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Fig. 1 An experimental model of S. aureus evolution in human macrophages. (A) Schematic representation of the long-term S. aureus THP-1 macrophage infection protocol. For each infection cycle, THP-1 macrophages were differentiated by PMA stimulation (as described in Materials and Methods) and infected with Lineages 1?4 of N315 or SH1000 for 1 h. Bacteria were kept under intracellular selective pressure by incubating the culture with a low dose of gentamicin for 48 h or 72 h. Antibiotics were then removed and the bacteria were allowed to escape and multiply in the extracellular space for 24 h. As a control, N315 and SH1000 Lineages 5 and 6 were grown in macrophage culture media alone. The bacteria were incubated at 37°C for 1 h and then incubated at 4°C for 48 h to 72h to mimic the growth restriction of the antibiotic step of Lineages 1?4. The culture was then moved to the 37°C incubator for 24 h. At the end of the cycle, the cell culture media was collected and used for a new infection cycle, bacterial quantification, and colony analysis in TSA and blood agar plates, and the remainder was stored at ?80°C in glycerol. Figure made using Servier Medical Images. (B and C) Amount of bacteria used for infection at the beginning of each infection cycle and recovered at the end of each cycle, after 24 h at 37°C without antibiotics. Lineages 1?4 evolved in the presence of THP-1 macrophages and Lineages 5?6 in macrophage culture media alone. Total of 38 cycles.