ABSTRACT Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated. Fusobacterium nucleatum (14 of 14), Porphyromonas gingivalis (12 of 14), and Tannellera forsythensis (8 of 14) were frequently detected. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms, P. gingivalis was immunohistochemically detected in all parts of the extraradicular biofilms. Positive reactions to anti- F. nucleatum and anti- T. forsythensis sera were found at specific portions of the biofilm. These findings suggested that P. gingivalis , T. forsythensis , and F. nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis.

ABSTRACT Porphyromonas gingivalis , an oral bacterium, might play a role in the pathogenesis or progression of adult periodontitis. In this study, we isolated from P. gingivalis a putative glycosyltransferase gene, designated gtfA , which had a consensus domain for glycosyltransferase in its N terminus. GtfA consisted of 248 amino acids and its predicted molecular mass was 28 kDa; however, as the molecular mass of endogenous GtfA protein was around 40 kDa, this suggested that GtfA had undergone some posttranslational modifications. To reveal the role of the gtfA gene in P. gingivalis , we established gtfA -deficient strains by allelic replacement. Morphologically, gtfA -deficient P. gingivalis lacked mature fimbriae. gtfA -deficient P. gingivalis also showed a very low ability for autoaggregation, and its ability to attach to epithelial cells was severely impaired. Thus, the results indicate that the gtfA gene is required for P. gingivalis autoaggregation as well as attachment to epithelial cells. These results suggest that GtfA might have an important role in the pathogenicity of P. gingivalis by regulating adhesion.

The participation of bacterial biofilms in the over-filled gutta-percha points associated with refractory periapical periodontitis has recently been reported. This study investigated the initial biofilm-forming ability of root canal isolates (Enterococcus faecalis, Streptococcus sanguis, Strep. intermedius, Strep. pyogenes, Staphylococcus aureus, Fusobacterium nucleatum, Propionibacterium acnes, Porphyromonas gingivalis and Prevotella intermedia) on gutta-percha points in vitro. Each bacterial strain was suspended in 100% cell culture medium or in culture medium containing 4.5, 45 or 90% (vol/vol) serum. The bacterial suspensions were then co-incubated anaerobically with gutta-percha points for 7 d. The gutta-percha points were processed for scanning electron microscopic observation and examined for biofilm presence and thickness. E. faecalis, Strep. sanguis, Strep. intermedius, Strep. pyogenes and Staph. aureus biofilms were generated on the surfaces of the specimens incubated in culture medium supplemented with 45 or 90% (vol/vol) serum. The E. faecalis and Strep. sanguis biofilms were significantly thicker than those of Strep. intermedius, Strep. pyogenes and Staph. aureus. No biofilms were detected on the specimens incubated with F. nucleatum, Prop. acnes, Porph. gingivalis and Prev. intermedia. These findings suggest that Gram-positive facultative anaerobes have the ability to colonize and form extracellular matrices on gutta-percha points, while serum plays a crucial role in biofilm formation.