The most common mechanisms of linezolid resistance involve mutations in the central loop of domain V of the 23S rRNA, or carrying the cfr gene implicated in the methylation of A2503 in the 23S rRNA of the large ribosomal subunit. This methylation affects the binding of at least three antimicrobial classes (phenicols, lincosamides, and streptogramin A), leading to a multi-drug resistant phenotype. The aim of our study was to investigate the rapid spread, in Italy, of staphylococci strains sharing diverse levels of resistance to linezolid, compared with the molecular mechanisms responsible for this resistance. Strains in study and antibiotic-resistance tests The sample analyzed was represented by 50 clinical Staphylococcus spp. isolates (45 S.epidermidis, 1 S.capitis, 3 S.hominis and 1 S.aureus) collected during 2009-2011, by broth dilution (MICs) and E-test methods. MICs for linezolid (Pfizer Pharma, Milan-Italy) and comparator agents, i.e. oxacillin, erythromycin, clindamycin, lincomycin, vancomycin, teicoplanin, chloramphenicol, quinupristin/dalfopristin (obtained as pure powders for analysis from Sigma Aldrich, Milan – Italy) and daptomycin (Novartis Pharma, Basel, Switzerland) were obtained using the microdilution method, in accordance with Clinical Laboratory Standards Institute guidelines (CLSI, 2012). The European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines were also used for comparison (http://www.eucast.org/clinical_breakpoints/ Version 2.0, valid from 2012-01-01). Polymerase chain reaction (PCR) of the resistance determinants Chromosomal extraction of whole genomic DNA was performed as previously described (Picher D. et al. 1989). All strains were screened for the presence of mecA (PBP2A), ermA, ermB, ermC, vga, vgb, vat, vatB, and cat genes. The (Staphylococcal Chromosomal Cassette) SCCmec cassettes were first determined by a multiplex-PCR protocol previously described, and assigned to the corresponding types. Furthermore, the results were confirmed by different multiplex-PCR protocols, focusing on the mec gene complex and the ccr gene complex. PCR and multiplex PCR amplifications were performed in a Biometra Personal Cycler. Molecular characterization (PFGE and MLST) Chromosomal extraction of whole genomic DNA, macrorestriction digestions with SmaI, for S.epidermidis, and ApaI, for S.hominis (New England Biolabs) and PFGE (CHEF-DR® II apparatus; Bio-Rad, CA) were obtained following a modified protocol previously reported (Bongiorno D., et a. 2010. J Antimicrob Chemother). Similarities among SmaI macrorestriction patterns were identified according to established criteria (Tenover F.C. et al. 1995). The comparison of similarities among groups for a long-term evaluation was performed by using the type strains already published, as internal standard. For the MLST scheme, PCR conditions and sequencing followed the instruction given at http.//sepidermidis.mlst.net. Numbers for alleles and STs (sequence-types) were assigned according to the S. epidermidis MLST database, the eBURST programme v3 was used to elaborate these data. Testing for the presence of the linezolid resistance mutations Domain V of the 23S rRNA gene was amplified by PCR using the primers V1-GCGGTCGCCTCCTAAAAG and V2-ATCCCGGTCCTCTCGTACTA, the amplicon size was 420 bp, it was treated with restriction enzyme NheI and analyzed by agarose gel electrophoresis to confirm the absence/presence of the most frequent mutation G2576T, which generates a new NheI site. Fragments were also sequenced for confirmation. Mutations in the domain V of the 23S rRNA, and in rplC, D and V genes-codifying respectively L3, L4 and L22 proteins, L3, L4 and L22 ribosomal proteins-or cfr-mediated linezolid resistance, were confirmed by PCR and sequence analysis (Blast alignment versus S. epidermidis ATCC 35984/RP62A) as described elsewhere (Bongiorno D., et al. J Antimicrob Chemother. 2010).