Data were subjected
to a statistical analysis using the Chi-square test (SPSS package, SPSS Inc, Chicago, IL, USA). Differences were considered significant SIS3 if P values were lower than 0.05. Phenotypic assays The hemolytic activity of the isolates was determined on Columbia agar supplemented with 5% horse blood (COH, bioMériux) after incubation at 37°C for 72 h following a procedure previously described . The ability of the isolates to form slime was assessed using the Congo Red agar assay (CRA) . The plates were incubated at 37°C for 24 h and, then, for additional 24 h at room temperature. Determination of MIC’s to antibiotics The determination of the MIC’s to several antibiotics commonly used against staphylococcal infections was evaluated by a microdilution method using the Sensititre plates BMS 907351 Staenc1F (Trek Diagnostic Systems, Cleveland, OH) following the manufacturer’s instructions. The antibiotics analyzed were: penicillin, ampicillin, amoxycillin-clavulanic acid, teicoplanin, chloramphenicol, erythromycin, mupirocin,
streptomycin, gentamicin, clindamycin, oxacillin, ciprofloxacin, fosfomycin, imipenem, nitrofurantoine, trimethoprim-sufamethoxazole, tetracycline, vancomycin, linezolid, quinupristin-dalfopristin and selleck chemicals llc rifampin. Data were submitted to the statistical analysis described above. Screening formecA gene and typing of the staphylococcal chromosome cassettemec(SSCmec) Presence of themecA gene was evaluated by PCR using primersmecA forward (5′-GGTCCCATTAACTCTGAAG-3′) andmecA reverse (5′-AGTTCTGCAGTACCGGATTTTGC-3′),
which results in a 1,040 bp fragment . The SCCmecwas subjected to a typing procedure , which implied the PCR amplification of theccrB gene followed by RFLP analysis using endonucleasesHinfI andBsmI. Presence ofmecAand SCCmectyping was confirmed using all the primers and conditions described by Zhang et al. . Acknowledgements This work was supported by the FUN-C-FOOD (Consolider-Ingenio www.selleck.co.jp/products/Adriamycin.html 2010) and AGL2007-62042 projects from the Ministerio de Educación y Ciencia (Spain). S. Delgado was the recipient of a postdoctoral fellowship from the same Ministry. We are grateful to H. Herrero and the Association “”Amamantar”" (Avilés, Asturias) for their collaboration in the collection of the milk samples analyzed in this study. Electronic supplementary material Additional file 1:PCR-RFLP of the ccr B gene using endonucleases Hinf I and Hinf I/ Bsm I. The figure provided shows the profiles of SCC mec types III and IV using the method of Yang et al. . In lanes 1 and 3ccrB amplicons are cut withHinfI whereas in lanes 2 and 4 the amplicons are cut withHinfI andBsmI. Lanes 1 and 2:S. epidermidisDF2LAB, SCCmectype III (537, 106 bp and 320, 174, 106 bp respectively); lanes 3 and 4:S. epidermidisV1LD1, SCCmectype IV (264, 227, 154 and 227, 171, 153, 93 bp respectively); M, molecular weight marker. (PDF 46 KB) Additional file 2:Multiplex tuf gene-based PCR assay for the specific identification of S. aureus and S.