Calf blood

The clinical strains MRSA 43484 and 88779 were isolated from patients with skin abscesses and were obtained from UNION therapeutics A/S. The laboratory strains of S. aureus used in this study (ATCC 29213, ATCC 25293, NCTC 8325, and JE2) were obtained from the Department of Veterinary Disease Biology at the University of Copenhagen. S. aureus RN4220 and its mutator derivative RN4220 ΔmutS were kindly provided by A. J. O'Neill (University of Leeds, UK).
All strains were cryopreserved in Brain Heart Infusion broth supplemented with glycerol 15% (v/v) at −80°C and cultivated aerobically at 37°C in nutrient agar base no. 2 supplemented with calf blood 5% (v/v) (Oxoid, Basingstoke, UK). All bacterial cultures were grown in Mueller–Hinton broth (MHB) (Oxoid, Basingstoke, UK). When appropriate, culture medium was supplemented with ATx201 (Niclosamide; CAS: 50‐65‐7), rifampicin, fusidic acid, mupirocin, and retapamulin (Sigma‐Aldrich) and the pH adjusted to different values with NaOH and HCl solutions of different concentrations (.1, 1, 2 and 5 M).

Isolates were grown overnight at 37 °C on blood agar (Columbia agar base [Oxoid, Hampshire, UK]) supplemented with 5% calf blood [SSI, Copenhagen, DK]). Single colonies were harvested directly from the agar plates and genomic DNA was purified using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The DNA libraries were generated using Nextera XT kit (Illumina Inc., San Diego, Ca) according to manufacturer’s instructions. Finally, Illumina’s MiSeq platform was used for paired-end DNA sequencing with a read length of 2 × 251 bp for all isolates except the seven isolates from the Danish poultry industry which were sequenced with a read length of 2 × 300 bp.

The faecal samples were thawed at 5 °C overnight. The 10–15 mL faecal samples were diluted in 5 mL phosphate-buffered saline (PBS) buffer and homogenized. Subsequently, 100 µL of the suspensions were spread onto Columbia agar with 5% calf blood (Oxoid, Basingstoke, UK) and 100 µL on MacConkey agar (Oxoid) and incubated at 37 °C. The Escherichia coli colonies were identified by MALDI-TOF, as described by Nonnemann et al. [60 (link)].