In the present study it has been shown that the reactive nitrogen species, peroxynitrite, can cause at least a 7.1-fold increase in the frequency of occurrence of drug-resistant mutants of Helicobacter pylori at a pathophysiological concentration (e.g. 1.0 μM) and in the presence of CLR. Furthermore, the CLR MIC of these resistant H. pylori strains increased by at least 250 times or higher in CLR susceptibility. In the 45 resistant strains, the modification of 23S rRNA A2142G was the predominant mutation (22/45), followed by A2143G (17/45) within the sequences of 23S rRNA. The other mutants were one each (1/45) in A2142T, and T2269G, and two each (2/45) in C2695G and T1944C, respectively. These results show that the inflammatory host reaction involving induction of reactive oxygen species (e.g. O
·−2 ), and the inducible form of nitric oxide synthase, is a significant cause of mutation via peroxynitrite formation, particularly in drug-resistant bacterial strains.
Key words drug resistant mutant, free radical, peroxynitrite, 23S rRNAGeneration of drug-resistant mutant pathogenic bacteria is a serious problem. In previous studies (1, 2), we have demonstrated greatly increased mutation frequency in a mouse model of influenza viral infection. During one 10-14 day disease-span of a mouse, a 6-to 7-fold increase in the occurrence of mutant virus compared with the non-diseased state was noted. This mutation is caused primarily by ONOO − (1-4), a product of an endogenous reaction between O ·− 2 and NO, which are generated by inflammatory leukocytes at sites of infection (1-7). ONOO − is a highly oxidative chemical which functions in the rapid oxidation or cleavage of nucleic acids, proteins and lipids (8). It is also, however, a strong nitrating agent, and thus 3-nitrotyrosine (9) and concomitant formation 2 in the presence of cytochrome P450 NADPH reductase, nitric oxide synthases, or cytochrome b5 reductase in a non-stoichiometric manner (10,11), that is, similar to a propagation reaction of lipid peroxidation.This result means that an inflammatory site is a "hot spot" of free radical generation. Also, we have published a more recent report showing that ONOO − in pathophysiological concentration (1-8 μM) greatly increases the mutation frequency in Salmonella typhimurium, as revealed by a modified Ames test in which no cytosolic cytochrome