Online ISSN : 1347-5320
Print ISSN : 1345-9678
ISSN-L : 1345-9678
A Novel Method of Antibacterial Evaluation Based on the Inhibition of Hydrogen Sulfide Producing Activities of Salmonella
–Using Copper as a Model Antibacterial Agent–
Yutaka MidorikawaMasaaki NakaiKaoru MidorikawaMitsuo Niinomi
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JOURNALS RESTRICTED ACCESS Advance online publication

Article ID: M2016007


A novel method for detecting antimicrobial activity using an innate property of the Salmonella bacteria, namely, the ability of Salmonella to produce hydrogen sulfide (H2S) was developed in this study. The validity of the method was evaluated by comparing the antibacterial activity of copper to that of aluminum. Salmonella was inoculated over the entire surface of deoxycholate hydrogen sulfide lactose (DHL) agar plates that included Ammonium ferric citrate (C6H8FeN). Approximately 25 μL of cupric chloride (CuCl2, 1% weight ratio) solution or aluminum chloride (AlCl3, 1% weight ratio) solution was added to the center of the medium. The surface of the medium was covered with polyethylene terephthalate (PET) films to induce an anaerobic state. Salmonella was cultured under anaerobic conditions at 310 K (37℃) for 86.4 ksec (24 h). The antibacterial activity of copper was determined by observing the medium surface color change due to iron sulfide (FeS) formation, which was caused by the production of H2S by Salmonella; blackness indicated presence of newly formed FeS. A quantitative evaluation of copper's antimicrobial activity was performed using a gradient of CuCl2 concentrations; results were compared with those of the present standard method, Kirby-Bauer disk diffusion method on the Mueller Hinton medium. Finally, in order to evaluate the antibacterial activity of metals, Salmonella was inoculated on DHL agar plates. Subsequently, Japanese coins (1-yen, 5-yen, 10-yen, 50-yen, 100-yen, and 500-yen coins) were placed on the agar and cultured at 310 K for 86 ksec. Salmonella cultured in the presence of AlCl3 produces black color, while no blackening is observed with CuCl2, suggesting that copper possesses an antibacterial property against Salmonella. CuCl2 suppresses H2S production by Salmonella, as copper ions form a transparent circle or ellipse (new halo) around the point at which CuCl2 had has been plated. The size of the new halo increases in direct proportion to the concentration of CuCl2. The halo is no longer visible at 0.034 mg of CuCl2 in our method, while the halo disappears with 4.34 mg of CuCl2 in the Kirby-Bauer test. Therefore, the present method is 129 times more sensitive than the standard method, suggesting increased usefulness and effectiveness in testing antibacterial activity. No FeS-dependent black circle is formed under any of the coins, with the exception of the 1-yen coin, which contains aluminum and no copper. Therefore, the copper-containing coins have an antibacterial effect.

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© 2016 The Japan Institute of Metals and Materials