Carbapenem MIC determination using broth dilution.

All Enterobacteriaceae isolates that showed resistance or intermediate level of susceptibility against meropenem (10 μg) were further investigated using standard broth microdilution. A 5-μg/mL concentration of meropenem stock solution was prepared in methanol and distilled water. A working solution of meropenem was prepared with a concentration of 64 μg/mL by adding 40 μL of stock solution to 3.96 mL of Mueller-Hinton broth 2 (Oxoid Ltd., UK). Using the 64-μg/mL working solution, desired working solutions with concentrations of 0.065, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 were prepared by 2-fold serial dilutions into the broth. From each working solution, 50 μL was transferred to a 96-well plate (Tarson microtest plate). A bacterial suspension adjusted to a 0.5 McFarland standard in 5 mL of Mueller-Hinton broth 2 was added to each well that contained meropenem working solution and incubated at 37°C for 16 to 20 h. The MIC was recorded as the lowest concentration of drug that completely inhibited visible growth. A cutoff value provided by CLSI was used to interpret the meropenem MIC result.