Bacterial growth and infection

The S. pneumoniae D39-GFP strain was grown on blood agar plates overnight at 37°C with 95% O₂/5% CO₂. Single colonies were picked from plates and grown in brain-heart infusion broth at 37°C with CO₂ until an OD 600 of 0.5 was reached. Bacteria were resuspended in saline and diluted to achieve appropriate dose with a 100-µl volume. For long-term infection and acute bacteria infection, doses of 2 × 10³ CFU or 10⁴ CFU and 5 × 10⁶ CFU were administered i.v., respectively. Generation of reporter bacteria for oxidation was performed as previously described (Surewaard et al., 2016). In brief, fresh streptococcal cultures were washed twice in saline, resuspended at 5 × 10⁸ CFU in 500 µl in carbonate, pH 8.3, buffered saline, and labeled for 30 min with 20 µg ml⁻¹ AF647 N-hydroxysuccinimide ester (Thermo Fisher Scientific) and 60 µg ml⁻¹ OxyBURST Green H2DCFDA SE (DMSO stock; Thermo Fisher Scientific) under vigorous agitation. Activation of OxyBURST was accomplished by adding 250 µl of 1.5 M hydroxylamine, pH 8.5, and incubating for 30 min on ice. Reporter bacteria were washed twice with PBS and injected i.v. into mice at 5 × 10⁶–10⁷ CFU.