) a significant gain in clinical attachment as well as increased volumetric bone fill of the defects 12 and 18 months following the various treatment modalities when compared to baseline (P < 0.05); and 2) no statistically significant differences among the tested treatment modalities and surgical debridement alone for all parameters (P > 0.05).

Several plasmids have been described in Actinobacillus actinomycetemcomitans, a gram-negative coccobacillus. Recently, the nucleotide sequence of pVT736-1, a cryptic plasmid of A. actinomycetemcomitans VT736, was determined. This plasmid possesses all the features necessary for rolling circle replication. The present study involved a transcriptional analysis of pVT736-1. Results of Northern (RNA) blot analyses and primer extension studies indicated that the two open reading frames identified in pVT736-1 are each preceded by at least one promoter. Expression of these promoters varied with growth phase. In addition, an antisense RNA (Cop RNA) appeared to control the synthesis of the putative replication protein. To our knowledge, this is the first rolling circle replicating plasmid isolated from a gram-negative organism that has been subjected to such detailed analysis.

Comparison of susceptibility data for Actinobacillus actinomycetemcomitans has been difficult because of the lack of standard susceptibility testing conditions. In this study, minimum inhibitory concentration to tetracycline was evaluated by comparing different media, air conditions and incubation times. Ten of 22 (45%) A. actinomycetemcomitans isolated from periodontally diseased sites grew on media supplemented with 4 micrograms per ml of tetracycline, but minimum inhibitory concentrations ranged from 0.125 to 8 micrograms/ml depending on the media and condition used. The best results were obtained with brain heart infusion agar (Difco Laboratories, Detroit MI) incubated in 5% CO2 for 48 h. Eighteen (82%) of the A. actinomycetemcomitans isolates hybridized with the Tet B determinant. The Tet B determinant was transferable between A. actinomycetemcomitans isolates as well as a Haemophilus influenzae recipient and appears to be associated with conjugative plasmids.

To develop targeted gene integration in the periodontal pathogen Actinobacillus actinomycetemcomitans, a ColE1-based, spectinomycin-resistant plasmid containing a segment of the leukotoxin gene was electroporated into strain JP2. In all of the stable spectinomycin-resistant transformants that arose, the plasmid had recombined into the genomic leukotoxin locus since ColE1-based vectors cannot replicate extrachromosomally in A. actinomycetemcomitans. Directed genomic integration was then used to construct a leukotoxin-negative strain by transforming the leukotoxin-producing strain JP2 with a ColE1-based plasmid containing an internal fragment of the leukotoxin gene. Cytotoxicity assays proved that these transformants had < 0.1% of the leukotoxin activity of the parental strain. These results demonstrate that integration-based approaches can be used for generating isogenic mutants in specific virulence genes in A. actinomycetemcomitans.

The 64-kDa protein to which about half the sera from patients with localized juvenile periodontitis and rapidly progressive periodontitis reacted strongly was purified from Actinobacillus actinomycetemcomitans Y4. Determination of the N-terminal sequence of the protein revealed that it was a GroEL-like protein. The DNA fragment containing the groEL gene of A. actinomycetemcomitans was amplified by polymerase chain reaction, and the groESL operon was cloned by using colony hybridization with the amplified fragment from A. actinomycetemcomitans chromosomal DNA. Sequence analysis revealed that structures of the operon and its products were typical in gram-negative bacteria. Rabbit polyclonal antibodies to the 64-kDa protein cross-reacted with approximately 65-kDa proteins of Haemophilus aphrophilus, Haemophilus influenzae, Haemophilus paraphrophilus, Escherichia coli and Eikenella corrodens but not with any cellular proteins of Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum. It is possible that antibodies reactive to the 64-kDa protein in periodontitis patients are induced by the cross-reactivity with the hsp60 proteins of other bacteria.

The periodontal pathogen, Actinobacillus actinomycetemcomitans, produces leukotoxin, a protein that specifically lyses host defense cells. The leukotoxin is similar in sequence and operon organization to the Escherichia coli alpha-hemolysin and other members of the RTX family of toxins. However, unlike the other RTX toxins, the A. actinomycetemcomitans leukotoxin is not secreted from the cell and instead remains associated with the outer membrane. Nonetheless, the A. actinomycetemcomitans Ikt operon contains two genes, IktB and IktD, that appear analagous to the toxin localization genes found in the other Gram-negative bacteria. Thus, to determine the roles of these putative transport genes in A. actinomycetemcomitans, we have used insertional mutagenesis to generate mutant strains lacking functional LktB and/or LktD. When either IktD or both IktB and IktD were inactivated, the level of detectable leukotoxin protein in the cell decreased significantly. However, the IktB and IktD mutations had no effect on the levels of leukotoxin RNA. Thus, the lack of LktB and LktD proteins must affect LktA synthesis post-transcriptionally. It is proposed that this is an indirect effect of leukotoxin mislocalization in IktB- and IktD- mutants. Finally, analysis of the mutants revealed that LktB and LktD are not essential for the formation of extracellular membrane vesicles in A. actinomycetemcomitans.

