Subgingival microbial associations in severe human periodontitis.

Human periodontitis is associated with a widely diverse and complex subgingival microbiota encompassing both gram-positive and gram-negative bacteria, facultative and anaerobic organisms, and possibly yeasts [1]. Symbiotic, antagonistic, and commensal interrelationships likely occur between the various microbial inhabitants of periodontal pockets and play an important role in determining the subgingival microbial composition of dental plaque and the clinical periodontal status. Previous cultural investigations of microbial interactions in human periodontitis were limited by the relatively small number of patients [2, 3] and periodontal organisms studied [4] and because periodontal site data were reported without accounting for subject-based factors [2]. The present study evaluated 13 cultivable periodontal taxa for positive and negative associations in mutual subject colonization of deep periodontal pockets in 1,255 adult patients with advanced periodontitis (710 females and 545 males; age range, 23-96 years; mean age, 52.4 years) who were treated at the graduate periodontal clinics of the University of Southern California School of Dentistry (Los Angeles) and private periodontal practices geographically distributed throughout the United States. Most of the study subjects had previously undergone various forms of periodontal therapy but had recurrent deepening of probing pocket depths. The patients selected were not medically compromised, had not received periodontal and/or antibiotic treatment within the preceding 3 months, and presented with at least three periodontal sites on separate teeth with probing pocket depths of at least 6 mm. Samples for microbial cultures were obtained, transported, and processed as previously described [4, 5]. In brief, following isolation and removal of supragingival deposits, three to five of the deepest periodontal pockets per patient were sampled, each with insertion of a single sterile fine paper point (Johnson & Johnson, Windsor, NJ). After placement for 10 seconds, the paper points were pooled into a vial containing 2.0 mL of anaerobically prepared and stored VMGA III transport medium [5] and six to eight glass beads 3 mm in diameter. On-campus samples were processed within 4 hours, and specimens from extramural centers were processed within 24-48 hours of sampling. VMGA III transport medium possesses a high-preservation capability for oral microorganisms during transit to the laboratory [5]. The sample vials were warmed to 35?C for 10 minutes before processing to liquefy the gelatin in the VMGA III transport medium. The sampled plaque organisms were then dispersed with a Vortex mixer (Scientific Industries, Bohemia, NY) at the maximal setting for 45 seconds, and serial 10-fold dilutions were carried out in VMG I anaerobic dispersion solution [5]. With use of a sterile bent glass rod, 0.1-mL aliquots of appropriate dilutions were plated onto prereduced enriched brucella blood agar-4.3%