Assessing the progression of chronic periodontitis using subgingival pathogen levels: a 24-month prospective multicenter cohort study.

BACKGROUND: The diagnosis of the progression of periodontitis presently depends on the use of clinical symptoms (such as attachment loss) and radiographic imaging. The aim of the multicenter study described here was to evaluate the diagnostic use of the bacterial content of subgingival plaque recovered from the deepest pockets in assessing disease progression in chronic periodontitis patients. METHODS: This study consisted of a 24-month investigation of a total of 163 patients with chronic periodontitis who received trimonthly follow-up care. Subgingival plaque from the deepest pockets was recovered and assessed for bacterial content of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans using the modified Invader PLUS assay. The corresponding serum IgG titers were measured using ELISA. Changes in clinical parameters were evaluated over the course of 24 months. The sensitivity, specificity, and prediction values were calculated and used to determine cutoff points for prediction of the progression of chronic periodontitis. RESULTS: Of the 124 individuals who completed the 24-month monitoring phase, 62 exhibited progression of periodontitis, whereas 62 demonstrated stable disease. The P. gingivalis counts of subgingival plaque from the deepest pockets was significantly associated with the progression of periodontitis (p < 0.001, positive predictive value = 0.708). CONCLUSIONS: The P. gingivalis counts of subgingival plaque from the deepest pockets may be associated with the progression of periodontitis.

Salivary pathogen and serum antibody to assess the progression of chronic periodontitis: a 24-mo prospective multicenter cohort study.

BACKGROUND AND OBJECTIVE: A diagnosis of periodontitis progression is presently limited to clinical parameters such as attachment loss and radiographic imaging. The aim of this multicenter study was to monitor disease progression in patients with chronic periodontitis during a 24-mo follow-up program and to evaluate the amount of bacteria in saliva and corresponding IgG titers in serum for determining the diagnostic usefulness of each in indicating disease progression and stability. MATERIAL AND METHODS: A total of 163 patients with chronic periodontitis who received trimonthly follow-up care were observed for 24 mo. The clinical parameters and salivary content of Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans were assessed using the modified Invader PLUS assay, and the corresponding serum IgG titers were measured using ELISA. The changes through 24 mo were analyzed using cut-off values calculated for each factor. One-way ANOVA or Fisher's exact test was used to perform between-group comparison for the data collected. Diagnostic values were calculated using Fisher's exact test. RESULTS: Of the 124 individuals who completed the 24-mo monitoring phase, 62 exhibited periodontitis progression, whereas 62 demonstrated stable disease. Seven patients withdrew because of acute periodontal abscess. The ratio of P. gingivalis to total bacteria and the combination of P. gingivalis counts and IgG titers against P. gingivalis were significantly related to the progression of periodontitis. The combination of P. gingivalis ratio and P. gingivalis IgG titers was significantly associated with the progression of periodontitis (p = 0.001, sensitivity = 0.339, specificity = 0.790). CONCLUSIONS: It is suggested that the combination of P. gingivalis ratio in saliva and serum IgG titers against P. gingivalis may be associated with the progression of periodontitis.

Porphyromonas gingivalis displays a competitive advantage over Aggregatibacter actinomycetemcomitans in co-cultured biofilm.

BACKGROUND AND OBJECTIVE: Biofilm formation occurs through the events of cooperative growth and competitive survival among multiple species. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are important periodontal pathogens. The aim of this study was to demonstrate competitive or cooperative interactions between these two species in co-cultured biofilm. MATERIAL AND METHODS: P. gingivalis strains and gingipain mutants were cultured with or without A. actinomycetemcomitans. Biofilms formed on glass surfaces were analyzed by crystal violet staining and colony counting. Preformed A. actinomycetemcomitans biofilms were treated with P. gingivalis culture supernatants. Growth and proteolytic activities of gingipains were also determined. RESULTS: Monocultured P. gingivalis strains exhibited a range of biofilm-formation abilities and proteolytic activities. The ATCC33277 strain, noted for its high biofilm-formation ability and proteolytic activity, was found to be dominant in biofilm co-cultured with A. actinomycetemcomitans. In a time-resolved assay, A. actinomycetemcomitans was primarily the dominant colonizer on a glass surface and subsequently detached in the presence of increasing numbers of ATCC33277. Detachment of preformed A. actinomycetemcomitans biofilm was observed by incubation with culture supernatants from highly proteolytic strains. CONCLUSION: These results suggest that P. gingivalis possesses a competitive advantage over A. actinomycetemcomitans. As the required biofilm-formation abilities and proteolytic activities vary among P. gingivalis strains, the diversity of the competitive advantage is likely to affect disease recurrence during periodontal maintenance.

