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

我们从临床标本中获得的难治性根尖周炎，根管充填材料过度填充，根尖牙碎片进行形态学检查。电镜观察发现9例根管充填材料根外区及根尖周表面有细菌生物膜覆盖，并有糖萼样结构。在根管充填材料的根外部位的生物膜中，丝状、长杆状和螺旋形细菌占优势，并且在一些部位也检测到从糖萼样结构迁移的增殖细胞。接下来，我们使用改良的Robbins装置（MRD）制备牙龈卟啉单胞菌生物膜，并将其与增殖细胞生物膜进行比较，使用基于由辅酶A酶-β-内酰胺酶相互作用产生的发光的ATP测定。细胞内ATP浓度的增加速率， ...更多信息 对数生长期牙龈卟啉单胞菌的生长速率为13.88 nmol/h，而14 d后成熟生物膜的生长速率为5.05 × 10 ~ 5 <-2>pmol/h，表明牙龈卟啉单胞菌在成熟生物膜中的生长速率约为其对数生长期的1/3.0 × 10 ~ 5。为了研究牙龈卟啉单胞菌生物膜的抗菌药物敏感性，我们采用ATP法检测了氯己定、盐酸米诺环素和甲硝唑对牙龈卟啉单胞菌生物膜的影响。用氯己定孵育一天后，ATP浓度降至1/1000，此后ATP浓度逐渐降低。与此相反，与盐酸米诺环素和甲硝唑孵育7天后，ATP浓度约为对照的一半。这些结果表明盐酸米诺环素和甲硝唑对牙龈卟啉单胞菌形成的生物膜没有显著的杀菌作用。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