Urine: midstream urine was obtained from young, male, healthy volunteers, frozen, thawed, pooled, sterile filtered (2 µm) and frozen again until usage to guarantee the usage of one single pooled batch of urine for all tests. The iron content of pooled urine, as determined in the central laboratory of the General Hospital of Vienna, was ∼ 0.05 mg/L/24 h.
CAMHB: CAMHB (Sigma–Aldrich, Steinheim, Germany) containing 17.5 g/L casein acid hydrolysate, 3 g/L beef extract and 1.5 g/L starch, 20–25 mg/L calcium, 10–12.5 mg/L magnesium and iron content ∼0.05 mg/L served as an additional reference medium for MIC testing as iron concentrations are similar to the physiological concentrations found in urine.
ID-CAMHB: the broth was produced as recommended by the EUCAST guidance document on cefiderocol BMD (latest version, December 2020) and as described in detail by Hackel et al.14 (link) In short, ID-CAMHB was prepared by adding 100 g of Chelex® 100 resin (Bio-Rad Laboratories, Hercules, CA, USA) to 1 L of freshly prepared CAMHB. The suspension was stirred for 2 h at room temperature (22°C), filtered using a 0.2 μm filter and cation adjusted as recommended.
The pH was set for all tested media (pooled urine, CAMHB and ID-CAMHB) with HCl or NaOH directly before the start of the experiments to values of 5, 7 and 8.
CAMHB: CAMHB (Sigma–Aldrich, Steinheim, Germany) containing 17.5 g/L casein acid hydrolysate, 3 g/L beef extract and 1.5 g/L starch, 20–25 mg/L calcium, 10–12.5 mg/L magnesium and iron content ∼0.05 mg/L served as an additional reference medium for MIC testing as iron concentrations are similar to the physiological concentrations found in urine.
ID-CAMHB: the broth was produced as recommended by the EUCAST guidance document on cefiderocol BMD (latest version, December 2020) and as described in detail by Hackel et al.14 (link) In short, ID-CAMHB was prepared by adding 100 g of Chelex® 100 resin (Bio-Rad Laboratories, Hercules, CA, USA) to 1 L of freshly prepared CAMHB. The suspension was stirred for 2 h at room temperature (22°C), filtered using a 0.2 μm filter and cation adjusted as recommended.
The pH was set for all tested media (pooled urine, CAMHB and ID-CAMHB) with HCl or NaOH directly before the start of the experiments to values of 5, 7 and 8.