The samples were analyzed via electrophoresis in 1% agarose gels (Agarose LE, Promega) using a 100 bp DNA ladder (Gibco/BRL Life Technologies,

Breda, The Netherlands). E. faecium strain ATCC 51559 (vanA + ) and E. faecalis strain ATCC® 51299 (vanB + ) were used as controls in the PCR experiments [24]. Table 1 Primers sequences used in this study Gene Primer Sequence (5′ to 3′) Size (bp) Reference vanA vanA-F CATGAATAGAATAAAAGTTGCAATA 1,030 ABT-263 solubility dmso (Clark et al., 1993) [23] vanA-R CCCCTTTAACGCTAATACGATCAA vanB vanB-F GTCACAAACCGGAGGCGAGGA 433 (Clark et al., 1993) [23] vanB-R CCGCCATCCTCCTGCAAAAAA esp Efm esp-F TTGCTAATGCTAGTCCACGACC 945 (Shankar et al., 1999) [25] esp-R GCGTCAACACTTGCATTGCCGA hyl Efm hyl-F

GAGTAGAGGAATATCTTAGC 661 (Rice et al., 2003) [14] hyl-R AGGCTCCAATTCTGT PCR screening for the esp and hyl genes DNA from bacterial cultures was extracted and amplified via PCR using primers for the esp Efm and hyl Efm genes (Table 1), generating bands of 954 bp and 661 bp, respectively [14, 25]. Molecular typing of VREF PFGE of the 12 VREF clinical isolates was carried out following the protocols of Morrison et al. [26, 27]. Briefly, the samples were digested with 50 U of SmaI (New England Biolab, Ipswich, MA, USA) for 4 h at 25°C. The digested plugs were separated via electrophoresis in 1% agarose gels (BioRad, Hercules, California, USA) using ultra-pure DNA agarose (BioRad, Hercules, California, USA), with 0.5X TBE as the running buffer in the CHEF MAPPER system (BioRad Laboratories, Hercules, California, 3-Methyladenine cell line USA), run at 6 V/cm at 14°C under two Protein Tyrosine Kinase inhibitor different linear ramped pulse times: 1 to 10 s for 16 h and 10 to 40 s for 22 h. A PFGE lambda ladder (New England Biolabs, Hertfordshire, England, UK) was used as a molecular

weight marker, and the gels were stained for 40 m with 0.5 mg/ml of ethidium bromide for visualization under UV light. The obtained banding patterns were initially interpreted via visual inspection according to the criteria specified by Tenover et al. [28]. Cluster analysis was performed with BioNumerics (Applied Maths, Inc., Austin, TX, USA) using the DICE correlation coefficient and the unweighted pair group mathematical average algorithm (UPGMA) as the grouping P-type ATPase method [29]. The PFGE pulsotypes of the 12 VREF clinical isolates were also genotyped through multilocus sequence typing (MLST) according to a standard protocol described by Homan et al. [17]. Fragments of seven housekeeping genes (atpA, ddl, gdh, purK, gyd, pstS and adk) were sequenced using a 3730xl DNA Analyzer (Applied Biosystems, Foster City, California, USA), thus obtaining their allelic profiles, and the STs for each unique allelic profile were designated on the basis of information from the MLST website (http://​efaecium.​mlst.​net).