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).
Table 1.
Hemolytic activities of α-hemolysin produced by S. aureus cultured with graded subinhibitory concentrations of thymol.
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.
Table 2.
Primers used for quantitative RT-PCR in the study.