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.

Actinobacillus actinomycetemcomitans (A.a.) can produce a potent leukotoxin that is thought to be involved in evasion of the host immune response. In order to understand the role of A.a. and its leukotoxin in the initiation and progression of periodontal disease, it is important determine how the expression of A.a. virulence factors might be regulated by the local periodontal micro-environment. To facilitate the measurement of leukotoxin levels, a leukotoxin-beta-galactosidase gene fusion was constructed and recombined into the chromosome of A.a. strain JP2 at the leukotoxin locus. The resulting strain, AAM17, produces beta-galactosidase under control of the leukotoxin promoter. It also produces leukotoxin, since integration of the gene fusion into the chromosome was designed to produce a duplication of the leukotoxin gene. This strain was used to measure the change in leukotoxin level in response to alterations in two environmental signals: iron concentration and oxygen tension. When AAM17 was grown in iron-limited media that did not alter growth rate but did increase the levels of other iron-regulated proteins, the levels of the leukotoxin-beta-galactosidase were similar to those found in AAM17 grown in iron-replete media. These results were confirmed in strains AAM17 and JP2 by leukotoxicity assays and RNA blots. Aerobic growth of AAM17 resulted in a three-fold decrease in leukotoxin beta-galactosidase activity compared with anaerobically grown cells. These results indicate that the A.a. leukotoxin is regulated by some of the environmental signals that may vary in the gingival crevice.

Genetic analysis of an Actinobacillus actinomycetemcomitans population consisting of 88 clinically well characterized Finnish isolates performed by multilocus enzyme electrophoresis confirmed that the five serotypes divide into two phylogenetic lineages, one comprising serotypes b and c and one comprising serotypes a, d, and e. There was no association between any subpopulation and the periodontal health status of the subject from whom the isolates originated, suggesting that the role of A. actinomycetemcomitans in periodontitis is largely opportunistic in the population examined. Southern blot analyses of genomic DNA digested with each of the restriction endonucleases MspI, RsaI, and TaqI revealed extremely limited genetic polymorphism of the structural leukotoxin gene, ltxA, and its associated promoter. All isolates hybridized to a 530-bp DNA fragment derived from the promoter region of the leukotoxin gene operon of a minimally leukotoxic A. actinomycetemcomitans strain. Deletion of the 530-bp sequence has been associated with significantly increased toxin production detected among isolates from patients with juvenile periodontitis in North America but was detected neither among the 88 isolates in the present collection analyzed nor among more than 60 strains in another population of northern European A. actinomycetemcomitans isolates analyzed previously.

Although extensive amino acid homology exists among the various Repeats in ToXin (RTX) family of bacterial cytolysins, the cellular and species specificities remain unique for individual toxins (i.e. Actinobacillus actinomycetemcomitans leukotoxin (LtxA) kills human monomyelocytes while a related toxin, Pasteurella hemeolytica leukotoxin (LktA) kills bovine lymphoid cells). To determine the Ltx domain responsible for species specificity, ltxA/lktA chimeric toxin genes were expressed in tandem with the ltxC gene under control of the P lambda promoter. The ability of lysates to kill either HL-60 (human) or BL-3 (bovine) cells was assessed by trypan blue exclusion. The critical area required for the chimeric toxins to recognize human target cells is a 253-amino acid fragment (residue 688-941) that contains the GGXGXDX(L[I[V[W[Y[F)X repeats. A panel of 12 neutralizing anti-LtxA monoclonal antibodies also recognized specificities within the 253-amino acid fragment. Epitope mapping of the monoclonal antibody panel showed that all antibodies bound to one of three sites on the LtxA molecule. One monoclonal recognized epitope A which was composed of LtxA residues 698-709 (KLDYYYTNKGFK), six antibodies recognized epitope B, a peptide composed of residues 746-757 (LIYGYDGDDRLY), whereas the remaining five monoclonals recognized epitope C, which is composed of residues 926-937 (DRARLKRQFELQ).

