Rapid identification of Actinomycetaceae and related bacteria.
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Identification of new isolates belonging to the family Actinomycetaceae requires extensive numbers of biochemical tests, supplemented with gas-liquid chromatography determination of fermentation end products and, often, analysis of cell wall composition. This paper describes the results of the testing of 162 strains of Actinomycetaceae and related taxa for 20 different enzymatic activities including phosphatases, esterases, aminopeptidases, and glycosidases. The results of all tests were read after 4 h of incubation. The results obtained in the study provide significant new information on the biochemical properties of these groups of bacteria. An identification scheme based upon 13 selected tests, which allow the identification of these groups of bacteria within 4 h, is proposed. (+info)
Blastogenic response of human lymphocytes to oral bacterial antigens: comparison of individuals with periodontal disease to normal and edentulous subjects.
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Cell-mediated immunity in humans to antigens derived from oral plaque bacteria was investigated by using the lymphocyte blastogenesis assay. Subjects with varying severities of periodontal disease including normal, gingivitis, periodontitis, and edentulous were compared. Mononuclear leukocytes were separated from peripheral blood and cultured with antigens prepared by sonication of Actinomyces viscosus (AV), Actinomyces naeslundii (AN), Veillonella alcalescens (VA), Leptotrichia buccalis (LB), Bacteroides melaninogenicus (BM), and homologous dental plaque (DP). The lymphocyte response of subjects with gingivitis or periodontitis was significantly greater than that of normal subjects to antigens of AV, AN, and DP, but did not differ from the response of edentulous subjects. Periodontitis subjects were significantly more reactive than edentulous and normal subjects in response to VA, LB, and BM. These findings suggest that the tested gram-negative bacteria and the host response they evoke are associated with advanced periodontal destruction. (+info)
Identification by PCR of Helicobacter pylori in subgingival plaque of adult periodontitis patients.
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The PCR was used to detect the presence of Helicobacter pylori in subgingival plaque samples from patients with adult periodontitis. Primers based upon the 16S ribosomal RNA (rRNA) gene sequence of H. pylori were used in a single round of PCR to amplify a 295-bp DNA fragment and the identity of the amplified products was confirmed by Southern blot hybridisation to a digoxigenin-labelled H. pylori probe. Further confirmation of product identity was obtained by DNA sequencing of a proportion of the amplified products. The assay was demonstrated to be specific for H. pylori with a lower limit of detection of 100 fg of bacterial genomic DNA. In all, 73 samples from 29 patients were analysed, of which 24 (33%) were H. pylori-positive by PCR; the proportion of patients carrying H. pylori in at least one sampled site was 38% (11 of 29). This is the first study to demonstrate the presence of H. pylori in the subgingival plaque of patients with adult periodontitis and indicates that, in this patient group at least, subgingival plaque may be a reservoir for H. pylori infection. (+info)
Antimicrobial susceptibility and composition of microcosm dental plaques supplemented with sucrose.
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The aims of this study were to evaluate the effects of repeated chlorhexidine gluconate (CHG) pulsing on the viability and bacterial composition of microcosm dental plaques derived from human saliva. The biofilms were grown on bovine enamel discs in a constant-depth film fermentor fed with an artificial saliva which was supplemented thrice daily with sucrose. The microcosm plaques had total viable anaerobic counts of 5 x 10(8) CFU per mm2 and consisted of 12% Actinomyces spp., 85% streptococci, and 0.2% Veillonella spp. When pulsed twice daily with 0.2% CHG, there was an immediate 1.3-log10 reduction in the total viable (anaerobic) count. However, as pulsing continued, the viable counts recovered, and after 4 days, the anaerobic count reached its pre-CHG-pulsing level, although the bacterial composition of the biofilms had changed. The results of this study show that twice-daily pulsing with 0.2% CHG over a 4-day period was ineffective at reducing the total anaerobic viable count of the biofilms but did alter their bacterial composition. (+info)
Inactivation of the gbpA gene of Streptococcus mutans alters structural and functional aspects of plaque biofilm which are compensated by recombination of the gtfB and gtfC genes.
