Phagocytosis and Killing of S. aureus by Neutrophils

Phagocytosis and killing of S. aureus by human neutrophils in presence of selected compounds was determined as described before^{47 (link)}. Polymorphonuclear cells (PMNs) were isolated from healthy human heparinized-defibrinated blood (Seralab Logistics) using Ficoll-Plaque PREMIUM (GE-Healthcare) according to manufacturer’s protocol and resuspended at a final concentration of 1 × 10⁷ PMNs ml⁻¹ in HBSS supplemented with human serum. S. aureus strains were cultured to the early stationary phase and 10 ml of culture were centrifuged, washed twice with sterile PBS and resuspended in Hank’s Balanced Salt Solution (HBSS) supplemented with human serum at a final concentration of 4 × 10⁵ bacteria ml⁻¹. Finally, 0.2 ml of PMNs solution was mixed with 0.2 ml of S. aureus solution and 600 μl of HBSS supplemented with human serum. Compounds were added at a final concentration of 5 μM. After incubation at 37 °C for 30 min, each sample was treated with gentamicin 100 mg ml⁻¹ and then 100 μl of each mixture were added to 1 ml of pH 11 solution. Finally, serial dilutions were plated on TSA to determine the number of colony-forming units (CFU) in presence of the different compounds. All data were referred to initial CFU number.