Menadione-catalyzed O-2(-) production by Escherichia coli cells: Application of rapid chemiluminescent assay to antimicrobial susceptibility testing

This study proposes a novel chemiluminescent assay of bacterial activity. L uminol chemiluminescence (LC) was amplified on addition of menadione to Esc herichia coli suspension, and it was effectively inhibited by addition of s uperoxide dismutase rather than catalase, This fact suggests that H2O2 prod uced from O-2(-) by Superoxide dismutase is decomposed by catalase of E, co li, NAD(P)H:menadione reductase activities in periplasm and cytosol corresp onded to the amplification of menadione-catalyzed LC, and outer and cytopla smic membranes were only slightly involved in the LC. The total activity an d V-max of NAD(P)H:menadione reductase in the cytoplasm were greater than t hose in the periplasm. A transient increase in menadione-catalyzed LC was o bserved in the exponential phase and the LC decreased in the stationary pha se during growth off. coli, Menadione-catalyzed LC was sensitive to antibio tic action. A decrease in menadione-catalyzed LC by the impairment of membr ane functions and by the inhibition of protein synthesis was observed at 5 min and 3 hr, respectively. These findings suggest the possibility that men adione-catalyzed luminol chemiluminescent assay is applicable to rapid anti microbial assay because LC is sensitive to the change in growth and cytotox ic events caused by antimicrobial agents.