Aggregation of human platelets by gingipain-R from Porphyromonas gingivalis cells and membrane vesicles.

The hypothesis that there is an association between periodontitis and cardiovascular disease suggests new lines of research on the mechanism whereby oral bacteria might exert systemic effects. This study was conducted to ascertain and quantitate the effect of Porphyromonas gingivalis on human platelets in vitro. A second related objective was to purify and identify the aggregating vector. Aggregation was measured by platelet turbidometry and gingipain-R was purified from P. gingivalis membrane vesicles by Sepharose 2B and hydroxyapatite chromatography. The in vitro aggregation of platelets requires that at least 1.0 x 10(4) cells be stirred with 1.35 x 10(8) platelets. The specific activity is substantially increased in the membrane vesicles that are shed by this bacterium. Aggregability was due to gingipain-R activity, a potent cysteine protease that was found to be highly concentrated in the membrane vesicle fraction. The enzyme was purified 18-fold in high yield from the membrane vesicles, and consists of two noncovalently linked proteins that migrate at 49 and 44 kDa on SDS-PAGE. Aggregation of platelets by gingipain-R was shown to be dose-dependent, and inhibited by leupeptin and arginine, but not by anti-thrombin III. This is the first report enumerating the specific number of cells and lowest concentration of membrane vesicles necessary to evoke a full human platelet response, and the first report to assign this activity to gingipain-R.

Effect of an essential oil-containing antiseptic mouthrinse on induction of platelet aggregation by oral bacteria in vitro.

BACKGROUND: With an increasing body of data suggesting an association between periodontitis and cardiovascular disease, studies have been conducted to elucidate potential mechanisms by which oral bacteria might exert systemic effects. 2 oral bacteria, Streptococcus sanguis and Porphyromonas gingivalis, have been shown to induce platelet aggregation in vitro. This study was conducted to determine the effect of treatment with an essential oil mouthrinse (Listerine Antiseptic) on the platelet-aggregating activity of these organisms. METHOD: Bacteria were grown under standard culture conditions. S. sanguis ATCC strain 10556 was exposed for 3 min to the essential oil mouthrinse at either full strength or a 1:1 dilution, while P. gingivalis FDC strain 381 was exposed to the essential oil mouthrinse at a 1:10 dilution. Positive control cells were treated with Hanks balanced salt solution (HBSS). Aggregation was measured using a recording platelet aggregometer. The assay of each organism in its respective mouthrinse dilution(s) or HBSS was repeated 5 times. RESULTS: In all cases, the HBSS-treated organisms induced platelet aggregation, with mean(+/-S.E.) lag times of 12.30 (+/-1.36) min and 11.36 (+/-0.58) min for P. gingivalis and S. sanguis, respectively. In contrast, treatment with the essential oil mouthrinse completely inhibited the platelet aggregating activity of P. gingivalis and of S. sanguis exposed to the 1:1 mouthrinse dilution in all assays; the aggregating activity of S. sanguis treated with full-strength mouthrinse was completely inhibited in 4 of 5 assays, and inhibited by 75% in the 5th, for a mean inhibition of 95 +/- 1.5%. CONCLUSION: This study provides additional evidence that the essential oil mouthrinse can interfere with bacterial cell surface-associated activities which may have clinical relevance.

Campylobacter rectus in human periodontitis.

Campylobacter rectus (formerly Wolinella recta) in periodontitis lesions was studied relative to age and sex distribution, relationship to disease-active periodontitis, response to periodontal debridement and in vitro antimicrobial susceptibility. Subgingival C. rectus was collected with paper points, transported in VMGA III and plated onto nonselective enriched brucella blood agar and Hammond's selective medium for C. rectus, both incubated anaerobically. C. rectus was recovered from 80% of 1654 periodontitis patients. Although the organism showed similar age and sex occurrence, its proportional recovery in culture-positive adults was inversely related to increasing age (r = 0.999, P < 0.001). The organism was positively associated (summary odds ratio = 2.95) with disease activity in a 24-month longitudinal study of 93 adult periodontitis patients on maintenance therapy. C. rectus decreased from 8.2% to 0.7% following local periodontal debridement of 20 culture-positive adult periodontitis patients. The organism exhibited high in vitro susceptibility to therapeutic levels of tetracycline hydrochloride, metronidazole, penicillin G and ciprofloxacin. These findings further delineate the epidemiology and potential pathogenic role of C. rectus in human periodontitis.

Enterococci in human periodontitis.

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

Peptostreptococcus micros in human periodontitis.

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

Subgingival microflora of advanced periodontitis in the Dominican Republic.

