Identification of periodontal disease-associated bacteria in the "plaque-free zone"

Background: Subgingival plaque bacteria live within a biofilm covered with glycocalyx, and little is known of the bacterial species associated with biofilm formation at the bottom of human periodontal pockets, the so-called "plaque-free zone" (PFZ). Methods: Seventy-seven extracted teeth from 56 patients with severe advanced adult periodontitis were examined. Porphyromonas gingivalis, Campylobacter rectus, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Treponema denticola, Prevotella nigrescens, and Actinomyces viscosus were examined by scanning immunoelectron microscopic techniques, using both secondary and back-scattered imaging, with rabbit antibodies specific for each bacteria. Results: Secondary electron images showed that rods, filaments, and spirochete-shaped bacteria formed small aggregates in the PFZ. Some of the bacteria were covered with an amorphous film-like structure. By back-scattered electron imaging, positive reactions with anti-P. gingivalis were found in 8 of 13 samples examined, and film-like structures coated several cells of 6 positive samples examined. Labeled cells with anti-C rectus, anti-T. denticola and anti-P. nigrescens were detected in 3 of 11, 5 of 10, and 1 of 8 samples examined. A. viscosus were found in 6 of 11 of the samples. A. viscosus tended to overlay the amorphous capsula and aggregate. F. nucleatum and A. actinomycetemcomitans were not detected in any samples examined. Conclusions: These findings indicated that P. gingivalis, C. rectus, T. denticola, P. nigrescens, and A. viscosus were present in the PFZ, and that some specified bacteria were possibly related to plaque-biofilm formation of subgingival plaque.