A novel gene required for natural competence in Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Natural competence is the ability of bacteria to take up extracellular DNA and incorporate it into their genomes. Some strains of Aggregatibacter actinomycetemcomitans, a critical periodontal pathogen, are naturally competent for transformation. However, information on natural competence genes is limited for this species. The aim of this study was to confirm the involvement of a novel gene identified near the fimbriae gene cluster in natural competence. MATERIAL AND METHODS: The functions of putative open reading frames (ORFs), designated AA00863-AA00865, in the Oralgen project database for A. actinomycetemcomitans strain HK1651, have not been determined. Using naturally transformable A. actinomycetemcomitans strains D7S-1 and ATCC29523, we created deletion mutants of homologous genes of these ORFs. Natural competence in the study strains was determined using an agar-based transformation frequency assay. RESULTS: Mutation of the AA00865 homolog, which we named urpA in A. actinomycetemcomitans strain D7S-1, resulted in the loss of natural competence, whereas mutations of the AA00864 and AA00863 homologs, located downstream of urpA gene, did not. Similar results were also observed in the mutants of A. actinomycetemcomitans ATCC29523. Complementation of the deleted sequence in the urpA mutant restored natural competence. CONCLUSION: The urpA gene is a novel gene required for natural competence in A. actinomycetemcomitans and does not exhibit significant homology with any natural competence genes previously identified in other bacterial species.

Adherence of Aggregatibacter actinomycetemcomitans via serotype-specific polysaccharide antigens in lipopolysaccharides.

INTRODUCTION: Gram-negative Aggregatibacter actinomycetemcomitans is recognized as an important periodontal pathogen. A striking property of this bacterium is its ability to form a tenacious biofilm adhering to abiotic surfaces. Both fimbrial and non-fimbrial adhesins are believed to be responsible for this ability. In our study, specific markerless mutants in the biosynthesis genes of cell surface polysaccharides were constructed with the Cre-loxP recombination system to identify non-fimbrial adhesin(s). METHODS: Non-fimbriated A. actinomycetemcomitans strain ATCC29523 (serotype a) was used to construct a deletion mutant of serotype-a specific polysaccharide antigen (SPA-a) in lipopolysaccharide (LPS). The LPS was purified through a polymyxin B column following phenol extraction, and verified by silver staining following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblot analysis using rabbit antisera raised against SPA-a. Strains were grown in broth for 2 days and examined for the adherence of bacterial cells on the glass surface. RESULTS: Strain ATCC29523 formed a thin film of bacterial growth on the glass surface. The deletion of SPA-a affected its ability to form this thin film. When this mutant was rescued with the wild-type SPA-a gene cluster, its adherence-positive phenotype was restored. CONCLUSION: SPA-a in the LPS molecule appears to promote the adherence of A. actinomycetemcomitans cells to abiotic surfaces.

Detection of interleukin-1 beta mRNA in rat periapical lesions.

Cells expressing interleukin-1 beta (IL-1 beta) mRNA were demonstrated by in situ hybridization in rat periapical lesions. A great number of osteoclasts with significant tartrate-resistant acid phosphatase activity were observed on the bone surfaces, and numerous IL-1 beta mRNA-expressing cells were widely distributed in the periodontal ligaments. IL-1 beta mRNA-expressing cells were mainly observed around the blood vessels in the vicinity of the bone resorption sites and occasionally found near the osteoblasts. Immunohistochemistry and enzyme histochemistry assays showed that IL-1 beta mRNA-expressing cells were not bone cells, but that they had the characteristic features of macrophages. These results suggested that macrophages may contribute to the production of IL-1 beta and play an important role in activation of osteoclastic bone resorption.

Porphyromonas gingivalis gingipain is involved in the detachment and aggregation of Aggregatibacter actinomycetemcomitans biofilm.

Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are major periodontal pathogens that cause several types of periodontal disease. Our previous study suggested that P. gingivalis gingipains secreted in the subgingival environment are related to the detachment of A.actinomycetemcomitans biofilms. However, it remains unclear whether arginine-specific cysteine proteinase (Rgp) and lysine-specific proteinase (Kgp) play different roles in the detachment of A. actinomycetemcomitans biofilm. The aim of this study was to investigate possible disruptive roles of Kgp and Rgp in the aggregation and attachment of A. actinomycetemcomitans. While P. gingivalis ATCC33277 culture supernatant has an ability to decrease autoaggregation and coaggregation of A. actinomycetemcomitans cells, neither the boiled culture supernatant of ATCC33277 nor the culture supernatant of KDP136 showed this ability. The addition of KYT-1 and KYT-36, specific inhibitors of Rgp and Kgp, respectively, showed no influence on the ability of P. gingivalis culture supernatant. The result of gelatin zymography suggested that other proteases processed by gingipains mediated the decrease of A. actinomycetemcomitans aggregations. We also examined the biofilm-destructive effect of gingipains by assessing the detachment of A. actinomycetemcomitans from polystyrene surfaces. Scanning electron microscope analysis indicated that A. actinomycetemcomitans cells were detached by P. gingivalis Kgp. The quantity of A. actinomycetemcomitans in biofilm was decreased in co-culture with P. gingivalis. However, this was not found after the addition of KYT-36. These findings suggest that Kgp is a critical component for the detachment and decrease of A. actinomycetemcomitans biofilms.

Regenerative effect of azithromycin on periodontitis with different levels of gingival inflammation: three case reports.

BACKGROUND: Azithromycin is an antibiotic belonging to the macrolides. Previous case reports showed that azithromycin has a regenerative effect on periodontal tissue in addition to improving periodontal gingival inflammation. Recently, we experienced three periodontitis cases, all of which showed severe bone loss. However, their gingival inflammatory signs differed greatly. The present case reports evaluated the regenerative effects of azithromycin on periodontitis sites with different clinical signs of gingival inflammation. METHODS: In Case 1, generalized chronic periodontitis with severe gingival inflammation was treated with azithromycin before periodontal treatment. In contrast, Case 2 presented with few clinical signs of gingival inflammation, but was treated with azithromycin prescribed within a day of scaling and root planing. In Case 3, teeth with moderate gingival inflammation were treated with azithromycin after a series of scaling and root planing. RESULTS: Remarkable alveolar bone growth, regardless of baseline gingival inflammation, was noted in all three cases. CONCLUSIONS: The use of adjunctive azithromycin in scaling and root planing may be effective for periodontal tissue regeneration. This property may be independent of the degree of baseline gingival inflammation.

The pga gene cluster in Aggregatibacter actinomycetemcomitans is necessary for the development of natural competence in Ca(2+) -promoted biofilms.

Natural competence is the ability of bacteria to incorporate extracellular DNA into their genomes. This competence is affected by a number of factors, including Ca(2+) utilization and biofilm formation. As bacteria can form thick biofilms in the presence of extracellular Ca(2+) , the additive effects of Ca(2+) -promoted biofilm formation on natural competence should be examined. We evaluated natural competence in Aggregatibacter actinomycetemcomitans, an important periodontal pathogen, in the context of Ca(2+) -promoted biofilms, and examined whether the pga gene cluster, required for bacterial cell aggregation, is necessary for competence development. The A. actinomycetemcomitans cells grown in the presence of 1 mm CaCl2 exhibited enhanced cell aggregation and increased levels of cell-associated Ca(2+) . Biofilm-derived cells grown in the presence of Ca(2+) exhibited the highest levels of natural transformation frequency and enhanced expression of the competence regulator gene, tfoX. Natural competence was enhanced by the additive effects of Ca(2+) -promoted biofilms, in which high levels of pga gene expression were also detected. Mutation of the pga gene cluster disrupted biofilm formation and competence development, suggesting that these genes play a critical role in the ability of A. actinomycetemcomitans to adapt to its natural environment. The Ca(2+) -promoted biofilms may enhance the ability of bacteria to acquire extracellular DNA.

