title: Real-time monitoring of the metabolic activity of periodontopathic bacteria.
abstract: 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.
authors.affiliation: 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.
authors.forename: Kazuko
authors.initials: K
authors.lastname: Ishiguro
authors.affiliation: Division of Oral Ecology and Biochemistry, Tohoku University Graduate School of Dentistry, Sendai, Japan.
authors.forename: Jumpei
authors.initials: J
authors.lastname: Washio
authors.affiliation: Division of Advanced Prosthetic Dentistry, Tohoku University Graduate School of Dentistry, Sendai, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Sasaki
authors.affiliation: Division of Oral Ecology and Biochemistry, Tohoku University Graduate School of Dentistry, Sendai, Japan. Electronic address: nobu-t@dent.tohoku.ac.jp.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Takahashi
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001431:Bacteriological Techniques / Q000379:methods*; D016967:Fusobacterium nucleatum / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D010078:Oxazines / Q000378:metabolism; D010518:Periodontitis / Q000382:microbiology*; D016966:Porphyromonas gingivalis / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D018713:Prevotella intermedia / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013295:Streptococcus mutans / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; C014180:resorufin
keywords: Antimicrobial substance; Bacterial metabolism; Fluorescent dye; Resazurin