Daptomycin

To ensure that the pharmacokinetic parameters in the animal models were similar to those in humans (34 (link)), Daptomycin (45 and 90 mg) was administered as a single s.c. dose, and 12 mg Daptomycin was also administered as a single i.v. dose, to healthy male Wistar rats, weighing ~330 g. The experiments were performed in triplicate. Blood samples were collected from the inferior vena cava at 0, 0.5, 1, 2, 3, 4, 5, 8, 12, and 24 h in heparinized tubes. Plasma was collected via centrifugation, and the samples were stored at −80°C until analysis.
A Daptomycin quantification assay was performed as previously described, using an ultra high performance liquid chromatography (UHPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) analysis (47 (link)). Daptomycin was purchased from Glentham Life Sciences (Corsham, UK), and ²H₅-Daptomycin trifluoroacetic acid was purchased from Alsachim (Illkirch Graffenstaden, France). All of the UHPLC-ESI-MS/MS analyses were performed using an Agilent 1290 UHPLC system coupled with an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).

One gram of homogenised faecal sample was added to 10 mL of sterile buffered peptone water (Biokar diagnostics) and plated onto SuperPolymyxin medium [19 (link)], an eosin methylene blue agar (Biokar diagnostics) supplemented with 3.5 μg/ml colistin (Sigma-Aldrich), 10 μg/ml daptomycin (Glentham Life Sciences), and 5 μg/ml amphotericin B (Glentham Life Sciences), and incubated for 24 h at 37°C. For each faecal sample, up to five colonies with a E. coli phenotype were isolated (i.e. metallic green sheen in SuperPolymyxin medium).

To ensure that the pharmacokinetic parameters in the animal models were similar to those in humans (34 (link)), Daptomycin (45 and 90 mg) was administered as a single s.c. dose, and 12 mg Daptomycin was also administered as a single i.v. dose, to healthy male Wistar rats, weighing ~330 g. The experiments were performed in triplicate. Blood samples were collected from the inferior vena cava at 0, 0.5, 1, 2, 3, 4, 5, 8, 12, and 24 h in heparinized tubes. Plasma was collected via centrifugation, and the samples were stored at −80°C until analysis.
A Daptomycin quantification assay was performed as previously described, using an ultra high performance liquid chromatography (UHPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) analysis (47 (link)). Daptomycin was purchased from Glentham Life Sciences (Corsham, UK), and ²H₅-Daptomycin trifluoroacetic acid was purchased from Alsachim (Illkirch Graffenstaden, France). All of the UHPLC-ESI-MS/MS analyses were performed using an Agilent 1290 UHPLC system coupled with an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).