, 2004; Marlinghaus et al., 2011). To impair adhesion due to fibrinogen this website binding, this isolate was selected for a knockout of the fbl gene by homologous recombination and the knockout mutant was named MB105 (Table 1). Fibrinogen binding was completely abolished in the MB105 mutant in contrast to their fibronectin-binding attributes (Fig. 1a and b). Clinical isolates of S. lugdunensis invaded the human bladder carcinoma cell line 5647 relative to the invasion

of S. aureus Cowan I, which was defined as 100%. The non-invasive S. carnosus TM 300 has been shown to have a relative invasiveness of 11.6%. Some clinical isolates of S. lugdunensis were internalized up to 6.7-fold compared with S. carnosus, which is equivalent to a relative invasiveness of 78% of that of S. aureus Cowan I (Fig. 2a). Clinical isolates of S. lugdunensis invaded the endothial cell line EA.hy 926. The invasion of S. aureus Cowan I into the cell

line EA.hy 926 was defined as 100%. The non-invasive S. carnosus TM 300 has been shown to have a relative invasiveness of 7.5% to that of S. aureus Cowan I. Some clinical isolates of S. lugdunensis were internalized up to 7.4-fold compared with S. carnosus, which find more is equivalent to a relative invasiveness of 55% of that of S. aureus Cowan I (Fig. 2b). The invasion of epithelial and endothelial cells as determined by the FACS-invasion assay was confirmed by characterizing the intracellular location of the bacteria. A previously described intra/extracellular staining method (Agerer et al., 2004) and TEM were thus used (Hamill et al., 1986). FITC-stained and biotin-labeled bacteria were submitted to the invasion experiment to stain extracellular bacteria. After invasion of cells, extracellular bacteria were stained with streptavidin-conjugated Alexa 647. Cells and bacteria (intra- and extracellular) were investigated by confocal microscopy as previously described (Agerer et al., 2004). Up to 10 FITC-stained bacteria were found in selected Tyrosine-protein kinase BLK planes of 5637 cells

(Fig. 3). To confirm the intracellular location of the bacteria by a third method, human urinary bladder carcinoma cell line 5637 treated with S. lugdunensis were submitted to electron microscopy. In TEM, S. lugdunensis was detected inside human urinary bladder carcinoma cells, surrounded by a phagosome-like membrane, similar to pictures described for invasive S. aureus (Sinha et al., 1999) and S. saprophyticus (Szabados et al., 2008) strains. Up to 20 bacteria per cell were found in selected eukaryotic cells (Fig. 4). Fibrinogen-binding adhesins have been described for a variety of bacteria (Palma et al., 2001). One might expect that adhesion to eukaryotic cells via binding to fibrinogen could supposedly promote invasion. Nevertheless, an effect of fibrinogen on the invasion of cells has not been described for S. aureus. The invasion of the clinical strains of S.