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Inactivation of the gbpA gene of Streptococcus mutans increases virulence in a gnotobiotic rat model and also promotes in vivo accumulation of organisms in which gtfB and gtfC have recombined to reduce virulence (K. R. O. Hazlett, S. M. Michalek, and J. A. Banas, Infect. Immun. 66:2180-2185, 1998). These changes in virulence were hypothesized to result from changes in plaque structure. We have utilized an in vitro plaque model to test the hypothesis that the absence of GbpA alters S. mutans plaque structure and that the presence of gtfBC recombinant organisms within a gbpA background restores a wild-type (wt)-like plaque structure. When grown in the presence of sucrose within hydroxyapatite-coated wells, the wt S. mutans plaque consisted primarily of large aggregates which did not completely coat the hydroxyapatite surface, whereas the gbpA mutant plaque consisted of a uniform layer of smaller aggregates which almost entirely coated the hydroxyapatite. If 25% of the gbpA mutants used as inoculum were also gtfBC recombinants (gbpA/25%gtfBC), a wt-like plaque was formed. These changes in plaque structure correlated with differences in susceptibility to ampicillin; gbpA plaque organisms were more susceptible than organisms in either the wt or gbpA/25%gtfBC plaques. These data allow the conclusion that GbpA contributes to S. mutans plaque biofilm development. Since the changes in plaque structure detailed in this report correlate well with previously observed changes in virulence, it seems likely that S. mutans biofilm structure influences virulence. A potential model for this influence, which can account for the gtfBC recombination compensating gbpA inactivation, is that the ratio of glucan to glucan-binding protein is a critical factor in plaque development. (+info)
Antiphosphorylcholine antibody levels are elevated in humans with periodontal diseases.
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Human immunoglobulin G2 (IgG2) serum concentrations and the IgG2 antibody response to Actinobacillus actinomycetemcomitans can be influenced by genes, by environmental factors such as smoking, and by periodontal disease status. Examination of the IgG2 response to phosphorylcholine (PC), a response thought to be mainly induced by the C polysaccharide of Streptococcus pneumoniae, suggested that periodontal disease status was also associated with this response. This prompted the hypothesis that PC is an important oral antigen associated with organisms in the periodontal flora and that anti-PC antibody is elevated as a consequence of periodontal disease. Subjects in various periodontal disease diagnostic categories in which attachment loss is exhibited were tested for anti-PC in serum. Those with adult periodontitis, localized juvenile periodontitis, generalized early-onset periodontitis, and gingival recession all had similar levels of anti-PC IgG2 serum antibody which were significantly greater than in the group of subjects with no attachment loss. Analysis of plaque samples from subgingival and supragingival sites in all diseases categories for reactivity with the anti-PC specific monoclonal antibody TEPC-15 revealed that a substantial proportion of the bacteria in dental plaque (30 to 40%) bear PC antigen; this antigen was not restricted to morphotypes resembling only cocci but was also present on rods and branched filamentous organisms. We found that S. mitis, S. oralis, and S. sanguis, as well as oral actinomycetes, including A. viscosus, A. odontolyticus, and A. israelii, incorporated substantial amounts of [(3)H]choline from culture media. Further analysis of antigens derived from these organisms by Western blot indicated that S. oralis, S. sanguis, A. viscosus, A. odontolyticus, and A. israelii contained TEPC-15-reactive antigens. The data show that many commonly occurring bacterial species found in dental plaque contain PC antigen and that immunization with plaque-derived PC antigens as a consequence of inflammation and periodontal attachment loss may influence systemic anti-PC antibody concentrations. (+info)
A clinical comparison of the efficacy and efficiency of two professional prophylaxis procedures in orthodontic patients.
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This study compared the efficacy and efficiency of two professional prophylaxis procedures in orthodontic patients performing different oral hygiene regimens: the air powder polishing system (APP), and the rubber cup and pumice (RCP) technique. Sixty-two patients were divided into two groups: group I included 40 subjects who did not use any chlorhexidine mouthwash and group II comprised 22 subjects who regularly rinsed with a chlorhexidine mouthwash (at a 0.12 per cent concentration) and showed increased tooth staining. Using a split-mouth experimental design, the buccal and lingual tooth surfaces were cleaned in half of the mouth by the APP and in the opposite half by the RCP technique. Tooth surfaces were scored before (PRE) and after (POST) the experimental procedures for the plaque index (PI), and for the presence of tooth staining. In addition, the treatment time required by each procedure was recorded. In test group I, significant reductions in the PI after APP and RCP were observed. Likewise, in test group II, both procedures significantly reduced the baseline PI values. In both experimental groups, the percentage of stained sites significantly decreased after APP and RCP, but in test group II, APP seemed to be more effective than RCP. In addition, APP required significantly less time than RCP to remove dental plaque and staining. These data show that both professional prophylaxis procedures are effective in orthodontic patients, with APP being the most time-efficient technique and the most effective method for removal of tooth staining. (+info)
Improved multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis.
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Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and four P. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100 B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species. A. actinomycetemcomitans, B. forsythus, and P. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays. (+info)