Lytic Enzyme Susceptibility Test

The bacterial cells were grown in 20 mL BHI broth to logarithmic stage (OD_600nm = 0.6), and harvested by centrifugation. The cell pellet was suspended in 20 mL of 4% SDS, and inactivated at 65°C for 2 h. The inactivated cell suspension was centrifuged, and the cells were washed with deionized water three times. The washed cell pellet was suspended with PBS to reach 0.4 OD_600nm, and then tested for susceptibility to commercially available lysostaphin (E. coli recombinant, Prospec, Ness-Ziona, Israel) or mutanolysin (recombinant, A&A Biotechnology, Gdynia, Poland) lytic enzyme. Lysostaphin and mutanolysin were added to 1 mL of the tested cell suspension at a final concentration of 8 μg/mL and 0.2 U/mL, respectively, followed by incubation in a plastic cuvette at 30°C at 120 rpm rotation. The reduction in OD by cell lysis was measured using a DU730 spectrophotometer (Beckman Coulter, CA, United States).