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Objectives Limited data exist regarding the role of agr dysfunction in reducing susceptibility to vancomycin in methicillin-susceptible Staphylococcus aureus (MSSA). This study investigated the clinical and molecular epidemiology of MSSA causing bacteraemia, with emphasis on the reduced susceptibility to vancomycin (RSV) phenotype (MIC ≥1.5 mg/L) and its relationship with agr dysfunction. Methods All MSSA bloodstream isolates obtained at our hospital during 2010 were analysed. Antimicrobial susceptibility was determined and time–kill experiments were performed for oxacillin. Multilocus sequence type and agr genotype were determined and DNA microarray analysis of virulence factors was performed. agr dysfunction was assessed phenotypically and by RT–PCR quantification of RNAIII. Results Of 84 MSSA, 55 (65.5%) exhibited the RSV phenotype, comprising 13 clonal complexes. agr II polymorphism was more prevalent in RSV than non-RSV isolates (41.8% versus 17.2%, P = 0.023) and average levels of RNAIII gene expression were higher in RSV than non-RSV isolates (ΔCt 4.05 ± 3.29 versus 1.5 ± 2.11, P = 0.005), implying greater agr dysfunction in RSV MSSA. Conclusions We demonstrated a correlation between RSV phenotype in MSSA and reduced agr expression, particularly in association with the agr II genotype. These results may help to understand the role of agr dysfunction in the increased mortality in MSSA infections. [ABSTRACT FROM PUBLISHER]