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...

The newly developed Rapid Lumi Eiken/IS60 (RL/IS60) system automatically determines MICs by detecting chemiluminescence produced in the reaction of a chemiluminescent probe and oxygen metabolites from living microorganisms. The present study evaluated this system for accuracy in antimicrobial susceptibility testing. Chemiluminescence intensities after 4 h of cultivation of clinically important strains were plotted against various concentrations of antimicrobial agents, which resulted in curves reflecting the levels of susceptibility. Sixty-percent inhibitory concentrations based on the susceptibility curves agreed with MICs determined by the reference microdilution method. When the MICs of antimicrobial agents for four quality control (QC) strains (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa) were determined by the RL/IS60 system, most (91.1%) of them were within the QC limits proposed by the National Committee for Clinical Laboratory Standards. The system was further assessed for a total of 162 clinical isolates, including E. coli, Citrobacter freundii, Enterobacter cloacae, Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis, Morganella morganii, P. aeruginosa, Haemophilus influenzae, S. aureus, coagulase-negative staphylococci, Enterococcus faecalis, Enterococcus faecium, and Streptococcus pneumoniae. Overall, there was 90.6% agreement between the RL/IS60 system and the reference microdilution method. Our results suggest that the RL/IS60 system provides rapid and reliable MICs of a variety of antimicrobial agents for clinical isolates as well as QC strains.The significant increase in the number of drug-resistant microorganisms emphasizes the great need for rapid and accurate methods for determining resistance to antimicrobial agents. Current standard methods, the broth microdilution (12) and disk diffusion tests (13), approved by the National Committee for Clinical Laboratory Standards (NCCLS), require more than 18 h before the final results are known. New methods are desired that could detect the biological activity in a short incubation time and provide MICs equivalent to those determined by the standard tests.The chemiluminescence assay, which detects photon emissions released from living organisms, is a sensitive method for monitoring viability. Chemiluminescence is produced in the reaction of oxygen metabolites generated during glycolysis with chemiluminescent probes such as luminol and lucigenin (2, 16). We previously showed the application of a luminol-mediated chemiluminescent assay to antimicrobial susceptibility testing for Escherichia coli (17). Chemiluminescence intensity increased during the exponential phase of growth. The reaction was enhanced by a catalyst, menadione. Antimicrobial agents (erythromycin, tetracycline, and oxytetracycline) inhibited chemiluminescence release from E. coli. The effect of antimicrobial agents was detectable within 1.5 h. The results suggested the usefulness of this assay for rapid susceptibility testing.Recen...

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