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A large clonal outbreak of multidrug-resistant CC17 ST17 Enterococcus faecium containing Tn5382 in a hospital in the north of Spain is described.We characterized vancomycin-resistant E. faecium isolates from 10 infected and 40 colonized inpatients from a single hospital by PFGE, multiple-locus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST). Genes encoding antibiotic resistance (ampicillin, aminoglycosides, macrolides, quinupristin/dalfopristin, quinolones, tetracycline) and putative virulence traits were analysed.All isolates showed highly similar PFGE profiles and were assigned to the type MT1 by MLVA and to ST17 (CC17) by MLST. The Tn5382 type identified in all isolates was linked to pbp5 and contained a 5 bp deletion and 10 point mutations within the intergenic vanSB–vanYB region. Other resistance genes identified were erm(B), mef(E), tet(M), ant(6′)-Ia, aph(3′)-IIIa and aac(6′)-Ie-aph(2′′)-Ia. All isolates carried the unexpressed tet(M) gene. The high level of ciprofloxacin resistance was attributable to the first described Gly-61 and Ile-80 mutations in ParC and the Tyr-83 or Arg-83 mutations in GyrA. All isolates contained esp. The presence of hyl was variable.A large clonal outbreak caused by multidrug-resistant CC17 E. faecium containing pbp5–Tn5382 is described. The persistence of this clone, which has been recovered from both hospital and community settings since 2005, and the possibility of transferring this Tn5382 to other epidemic ampicillin-resistant clonal types currently circulating in Spain might contribute to increasing the prevalence of vancomycin-resistant enterococci in our area. This study constitutes the first description of mef(E) in E. faecium.