Menadione-catalyzed O2- production by Escherichia coli cells: application of rapid chemiluminescent assay to antimicrobial susceptibility testing.
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 H2O2 produced from O2 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 menadione-catalyzed 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.
Yamashoji
S
S
Kobe Gakuin Women's College, Hyogo, Japan.
Manome
I
I
Ikedo
M
M
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D002384:Catalysis; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008165:Luminol / Q000378:metabolism / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000494:pharmacology; D024483:Vitamin K 3 / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D007202:Indicators and Reagents; D009249:NADP; D008165:Luminol; D024483:Vitamin K 3; D006861:Hydrogen Peroxide; D002374:Catalase; D013482:Superoxide Dismutase; D010100:Oxygen