Lysostaphin solution

Strains were grown overnight on Trypticase Soy Agar (BD Biosciences, Sparks, MD) at 37°C to obtain isolated colonies. Individual colonies were selected to start an overnight culture of Trypticase Soy Broth (BD Biosciences, Sparks, MD). After 12–18 hours of growth, 750 μl of the liquid culture was pelleted and the supernatant was removed. The pelleted cells were stored at -80°C until DNA extraction. To isolate DNA, each pellet was resuspended in 200 μl 1x Phosphate Buffered Saline with 0.2 M EDTA. To lyse the cells, the following were added to each suspension: 12 μl of Lysozyme solution (Sigma, St. Louis, MO), 1 μl RNase (Roche, Mannheim, Germany), 7.5 μl Lysostaphin solution (Sigma, St. Louis, MO), and 7.5 μl Mutanolysin solution (Sigma, St. Louis, MO). The cells were then incubated for 1 hour at 37°C. Forty microliters of Proteinase K (Roche, Mannheim, Germany) was added and the suspension was incubated overnight at 55°C. The following day, a Roche High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) was used to isolate DNA according to the manufacturer’s protocol. The Elution Buffer containing DNA samples was centrifuged for 5 minutes at 8000xg to remove visible debris. The supernatant was transferred into a clean 1.5 mL tube and stored at 4°C until further analysis.

Bacterial isolates were cultivated overnight according to the propagation procedure of the supplier (Microbiologics, MN, United States). Automated DNA extraction from Gram-positive and Gram-negative bacteria was performed on a QIAsymphony instrument (QIAGEN, Hilden, Germany) using the QIAsymphony DSP DNA Kit (QIAGEN). Modifications of the manufacturers standard protocol included: for extraction from S. aureus addition of lysostaphin solution (5 U per sample; Sigma-Aldrich, St. Louis, MO, United States); lysis of Gram-positive bacteria on device (37°C/shaking at 900 rpm for 1 h); a third wash step to enhance purification for extraction of Gram-positive and Gram-negative bacteria. Each independent DNA extraction contained a no template control (NTC) containing molecular grade water only.