Trypticase soy agar 2 with 5 sheep blood

Semi-quantitative assays in specific media were used to determine enzymatic activities. To perform these assays, 5 µl of the respective overnight bacterial cultures were spotted onto the following three types of agar plates. (i) Milk agar was used to determine protease activity due to the hydrolysis of casein. The modified recipe used in this study contained 0.5% tryptone, 0.25% yeast extract, 0.1% dextrose, 1% skim milk powder, and 1.25% agar. The bacteria were incubated on these plates for 24 h at 30 °C. (ii) Trypticase Soy Agar II with 5% sheep blood (BD, Heidelberg, Germany) was used to test for hemolysis (24 h at 30 °C). (iii) Egg yolk agar plates (Hardy Diagnostic, Santa Maria, CA, USA) were used to evaluate lecithinase activity (72 h at 30 °C). Lecithinase activity was quantified after taking pictures with a tabletop scanner and analyzing these images using ImageJ^{75 (link)}. Values are given according to the formula (area of bacteria + area of precipitate) − (area of the bacteria) = area of precipitate [cm²].

Staphylococcus aureus strain N315 (ATCC 29213) obtained from BEI Resources (Catalog No. NR-45898), Manassas, VA, United States (Biodefense and Emerging Infections Research Resources)¹ served as the parental DAP-S strain and was propagated in Mueller Hinton Broth (MHB) (BD BBL^TM) and frozen in 15% glycerol in 1 ml aliquots for further use. Daptomycin resistant isolates were derived from the parent N315 strain which was consistently exposed to increasing doses of daptomycin in MHB in a bioreactor or in serial passaged flask culture. For every experiment, blood agar plates (Trypticase Soy Agar II with 5% sheep blood, BD Biosciences) were streaked with bacteria from frozen glycerol stocks. After overnight incubation at 37°C with 5% CO₂, a single colony was used to seed the bioreactor or flask culture populations. Three bioreactor replicates (populations A–C) and one serial passaged flask culture experiment (population P) were performed with populations B and C being exposed to 0, 6, 10, and 14 μg/ml daptomycin and populations A and P being exposed to 6 and 10 μg/ml of daptomycin.