Subinhibitory concentrations of thymol reduce enterotoxins A and B and alpha-hemolysin production in Staphylococcus aureus isolates

Abstract

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., alpha-hemolysin and enterotoxins) by S. aureus.

Methodology/principal findings: Secretion of alpha-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 alpha-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 alpha-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 alpha-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 alpha-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 beta-lactams and glycopeptide antibiotics, which induce expression of alpha-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.

Figure 1. Growth curve for S. aureus ATCC 29213 (A) and MRSA strain 2985 (B).

(◊), untreated S. aureus; (•), S. aureus cultured with 8 µg/ml thymol; (*), S. aureus cultured with 16 µg/ml thymol; (×), S. aureus cultured with 32 µg/ml thymol; (△), S. aureus cultured with 64 µg/ml thymol; (□), S. aureus cultured with 128 µg/ml thymol. Values are the averages of three independent experiments. * represents p<0.05.

Figure 2. Western blot analysis of α-hemolysin, SEA and SEB production.

Both strain ATCC 29213 (A) and MRSA 2985 (B) were cultured with graded subinhibitory concentrations of thymol to an OD₆₀₀ of 2.5. Supernatants were subjected to SDS-PAGE. After transfer to polyvinylidene fluoride membranes, proteins were stained with the indicated antibodies against α-hemolysin, SEA and SEB. Horseradish peroxidase-conjugated goat anti-rabbit antiserum was used as secondary antibody, and the blots were developed using the ECL substrate (GE Healthcare, UK).

Figure 3. TNF release from splenocytes stimulated with supernatants of S. aureus.

Spleen cells were stimulated with supernatants of S. aureus previously grown to an OD₆₀₀ of 2.5 in the absence or presence of graded subinhibitory concentrations of thymol in RPMI-1640. After 16 h of stimulation, TNF release was measured by ELISA. Values represent the mean ± SD for three independent experiments. * indicates statistically significant reduction in TNF release (p<0.05).

Figure 4. Relative expression of hla (A), sea (B), seb (C) and agrA (D) in S. aureus.

S. aureus ATCC 29213 was cultured with different subinhibitory concentrations of thymol to the post-exponential growth phase (t = 240 min). Transcript levels were monitored by quantitative RT-PCR as described in the Materials and Methods. The drug-free culture was used as calibrator. Values represent the mean ± SD for three independent experiments. * represents p<0.05 and ** represents p<0.01.