2.2.5. Genotypic characterization of isolates
As a prerequisite, DNA was extracted using the DNeasy Blood and Tissue kit using lysostaphin to obtain bacterial lysis. Hence, isolates were genotypically confirmed as S. aureus by means of multiplex-PCR and simultaneous detection of
23S rDNA and nuc .
Strains were further characterized by spa-typing according to Harmsen et al. (2003), and in some cases multi-locus-sequence-typing (MLST) . In addition, a commercially available microarray kit (Identibac S. aureus Genotyping, Alere Technologies GmbH, Jena, Germany) was applied.
This array covers 333 target sequences corresponding to approximately 185 distinct genes and their allelic variants.
These include among others species-specific controls, genes encoding for relevant antibiotic resistance determinants
and virulence factors (SE encoding genes) as well as agr group and capsule typing markers.
The array was performed according to the manufacturer's instructions and analysis of the array profiles based on
the presence or absence of the enquired genes was done with Bionumerics Software