Resistance development profiling of piperacillin in combination with the novel β-lactamase inhibitor BLI-489

To evaluate development of resistance to the piperacillin/BLI-489 combination. 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 beta-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 medium for 10 days. 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 < 1.0 x 10(-9). Two AmpC-producing Pseudomonas aeruginosa strains had a mutation frequency of 6.52 x 10(-6) and 1.0 x 10(-7); a beta-lactamase-negative P. aeruginosa strain had a mutation frequency of 2.68 x 10(-8). 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). 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.