Determining the minimum inhibitory concentrations

Minimum inhibitory concentrations (MIC) for ciprofloxacin, tobramycin and meropenem were obtained using E-test strip diffusion and micro broth dilution methods. E-test plastic strips (Liofilchem, Italy) that have a predefined gradient of antibiotic concentration [25]. A 0.5 McFarland standard suspension was inoculated onto Mueller-Hinton agar plates. Up to four E-test strips were placed on each plate following the manufacturer’s instructions (Liofilchem, Italy). The concentrations measured were: ciprofloxacin: 0.002–32 μg/mL, tobramycin: 0.016–256 μg/mL, and meropenem: 0.002–32 μg/mL. For the micro broth dilution method, we diluted overnight cultures of P. aeruginosa isolates (1:50) in fresh LB media, which we allowed to grow to exponential phase (OD600 = 0.4) and then diluted to 2–8 10⁵ colony forming units (CFU) per mL in 96-well polypropylene plates with or without series of concentrations of the three antibiotics tested. The plates were incubated at 37°C and after 20–24 hours the highest concentration where no visible growth was observed was noted as the MIC value. The 10x-sub MIC values used in the present study for the three tested strains were the following: PAO1 (Cipro = 0.0125 μg/mL; Tobra = 0.0125 μg/mL, Mero = 0.05 μg/mL), Pae85 (Cipro = 0.0125 μg/mL; Tobra = 0.1 μg/mL, Mero = 0.025 μg/mL) and Pae112 (Cipro = 0.025 μg/mL; Tobra = 0.05 μg/mL, Mero = 0.025 μg/mL). The applied standardized MIC protocol is based on Wiegand et al. 2008 [26].