Sheep blood

Types 4 and 6A strains used in the study are described in Table 1. Clinical isolates were confirmed as Spn by sensitivity to ethylhydrocupreine hydrochloride (optochin) and typed using specific antisera (Statens Serum Institute). Pneumococci were grown statically in tryptic soy (TS) broth (Becton, Dickinson) at 37°C. Upon reaching the desired OD_620nm of ~1.0, cells were washed and diluted in sterile phosphate-buffered saline (PBS) for inoculation. For quantitative culture, serial dilutions were plated on TS broth agar supplemented with an appropriate antibiotic and either 5% sheep blood or catalase (6,300 U/plate; Worthington Biochemical Corporation) and incubated overnight at 37°C with 5% CO₂.
Colonies expressing different amounts of CPS were selected by visual screening using microscopy with oblique, transmitted illumination as described [16 (link)]. Mutants were confirmed by whole-genome sequencing. A corrected mutant (P2752) of the cpsE mutation in P385 was constructed by transforming P385 as previously described [26 (link)] with the cps PCR product of P376 using primers 5’- ACC ATT GTC TCT ACC TCT CAC -3’ and 5’- CGG AAT TCC TGT AAT TGA TGT CAT -3’. The transformation was verified using sequencing primer 5’- GAA GAT TCT CCT ACT TAC AGC AAC -3.

Unless otherwise specified, pneumococcal strains (described in Table S1) were grown statically in tryptic soy (TS) broth (Becton, Dickinson) at 37°C. Upon reaching the desired optical density of 1.0 at 620 nm, cells were washed and diluted in sterile phosphate-buffered saline (PBS) for inoculation. The diluted strains were mixed together at the desired density to generate inocula containing equal proportions of strains. For quantitative culture, serial dilutions were plated on TS broth–streptomycin (200 μg/mL) agar supplemented with either 5% sheep blood or catalase (6,300 U/plate; Worthington Biochemical Corporation) and incubated overnight at 37°C with 5% CO₂. To compare growth in nutrient conditions, capsule-switch mutants were grown in Brain Heart Infusion medium containing fetal bovine serum as previously described (29 (link)).