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 bi ofilm 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 advance d adult periodontitis were examined. Porphyromonas gingivalis, Campylobacte r rectus, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Tr eponema denticola, Prevotella nigrescens, and Actinomyces viscosus were exa mined by scanning immunoelectron microscopic techniques, using both seconda ry and back-scattered imaging, with rabbit antibodies specific for each bac teria. Results: Secondary electron images showed that rods, filaments, and spiroch ete-shaped bacteria formed small aggregates in the PFZ. Some of the bacteri a were covered with an amorphous film-like structure. By back-scattered ele ctron imaging, positive reactions with anti-P. gingivalis were found in 8 o f 13 samples examined, and film-like structures coated several cells of 6 p ositive samples examined. Labeled cells with anti-C rectus, anti-T. dentico la and anti-P. nigrescens were detected in 3 of 11, 5 of 10, and 1 of 8 sam ples examined. A. viscosus were found in 6 of 11 of the samples. A. viscosu s 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. den ticola, P. nigrescens, and A. viscosus were present in the PFZ, and that so me specified bacteria were possibly related to plaque-biofilm formation of subgingival plaque.