. . Table 2. Oligonucleotides used in this study. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... Oligonucleotide Sequence . .... clfA-F 59-GATTCTGACCCAGGTTCAGA ... clfA-R 59-CTGTATCTGGTAATGGTTCTTT . ... clfB-F 59-ATGGTGATTCAGCAGTAAATCC .... clfB-R 59-CATTATTTGGTGGTGTAACTCTT ... sdr-F 59-GTAACAATTACGGATCATGATG . ... sdr-R 59-TACCTGTTTCTGGTAATGCTTT . ... spa-F 59-AGCACCAAAAGAGGAAGACAA ... spa-R 59-GTTTAACGACATGTACTCCGT . ... ssp-F 59-ATCMATTTYGCMAAYGATGACCA . ... ssp-R 59-TTGTCTGAATTATTGTTATCGCC .... 16S rRNA-F 59-GAAAACTTGAGTGCAGAAGAGGAAAGTGG ... 16S rRNA-R 59-GATGTCAAGATTTGGTAAGGTTCTTCGCG . . .. . doi:10.1371/journal.pone.0001409.t002 Transmission electron microscopy Infected or non-infected adherent hMDMs cultured on 6-well plates, were trypsinized with 10% trypsin-EDTA (Sigma). After 10 minutes at 37uC, the cells were washed with ice-cold PBS and pelleted at 4uC. The cells were then fixed with Karnovsky solution (2% paraformaldehyde, 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer, pH 7.4, 30 min at room temperature, washed in the same buffer lacking the fixative and post-fixed in 2% osmium tetraoxide for 1h [87]. The pellets were dehydrated through serial incubation in graded series of ethanol solutions (30%, 60%, 90%, and 100%) and finally embedded in Epon. The sections were counter-stained with uranyl acetate, lead citrate and examined with a Zeiss electron microscope. MLVF (Multiple-locus VNTR fingerprinting) and PCR analysis Total DNA of the isolates for PCR and MLVF typing were performed as described by Sabat et al. [56]. For DNA extraction, cell lysates were resuspended in 100 ml of lysis buffer (6 mM Tris- HCl, pH 7.6, 1 M NaCl, 0.1 M Na2EDTA, 0.5% Brij 58, 1% N- REFERENCES 1. Archer GL (1998) Staphylococcus aureus: A well-armed pathogen. Clin Infect Dis 26: 1179–1181. 2. Lowy FD (1998) Staphylococcus aureus infections. 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The primers were designed based on the completed and unfinished S. aureus genomes available from The Institute for Genomic Research (http://www.tigr.org) (COL strain), the University of Oklahoma (http://www.genome.ou.edu) (strain 8325), the Sanger Institute (http://www.sanger.ac.uk) (strains MRSA252 and MSSA476), and Juntendo University (http://www.juntendo.ac.jp) (strains Mu50 and N315). Multiplex amplifications were performed in mixtures containing 1.5 mM MgCl2, 0.2 mM dNTPs and 0.05 U of Taq recombinant polymerase per ml. The final reaction volume of 20 ml contained 0.5 mM each of clfB-F and clfB-R, 1 mM each of clfA-F, clfA-R, sdrCDE-F, sdrCDE-R, spa-F, spa-R, sspA-F and sspA-R as well as 1 ml template DNA. The amplification of DNA fragments was performed with an initial denaturation (94uC, 5 min), followed by 20 cycles of denaturation (94uC, 30 sec), annealing (55uC, 30 sec) and extension (72uC, 30 sec) with a single final extension step (72uC, 5 min). 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