Molecular Characterization of Staphylococcus Isolates

Genomic DNA was extracted using the Wizard Genomic DNA preparation kit (Promega, Madison, WI). The staphylococcus protein A (spa) gene was amplified as described [29 (link), 30 (link)]. The detection of the mecA and PVL genes was determined by PCR [31 (link), 32 (link)]. spa types were assigned using Ridom® StaphType software (version2.2.1; Ridom GmbH, $\mathrm{W}\ddot{\stackrel{-}{\mathrm{u}}}\mathrm{r}\mathrm{z}\mathrm{b}\mathrm{u}\mathrm{r}\mathrm{g}$ , Germany). The Based upon Repeat Pattern (BURP) algorithm was used to group spa types into genetic clusters based on their genetic proximity [33 (link)]. A selected number of isolates were subjected to multilocus sequence typing (MLST) as described previously [34 (link)]. The most common, unique, and/or isolates of interest were chosen for MLST (e.g., those which were present in high numbers, low numbers, or unique in some manner including a novel spa type). Sequence types (STs) were assigned using organism specific MLST database (https://pubmlst.org/saureus/). PHYLOViZ software v2.0 was used to analyze Global optimal eBURST of STs and to draw minimum spanning tree and relatedness of STs as described by Francisco et al. [35 (link), 36 (link)]. Positive (USA 300) and negative controls (reaction mixture without DNA template) were used in mecA, PVL, and spa PCR.