Cloning of fnbB gene fragments Generic primers, corresponding to conserved DNA encoding the signal sequence and fibronectin binding domain 2, were designed from conserved sequences in fnbB genes from publicly available S. aureus genomes. PCR products were cleaved with BamHI restriction sites incorporated into the primers, ligated to BamHI-cleaved pBluescript DNA and transformed into E. coli. The cloned fnbB gene fragments were sequenced using T3 and T7 primers by GATC Biotech AG (Germany).

DNA hybridisation using fnbB type-specific probes DIG-labelled isotype-specific probes were synthesised by PCR. Primers were designed to amplify a small region of DNA (~300 bp) in the N3 sub-domain of isotypes I-VII. The PCR products were labelled by incorporating DIG-labelled dNTPs (Roche). Five ng of DNA encoding the A domain of FnBPB from clinical this website isolates was spotted onto positively charged nylon membranes (Roche) and allowed to air-dry. Membranes were incubated for 5 min on blotting paper soaked in denaturation selleckchem solution (1.5 M NaCl, 0.5 M

NaOH), 5 min in neutralization solution (1.5 M NaCl, 1 M Tris-HCl, pH 7.4), and finally check details for 15 min on blotting paper soaked with 2× SSC solution (300 mM NaCl, 30 mM tri-sodium citrate). DNA was fixed on the membranes by incubation at 120°C for 30 min. Membranes were incubated for 2 h at 68°C in pre-hybridization solution (5× SSC, 0.1% w/v N-lauroylsarcosine, 0.02% w/v SDS and 1× Blocking Reagent (Roche). DIG-labelled probes were denatured by heating at 95°C for 10 min, diluted in pre-hybridization solution and incubated with nylon membranes for 18 h at 68°C. Levetiracetam Following hybridization, the membranes were washed twice with 2× SSC/0.1% w/v SDS at room temperature followed by two washes with 0.5× SSC/0.1% w/v SDS at 68°C for 20 min. Membranes were equilibrated for 30 min in maleic acid buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5), and

bound DIG-labelled probes were detected by incubation for 30 min with alkaline phosphatase-conjugated anti-DIG antibody (Roche) diluted 1:10,000 in maleic acid buffer. After washing twice with maleic acid buffer containing 0.3% v/v Tween 20, the chemiluminescence substrate CSPD (Roche) was used to detect bound anti-DIG antibodies and membranes were exposed to X-OMAT UV Plus Film (Kodak). Bioinformatic and phylogenetic analysis of FnBPB A domain isotypes Protein sequences were aligned in pairwise combinations to calculate amino acid identity using the ExPASY SIM alignment tool http://​www.​expasy.​org/​tools/​sim-prot.​html. The concatenated MLST allele sequences of S. aureus strains were downloaded from the MLST database http://​saureus.​mlst.​net/​.