Cell-free extracts of strains belonging to the 5 serotypes of A. actinomycetemcomitans were screened for several enzymes. Enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt and the TCA cycle were present. Of these glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) were the most readily detected and stable. MDH and G6PDH retained more than 50% of their activities at alkaline pHs (10-11) for up to 6 h and 3 h at 25 degrees C, respectively, while at pH 6.5, 50% of their activities were lost within 2-3 h. The Km for malate oxidation catalysed by MDH was 5.8 x 10(-4) M while that for glucose-6-phosphate oxidation was 2.0 x 10(-4) M. The pH optima for MDH and G6PDH oxidation activities were 10 and 9.5, respectively. Among the 5 designated serotypes of A. actinomycetemcomitans three groups were delineated by multilocus enzyme electrophoresis using MDH and G6PDH.

The Actinobacillus actinomycetemcomitans population consists of a large number of clones among which the ubiquitous leukotoxin gene operon appears very homogeneous. Population genetic analyses performed by multilocus enzyme electrophoresis together with DNA fingerprinting and analyses of genomic DNA restriction fragment length polymorphisms (RFLP) on 97 strains isolated over a period of 45 years revealed that each of the serotypes a, b, c, d and e comprise genetically isolated subpopulations and that successful horizontal transfer of genomic DNA between strains of different serotypes appears to be extremely rare in vivo. In contrast, recombination between strains of the same serotype in general appears to take place in nature. The results provide evidence that non-serotypeable strains are serotype antigen-deficient variants originating from strains of the known serotypes. Serotype b and c strains may contain transmittable DNA sequences not found in strains of the other serotypes.

A geographically homogeneous population of 83 subjects, from 21 families with localized juvenile periodontitis (LJP), and 35 healthy control subjects was monitored, over a 5-year period, for the presence of the periodontal pathogen Actinobacillus actinomycetemcomitans. Restriction fragment length polymorphism (RFLP) analysis was used to monitor the distribution of genetic variants of this bacterium in LJP-susceptible subjects that converted from a healthy to a diseased periodontal status. A. actinomycetemcomitans was cultured from 57% of the LJP family members accessioned into the study. Nine of 36 LJP-susceptible subjects, in seven families, developed signs of periodontal destruction. All but one of these conversion subjects harbored A. actinomycetemcomitans. Bacterial variants representative of a single RFLP group (II) showed the strongest correlation with conversion (P < 0.002). Six of nine conversion subjects were infected with A. actinomycetemcomitans from this group. RFLP group II variants also prevailed in 8 of 22 probands but were absent in the 35 healthy control subjects. In contrast to the selective distribution of group II variants is diseased individuals, variants belonging to RFLP groups XIII and XIV were found exclusively in the control subjects. Thus, the use of RFLP to type clinical isolates of A. actinomycetemcomitans has resulted in the identification of genetic variants that predominate in LJP and health. These results indicate that studies concerned with the pathogenicity of this bacterium in LJP should be focused on the group II variants.

This clinical study evaluated the survival rates of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia in periodontal pockets following scaling and root planing and intra-pocket irrigation with antimicrobial agents in patients with moderate and severe periodontitis. The number of target organisms was determined utilizing DNA probes. Adult periodontitis patients were selected on the basis that the subgingival flora contained at least one of the target organisms. Forty-eight (48) inflamed pockets > or = 5 mm in depth with probing attachment loss and containing at least one of the target species were then selected in 7 adult patients who harbored these bacteria. Following baseline clinical and bacterial examination, all patients received thorough scaling and root planing. In addition, 1 or 2 teeth in each patient which harbored the target flora at baseline were randomly assigned to each of the following 4 treatment modalities: 1) control group, no irrigation; 2) saline group, irrigation with 2 cc of physiologic saline; 3) tetracycline group, irrigation with 2 cc of aqueous tetracycline hydrochloride, 50 mg/ml (5%); and 4) chlorhexidine group, irrigation with 2 cc 0.12% chlorhexidine. All selected sites (5 to 8 per patient) were nonadjacent teeth. Clinical parameters and microbial analysis were recorded again at one week, and one month post-irrigation. The survival rate of the target microorganisms was determined and the effect of irrigation with antimicrobial agents on this microflora was compared with the control groups (1 and 2).(ABSTRACT TRUNCATED AT 250 WORDS)

This study introduces a strategy to identify and produce sequences useful as genetic markers, or native genetic probes for DNA-DNA hybridization in bacterial strains where the genetics is not well described. Actinobacillus actinomy-cetemcomitans (A.a.) was used as an example. Fifty ng genomic DNA from A.a. ATCC 33384 and Haemophilus aphrophilus ATCC 33389 was amplified in a thermocycler using a single 10-mer primer. The PCR products were separated by electrophoresis on a 1% submarine agarose gel containing ethidium bromide and visualized by UV illumination, and the strain-specific amplitypes were compared. DNA from two bands, 0.9 and 4 kb, unique for the A.a. strain, was cut out, amplified under high stringency with the same primer and labeled by replacing 33.3 microM dTTP with digoxigenin-labeled dUTP in the reaction mixture. The labeled probe was then repeatedly used for hybridization to DNA from various A.a., H. aphrophilus, and other bacterial strains of the Pasteurellaceae family. The results showed that the 0.9-kb probe detected all A.a. tested, and distinguished it from other closely related bacterial species. We conclude that the described strategy is useful for identifying and selecting genetic sequences useful as genetic markers in A.a.

