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Background  Targeting bacterial virulence factors is now gaining interest as an alternative strategy to develop new types of anti-infective agents. It has been shown that thymol, when used at low concentrations, can inhibit the TSST-1 secretion in  Staphylococcus aureus . However, there are no data on the effect of thymol on the production of other exotoxins (e.g., α-hemolysin and enterotoxins) by  S. aureus .    Methodology/Principal Findings  Secretion of α-hemolysin, SEA and SEB in both methicillin-sensitive and methicillin-resistant  S. aureus  isolates cultured with graded subinhibitory concentrations of thymol was detected by immunoblot analysis. Hemolysin and tumor necrosis factor (TNF) release assays were performed to elucidate the biological relevance of changes in α-hemolysin, SEA and SEB secretion induced by thymol. In addition, the influence of thymol on the transcription of  hla ,  sea , and  seb  (the genes encoding α-hemolysin, SEA and SEB, respectively) was analyzed by quantitative RT-PCR. Thymol inhibited transcription of  hla ,  sea  and  seb  in  S. aureus , resulting in a reduction of α-hemolysin, SEA and SEB secretion and, thus, a reduction in hemolytic and TNF-inducing activities.    Conclusions/Significance  Subinhibitory concentrations of thymol decreased the production of α-hemolysin, SEA and SEB in both MSSA and MRSA in a dose-dependent manner. These data suggest that thymol may be useful for the treatment of  S. aureus  infections when used in combination with β-lactams and glycopeptide antibiotics, which induce expression of α-hemolysin and enterotoxins at subinhibitory concentrations. Furthermore, the structure of thymol may potentially be used as a basic structure for development of drugs aimed against these bacterial virulence factors.

Subinhibitory Concentrations of Thymol Reduce Enterotoxins A and B and α-Hemolysin Production in Staphylococcus aureus Isolates