MyD88 is essential for alveolar bone loss induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide in mice.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacteria highly associated with localized aggressive periodontitis. The recognition of microbial factors, such as lipopolysaccharide from A. actinomycetemcomitans ((Aa)LPS), in the oral environment is made mainly by surface receptors known as Toll-like receptors (TLR). TLR4 is the major LPS receptor. This interaction leads to the production of inflammatory cytokines by myeloid differentiation primary-response protein 88 (MyD88) -dependent and -independent pathways, which may involve the adaptor Toll/interleukin-1 receptor-domain-containing adaptor inducing interferon-ß (TRIF). The aim of this study was to assess the involvement of MyD88 in alveolar bone loss induced by (Aa)LPS in mice. C57BL6/J wild-type (WT) mice, MyD88, TRIF or TRIF/MyD88 knockout mice received 10 injections of Aa LPS strain FDC Y4 (5 µg in 3 µl), in the palatal gingival tissue of the right first molar, every 48 h. Phosphate-buffered saline was injected in the opposite side and used as control. Animals were sacrificed 24 h after the 10th injection and the maxillae were removed for macroscopic and biochemical analyses. The injections of Aa LPS induced significant alveolar bone loss in WT mice. In the absence of MyD88 or TRIF/MyD88 no bone loss induced by (Aa)LPS was observed. In contrast, responses in TRIF(-/-) mice were similar to those in WT mice. Diminished bone loss in the absence of MyD88 was associated with fewer TRAP-positive cells and increased expression of osteoblast markers, RUNX2 and osteopontin. There was also reduced tumor necrosis factor-α production in MyD88(-/-) mice. There was less osteoclast differentiation of hematopoietic bone marrow cells from MyD88(-/-) mice after (Aa)LPS stimulation. Hence, the signaling through MyD88 is pivotal for (Aa)LPS-induced osteoclast formation and alveolar bone loss.

Clonal diversity and stability of subgingival Eikenella corrodens.

Eikenella corrodens is a commensal subgingival bacterium commonly found in both periodontally nondiseased and diseased subjects. The present study examined the clonal diversity and stability of subgingival E. corrodens over time. Ninety-five subjects were enrolled at the baseline examination, including 44 periodontally nondiseased subjects and 51 subjects with aggressive periodontitis. Twenty-two nondiseased subjects and 11 subjects with aggressive periodontitis were subsequently reexamined after an average interval of 14 months. Two subgingival plaque samples were obtained from each subject to determine the total cultivable bacteria. In addition, multiple E. corrodens isolates from each sample were recovered for clonal analysis by arbitrarily primed PCR. The mean numbers of distinct E. corrodens clones harbored by nondiseased subjects and subjects with aggressive periodontitis were 1.3 and 3.0, respectively. Thirty-nine percent of the nondiseased subjects and 63% of the subjects with aggressive periodontitis harbored multiple clones of E. corrodens. The numbers of distinct E. corrodens clones increased significantly (Mann-Whitney ranking test, P < 0.05) in sites from patients with aggressive periodontitis, in sites with pocket depths of 4 mm or greater, in sites with a clinical attachment loss of 2 mm or greater, and in sites coinfected with Porphyromonas gingivalis. Comparison of E. corrodens clones recovered at the baseline and those recovered at the follow-up examination showed that E. corrodens colonization was not stable. Thirty-eight of the 66 follow-up samples (58%) showed a complete change (including de novo colonization of the sites or complete elimination of the organism from the sites) of the subgingival E. corrodens clonal types between the baseline and the follow-up examinations. Our results suggest a complexity of subgingival microbiota not seen previously.

Colorimetric microtiter plate based assay for detection and quantification of amplified Actinobacillus actinomycetemcomitans DNA.

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.

Clonal distribution of natural competence in Actinobacillus actinomycetemcomitans.

The competence for natural transformation was investigated in 67 Actinobacillus actinomycetemcomitans strains. The transformation assays were performed with both cloned DNA fragments and chromosomal markers of A. actinomycetemcomitans. Competence was found in 12 of 18 serotype a strains, 0 of 21 serotype b strains, 0 of 14 serotype c strains, 3 of 6 serotype d strains, 3 of 4 serotype e strains, 0 of 3 serotype f strains, and 0 of 1 nonserotypeable strain. The transformation frequencies varied from 5 x 10(-3) to 4 x 10(-6) (median 1.5 x 10(-4)). The distribution pattern of natural competence is concordant with the major clonal lineages of A. actinomycetemcomitans. Serotype a strains are predominantly competent for transformation, while serotypes b and c strains are apparently non-competent.