agr, toxin and MSCRAMM characterization
Genomic DNA, used as a template for PCR amplification, was extracted with QIAamp®DNA Mini Kit (Cat No. 51306, Qiagen) following the manufacturer’s instructions, with some modifications. Briefly, a bacterial suspension was centrifuged and the pellet was resuspended in 200ul physiological saline solution 0.9% and subjected to freezing and thawing twice. After centrifugation the bacteria pellet was resuspended in 180µl of enzyme solution: 20 mg/mL lysozyme (cat No. 10837059001 Sigma-Aldrich-Merck KGaA, Darmstadt, Germany) and 100 μg/ml lysostaphin (cat. No. L7386-15MG, Sigma-Aldrich-Merck KGaA) in TE buffer pH 8.0 (cat.No. AM9849 Ambion, Invitrogen). After these changes, the indications provided by the manufacturer were followed.
Toxin and MSCRAMM genes included in the study and listed in table S1 were tested as previously described (Stefani et al. , 2009). Theagr locus was typed using a multiplex PCR assay (Gilot et al. , 2002). PCR for the cna gene was performed used the following primers in 5’-3’: F- GGAAAACGACCAACTGAAATCAAAG, R- TCTGGCGTATATTTATTCGTCACAATC. PCR was performed at 57°C, the product size was 239bp, strain MW2 was used as internal control.
PCR amplification was carried out in a Veriti Thermal Cycler (Applied Biosystems, ThermoFisher, Italy) in a total volume of 25µl containing 2× Multiplex PCR Master Mix (cat. No. BR0200804, biotechrabbit GmbH, Hennigsdorf, Germany), and 10ng template DNA.