A study of the predominant subgingival microflora was carried out in 24 periodontitis patients, 18 to 60 years of age, in Santo Domingo, Dominican Republic. Paper point sampling, transport in VMGA III, and conventional microbiological techniques were utilized. Direct microscopic examination revealed that cocci and nonmotile organisms made up 85% of the total organisms and spirochetes as little as 3%. Nonselective culturing showed Gram-negative organisms to constitute 53% of total isolates. Fusobacterium nucleatum averaged 15%, black-pigmented anaerobes 7%, and Peptostreptococcus micros 10% of the cultivable microflora. Enteric rods and acinetobacter species were recovered from 16 patients and comprised 23% of the cultivable flora. Enterobacter cloacae occurred in 8 patients, Klebsiella oxytoca in 3 patients, and 7 other species in 10 patients. Parallel studies have found a significantly lower prevalence of enteric rods in advanced periodontitis patients in the USA. In conclusion, fewer spirochetes and markedly more enteric rods seem to inhabit adult periodontitis lesions in Santo Domingo patients compared to those in USA. High levels of subgingival enteric rods in periodontitis patients in Santo Domingo may have important prophylactic and therapeutic implications.

Clinical and microbiological findings on newly inserted hydroxyapatite-coated and pure titanium human dental implants.

The clinical and microbiologic features of 30 hydroxyapatite-coated root-form endosseous dental implants (Tri-Stage) were compared to 10 similar pure titanium implants without hydroxyapatite coatings. In 7 of 9 partially edentulous patients studied, pure titanium fixtures were placed adjacent to hydroxyapatite-coated implants. Implants in the maxilla were submerged beneath mucosal tissues after implant placement for a minimum of 6 months, and in the mandible for at least 4 months. All patients were prescribed short-term beta-lactam antibiotic therapy after fixture placement, and 8 of 9 used chlorhexidine mouthrinses after fixture exposure. Clinical and microbiological examination was carried out 7-10 months after fixed prosthetic loading of the implants. Clinical measurements included the gingival index, plaque index, bleeding on probing and peri-implant probing depths determined with the Florida Probe system. Subgingival microbial samples were collected with paper points and transported in VMGA III. Specimens were examined by direct phase-contrast microscopy and were plated onto nonselective and selective culture media for anaerobic and aerobic incubation. No significant mean clinical or microbiological differences were found between the implant types, although one hydroxyapatite-coated implant exhibited deep probing depths, bleeding on probing and marked radiographic crestal bone loss. Streptococcus sanguis and Streptococcus mitis were the most predominant organisms recovered from clinically stable implants, whereas high proportions of Fusobacterium species and Peptostreptococcus prevotii were isolated from the ailing hydroxyapatite-coated implant. One or more implants in 8 of the study subjects yielded enteric rods, pseudomonads, enterococci or staphylococci. The prognosis of implants with varying early microbiotas needs to be established in longitudinal studies.

Microbial aspects of failing osseointegrated dental implants in humans.

The subgingival microflora of 18 failing implants were examined for pathogenic periodontal microorganisms. Peptostreptococcus micros was recovered from 6 failing implants, Wolinella recta from 6, Fusobacterium species from 5, Candida albicans from 5, and Bacteroides intermedius from 4. Enteric rods or pseudomonads constituted a significant part of the microflora in 5 failing implants. Actinobacillus actinomycetemcomitans, nonpigmented Bacteroides species, Capnocytophage species, and staphylococci were detected in a few implant failures. The present study showed that a complex microflora comprising oral as well as primarily non oral organisms, and bacteria as well as yeasts, can be associated with failing implants. This great diversity in microbial composition and antimicrobial susceptibility among "peri-implantitis" isolates suggest that antimicrobial therapies for implant failures should not be implemented without a prior comprehensive microbiological analysis.

Microbiological study of HIV-related periodontitis.

The subgingival microbiota in 14 persons with HIV-periodontitis was examined. Subgingival plaque samples were collected with paper points, transported in VMGA III, and plated on anaerobic enriched brucella blood agar and various selective media. HIV-periodontitis sites revealed Actinobacillus actinomycetemcomitans, Wolinella recta, Peptostreptococcus micros, and Bacteroides intermedius, each averaging 7% to 16% of the cultivable subgingival flora in positive patients. High levels of spirochetes also were detected in diseased sites with phase-contrast microscopy. Low levels of Candida albicans or enteric Gram-negative rods were recovered in the subgingival flora in 7 HIV-periodontitis patients or Bacteroides fragilis, Fusobacterium necrophorum, Fusobacterium varium, and Eubacterium aerofaciens were recovered in 8 patients. These findings suggest that the major components of the subgingival microbial flora in HIV-periodontitis are similar to those associated with adult periodontitis in systemically healthy persons. However, HIV-periodontitis lesions also may contain organisms which are rarely found in common types of periodontitis. The etiological significance of specific periodontal organisms in HIV-periodontitis awaits further longitudinal study.

Age and sex relationships of superinfecting microorganisms in periodontitis patients.

The occurrence by age and sex of subgingival enteric rods and pseudomonads, yeasts, and staphylococci was studied in 3075 "refractory" periodontitis patients referred for microbiological analysis. Each subject contributed a pooled subgingival sample obtained from 3 deep periodontal pockets with paper points. Selective and nonselective media and commercial identification kit systems were used for microbial isolation and speciation. Females constituted about 60% of the study subjects, and almost one-third of all patients were in their forties. Females (47.3%) showed a higher prevalence of the study organisms than males (43.9%). Older females (15.9%) and males (15.3%) revealed significantly higher prevalences of enteric rods and pseudomonads than younger individuals (10.9%), and older infected females yielded significantly higher viable counts than younger infected females. The sexes demonstrated a similar prevalence of staphylococci (about 28%), but younger infected females and males showed significantly higher viable counts than older infected individuals. No sex or age relationships were found for yeasts (about 14% of individuals infected). The high level of subgingival enteric rods and pseudomonads in some individuals may be important in the pathogenesis of geriatric and other forms of periodontitis and may have therapeutic implications.

