Ishiguro Kazuko, Washio Jumpei, Sasaki Keiichi, Takahashi Nobuhiro
Division of Oral Ecology and Biochemistry, Tohoku University Graduate School of Dentistry, Sendai, Japan; Division of Advanced Prosthetic Dentistry, Tohoku University Graduate School of Dentistry, Sendai, Japan.
Division of Oral Ecology and Biochemistry, Tohoku University Graduate School of Dentistry, Sendai, Japan.
J Microbiol Methods. 2015 Aug;115:22-6. doi: 10.1016/j.mimet.2015.05.015. Epub 2015 May 15.
Bacterial metabolic activity is associated with the onset and progression mechanisms of oral biofilm-mediated disease; however, at present no method to monitor bacterial metabolism exists, especially for periodontopathic bacteria. Therefore, we aimed to establish a novel method for monitoring the metabolic activity of periodontopathic bacteria, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Fusobacterium nucleatum (Fn), as well as Streptococcus mutans (Sm) for comparison. The method is based on the dye resazurin, which is converted to the fluorescent molecule resorufin by reducing molecules derived from bacterial metabolism. Additionally, the effects of antimicrobial substances on bacterial metabolic activity were evaluated using this method. When bacterial suspensions were incubated with tryptone, glutamate, aspartate or glucose in the presence of resazurin, the fluorescence intensity increased over time by these bacterial metabolic reactions, indicating that this method can be used to monitor the metabolic activity of periodontopathic bacteria. Chlorhexidine showed the 50% inhibitory concentration (IC50) of 15-49 μg/ml for tryptone metabolism by Pg, Pi, and Fn, and 7.1-18 μg/ml for glucose metabolism by Pi and Sm. The IC50s for cetylpyridinium chloride and sodium dodecyl sulfate were 0.8-2.1 and 28-44 μg/ml, respectively for all bacteria examined. Fluoride had no effect except the IC50 of 640 μg/ml for Sm, while minocycline hydrochloride had no effect on any of the bacteria. The present study established the method for real-time monitoring of the metabolic activity of periodontopathic bacteria, and the method might be useful for evaluating the effects of antimicrobial substances on the bacterial metabolic activity.
细菌代谢活性与口腔生物膜介导疾病的发生和进展机制相关；然而，目前尚无监测细菌代谢的方法，尤其是针对牙周病原菌。因此，我们旨在建立一种新方法，用于监测牙周病原菌牙龈卟啉单胞菌（Pg）、中间普氏菌（Pi）、具核梭杆菌（Fn）以及变形链球菌（Sm，用于对比）的代谢活性。该方法基于刃天青染料，其通过细菌代谢产生的还原分子转化为荧光分子试卤灵。此外，使用该方法评估了抗菌物质对细菌代谢活性的影响。当细菌悬液在刃天青存在的情况下与胰蛋白胨、谷氨酸、天冬氨酸或葡萄糖一起孵育时，这些细菌代谢反应会使荧光强度随时间增加，表明该方法可用于监测牙周病原菌的代谢活性。洗必泰对Pg、Pi和Fn的胰蛋白胨代谢显示出50%抑制浓度（IC50）为15 - 49μg/ml，对Pi和Sm的葡萄糖代谢显示出7.1 - 18μg/ml。对于所有检测的细菌，十六烷基氯化吡啶和十二烷基硫酸钠的IC50分别为0.8 - 2.1μg/ml和28 - 44μg/ml。除了对Sm的IC50为640μg/ml外，氟化物无作用，而盐酸米诺环素对任何细菌均无作用。本研究建立了实时监测牙周病原菌代谢活性的方法，该方法可能有助于评估抗菌物质对细菌代谢活性的影响。
