Intracellular Survival Assay for Pneumococcal Persistence

The intracellular survival assays of pneumococci were performed as reported previously (15 (link), 16 (link)). For the treatment with NAC (Sigma A9165), A549 cells were incubated with 5 mM NAC for 3 h, while Raw 264.7 cells were incubated with 10 mM NAC for 1 h. The NAC treatment was carried out in parallel to the bacterial infection protocol, as described (17 (link)). Apoptosis/necrosis was quantified by flow cytometry using a propidium iodide labeling kit (ThermoFisher). The cell infection scheme was carried out as described in Fig. S7.
To analyze the emergence of FQ-persistent pneumococci, the bacterial-infected A549 and Raw 264.7 cells were cultured in DMEM-1% FBS-6μg/mL levofloxacin. The pneumococci-infected PLB-985 and PLB-985-KO cells were cultured in RPMI 1640/1% SFB/1,3% DMSO/6 μg/mL levofloxacin. Cell cultures were incubated at 37°C and 5% CO₂ at different time points. Cells were lysed by centrifugation for 10 min at 15,000 g and the bacterial pellet was resuspended in BHI. The number of internalized bacteria was quantified after serial dilutions of lysates and plating on blood agar plates. The number of surviving bacteria obtained at t0 was defined as 100% survival in all the cases, and the data obtained at different time points were used to calculate the respective percentages of FQ persisters.