In the environment, there are numerous mutagens that may affect microorganisms genes. It is well known that mutagens induce mutations in microorganisms and change their susceptibility to bactericidal

drugs (Posnick & Samson, 1999; Takechi et al., 2006). However, bacteria have different susceptibility to the action of mutagens even within a same serotype, such as differences among Salmonella Typhimurium strains, tester strains of Ames test (Levin et al., 1982; Gee et al., 1994; Jemnitz et al., 2004). Therefore, we considered that susceptibilities to mutagens must be investigated in clinically important bacteria as to the emergence of drug resistance. We were unable, however, to find any reports describing whether or how these mutagens induce mutations in clinically important microorganisms, or if the induced mutations might confer resistance to antibacterial agents. Pseudomonas aeruginosa infects ABT-199 purchase immunosuppressed patients and causes high mortality in hospitalized patients (Stover et al., 2000).

It is unique because it possesses intrinsic resistance to a variety of antimicrobial agents (Chen et al., 2008). Ciprofloxacin (CPFX), a new quinolone antibacterial agent that inhibits type II topoisomerases, has been effective in treating P. aeruginosa infections. The emergence, however, of CPFX-resistant P. aeruginosa with mutations in gyrA/gyrB/parC/parE, find more which encode gyrase or topoisomerase IV, has been reported (Jalal & Wretlind, 1998; Akasaka et al., 2001; Mouneimne et al., 1999; Wydmuch et al., 2005). Essential for treating tuberculosis, rifampicin (Rif) is an inhibitor of RNA polymerase (Campbell et al., 2001). In Phospholipase D1 recent years, however, Rif-resistant Mycobacterium tuberculosis has become a serious worldwide clinical problem. Resistance to Rif is conferred by mutations in the rpoB gene, which encodes the β-subunit of RNA polymerase (Mariam et al., 2004). How Rif-resistant bacteria acquire mutations in the rpoB gene is not known. We designed this study to clarify whether and how mutagens in the environment and drugs are involved in the evolution of drug-resistant microorganisms. We exposed

P. aeruginosa to environmental mutagens. Then, we calculated the incidence of Rif- and CPFX-resistant P. aeruginosa and analyzed the gene sequences for rpoB and gyrA/gyrB/parC/parE. We found that environmental mutagens and an anticancer drug induce drug resistance in P. aeruginosa, and that the mutation spectra are similar to clinically isolated samples of drug-resistant P. aeruginosa. Ethylmethansulfonate (EMS) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) were obtained from Sigma-Aldrich, (St. Louis, MO). N-nitroso-N-methylurea (MNU) and benzopyrene (BP) were obtained from Wako (Kyoto, Japan). N-nitrosonornicotine (NNN) was obtained from Toronto Research Chemicals Inc. (Ontario, Canada) and 1,6-dinitropyrene (1,6-DNP) from AccuStandard (New Haven, CT). MNU, 1,6-DNP, and NNN were each dissolved in DMSO.