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

我们对难治性根尖周炎、根管填充物过度填充和根尖牙碎片拔牙的临床标本进行了形态学检查。电镜观察11个样品中有9个在根管填充物根外区和根尖周根表面检测到覆盖糖萼样结构的细菌生物膜。管状填充物根外部位的生物膜中以丝状、长棒状和螺旋体状细菌为主，部分部位也有糖萼样结构迁移的浮游细胞。接下来，我们利用改良的罗宾斯装置（MRD）制备了牙龈卟啉单胞菌生物膜，并利用基于荧光素-荧光素相互作用产生的发光的ATP测定，研究了生物膜的形态和生物活性。细胞内ATP浓度的增加速率，在对数期浮游细菌的生长速率为13.88 nmol/h，而成熟生物膜在14天后的生长速率为5.05 × 10^<-2> pmol/h，表明成熟生物膜中牙龈单胞菌的生长速率约为浮游生物的1/3.0 × 10^5。MRD循环7天后观察到生物膜的形成，这与生物膜的形态变化和细胞内ATP浓度的增加有关。为了研究牙龈假单胞菌生物膜的抗菌敏感性，我们采用ATP法研究了氯己定、盐酸米诺环素和甲硝唑等抗菌药物对牙龈假单胞菌生物膜的抗菌作用。氯己定孵育1 d后，ATP浓度降至1/1000，此后ATP浓度逐渐降低。与盐酸米诺环素和甲硝唑孵育7 d后，ATP浓度约为对照组的一半。结果表明，盐酸二甲胺四环素和甲硝唑对牙龈假单胞菌形成的生物膜没有明显的杀菌作用。少

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