ND, not determined, -, negative PCR amplification.

Table 2 Characteristics and bla locus allotypes of MSSA strains used in this studya) Clonal complex b) MLST (ST) PFGE type Strain Origin Isolation date bla locus alleles             blaZ blaI blaR1   1 G IPOP38 PRI-724 datasheet Portugal 2001 6 2 10 1 188 L IPOP58 Portugal 2001 6 2 10   573 M HSJ109 Portugal 1995 6 2 10 5 5 C HSA29 Portugal 1992-1993 11 4 7   5 C IPOP41 Portugal 2001 6 3 6 8 8 J IPOP65 Portugal 2001 8 MRT67307 chemical structure 2 ND   615 E IPOP32 Portugal 2001 9 1 4 9 9 D HSJ122 Portugal 1995 12 1 12 10 10 Q DCC300 Portugal 1996-1997 9 1 5 12 12 X HSJ130 Portugal 1995 3 3 6   12 X Draftees728 Portugal 1996-1997 1 1 1 15 15 K HSA9 Portugal 1992-1993 6 9 ND 20 20 N HSA47 Portugal 1992-1993 6 8 11 22 22 T Draftees721 Portugal 1996-1997 6 3 5 25 25 S HSA76 Portugal 1992-1993 1 1 1   30 A IPOP37 Portugal 2001 13 1 1 30 34 B IPOP24 Portugal 2001 6 ND ND   34 B IPOP34 Portugal 2001 1 1 ND   NA B IPOP26 Portugal 2001 1 ND ND 45 45 H HSA19 Portugal 1992-1993 6 2 10   45 H IPOP56 Portugal 2001 6 ND ND 97 97 P IPOP50 Portugal 2001 6 ND ND 121 121 F IPOP44 Portugal 2001 10 1 5 Singleton 580 R DCC1185 Portugal SB-715992 in vitro 1996-1997 1 1 1 a) MSSA strains have been previously characterized by PFGE and MLST[62]. b) Clonal complexes were determined using the E-burst software http://​saureus.​mlst.​net/​eburst/​database.​asp, last accessed on

June 04, 2009. NA, not available; ND, not determined. Media and

growth conditions Strains were grown overnight at 37°C on Fludarabine price tryptic soy agar or tryptic soy broth under aerobic conditions. DNA isolation Total DNA was prepared using the Wizard genomic DNA preparation kit (Promega, Madison, WI, USA), according to the manufacturer’s recommendations, except for the addition of lysostaphin at 0.5 mg/mL and RNase at 0.3 mg/mL for the lysis step. DNA amplification and sequencing The allelic variation on the β-lactamase locus was evaluated by sequencing internal fragments of blaZ and its transcriptional regulators, blaI and blaR1, amplified by PCR. Based on the available sequence at GenBank (accession number: X52734) for Tn552 of S. aureus, three pairs of primers were designed as follows (5′ → 3′): blaZ F1, GAT AAG AGA TTT GCC TAT GC; blaZ R1, GCA TAT GTT ATT GCT TGA CC; blaI F1, GCA AGT TGA AAT ATC TAT GG; blaI R1, GAA AGG ATC CAT TTT CTG TAC ACT CTC ATC; blaR1 F1, CAT GAC AAT GAA GTA GAA GC; and blaR1 R1, CTT ATG ATT CCA TGA CAT ACG. The predicted amplicon sizes were 533 bp for blaZ, 484 bp for blaI and 537 bp for blaR1. PCR was performed in a T1 Thermocicler (Biometra) with the following conditions: 94°C for 4 min; 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min; and a final extension at 72°C for 10 min.