Ten clinical MP isolates from CF patients at an academic medical center were tested for susceptibility, and time–kill analyses were performed with ceftolozane/tazobactam and the following SOC antimicrobials: ceftazidime, cefepime, meropenem, ciprofloxacin, tobramycin, and polymyxin B. This study was approved by the Institutional Biosafety Committee. This article does not contain any new studies with human or animals performed by any of the authors. This study does not meet the definition of a clinical trial, and so was not registered at ClinicalTrials.gov.
Ceftolozane powder was obtained from Merck (Kenilworth, NJ, USA). Tazobactam (Sigma-Aldrich, St Louis, MO, USA), ceftazidime (Sagent Pharmaceuticals, Schaumburg, IL, USA), cefepime and meropenem (Sandoz, Princeton, NY, USA), ciprofloxacin and tobramycin (Hospira, Lake Forest, IL, USA), and polymyxin B (XGen Pharmaceuticals, Big Flats, NY, USA) were obtained commercially.
MICs were determined with Etest according to standard procedures and read by two individuals. If there was a difference between the readings, a third individual analyzed the result. If there was a difference in MIC of more than a half dilution, the Etest was repeated.
Time–kill experiments were performed in duplicate in Mueller–Hinton broth (BBL; Becton, Dickinson, Sparks, MD, USA) in 24-well macrowell plates as previously described . The plate was filled with 100 µL of antibiotic stock solution, 200 µL of a 1:10 dilution of a 1.75 McFarland-standard organism suspension for a target bacterial inoculum of 106 colony-forming units (CFU) per milliliter, and sufficient volume of Mueller–Hinton broth for a total volume of 2 mL. Sample aliquots of 100 µL were obtained from each well at 0, 4, 8, and 24 h, and were serially diluted in cold 0.9% sodium chloride. Bacterial counts were determined with a Whitely automatic spiral plater (Don Whitely Scientific, Shipley, UK). The lower limit of detection for time–kill studies was 101 CFU/mL. Plates were incubated at 37 °C for 24 h, at which time colony counts were performed with a ProtoCOL colony counter (Synoptics, Frederick MD, USA). All strains were tested against ceftolozane/tazobactam (1.5 and 3 g), ceftazidime (2 g), cefepime (2 g), ciprofloxacin (400 mg), meropenem (1 g), tobramycin (25 mg/L), and polymyxin B (1.25 mg/kg) with use of free physiologic peak concentrations. The physiologic free peak concentrations were 60/12.6, 120/25.2, 151, 112, 3.2, 110, 25, and 2.1 mg/L, respectively [13,14,15,16,17,18]. Time–kill curves were generated by plotting mean colony counts (log10 CFU/mL) versus time to compare 24-h killing effects (Fig. 1). Bactericidal activity was defined as a 3 log10 CFU/mL reduction or greater from the baseline.
Differences in log10 CFU per milliliter were analyzed by analysis of variance with Tukey’s post hoc test. P < 0.05 was considered significant. All statistical analyses were performed with IBM SPSS Statistics (version 24.0; IBM, Armonk, NY, USA).