Staphtype software version 2

DNA extraction: Strains were grown overnight in tryptic soy broth at 37 °C. For PCR analysis, genomic DNA was extracted using the DNeasy tissue kit (Qiagen, Hilden, Germany), and lysostaphin (100 mg/L; Sigma, Taufkirchen, Germany) for bacterial lysis.
PCR for resistance genes: PCR analysis was performed for mecA and mecC in order to confirm MRSA as previously described [36 (link)].
Spa-typing, BURP and MLST: All S. aureus isolates were subjected to spa-typing as previously described [36 (link)]. Related spa-types (costs ≤ 4) were grouped into spa-clonal complexes (spa-CC) by use of the BURP algorithm (Ridom StaphType software version 2.2.1, Ridom, Münster, Germany). The spa-CCs were allocated to MLST CCs (Ridom SpaServer—Spa-types; database of the German National Reference Center for Staphylococci and Enterococci). Subsets of the isolates were subjected to MLST as described elsewhere [36 (link)].

MRSA status was confirmed by PCR for mecA at the RKI [30 (link)]. Spa typing of the MRSA isolates was performed as described by Harmsen et al. [31 (link)]. All spa types were assigned with Ridom StaphType software version 2.2.1 (Ridom GmbH, Wurzburg, Germany). PCR for luk-PV was performed as described previously [32 (link)]. Multi-locus sequence typing (MLST) was performed for each representative isolate for each spa type according to Enright et al. [33 (link)]. Primers used for MLST correspond to the protocol as described previously, with the exception of the forward primer for tpi; we used the sequence 5-GCAT TAGCAGATTTAGGCGT-3. Assignment to sequence types was performed by means of the MLST net database (http://www.mlst.net/submissions/default.asp).

Using the primers listed in Table 8, all the investigated MRSA isolates (n = 34) were subjected to a PCR assay for amplification of the polymorphic repeat region (X region) of the spa gene as described elsewhere^{90 (link)}. The spa amplicons were then purified using a Qiagen DNA purification kit (Qiagen GmbH, Hilden, Germany) and sequenced by Macrogen® (Seoul, South Korea) using capillary electrophoresis. spa typing was conducted as described by Harmsen et al.^{91 (link)}, and the resulting spa types were then clustered into related spa clonal complexes (spa-CCs) using the BURP algorithm implemented in the Ridom StaphType software version 2.2.1 (Ridom GmbH, Würzburg, Germany). The default parameters of the BURP algorithm (exclusion of spa types shorter than 5 repeats and clustering of spa types if cost is less or equal to 5) were applied in this analysis, in order to prevent the formation of spa clusters that are too large or non-specific^{92 (link)}. The spa type that could not be assigned to a cluster was considered as a singleton. Due to the high concordance between spa typing and MLST^{24 (link)}, the MLST clonal complexes (CC) corresponding to the respective spa-CCs were deduced from the data on the Ridom SpaServer (http://spaserver.ridom.de, last accessed on 15 November 2016) and by literature search.

All S. aureus isolates were screened for the presence of Panton Valentine Leucocidin (PVL) by PCR as previously described [22 (link)]. Spa-typing of S. aureus isolates was performed as previously described by Harmsen et al. [23 (link)]. DNA sequences were determined using an ABI Prism 3130 genetic analyser (Applied Biosystems, Foster City, USA). Spa-types were assigned using the Ridom Staph Type software version 2.2.1 (Ridom GmbH, Würzburg, Germany) [23 (link)]. Multilocus Sequence Typing (MLST) was performed on a subset of isolates (n = 70) as described by Enright et al. [24 (link)].