Analysis of bacterial biofilms as a virulence factor for refractory periapical periodontitis

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We morphologically examined the clinical specimens obtained from extracted teeth with refractory periapical periodontitis, canal filling materials excessively filled, and apical tooth fragment. In 9 of 11 samples, bacterial biofilms covered with glycocalyx-like structure were detected at the extraradicular area of canal filling materials and periapical root surface in electron microscopic observation. Filaments, long rods and spirohete-shaped bacteria were predominant in the biofilms at extraradicular sites of canal filling materials and planktonic cells emigrated from the glycocalyx-like structure, were also detected in several sites.Next, we generated the Porphyromonas gingivalis biofilms by use of modified Robbins device (MRD), and we investigated the biofilms morphologically as well as biological activity compared to their planktonic counterparts, using ATP assay based on luminescence generated by luciferase-luciferin interaction. The rate of increase of intracellular ATP concentra … More tion of the planktonic bacteria at log phase was 13.88 nmol/h, while that of mature biofilms after 14 days were 5.05 X 10^<-2> pmol/h, and these results indicated that growth rate of P.gingivalis in mature biofilms were about 1/3.0 X 10^5 of their planktonic counterparts. Formation of biofilms was observed 7 days after circulation in MRD and there was co-relation with the morphological changes of biofilms and increase of intracellular ATP concentration.To investigate antimicrobial susceptibility of P.gingivalis biofilms, we examined the effects of antimicrobial agents such as chlorhexidine, minocycline-hydrochloride and metronidazole against P.gingivalis biofilms, using ATP assay. ATP concentration was decreased to 1/1000 after incubation with chlorhexidine for a day, and thereafter ATP concentration were gradually decreased. In contrast, ATP concentration after 7 days incubation with minocycline-hydrochloride and metronidazole, were about a half of that of control. These results indicate that minocycline-hydrochloride and metronidazole have no significant bactericidal effects against P.gingivalis formed biofilms. Less