Menadione‐Catalyzed O₂^– Production by Escherichia coli Cells: Application of Rapid Chemiluminescent Assay to Antimicrobial Susceptibility Testing

Abstract

This study proposes a novel chemiluminescent assay of bacterial activity. Luminol chemiluminescence (LC) was amplified on addition of menadione to Escherichia coli suspension, and it was effectively inhibited by addition of superoxide dismutase rather than catalase. This fact suggests that H202 produced from O 2 -by superoxide dismutase is decomposed by catalase of E. coli. NAD(P)H:menadione reductase activities in periplasm and cytosol corresponded to the amplification of menadione-catalyzed LC, and outer and cytoplasmic membranes were only slightly involved in the LC. The total activity and Vmax of NAD(P)H:menadione reductase in the cytoplasm were greater than those in the periplasm. A transient increase in menadione-catalyzed LC was observed in the exponential phase and the LC decreased in the stationary phase during growth of E. coli. Menadione-catalyzed LC was sensitive to antibiotic action. A decrease in menadione-catalyzed LC by the impairment of membrane functions and by the inhibition of protein synthesis was observed at 5 min and 3 hr, respectively. These findings suggest the possibility that menadionecatalyzed luminol chemiluminescent assay is applicable to rapid antimicrobial assay because LC is sensitive to the change in growth and cytotoxic events caused by antimicrobial agents.Key words: Menadione, NAD(P)H:menadione reductase, O2 -, Chemiluminescence Current dilution and disk antimicrobial susceptibility tests require more than 18 hr to obtain results (11, 12) because these tests depend on visualization of cell growth in broth or on agar medium. Various modified tests have been proposed for reduction of the evaluation time. For example, detection of specific enzyme activities in bacteria can be performed optically with ftuorogenic or chromogenic substrates (8, 9), and the concentration of ATP in bacteria can be determined by bioluminescent assay (3, 10). These sensitive methods have led to greater accuracy and faster detection of viable bacterial cells. However, these methods are not popular in many laboratories because chromogenic and ftuorogenic reactions depend on reaction conditions of the media including pH, temperature, ion concentrations and enzyme inhibitors. The bioluminescent assay of ATP has not become an established method because the concentration of ATP is changeable during cell lysis and extraction of ATP (6).*Addresscorrespondence to Dr. Shiro Yamashoji, Kobe Gakuin Women's College, 27-1 Hayashiyama-cho, Nagata-ku, Kobe, Hyogo 653--0861, Japan. Fax. 078-641-8864. 333In a previous paper, we proposed a rapid chemiluminescent assay for the determination of viable cell density of yeast cells (13,22,23), while Peters et al proposed a lucigenin chemilurninescent assay for continuous determination of intracellular active oxygen metabolites in Escherichia coli (16). These chemiluminescent assays depend on menadione-catalyzed production of active oxygen species by viable yeast or bacterial cells, but our chemiluminescent assay and Peters' assay suggest extracellular and intracellu...

Evaluation of a Novel Automated Chemiluminescent Assay System for Antimicrobial Susceptibility Testing

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A rapid antimicrobial susceptibility test based on chemiluminescence assay and its application to screening of genotypes in vancomycin-resistant enterococci

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