We used 13 Enterobacteriaceae isolates harboring blaIMP–1 (4 Enterobacter hormaechei, 5 Escherichia coli, and 4 Klebsiella pneumoniae) provided by Toho University, and 13 Enterobacteriaceae isolates harboring blaIMP–6 (8 E. coli and 5 K. pneumoniae) obtained in our previous CRE surveillance in Osaka, Japan . E. hormaechei isolates belonging to Enterobacter cloacae complex were included in this analysis as one of blaIMP–1 representative carriers, which were detected in an outbreak reported in Tokyo, Japan . Through the experiments explained below, we used meropenem trihydrate (Tokyo Chemical Industry, Japan, Tokyo) and cefmetazole sodium salt (Sigma-Aldrich, Saint Louis, MO, United States) as antibiotic agents.
We determined minimum inhibitory concentrations (MICs) of MEM and CMZ by the broth microdilution method based on the Clinical and Laboratory Standards Institute (CLSI) document M07-A10 . Bacterial growth was evaluated by visible observation for turbidity. Briefly, we inoculated 5 × 105 colony forming units (CFU)/mL of bacterial suspension into cation-adjusted BBL™ Mueller Hinton II Broth (Becton, Dickinson and Company, Sparks, MD, USA) and incubated at 35°C in ambient air for 18 h. MIC was defined as the lowest concentration of the tested antimicrobial that completely inhibited the growth of bacteria.
Details of the checkerboard method and time-killing assay have been presented previously . In the checkerboard method, synergistic effect between MEM and CMZ against IMP–1 or IMP–6 producers was quantified by calculating the fractional inhibitory concentration (FIC) index. FIC index value ≤0.5 was defined as synergy, >0.5 to ≤4.0 as indifferent, and >4.0 as antagonistic. The assay was performed in duplicate. In case a synergistic effect was observed, MEM MIC fold-reduction was determined based on the lowest FIC index.
We conducted time-killing assays using the following two E. coli isolates: TUM13773 carrying blaIMP–1 and E109 carrying blaIMP–6. During these assays, each isolate was incubated in Muller-Hinton II broth devoid of antibiotic (control) and with MEM, CMZ, or MEM/CMZ combination at the concentration of 25% of the MIC for individual isolates. CFUs of bacteria at 3, 6, 9, and 24 hours after beginning the treatment were counted. The averages of CFUs were calculated from duplicated assays. We defined an efficacy of the combination therapy as bactericidal when ≥ 3 log10 CFU/mL reduction compared to the initial bacterial count was observed.