Actinobacillus actinomycetemcomitans and Bacteroides intermedius in human periodontitis: age relationship and mutual association.

This study examined age relationships and mutual interrelationships between cultivable Actinobacillus actinomycetemcomitans and Bacteroides intermedius in 1624 periodontitis patients, 15 to 89 years of age. Each subject contributed a pooled subgingival sample, obtained from 3 deep periodontal pockets with paper points. A. actinomycetemcomitans occurred with higher prevalence (74%) and mean recovery (7% in culture-positive patients) in patients less than 25 years old than in adult and geriatric patients (prevalence about 31%; mean recovery about 1%). The organism was detected in 85% of localized juvenile periodontitis patients. B. intermedius was recovered from 45% of the study subjects, averaged about 7% of total isolates in positive patients, and showed no predilection for any age group. As determined from predicted and observed values for A. actinomycetemcomitans and B. intermedius, occurring alone and in combination, no synergistic or antagonistic relationships between the organisms could be delineated with respect to subgingival colonization. The therapeutic implication of these findings is discussed.

In vitro antimicrobial sensitivity of enteric rods and pseudomonads from advanced adult periodontitis.

The prevalence and in vitro antimicrobial sensitivity of isolates of enteric rods and pseudomonads was examined in 844 adult periodontitis patients. These organisms were recovered from 13.5% of the study subjects. Ciprofloxacin exhibited the highest inhibitory activity of the 14 oral antimicrobial agents tested. Beta-lactam antibiotics were largely ineffective, and tetracycline failed to inhibit most Pseudomonas species. In vitro sensitivity data suggest that a subgingival microbiota comprising mainly streptococci would result from therapy that combined ciprofloxacin and metronidazole. Since streptococci may inhibit the growth of several putative periodontal pathogens, populations of "beneficial" streptococci in the periodontal pocket might constitute a very attractive therapeutic outcome. Controlled clinical studies are needed to clarify the possible role of ciprofloxacin in the treatment of destructive periodontitis.

Prevalence and antimicrobial susceptibility of Enterobacteriaceae, Pseudomonadaceae and Acinetobacter in human periodontitis.

This study examined the occurrence of non-oral gram-negative facultatively anaerobic rods in advanced adult periodontitis. Speciation and in vitro antimicrobial susceptibility testing was performed using the MicroScan micromethod system. A total of 42 taxa of Enterobacteriaceae, Pseudomonadaceae and Acinetobacter were isolated from 427 of 3,050 (14.0%) patients. In 159 (5.2%) patients, these organisms comprised more than 5% of the cultivable subgingival microflora. Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Klebsiella oxytoca and Enterobacter agglomerans accounted for more than 50% of all strains isolated. Virtually all study strains demonstrated high in vitro susceptibility to ciprofloxacin, but exhibited variable susceptibility patterns to 18 other antimicrobial agents tested. In 3 "refractory" periodontitis patients heavily infected with enteric rods, systemic ciprofloxacin therapy (500 mg BiD for 10 days) led to resolution of the subgingival infections and improved clinical periodontal status. The present findings indicate that 5% of severe periodontitis lesions may harbor high levels of non-oral, gram-negative, facultatively anaerobic rods. Systemic ciprofloxacin appears to be capable of eradicating these potential pathogens from deep periodontal pockets.

Staphylococci in human periodontal diseases.

The occurrence of subgingival staphylococci was determined in 506 individuals with advanced adult periodontitis, 108 with early-onset periodontitis, 13 with localized juvenile periodontitis, 18 with gingivitis, and 13 with 20 failing osseointegrated titanium dental implants. Subgingival samples were collected with paper points and transported in VMGA III. The bacterial samples were plated on Staphylococcus 110 medium which was incubated in 10% CO2, and on enriched brucella blood agar, which was incubated anaerobically. Staphylococcal isolates from 94 adult periodontitis subjects were speciated using the API STAPH Trac micromethod kit system and the Bacto Staph latex agglutination test for coagulase activity. Staphylococcus epidermidis comprised 45.8% and Staphylococcus aureus 22.3% of total staphylococcal isolates. At 1 microgram/ml, in vitro resistance by staphylococci was found to tetracycline (14.4% of isolates), penicillin (4.9%), erythromycin (12.1%), and metronidazole (31.9%). Subgingival staphylococci were isolated from approximately 50% of gingivitis and periodontitis patients. No statistically significant differences were found between these patient groups in the prevalence or mean proportions of staphylococci recovered. "Periimplantitis" lesions exhibited significantly higher proportions of staphylococci (15.1%) than gingivitis (0.06%) or periodontitis (1.2%) lesions. Staphylococci may play a role in some failing osseointegrated dental implants.