Time-kill synergy experiments.

Time-kill studies were performed using strain BIDMC 32, a KPC3-producing K. pneumoniae isolate, for all combinations that had demonstrated synergy against this strain in the checkerboard array. For combinations not synergistic against BIDMC 32 in the checkerboard array, a strain against which they had demonstrated synergy was chosen instead. Daptomycin plus colistin, which had not demonstrated synergy against any isolates in checkerboard studies, was not tested by the time-kill method. Antibiotic stocks were prepared as described above and diluted in 10 ml of CAMHB in 25- by 150-mm glass round-bottom tubes to the desired starting concentrations. For tigecycline, CAMHB was prepared fresh on the day of testing (44, 48). Concentrations were chosen on the basis of the FIC_I-MIN results from checkerboard array studies. If the concentrations tested did not demonstrate synergy in the time-kill study, the concentration of the noncolistin antibiotic was doubled and the experiment was repeated; if synergy was not demonstrated under these circumstances, no further concentrations were tested.

To prepare a starting inoculum for the time-kill studies, 100 μl of a 0.5 McFarland standard suspension of colonies from an overnight plate was added to 5 ml of CAMHB and incubated on a shaker in ambient air at 35°C until it reached log-phase growth (approximately 4 h). The culture was then adjusted to the turbidity of a 1.0 McFarland standard in CAMHB, and 100 μl was added to each of the antimicrobial solutions. A growth control and a negative control were run in parallel with each experiment. Cultures were incubated on a shaker in ambient air at 35°C.

Aliquots from the culture were removed at 0, 1, 2, 4, 6, and 24 h. A 10-fold dilution series was prepared in 0.9% sodium chloride. A 10-μl drop from each dilution was transferred to a Mueller-Hinton plate (Thermo Fisher, Waltham, MA) (54, 55) and incubated overnight in ambient air at 35°C. The colonies within each drop were counted; drops containing 3 to 30 colonies were considered countable. For countable drops, the cell density of the sample was calculated; if more than one dilution for a given sample was countable, the cell density of the two dilutions was averaged. If no drops were countable, the counts for consecutive drops above and below the countable range were averaged. The limit of detection was 300 CFU/ml. The antibiotic carryover effect was not observed at the concentrations tested. A combination was considered synergistic if it resulted in a ≥2-log₁₀ reduction in the number of CFU per milliliter at 24 h compared to the number of CFU per milliliter achieved with the most active agent alone. Bactericidal activity was defined as a reduction of ≥3 log₁₀ CFU/ml at 24 h compared to the starting inoculum (39, 56).