4.6. Long-Term Persistence

The human osteoblasts cell line hFOB 1.19 (ATCC CRL-11372) was cultivated, following the manufacturer’s indications, and it was infected with different S. aureus strains (Table S5). Briefly, osteoblasts cells were infected with a MOI (multiplicity of infection) of 50. After 1.5 hr, cells were washed with PBS, and lysostaphin (20 μg/mL) was added for 30 min, to lyse all extracellular or adherent staphylococci, and then fresh culture medium with Penicillin and streptomycin were added to the cells. The washing, the lysostaphin, and medium-exchange steps were repeated every two days, to remove all of the extracellular staphylococci. To detect live intracellular bacteria at different time points post-infection (p.i.), host cells were lysed in H₂O, and the number of colony-forming units (CFU) was determined by serial dilutions on blood agar. The colony phenotypes were analyzed by a Colony Counter Shuett (Biosys, Karben, Germany). SCVs were colonies with a diameter < 0.6 mm.