Hollow-fiber infection model.

The HFIM was assembled as described previously using FiberCell Systems polysulfone cartridges (C2011) in all experiments and conducted over 7 days (32, 33). One HFIM experiment was conducted for each dosing regimen and isolate combination with an initial bacterial concentration of 1 × 10⁸ CFU/ml.

Unbound amikacin blood exposures were simulated using the pharmacokinetic model derived by Romano et al., assuming an 80-kg patient with sepsis, a creatinine clearance of 100 ml/min, and 17% protein binding (34, 35). Amikacin dosing regimens of 15, 25, and 50 mg/kg once daily infused over 30 min were tested. High 50 mg/kg doses were also tested given that these doses have been previously used clinically (36). The ELF amikacin concentrations and resultant half-life in the HFIM apparatus were approximated using previous aminoglycoside ELF:serum ratios in conjunction with the established concentration-time curves for the blood amikacin exposure (14, 37, 38). In brief, the estimated unbound plasma concentration of amikacin was multiplied by the average ELF:serum penetration ratio (0.12, 0.3, 0.85, and 1.14) identified for other aminoglycosides (gentamicin and tobramycin) at the corresponding time points (0.5, 1, 2, and 4 h) (14, 37, 38). The ELF half-life (1.92 h) was derived from a noncompartmental analysis of the resultant concentration-time curve over the course of 24 h, which approximates that identified previously (39, 40). A mucin-bound fraction of 50% was assumed, representing a likely worst-case scenario (26). An ELF amikacin exposure following an intravenous dose of 30 mg/kg once daily administered over 30 min was simulated.

Samples were periodically removed from the central compartment outlet at 0.25, 0.5, 0.45, 1, 2, 3, 4, 6, 8, 10, 12, 24, 25, 30, 36, 48, 49, 54, 60, 72, 73, 78, 84, 96, 120, 144, 145, and 156 h to determine the amikacin concentration for pharmacokinetic analysis. As the central compartment contents rapidly equilibrate with the hollow-fiber cartridge, the concentrations obtained in the central compartment reflect that in the hollow-fiber cartridge. Bacterial quantification was performed with periodic sampling at 0, 2, 4, 6, 8, 11, 24, 35, 48, 59, 72, 96, 120, 144, and up to 168 h from the cartridge extracapillary space. Samples were washed twice in phosphate-buffered saline to minimize antibiotic carry-over. A 100-μl aliquot of an appropriately diluted bacterial suspension was manually plated onto CaMH agar and amikacin-containing CaMH agar (4-fold baseline isolate MIC). The limit of quantification was 2-log₁₀ CFU/ml.