Columbia blood agar base plates

S. suis strains S735-pCOM1-orf2 [22] and S735-pCOM1 [18], which are both derivatives of strain S735 [18] and strain 10 [23] have been described. S735 is a weakly virulent isolate of serotype 2. When orf2 (locus tag SSU0135 in P1/7 genome) from the virulent S. suis strain 10 was expressed from plasmid pCOM1-orf2 in strain S735 (S735-pCOM1-orf2), the virulence of the strain was increased drastically [22]. Bacterial strains were grown overnight at 37°C and 5% CO₂ on Columbia blood base agar plates (Oxoid) containing 6% (vol/vol) horse blood and 1 µg ml⁻¹ of erythromycin for plasmid maintenance. To grow the bacteria in liquid culture, one bacterial colony collected from blood base agar plates was propagated in Todd-Hewitt Broth (THB, Oxoid) supplemented with 1 µg ml⁻¹ erythromycin and incubated overnight in 100-ml culture bottles in the same conditions. The culture was then diluted in THB to an optical density of 600 nm of 0.1 and incubated till exponential phase. To grow the bacteria in host body fluids, porcine serum (VWR), and heparinized fresh blood derived from 3–5 weeks old piglets (WBVR) were used. Shortly, THB cultures at logarithmic phase were diluted to ∼10⁷ colony-forming units (CFU) ml⁻¹, inoculated in 100% normal porcine serum or whole blood, and incubated at 37°C. Bacterial load was monitored by CFU counting using Colombia agar plates.

S. suis strain 10 was previously described [17 (link)]. Bacteria were grown at 37°C and 5% of CO₂ on Columbia blood base agar plates (Oxoid) containing horse blood 6% (vol/vol) and supplemented with 100 µg/ml of spectinomycin when appropriate for a selection of transformants. For bacterial liquid cultures, one colony collected from solid culture was propagated in Todd-Hewitt Broth (THB, Oxoid) and incubated overnight in 100 ml-culture bottles under the same conditions.
For growth under in vivo mimicking conditions, overnight cultures were diluted in fresh THB supplemented with 0.5 mM 2,2-Bipyridyl (Bipyridyl), 10 mM sodium fluoride or 3 mM H₂O₂, incubated in a Bioscreen C instrument (Thermo Scientific) at 34°C, 37°C, or 42°C and the optical density at 600 nm was monitored every 20 min. For studying the growth of the bacteria in different host fluids, overnight THB cultures were diluted to around 10⁶ CFU/ml in Dulbecco’s phosphate-buffered saline, and bacteria were inoculated in fresh porcine blood, 100% commercial porcine serum (Sigma-Aldrich) or porcine cerebrospinal fluid (CSF) and incubated at 37°C and 5% of CO₂. Blood and CSF were collected from healthy 3–6 -week-old specified pathogen-free piglets from a high health farm, as piglets are most at risk for streptococcal suis infection at 4–10 weeks. Bacterial survival was monitored by CFU counting using solid agar plates.

Campylobacter jejuni NCTC 11168 (ATCC 700819), S. typhimurium SL1344, EIEC (ATCC 43893), and L. monocytogenes (ATCC 19114) were acquired from the Culture Collection of Food Microorganisms of Jiangnan University (Wuxi, China). Columbia blood agar base plates (Oxoid, United Kingdom) supplemented with 5% sterile sheep blood and C. jejuni selective supplement (Oxoid) were used to culture C. jejuni strains under microaerophilic conditions (5% O₂, 10% CO₂, 85% N₂) for 48 h at 37°C. S. typhimurium, EIEC and L. monocytogenes were cultured with brain-heart infusion broth (Haibo, China) for 24 h at 37°C.