Time-kill curves were performed for Ω76 (4 and 32 mg/liter), colistin (5 mg/liter), and an untreated control, against A. baumannii (P1270), in whole human blood.

An A. baumannii (P1270) culture was prepared in 10 ml of Mueller Hinton medium supplemented with meropenem (8 mg/liter) grown at 37°C/24 hours at 180 rpm. Whole human blood was freshly collected from D.N. via venipuncture in EDTA Vacutainer tubes (purple top). Approximately 107 CFU from this culture (33 μl), corresponding to the in vivo infective dose, was added to human blood such that the total volume was 1 ml (tube A). Separate experiments were performed to estimate CFU reductions at short time points (0, 2, 4, 6, 8, and 10 min) and long time points (0, 10, 20, 30, 40, 50, and 60 min). In all cases, Ω76 [32 mg/liter: 1.6 μl from a stock (20 mg/ml) in DW; 4 mg/liter: 2 μl from a stock (2 mg/ml) in DW] or colistin [0.5 μl from a stock (10 mg/ml) in DW] was only introduced to tube A after the 0 time point.

Long time points: Once a time point was reached, 100 μl was pipetted from tube A into 100 μl of 2 M NaCl (tube B, hypertonic solution to suspend small-molecule leakage). Ten microliters from tube B was diluted into 990 μl of a 1 M NaCl solution (tube C). Plating was performed immediately on Mueller Hinton agar supplemented with meropenem (8 mg/liter), preferably ending in 3 min. Tenfold dilutions were used for plating as follows: 100 μl from tube B → plate 1, 10 μl from tube B → plate 2, 100 μl from tube B → plate 2, 100 μl from tube C → plate 3, 10 μl from tube C → plate 4, 1 μl from tube C → plate 5.

Short time points: For all time points, 100 μl was pipetted from tube A into 100 μl of 2 M NaCl in 50% glycerol (tube B′, hypertonic solution to suspend small-molecule leakage, glycerol as a cryoprotectant). This tube was immediately flash-frozen in liquid nitrogen and stored at −80°C to completely stop AMP action. Further dilutions were performed for only after all time points were flash-frozen and safely stored. For each time point, tube B′ was thawed and 10 μl was immediately diluted in 990 μl of 1 M NaCl solution (tube C′). Plating was performed immediately, using the same protocol described previously for long time points. All the steps described here have also been illustrated in fig. S15.

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