The microbial colonization of expanded polytetrafluoroethylene membrane by putative periodontopathogens at 3 minutes of intraoral manipulation was determined in 42 patients with 42 mandibular posterior two‐ to three‐wall defects. Twenty patients exhibited no periodontal pockets of ≥ 5 mm, other than the study site, and low levels of pathogens (group A). Twenty‐two patients revealed multiple periodontal pockets of 5 mm or more and numerous pathogens (group B). Within the preceding 3 months of regenerative surgery, group A patients had received apically positioned flap surgery with osseous recontouring (except for the study site), and group B patients had been enrolled in a non‐surgical maintenance program. The subgingival microbiota was examined prior to regenerative therapy, and the membrane microbiota was examined at 3 minutes and at the time of removal at 6 weeks by culture, DNA probes, and phasecontrast microscopy. The mean initial defect depth was 7.4 mm for group A and 7.2 mm for group B. At 6 months, the difference in mean clinical attachment gain was statistically significant (P < 0.001; group A: 3.4 mm; group B: 1.4 mm). At 3 minutes, putative pathogens were detected in seven (16.7%) membranes in group B (group Binfected), and the associated sites gained only 0.6 mm in clinical attachment at 6 months. Clinical attachment gain was modeled as a linear function of the explanatory variables (r2 = 86%). The presence of Porphyromonas gingivalis detected by DNA probe at 3 minutes was associated with 1.5 mm less expected gain (P = 0.0002). Total microbial counts and the percentage of Peptostreptococcus micros and Capnocytophaga species at baseline, and of motile rods on the membrane surface facing the gingiva at 6 weeks, were statistically significant negative predictors of clinical attachment. For each week the membrane remained covered, an additional 0.5 mm gain could be expected (P = 0.002); and for every 10 sites that exhibited bleeding on probing, the clinical attachment gain was 0.6 mm less at the site of regeneration (P < 0.0001). The present results showed that putative pathogens may colonize membranes within 3 minutes of intraoral manipulation. The patient group treated with periodontal osseous surgery revealed the lowest levels of periodontal pathogens in the membranes and exhibited the most gain in clinical attachment. J Periodontol 1996;67:694–702.
The usefulness of a digoxigenin-labeled genomic DNA probe for the detection of subgingival Bacteroides forsythus was examined. In addition, the arbitrarily primed polymerase chain reaction (AP-PCR) was used to delineate the genetic diversity of B. forsythus periodontal isolates. The DNA probe detected 10(3) B. forsythus cells and yielded a strong signal at 10(4) cells. It reacted with B. forsythus ATCC 43037T and 44 clinical isolates and showed no detectable reactivity with 75 strains of 24 other oral microbial species. In comparison to culture, the DNA probe in a dot-blot method demonstrated a sensitivity of 88.8% and a specificity of 38.4% (accuracy, 72.5%). By colony-blotting on primary plates, a sensitivity of 98.1% and a specificity of 53.8% (accuracy, 82.5%) were obtained. B. forsythus was detected in 449 (73.1%) of 614 periodontitis patients. The occurrence of the organism was closely associated with Porphyromonas gingivalis, both species being present in 54.8% and absent in 22.2% of 270 study samples. AP-PCR identified 24 B. forsythus genotypes among 27 test strains. This study demonstrated the utility of a non-radioactive genomic probe for direct detection of B. forsythus in subgingival specimens. The species showed a considerable degree of genetic diversity. DNA analysis may help to determine the role of B. forsythus in periodontal disease and its mode of transmission among exposed individuals.
