Atcc 6303

Pneumococcal pneumonia was induced by either serotype 2 S. pneumoniae (D39), a non-encapsulated mutant strain (isogenic capsule locus(cps) deletion mutant D39Δcps) of D39 [11 (link),12 (link)] or a serotype 3 pneumococcal strain (ATCC 6303, American Type Culture Collection, Manassas, VA). All bacterial strains were grown for 3–6 hours to midlogarithmic phase at 37°C in Todd-Hewitt broth (Difco, Detroit, MI), supplemented with yeast extract (0.5%). Bacteria were harvested by centrifugation at 4000 rpm, and washed twice in sterile isotonic saline. Next, mice (n = 8 per strain for each time point), were inoculated with 10⁷ colony forming units (CFU) D39, 10⁸ CFU D39Δcps or 5 x 10⁴ CFU serotype 3 S. pneumoniae per mouse in a 50 μl saline solution and sacrificed 6, 24 or 48 hours thereafter as described [9 (link),12 (link)]. All mice survived to the predefined endpoint in all experiments. Collection and handling of samples were done as described [9 (link),12 (link)]. In brief, blood was drawn into heparinized tubes and organs were removed aseptically and homogenised in 4 volumes of sterile isotonic saline using a tissue homogenizer (Biospec Products, Bartlesville, UK). To determine bacterial loads, ten-fold dilutions were plated on blood agar plates and incubated at 37°C for 16 hours.

Streptococcus pneumoniae ATCC 6303 (a type 3 strain) was obtained from the American Type Culture Collection (ATCC). For in vivo challenges, frozen bacterial stocks of S. pneumoniae were streaked out on Trypticase Soy Agar II (TSA-II, BD Biosciences) plates containing 5% sheep blood. A single colony was inoculated into 5 ml of Todd-Hewitt broth with 0.5% added yeast extract (THY; both from Sigma Aldrich), and grown at 37°C with 5% CO₂ without shaking until bacteria reached an OD of 0.45–0.55 at 600 nm, representing ~10⁸ CFU/ml. Bacteria were spun down and resuspended in Dulbecco's Phosphate Buffered Saline (DPBS, Fisher Scientific), then diluted to the appropriate intended inoculum (7 × 10⁴-1.5 × 10⁶ CFU per mouse). 20 μl of bacterial suspension was inoculated per nare (40 μl total per mouse). The inoculum was verified after serial dilution in DPBS and plating on TSA-II plates with 5% sheep blood, incubated overnight at 37°C with 5% CO₂.

Pneumococci were maintained and heat-killed as described previously [33 (link)].
The pneumococcal strains ATCC 6301, ATCC 6303, ATCC 6305, ATCC 6314, ATCC 6319, ATCC 6323, ATCC 6326 and ATCC BAA-334 (referred to as TIGR4 in this study) were obtained from American Type Culture Collection (ATCC), USA. The corresponding serotypes according to ATCC are 1, 3, 5, 14, 19, 23, 26 and 4, respectively. D39 (NCTC 7466) and A66 (NCTC 7978) were sourced from National Collection of Type Cultures, United Kingdom, and are of serotypes 2 and 3, respectively. R36A (unencapsulated variant of D39) was procured from ATCC. The strains were chosen based on their serotype, virulence status in mouse models and the fact that the strains have been used by several investigators in the field to do similar in vitro or in vivo experiments.