8); and iii) in a chemically defined “synthetic CF sputum medium” (SCFM), that mimics the nutritional composition of CF sputum [24]. SCFM was prepared by using Casamino Acids Vitamin Assay (BD Difco) mixture containing each amino acid at concentration not significantly different from that originally described by Palmer and co-workers [24], except for a reduced amount of glycine and ornithine, which were therefore added from ad hoc prepared stock solutions to reach their required concentration. Susceptibility Ruxolitinib testing MICs and MBCs were determined by microdilution technique, in accordance with CLSI M100-S20 protocol [39], with some modifications.

Briefly, serial two-fold dilutions (64 to 0.12 μg/ml) of each AMP and Tobramycin (Sigma-Aldrich

S.r.l.; Milan; Italy) were prepared in SCFM at a volume of 100 μl/well in VS-4718 clinical trial 96-well microtiter plates (Bibby-Sterilin Italia S.r.l.; Milan, Italy). Each well was then inoculated with 5 μl of a standardized inoculum, corresponding to a final test concentration of about 0.5-1 × 105 CFU/well. After incubation at 37°C for 24 h, the MIC was read as the lowest concentration of the test agent that completely inhibited visible growth. To measure the MBC, 100 μl of broth from clear wells were plated on MHA plates, and incubated at 37°C for 24 h. MBC was defined as the lowest concentration of the test agent killing of at least 99.99% of the original inoculum. To evaluate the impact of “CF-like” Liothyronine Sodium experimental conditions on the antimicrobial activity of AMPs and Tobramycin, a set of PFGE-unrelated isolates representative for different levels of susceptibility to Tobramycin (4 P. aeruginosa, 3 S. maltophilia, and 4 S. aureus) was also tested for MIC and MBC values determined under standard CLSI-recommended conditions (i.e., aerobic atmosphere,

cation-adjusted Mueller-Hinton broth, and pH 7.2). Time-killing assay Kinetics of AMPs’ and Tobramycin’ activity was evaluated by using the broth macrodilution method against three representative isolates within each tested species. Briefly, the standardized inoculum (1×105 CFU/mL) was exposed to the test agent at 1xMIC in SCFM, and incubated at 37°C. After 10 min, 30 min and 1, 2, and 24-h of incubation, aliquots of each sample were diluted and plated onto MHA, then the KU 57788 viable counts determined after 24-h of incubation at 37°C. Killing curves were constructed by plotting the log CFU/mL versus time. Synergy testing The activity of each AMP combined to Tobramycin against CF strains was evaluated by checkerboard technique by using 96-well polystyrene microplate (Kartell S.p.A., Noviglio, Milan, Italy). Briefly, concentrations of multiple compounds (range: 64–0.