Haemolytic assays were carried out as previously described [30 (link)]. Briefly, whole blood was washed twice with phosphate-buffered saline (PBS) and resuspended in PBS to obtain 8% (wt/vol.). Blood was used within the first hour. To prepare the supernatants, five isolated colonies of each strain (ST121: Jwt, CEU268, CEU890 and ST96: DL9, CEU852, CEU886) were sown in 5 mL of TSB and kept at 37 °C for 6 h with shaking. All the cultures were centrifuged for 10 min at 3000 RPM and the supernatant was filtered. Then 100 μL of 8% red blood cells (RBC) were mixed with 100 μL of the different dilutions of the culture supernatants (100%, 50%, 25% and 10%) in 1.5 mL tubes, which resulted in 4% of the final RBC. The mixture was incubated at 37 °C for 30 min and centrifuged for 2 min at 7 K RPM in a minispin centrifuge (Eppendorf, Hamburg, Germany). Next 150 μL of supernatants were transferred to ELISA plates (Sigma-Aldrich, Missouri, USA) without disturbing the pellet. OD 450 nm was read to determine the haemolysis capacity of strains ST121 and ST96. For the positive control, RBC were mixed with 20% Triton X-100. For negative control, RBC were mixed with PBS. This protocol was carried out using three strains of each ST type to compare the virulence between strains ST121 and ST96.
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