However, this phenomenon has only been evaluated on a limited number of strains [12–16]. Therefore, the objective of this study was to further explore the “seesaw effect” in 150 clinical strains with varying susceptibilities. Additionally, eight selleck kinase inhibitor strains were utilized in time–kill studies to determine if the response to CPT was affected by changing glyco- or lipopeptide NSC23766 in vivo susceptibilities in isogenic strain pairs. Materials and Methods Bacterial Strains A total of 150 clinical MRSA strains from the Anti-infective Research Laboratory (Detroit, MI,
USA) collected between 2008 to 2012 were chosen for evaluation of the “seesaw effect”. All strains were randomly chosen clinical blood isolates. Additionally, four isogenic strain pairs were selected for further evaluation of these antibiotics in time–kill curves to compare differences in kill between parent and reduced selleck chemicals llc susceptibility
to VAN mutant isolates. Antimicrobials Ceftaroline (Teflaro®) powder was provided by Forest Laboratories, Inc. (New York, NY, USA). DAP (Cubicin®) was purchased commercially from Cubist Pharmaceuticals (Lexington, MA, USA). VAN and TEI were purchased commercially from Sigma Chemical Co. (St. Louis, MO, USA). Media Due to the calcium-dependent mechanism of DAP, MHB was supplemented with 50 mg/L of calcium and 12.5 mg/L of magnesium for all experiments. Colony
counts were determined using tryptic soy agar (TSA) (Difco, heptaminol Detroit, MI, USA). Susceptibility Testing Minimum inhibitory concentrations (MIC) for all study antimicrobials were determined by Etest methods according to the manufacturer’s instructions. Additionally, broth microdilution MICs were performed in duplicate at 1 × 106 according to Clinical and Laboratory Standards Institute (CLSI) guidelines for isogenic strain pairs as a comparison/validation of MICs determined by Etest methodology . All samples were incubated at 37 °C for 18–24 h. The following MIC data were determined for each tested antimicrobial: average MIC, MIC50, and MIC90. These MIC data were analyzed by linear regression to derive correlations coefficients between agents. In Vitro Time–Kills Four isogenic strain pairs were chosen as representative strains for evaluation in time–kill curves. Briefly, macro-dilution time–kill experiments were performed in duplicate using a starting inoculum of approximately 1 × 106 CFU/mL as previously described [17–19]. The 24-well culture plate was utilized with 100 μL of antibiotic stock solution, 200 μL of a 1:10 dilution of a 0.5 McFarland standard organism suspension, and sufficient volume of CAMHB for a total volume of 2 mL. Sample aliquots (0.1 mL) were removed over 0–24 h and serially diluted in cold 0.9% sodium chloride.