Objectives: To evaluate development of resistance to the piperacillin/BLI-489 combination. Methods: BLI-489 was used at a constant concentration of 4 mg/L. Spontaneous mutation frequency was measured on piperacillin/BLI-489-containing agar plates. Five b-lactamase-producing strains were exposed to a serial dilution of piperacillin/BLI-489, and the highest concentration allowing growth was used to inoculate subsequent serial passage for 10 days. Mutation stability was monitored in drug-free

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... m for 10 days. Results: Escherichia coli (OXA-3, OXA-7, ACT-1, SHV-1 or none), Salmonella enterica serovar Typhimurium (CTX-M-5), Klebsiella pneumoniae (SHV-1 and SHV-5) and Enterobacter cloacae (AmpC) had a spontaneous mutation frequency of 1.0Â10 29 . Two AmpC-producing Pseudomonas aeruginosa strains had a mutation frequency of 6.52Â10 26 and 1.0Â10 27 ; a b-lactamase-negative P. aeruginosa strain had a mutation frequency of 2.68Â10 28 . The mutant prevention concentration (MPC) was 32 mg/L. During serial passages, the MIC increased 64-and 128-fold for S. enterica serovar Typhimurium (CTX-M-5) and E. cloacae (AmpC), respectively, to !512 mg/L. The MIC reverted to 64 mg/L after serial passages in drug-free medium. The MICs increased only 4-fold for K. pneumoniae (SHV-1 and SHV-5), E. coli (OXA-3) and E. coli (SHV-1). Conclusions: Piperacillin/BLI-489 demonstrated a low probability of spontaneous resistance development in vitro for all of the strains tested with the exception of P. aeruginosa. The MPC value for all strains was 32 mg/L. Resistance developed during serial passage for two of the five strains tested; however, this resistance phenotype was unstable as MIC values reverted to 64 mg/L after propagation in drug-free medium.