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 15-49μg/ml for tryptone metabolism by Pg, Pi, and Fn, and 7.1-18μg/ml for glucose metabolism by Pi and Sm. The IC50s for cetylpyridinium chloride and sodium dodecyl sulfate were 0.8-2.1 and 28-44μg/ml, respectively for all bacteria examined. Fluoride had no effect except the IC50 of 640μg/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.
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