Clinical and microbiological features of periodontal healing in barrier membrane‐treated sites were determined in a randomized clinical trial. The study included 10 patients with advanced adult periodontitis and a minimum of one set of similar 2 to 3 wall intraosseous periodontal lesions with no furcation involvement. In each patient, one periodontal lesion was treated with a biodegradable membrane and a contralateral lesion with a nonresorbable barrier membrane. Within the preceding 3 months of regenerative therapy, all patients received full mouth osseous surgery except for the sites for regeneration, were instructed in oral hygiene, and were prescribed systemic ciprofloxacin and metronidazole (250 mg of each, TID, 8 days), starting 7 days before membrane placement. At baseline and at 6 months postsurgery, probing depth and clinical attachment level were assessed in each study site. The subgingival presence of suspected periodontal pathogens was determined by non‐selective and selective culture and by DNA probe analyses, and of human cytomegalovirus (HCMV) and Epstein‐Barr virus type 1 (EBV‐1) by a nested‐polymerase chain reaction detection method. At baseline, the barrier‐treated sites did not differ significantly in clinical and microbial parameters. Mean baseline probing depth was 7.8 ± 1.1 mm for bioabsorbable and 7.9 ± 1.3 mm for nonresorbable barrier‐treated sites. At 6 months, sites treated with bioabsorbable barrier revealed 4.6 ± 1.7 mm gain of clinical attachment (range: 1 to 7 mm) and sites treated with nonresorbable barrier 4.2 ± 2.0 mm (range: 1 to 8 mm). The 11 barrier‐treated sites that harbored 10% or less bacterial pathogens and were free of HCMV and EBV‐1 averaged significantly more clinical attachment gain than the 9 sites that yielded more than 10% bacterial pathogens and/or test viruses (5.6 mm versus 3.0 mm; P = 0.005). The present data suggest bioabsorbable and nonresorbable barriers provide similar clinical healing of 2 to 3 wall intraosseous periodontal lesions, emphasize the importance of controlling bacterial pathogens prior to and during periodontal healing, and point to the possible detrimental role of HCMV and EBV‐1 in periodontal repair. J Periodontol 1998;69:445–453.
Dialister pneumosintes is a nonfermentative, anaerobic, gram-negative rod that grows with small, circular, transparent, shiny, smooth colonies on blood agar. Even though D. pneumosinteshas been recovered from deep periodontal pockets, little is known about the relationship between the organism and destructive periodontal disease. This study describes a rapid PCR method to identify D. pneumosintes in periodontal samples. The PCR identification method detected as little as 10 pg of D. pneumosintes DNA or about 1 to 10 cells without nonspecific amplification of various periodontopathic bacteria. Twelve of 22 subgingival samples from adult periodontitis lesions yielded D. pneumosintes either by culture or by PCR identification. In culture-positive samples, D. pneumosintes averaged 3.9% (0.001 to 10.8%) of total isolates. Studies are needed to delineate virulence factors of D. pneumosintes pertinent to periodontal disease.
Anaerobic culture is employed routinely in the primary isolation of periodontal pathogenic bacteria. However, little or no data exist on the relative abilities of the Coy anaerobic chamber (Coy Laboratory Products, Grass Lake, Mich.), the GasPak (Becton Dickinson Microbiology Systems, Cockeysville, Md.), and the AnaeroPack (Mitsubishi Gas Chemical America, Inc., New York, N.Y.) systems to grow important periodontal species, including Porphyromonas gingivalis,Prevotella intermedia/nigrescens, Bacteroides forsythus, Eubacterium species,Campylobacter species, Fusobacterium species, and Peptostreptococcus micros. A total of 78 specimens from advanced periodontitis lesions were collected anaerobically, plated on enriched blood agar medium, and incubated at 35°C for 5 to 7 days in each anaerobic culture system. The three culture systems were equally efficient in isolating Porphyromonas gingivalis andPrevotella intermedia/nigrescens. The Coy anaerobic chamber yielded the highest proportional recoveries ofCampylobacter (P = 0.0001; nonparametric analysis of variance) and Eubacterium (P = 0.009). The Coy anaerobic chamber and the GasPak system demonstrated higher proportional recoveries of Bacteroides forsythus(P = 0.0006) and Peptostreptococcus micros(P = 0.0001) than the AnaeroPack system. The AnaeroPack system was most efficient in growingFusobacterium species (P = 0.0001). Overall, the Coy anaerobic chamber and the GasPak system showed the highest proportional recoveries of putative periodontal pathogens, but the recoveries by the various anaerobic test systems varied considerably from sample to sample.
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