A 16S rRNA-based polymerase chain reaction (PCR) detection method was used to determine the prevalence of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia. Prevotella nigrescens and Treponema denticola in subgingival specimens of 50 advanced periodontitis, 50 adult gingivitis and 50 pediatric gingivitis subjects. The optimal PCR conditions were determined for each study species. Agarose gel electrophoresis of PCR products from each study species revealed a single band of the predicted size. Restriction enzyme digestion of amplicons confirmed the specificity of the amplification. PCR detection limits were in the range of 25-100 cells. No cross-reactivity with other oral micro-organisms or nonspecific amplification was observed. The prevalence by PCR in advanced periodontitis, adult gingivitis and pediatric gingivitis subjects was 30%, 14% and 14% for A. actinomycetemcomitans, 86%, 18% and 8% for B. forsythus, 74%, 52% and 78% for C. rectus, 80%, 70% and 66% for E. corrodens, 70%, 10% and 14% for P. gingivalis, 58%, 12% and 18% for P. intermedia, 52%, 20% and 22% for P. nigrescens, and 54%, 16% and 16% for T. denticola, respectively. The prevalence was higher in the advanced periodontitis group than in both adult gingivitis and pediatric gingivitis for A. actinomycetemcomitans, B. forsythus, P. gingivalis, P. intermedia, P. nigrescens and T. denticola at P < 0.01, and for E. corrodens at P < 0.05. The prevalence of C. rectus was significantly higher in the advanced periodontitis group than in the adult gingivitis group at P < 0.01. Matching results between PCR and culture occurred in 28% (B. forsythus) to 71% (A. actinomycetemcomitans) of the samples; the major discrepancy occurred in the PCR-positive/culture-negative category. Matching results between PCR and DNA probe methods were found in 84% of the subjects (B. forsythus) and 70% (P. gingivalis). Odds ratio analysis revealed statistically significant positive associations between 17 of the 28 possible combinations (P < 0.01). This study demonstrated the utility of a 16S rRNA-based PCR detection method for identifying important subgingival microorganisms. The results indicated a strong association between the study species and periodontitis. Several previously unreported symbiotic relationships were found between the 8 species tested.

This volume of Periodontology 2000 represents the 25th anniversary of the Journal, and uses the occasion to assess important advancements in periodontology over the past quarter-century as well as the hurdles that remain. Periodontitis is defined by pathologic loss of the periodontal ligament and alveolar bone. The disease involves complex dynamic interactions among active herpesviruses, specific bacterial pathogens and destructive immune responses. Periodontal diagnostics is currently based on clinical rather than etiologic criteria, and provides limited therapeutic guidance. Periodontal causative treatment consists of scaling, antiseptic rinses and occasionally systemic antibiotics, and surgical intervention has been de-emphasized, except perhaps for the most advanced types of periodontitis. Plastic surgical therapy includes soft-tissue grafting to cover exposed root surfaces and bone grafting to provide support for implants. Dental implants are used to replace severely diseased or missing teeth, but implant overuse is of concern. The utility of laser treatment for periodontitis remains unresolved. Host modulation and risk-factor modification therapies may benefit select patient groups. Patient self-care is a critical part of periodontal health care, and twice-weekly oral rinsing with 0.10-0.25% sodium hypochlorite constitutes a valuable adjunct to conventional anti-plaque and anti-gingivitis treatments. A link between periodontal herpesviruses and systemic diseases is a strong biological plausibility. In summary, research during the past 25 years has significantly changed our concepts of periodontitis pathobiology and has produced more-effective and less-costly therapeutic options.

This article describes the subgingival microflora of the healthy periodontium, gingivitis, advanced adult periodontitis, and juvenile periodontitis. A total of seven to nine subjects were examined in each of the four periodontal clinical entities listed. The individual bacteriological samples included material from the base of a single periodontal pocket. The sampling, the treatment of the samples, and the bacteriological cultivations were carried out using continuous anaerobic techniques. Briefly, the healthy gingival sulcus harbored a scant microflora dominated by Gram‐positive organisms (85%), usually Streptococcus and facultative Actinomyces species. The development of gingivitis was accompanied by a marked increase in the total number of Gram‐negative organisms. Fusobacterium nucleatum, Bacteroides melaninogenicus ss. intermedius, Haemophilus species, and other Gram‐negative organisms comprised about 45% of the total gingivitis isolates. Streptococcus and facultative and anaerobic Actinomyces species constituted the majority of the Gram‐positive gingivitis isolates. The micro‐flora of advanced adult periodontitis was comprised mainly of Gram‐negative anaerobic rods (about 75%), B. melaninogenicus ss. asaccharolyticus and F. nucleatum being the most predominant isolates. The deep pocket microflora in juvenile periodontitis was also made up mainly of Gram‐negative organisms (about 65%), but was of a nature different from that of adult periodontitis, being predominated by isolates of Bacteroides species and other organisms of unknown species. The present article also concerns factors of importance for the colonization of Gram‐negative anaerobic rods in the oral cavity and periodontal pockets. In vitro experiments showed that cells of B. melaninogenicus ss. asaccharolyticus and other Gram‐negative organisms attached in high numbers to epithelial cells, hydrosyapatite (HA) surface, and Gram‐positive bacteria when suspended in phosphate‐buffered saline; however, the bacterial attachment to epithelial cells and HA was strongly inhibited in the presence of human saliva and serum. In contrast, saliva and serum had little effect upon the attachment of Gram‐negative bacteria to Gram‐positive bacterial cells. These findings agreed well with data from an in vivo study, in which streptomycin‐labeled cells of B. melaninogenicus ss. asaccharolyticus were introduced into the mouth of two volunteers. A significantly higher number of B. melaninogenicus cells was recovered from dental plaque than from the other oral surfaces studied. The present series of studies has pointed to certain Gram‐negative organisms as potential pathogens in rapidly progressing periodontal lesions. The available data on oral microbial ecology suggest that the presence of dental plaque containing Gram‐positive organisms may be essential for the attachment and colonization of several Gram‐negative species after their initial introduction into the mouth and the periodontal pocket area. The clinical relevance of these findings is discussed.

A selective medium, TSBV (tryptic soy-serum-bacitracin-vancomycin) agar, was developed for the isolation of Actinobacillus actinomycetemcomitans. TSBV agar contained (per liter) 40 g of tryptic soy agar, 1 g of yeast extract, 100 ml of horse serum, 75 mg of bacitracin, and 5 mg of vancomycin. The TSBV medium suppressed most oral species and permitted significantly higher recovery of A. actinomycetemcomitans than nonselective blood agar medium. The distinct colonial morphology and positive catalase reaction of A. actinomycetemcomitans easily distinguished this bacterium from Haemophilus aphrophilus, Capnocytophaga species, and a few other contaminating organisms. With the TSBV medium, even modestly equipped laboratories will be able to isolate and identify A. actinomycetemcomitans from clinical specimens.

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