Blood agar base bab

The bacterial strains and plasmids used are listed in Additional file 1. We used B. suis bv2 CITA 198 (herein Bs2WT) because, although Bs2WT and the B. suis bv2 reference strain (B. suis bv2 Thomsen) have the same PCR-RFLP pattern [3 (link)], the former shows a virulence pattern in mice typical of B. suis bv2 field strains and the latter is attenuated (Additional file 2).
Bacteria were routinely grown either in standard tryptic soy broth (TSB; Scharlau, Barcelona, Spain) or TSA (TSB supplemented with agar [Pronadisa, Laboratorios Conda, Spain]) at 37 °C. For the studies in mice, vaccines and challenge strains were grown on Blood Agar Base (BAB; Oxoid, UK). When needed, media were supplemented with 5% sucrose, diaminopimelic acid (DAP [Sigma]; 1 mM), 0.2% activated charcoal, kanamycin (Km) at 50 µg/mL or at 35 µg/mL, ampicillin (Amp) at 100 µg/mL and/or chloramphenicol (Cm) at 20 µg/mL (all from Sigma). The lactate-glutamate-glycerol-vitamins synthetic medium of Gerhardt’s [35 (link)] was supplemented with 1 mM methionine (mGSM) (this amino acid is required for growth of some Brucella strains in synthetic media [32 (link)] including Bs2WT (Zúñiga-Ripa, unpublished observations). All strains were stored at − 80 °C in skimmed milk (Scharlau, Barcelona, Spain) or in TSB supplemented with 0.5% yeast extract (Pronadisa, Laboratorios Conda, Spain) (TYSB) and 7% dimethylsulfoxide.

The strains used in this study are listed in Table S1. S. pneumoniae D39 was grown microaerobically at 37°C either in brain heart infusion broth (BHI), blood agar base (BAB) (Oxoid, UK) supplemented with 5% (v/v) defibrinated horse blood, or in chemically defined medium (CDM) supplemented with 55 mM of the selected sugar (Yesilkaya et al., 2006 (link), 2008 (link)). For anaerobic growth, an anaerobic cabinet was used. Escherichia coli was grown in Luria broth with shaking at 37°C, or in Luria agar (Oxoid). Growth was monitored by measuring the OD600, or by determining colony counts. Growth rates (μ) were calculated through linear regressions of the plots of ln(OD600) vs. time during the exponential growth phase. Spectinomycin, tetracycline, and kanamycin were added at 100, 15, and 250 μg/mL, respectively, for pneumococcal cultures, and for E. coli ampicillin and kanamycin were used at 100 and 50 μg/mL, respectively.