Determination of anti-staphylococcal activity of thymoquinone in combinations with antibiotics by checkerboard method using EVA capmat™ as a vapor barrier

Thymoquinone (Tq) has been reported to potentiate the in vitro growth-inhibitory activity of some antibiotics especially against Staphylococcus aureus. However, it has been shown that Tq vapors can affect the results of susceptibility testing by standard broth microdilution method. Therefore, we made a comparative experiment with and without ethylene vinyl acetate cap mats (EVA capmat™) on microplates. The results showed significant differences in the minimum inhibitory concentration values and proved this capmat as an effective vapor barrier. Therefore further experiments focused on the in vitro anti-staphylococcal combinatory effect of Tq with oxacillin, penicillin, and tetracycline against various S. aureus strains have been performed by checkerboard method using EVA capmat™. The combined effect was evaluated according to the sum of fractional inhibitory concentrations (ΣFIC). Synergy was obtained for combination with oxacillin against 3 (ΣFIC 0.263-0.450), with penicillin against 1 (ΣFIC 0.466) and with tetracycline against 2 strains tested (ΣFIC 0.400-0.475). Our results confirm previous reports on the Tq enhancement of anti-staphylococcal activity of antibiotics. Moreover, this is the first report on Tq synergy with oxacillin and penicillin against S. aureus. Our experiments also showed that Tq vapors can affect evaluation of combined effect by checkerboard assay, whereas the use of EVA capmat™ can avoid this.