Antibiotics MIC Determination Microdilution broth method in 96 microwell plates (TPP, Switzerland) was used to estimate the antibiotic’s susceptibility. Two-fold dilutions of antibiotics in brucella broth (acumedia, Michigan, USA) prepared in wells were inoculated with 106 CFU of bacteria with final volume of 0.2 ml in each micro-well Inhibitors,research,lifescience,medical plate, and incubated for 48 hours at 37°C. The MIC was expressed as the lowest concentration that completely inhibited visual growth. Furthermore, the lowest concentration inhibiting 90% of visual growth was considered as MIC90. MIC testing was BGJ398 manufacturer performed according to the recommendations of the clinical laboratory standards institute.31

The concentrations assayed for each antibiotic ranged from 0.064 to 128 μg/ml. The Inhibitors,research,lifescience,medical absorbance was determined at 590 nm (Thermo-lab Systems Reader, Finland). All tests were performed in triplicate and then averaged. The antibiotics investigated included levofloxacin, ofloxacin, sparfloxacin, ciprofloxacin and doxycycline, along with a blank test containing no antibiotics. Determination of MIC of Essential Oils Microdilution broth susceptibility assay was performed using three replicates Inhibitors,research,lifescience,medical of each serial dilutions of essential oil prepared in brucella® broth medium in 96-well microtiter plates.32 The concentrations of each essential oil in serial dilutions ranged from 0.75

to 100 µl/ml. The content of each well was supplemented with 100 μl of freshly grown bacterial Inhibitors,research,lifescience,medical culture containing 106 CFU/ml in brucella broth. The assay included positive control without essential oil and negative control lacking bacteria under the same conditions. The plate was incubated with shaking for 24 h at 37°C. The MIC was expressed as the lowest concentration that completely inhibited visual growth. Moreover, MIC90 was the lowest concentration that inhibited Inhibitors,research,lifescience,medical 90% of visual growth with absorbance at 590 nm. Essential Oil-Antibiotic Combination Effect Two B. melitensis isolates were employed to evaluate the additive effects of various concentrations

of T. syriacus essential Vasopressin Receptor oil on the MIC of levofloxacin. MIC was determined as described above. Two dilutions containing 3.125 and 6.250 µl/ml, of T. syriacus essential oil were then added to the 96-well microtiter plates to determine the MIC. The lowest concentration of levofloxacin that completely inhibited visual growth in presence of essential oil was recorded as the MIC. Results On the basis of the primary screening results (table 2), O. syriacum and T. syriacus essential oils showed a good antibacterial activity against B. melitensis. Whereas, no antibacterial activity was demonstrated by the essential oils of R. officinalis L., S. palaestina Benth, M. piperia and L. stoechas L (data not shown). In addition, B.