The purposes of this study were two-fold: to compare the DNA probe and enzyme linked immunosorbent assay (ELISA) microbial identification tests and correlate the levels of microorganisms with adult periodontitis. A single plaque sample were taken from each of 2 sites in 52 patients. Twelve of these patients were also sampled during and after treatment. The experimental site had clinical indicators of disease (bleeding on probing, probing and attachment loss of > or = 6 mm) and the contralateral site (control) was clinically healthy. A total of 176 plaque samples were collected, divided, processed, and sent for both types of quantitative microbial analyses. All of these samples were used to compare the DNA probe and ELISA methods while only the initial 104 pretreatment sites were used to correlate microorganisms/method with clinical indicators of adult periodontitis. DNA probes were used to assay for A. actinomycetemcomitans, P. gingivalis, P. intermedia, E. corrodens, F. nucleatum, T. denticola, and C. rectus. An ELISA utilizing monoclonal antibodies was used to assay for P. gingivalis, E. corrodens, T. denticola, and C. rectus. Comparison of the two methods revealed that the ELISA test identified P. gingivalis and C. rectus significantly more often than the DNA probe method and that T. denticola was detected more frequently with the DNA probe. The sensitivities and specificities varied widely among organisms and by test. P. gingivalis, as identified by ELISA, had the highest degree of sensitivity and specificity (0.90 and 0.82 respectively) to clinical indicators of adult periodontitis.

By using PCR, Actinobacillus actinomycetemcomitans strains were identified directly from plaque samples without the need to isolate or culture bacteria. DNA fragments were generated by a nested, two-step PCR amplification of the ribosomal spacer region between the 16S and 23S rRNA genes. For the first amplification, primers homologous to sequences common to all bacterial species were used. This was followed by a second amplification with primers specific to A. actinomycetemcomitans. The ribosomal DNA spacer region was amplified from as few as 10 bacterial cells within a total population of 10(8) cells (0.00001%), and cross-reactivity between species was not observed. DNA fragments specific for Porphyromonas gingivalis were generated from the same samples by using a P. gingivalis-specific primer, and equivalent sensitivity and specificity were observed. A. actinomycetemcomitans was detected in 60% and P. gingivalis was detected in 79% of 52 subjects tested. Sequence analysis of the spacer region DNA fragment for A. actinomycetemcomitans gave precise strain identification, producing unique sequences for seven reference strains and identification of nine plaque-derived isolates. A phylogenetic tree based on quantitative sequence relationships was constructed. Two-step PCR amplification directly from plaque samples combined with sequence analysis of the ribosomal DNA spacer region provides a sensitive assay for detection and strain identification of multiple species directly from a single plaque sample. This simplified approach provides a practical method for large-scale studies on the transmission and pathogenicity of periodontitis-associated bacteria.

Actinobacillus actinomycetemcomitans (Aa) has been implicated in most cases of localized juvenile periodontitis and some cases of severe adult periodontitis and refractory periodontitis. The Aa leukotoxin plays an important role in the pathogenesis of Aa associated periodontal disease. Rapid detection of Aa in a periodontal pocket is hampered by the slow growth and fastidious nature of this bacterium. In this study, we developed a rapid, sensitive, nonradioactive polymerase chain reaction (PCR) to identify a unique As sequence directly from subgingival plaque samples. Two oligonucleotide primers derived from DNA sequences of the leukotoxin gene were used in the PCR. The Aa-specific DNA fragments were analyzed by agarose gel electrophoresis and then visualized under 302 nm ultraviolet light after staining with ethidium bromide. In the 12 subgingival plaque samples screened, the Aa-specific sequences were found in five out of nine sites with periodontitis. No Aa-specific sequence was found in three healthy sites. The specificity of the amplified DNA fragments was confirmed by direct DNA sequencing. These results indicated that the PCR technique should assist in the rapid detection of Aa in subgingival plaque samples. Moreover, combined with direct DNA sequencing, this method can be used to study the molecular epidemiology of this periodontal pathogen.