Broth microdilution susceptibility testing.

Bacteria were grown for 18 to 20 h on BBL Trypticase soy agar with 5% sheep blood (Becton Dickinson, Franklin Lakes, NJ) at 37°C and then subcultured for study of F₂ generation colonies. MICs were determined by broth microdilution, using the same inoculum for all antimicrobials, following CLSI guidelines (33). Antimicrobial concentrations tested ranged from 0.0018 to 128 μg/ml, in 2-fold serial dilutions, using a constant concentration of 4 μg/ml of relebactam and tazobactam when combined with imipenem or ceftolozane, respectively. Plates were incubated for 16 to 20 h at 37°C, and the MIC was recorded as the first well with no growth. The MIC₅₀/MIC₉₀, cumulative MICs, and percent of isolates that were susceptible, intermediate, and resistant for each antimicrobial agent were interpreted using CLSI breakpoints (33).