Staphtype

Spa typing was performed using PCR followed by Sanger sequencing, as previously described [24 (link)]. Assignment of spa type Based Upon Repeat Pattern (BURP) analysis for determination of spa clonal complex (spa-CC) was performed using Ridom StaphType. The Ridom SpaServer was used to predict the multi-locus sequence types (STs) as described previously [25 (link)]. Phylogenetic trees were constructed using RAxML Tree, Geneious Version 2022.2, (https://www.geneious.com).

The spa typing was performed as previously described (32 (link)), and the individual spa types were assigned by using the Ridom StaphType software package, version 2.2.1 (Ridom, Germany). They were clustered into spa clonal complexes by using the based upon repeat pattern (BURP) algorithm. The multilocus sequence typing STs and/or clonal complexes (CC) were either inferred by using the SpaServer database (http://spaserver.ridom.de) or derived from previously published studies.
SCCmec types I to VI were determined by using multiplex PCR (33 (link)), and the subtyping of the IVa to IVd and IVg to IVh types was performed (34 (link), 35 (link)).

Spa typing, based on the amplification of the variable X region of the protein A gene, was performed as described previously [75 (link)]. After sequencing, the spa type was assigned using the Ridom StaphType software version 2. 2. 1 (Ridom GmbH, Würzburg, Germany) and the Ridom SpaServer (SpaServer.ridom.de/">https://SpaServer.ridom.de/; accessed on 25 May 2020).

The X region of the spa gene in each MRSA was amplified using PCR (Supplementary Table) (16 (link)). The amplified products were then sequenced and analyzed with the Ridom StaphType software (version 1.4; Ridom, GmbH, Wurzburg, Germany).² This software automatically identified the repeat profile and the spa type for each isolate.

The X region of the spa gene of each MRSA isolate was amplified by PCR and the amplified products were sequenced. Variation of short repeated regions in the spa gene was analyzed through Ridom StaphType software (Ridom GmbH, Würzburg, Germany) [20 (link)].

Positive S. aureus isolate genomic DNA was isolated using Wizard Genomic DNA preparation kit (Promega, Madison, WI). Polymerase chain reaction (PCR) was used to amplify the presence of methicillin resistance gene (mecA) and PVL genes (lukS, lukF) [24 (link), 25 (link)]. Furthermore, Staphylococcus protein A (spa; FOR 5’-GAACAA-CGTAACGGCTTCATCC-3′ and REV 5’-CAGCAGTAGTGCCGTTTGCCT) was used for molecular typing [26 (link)–28 (link)]. Ridom StaphType software was used to assign spa types (v2.2.1; Ridom GmbH, Wurzburg, Germany). The Based upon Repeat Pattern (BURP) algorithm was used to group spa types based on their genetic proximity [29 (link)], as well as Bionumerics software (version 7.6.2). Only spa typing was conducted, since previous studies have found high congruence and discriminatory power compared to MLST sequence data [28 (link), 30 (link), 31 ]. A positive (USA300) and negative control were used for all biochemical and molecular assays.

Spa-typing was carried out by PCR and amplicon sequencing in all S. aureus isolates. The spa sequences were analyzed using Ridom Staph-Type software version 1.5.21 (Ridom GmbH, Münster, Germany). Multilocus sequence typing (MLST) was performed on S. aureus and S. pseudintermedius isolates, as well as on a selected S. epidermidis isolate (Bannoehr et al., 2007 (link); Ruiz-Ripa et al., 2019 (link)). All isolates carrying the mecA gene were subjected to SCCmec typing by multiplex PCRs, and agr-typing was characterized following standard methodology in all S. aureus and S. pseudintermedius isolates (Zhang et al., 2005 (link); Perreten et al., 2010 (link)).