Todd hewitt broth

The samples were processed as recommended by the American Society for Microbiology’s guidelines^{24 (link)}. Briefly, after incubating the swabs in Todd-Hewitt broth (Thermo Scientific™, Singapore) aerobically at 37 °C for 18–24 h, 10 μl of each broth was subcultured on Columbia agar plates with 5% sheep blood (Oxoid, Singapore). The plates were incubated for 24 h at 37 °C in 5% CO₂. If GBS was not detected, the blood agar plate was incubated and examined after 48 h. All suspected GBS appeared as either beta-haemolytic or nonhemolytic, and Gram-positive cocci and catalase-negative cocci were taken for the CAMP (Christie–Atkins–Munch-Peterson) test. Streptococcus pyogenes ATCC 19615, Streptococcus agalactiae ATCC12386, and Staphylococcus aureus ATCC 25923 (Thermo Scientific™, Singapore) were the controls. All colonies that yielded a positive CAMP were considered GBS. Positive CAMP test results were confirmed using the Streptex™ Latex Agglutination test (Thermo Scientific™, Singapore) according to the manufacturer’s instructions. This latex agglutination test provides a complete solution for the isolation and differentiation of Lancefield Groups A, B, C, D, F, and G.

Group A streptococcus serotype M49 strain 591 was kindly provided by R. Lütticken (Aachen, Germany). All GAS strains were cultured in chemically defined medium (CDM) (van de Rijn and Kessler, 1980 (link)) or Todd-Hewitt broth (Thermo Fisher Scientific, Darmstadt, Germany) supplemented with 0.5% yeast extract (Thermo Fisher Scientific, Darmstadt, Germany) (THY), as indicated, at 37°C with a 5% CO₂/20% O₂ atmosphere. Escherichia coli strain DH5α (Gibco BRL, Eggenstein, Germany) was used as host for the construction, proliferation, and storage of recombinant plasmids. All E. coli strains were cultured in Lennox L Broth Base (Thermo Fisher Scientific, Darmstadt, Germany). For selection, antibiotics were added at the appropriate concentrations.

Nine S. suis isolates were evaluated in this study (SRD478, ISU2414, ISU2514, ISU2614, ISU2714, ISU2812, ISU2912, ISU1606, ISU2660) alongside a reference isolate (P1/7) with known virulence (Table 1). S. suis isolates were routinely grown at 37°C in Todd-Hewitt broth (Thermo Fisher Scientific Inc., Waltham, MA, United States) supplemented with 0.2% yeast extract (MilliporeSigma, St. Louis, MO, United States) (THY) and 5% filtered heat-inactivated horse serum (MilliporeSigma, St. Louis, MO, United States) (THY+) or on tryptic soy agar (TSA) containing 5% sheep blood (Becton, Dickinson and Co., Franklin Lakes, NJ, United States).

A total of 106 S. suis isolates obtained from swine samples collected within the US and submitted to the University of Minnesota Veterinary Diagnostic Laboratory between 2015 and 2017 were selected for the project (Supplementary Table 1). Frozen stocks (−80°C in 30% glycerol) of S. suis isolates were streaked onto tryptic soy agar containing 5% sheep blood (Becton, Dickinson and Co. Franklin Lakes, NJ) and grown overnight aerobically at 37°C with 5% CO2. Single colonies were used to inoculate Todd-Hewitt broth (Thermo Fisher Scientific Inc., Waltham, MA) supplemented with 0.2% yeast extract (MilliporeSigma, St. Louis, MO) and 5% filtered heat-inactivated horse serum (MilliporeSigma, St. Louis, MO). Broth cultures were grown aerobically at 37°C overnight in a shaking incubator (250 rpm).

S. mutans UA159 strain (ATCC 700610) was used in this work. Todd–Hewitt broth (Thermo Fisher Scientific Inc., Mississauga, ON, Canada) supplemented with 0.3% (w/v) yeast extract (THYE) was used for bacterial cultivation. S. mutans cells were cultivated at 37 °C with 5% CO₂. A nonpolar allelic replacement technique was used to generate the deletion mutants in the UA159 wild-type (WT) strain [20 (link)]. For the construction of the vector for toxin overexpression, the full-length coding region of the relE40 gene was PCR-amplified using UA159 genomic DNA and a PCR product cloned under the control of the constitutive promoter P₂₃ into the E. coli Streptococcus shuttle plasmid pIB166 [21 (link)] harboring a chloramphenicol resistance cassette. The construct was then transferred into the UA159 WT strain via natural transformation [22 (link)].

Transformations were carried out as previously described (7 (link)). Briefly, mid-logarithmic-phase cultures of the recipient strain were diluted 1:20 in competence medium (Todd-Hewitt broth; Oxoid, Basingstoke, United Kingdom) containing 1 mM calcium chloride (Sigma Aldrich Ltd., Dorset, United Kingdom), 0.2% bovine serum albumin (BSA; Sigma Aldrich Ltd., Dorset, United Kingdom), and 100 ng/ml competence-stimulating peptide 1 (CSP1; Mimotopes, Clayton, Victoria, Australia). Donor DNA was added at various concentrations to 500-µl aliquots of a competent cell suspension. Transformation reaction mixtures were incubated for 3 h at 37°C, and then 20-μl and 200-μl volumes were spread onto selective agar plates. Viable counts were determined in parallel to allow estimation of the transformation frequency.