Bacteriophage P68 was purchased from the Félix d’Hérelle Reference Center for Bacterial Viruses (Université Laval, Québec, Canada). The propagation strain of S. aureus RN4220 ΔtarM (16) was provided by A. Peschel from the Department of Infection Biology, University of Tübingen. Phage P68 was propagated on S. aureus RN4220 ΔtarM grown at 37°C in meat peptone broth [13 g of nutrient broth CM1 (Oxoid), 3 g of yeast extract powder L21 (Oxoid), and 5 g of peptone L37 (Oxoid) were dissolved in distilled water to a final volume of 1000 ml, and the pH was adjusted to 7.4 using 10 M NaOH].

Phage lysate from 300 ml of bacterial culture was centrifuged at 5000g for 30 min at 4°C. The resulting supernatant was filtered through a 0.45-μm polyether-sulfone syringe filter (Techno Plastic Products, Switzerland) to remove bacterial debris. Phages were pelleted by centrifugation at 64,000g for 2.5 hours at 4°C. The resulting pellets were resuspended in 350 μl of phage buffer [50 mM tris (pH 8.0), 10 mM CaCl2, and 10 mM NaCl] by mild shaking overnight at 5°C. The resulting solution was mixed with an equal amount of chloroform by gently inverting the tube 10×. The mixture was centrifuged at 3000g for 10 min at room temperature. The aqueous fraction from the chloroform mixture was overlaid onto a preformed CsCl step density gradient (1 ml of each 1.45 g/ml, 1.50 g/ml, and 1.70 g/ml of CsCl in phage buffer) and centrifuged at 194,000g for 4 hours at 12°C using an SW55Ti rotor (Beckman Coulter). Phage particles forming a visible band were collected with an 0.8-mm gauge needle and syringe. Cesium chloride was removed from the phage-containing fraction by dialysis against a 5000× excess of phage buffer at 4°C overnight using Visking dialysis tubing type 8/32″, 0.05 mm thick (part no. 1780.1, Carl Roth, Germany).

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