Lactate-Dependent Killing of Escherichia coli by Nitrite plus Hydrogen-Peroxide: A Possible Role of Nitrogen Dioxide

Abstract

The killing of Escherichia coli by nitrite plus hydrogen peroxide was observed in lactate, but not in phosphate or acetate. Although nitrite or hydrogen peroxide alone caused a slight decrease in bacterial survival, nitrite plus hydrogen peroxide killed bacteria synergistically in time-, dose-, and pH-dependent manners. The killing was increased with decreasing pH. The plot of viable cells versus [nitrous acid] was linear, Among the hydroxyl radical scavengers used, only benzoate and formate at concentrations higher than that of lactate inhibited the killing by nitrite plus hydrogen peroxide, whereas dimethyl sulfoxide enhanced it. The generation of peroxynitrous acid during the reaction of nitrite and hydrogen peroxide was confirmed by the formation of malondialdehyde using deoxyribose as a hydroxyl radical-like oxidant detector. The nitration of glycyl-tryosine was observed only in lactate buffer, but not in phosphate and acetate buffers. Benzoate and formate inhibited the nitration, whereas dimethyl sulfoxide and ethanol enhanced it. No evidence for the formation of nitric oxide and superoxide during the reaction of nitrite and hydrogen peroxide was found. These data suggest that nitrogen dioxide from the decomposition of peroxynitrous acid or secondary oxidants formed from the reaction of peroxynitrous acid with lactate is responsible for the lactate-dependent killing of E. coli induced by the reaction of protonated nitrite and hydrogen peroxide.