The data indicated that the MIC for nitrofurantoin STI571 was approximately 3:g/ml for all strains tested (data not shown). When plates were incubated for an additional 24 hrs, a small number of colonies arose, and these were presumptive nitrofurantoin-resistant mutants. By comparing the number of colonies found on an agar

plate after 48 hours incubation in the presence of nitrofurantoin to the number of colonies obtained when a similar aliquot was inoculated onto media lacking the antimicrobial agent, we were able to calculate the spontaneous buy GSI-IX mutation frequency to resistance to this agent. The data (Fig. 1) indicate that the mutation frequency associated with this antimicrobial agent varied about 10 fold among strains, with FA1090 being the least mutable among the strains tested, and MS11 being the most mutable. However, since it was possible to isolate mutants that readily grew on media containing levels of nitrofurantoin above the MIC, we hypothesized that the mutation responsible for this phenotype was in the coding sequence for the putative gonococcal nitroreductase gene. Figure 1 Spontaneous mutation frequency of various lab strains of N. gonorrhoeae. Mutation frequencies were determined BKM120 mouse by counting the number of colonies arising on the GCK +

Nitrofurantoin (3 μg/mL) plates after 48 hours of incubation at 37°C, 6% CO2, and dividing this number by the number of colonies arising on the GCK plates after 48 hours of incubation at 37°C, 6% CO2. Data represents

experiments done in triplicate, with error bars representing standard error. Identification of potential nitroreductase genes E. coli possesses two nitroreductases that can reduce these nitro-aromatic compounds; nfsA and nfsB, plus a nitroreductase activity encoded by a gene that has yet to be identified [18, 24]. Therefore, it is possible that GC may possess additional cAMP genes that confer nitroreductase activity. In E. coli, resistance to these nitro-aromatic antimicrobial agents occurs in a step-wise manner. A mutation that knocks out the function of NfsA raises the MIC about three fold. A second mutation that knocks out the function of NfsB increases resistance to about 10 times the MIC of wild-type strains [18, 24]. All attempts to isolate second-step mutants in N. gonorrhoeae were unsuccessful, indicating that this species only contains a single functional nitroreductase, or that the additional nitroreductases were insensitive to nitrofurantoin. Since two nitroreductases have been identified in E. coli, nfsA and nfsB [30, 31], we used the amino acid sequence for these two gene products to search the gonococcal translated genomic DNA sequence database. No significant similarity was found to nfsA. However, an ORF encoding a protein with some similarity to nfsB was found.