This

was serially diluted in two-fold steps (1 mL: 1 mL) to create the desired antibiotic range; each tube containing twice the ultimate concentration of drug in 1 mL of broth. An additional tube containing 1 mL of broth without drug is also prepared as the growth control. For testing S. aureus, the CA-MHB was supplemented with additional mTOR inhibitor NaCl to a final concentration of 2% (w/v) in order to enhance the methicillin resistant phenotype, if present, when testing for susceptibility against oxacillin [6, 22]. Freshly grown colonies of the microorganism to be tested were suspended in a 0.9% saline solution and adjusted to a 0.5 McFarland standard. This bacterial suspension was further diluted in CA-MHB 1:150-fold and 1 mL of this secondary suspension was added to each broth containing antibiotic. This produces a series of 2 mL cultures containing the desired range of antibiotic in which each culture contains approximately 5.0E + 05 CFU/mL of bacteria. The inoculation concentration was verified by removing a 0.01 mL aliquot from the growth control culture, diluting it 1000-fold in 0.9% saline solution and directly plating 0.1 mL for CFU enumeration. The cultures were incubated Copanlisib concentration at 35 ± 2°C, shaking at 350 rpm for 20–24 hours. The MIC of the drug/bacteria combination is determined as the culture containing the lowest concentration of antibiotic which fully

inhibits the propagation of the culture (no visual turbidity) after the incubation period. Time course sampling of the AST cultures and ETGA substrate conversion The experimental design of the study is shown

in Figure 1. After inoculation of each macrodilution broth with 4��8C approximately 5.0E + 05 CFU/mL of bacteria, at 0, 2, 4, 6, and 22 hours (the overnight incubation) a 0.01 mL aliquot was removed from each culture and diluted 1:10 in nuclease free water (Life Technologies, Carlsbad, CA). If the sample was taken from a turbid culture after 22 hours of incubation, the sample was diluted 1:1E + 04 in nuclease free water by serial dilution. From each diluted sample, 0.01 mL was removed and placed into a 1.5 mL screw-capped tube containing glass beads and 0.05 mL of ETGA reaction solution. The bead-mill tubes were subsequently milled for bacteria lysis, incubated at 37°C for 20 minutes followed by 95°C for 5 minutes (to terminate the reaction), spun down, and stored at -20°C prior to analysis. At the final time point, ETGA reagent and positive controls [21] were performed alongside the samples. Figure 1 Experimental design of the study. On day one, the macrobroth AST is assembled. At the indicated time points, an aliquot is removed from each broth and diluted ten-fold. A portion of the diluted sample is subjected to bead milling for bacterial lysis, and incubated for ETGA substrate conversion. Once processed, the samples are stored at -20°C prior to analysis. On day two, the MIC of the AST is determined by visual turbidity.