4.4. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Determination by Broth Microdilution and Resazurin Colorimetric Assay

MICs and MBCs of all antimicrobial agents against B. pseudomallei (strain 1026b) and E. coli (O157: H7, used as a comparative reference bacteria) were performed by the broth microdilution method recommended by the Clinical and Laboratory Standards Institute [63] and described by Irazazabal, et al. [64]. Briefly, bacterial cultures were adjusted in MHB to McFarland 0.5 turbidity standard (to reach 1–5 × 10⁵ CFU/mL). The equal volume of each antibacterial agent (50 µL) at a final concentration of 4, 8, 16, 32, 64, 128, 256, 512 µg/mL and bacterial solution (50 µL) were added in each well of the 96-well plate. Ceftazidime antibiotic was used as a positive control. The plate was then incubated in an incubator at 37 °C for 18–24 h. MIC and MBC determination in different strains of B. pseudomallei (1026b, H777 and 316c) with broth microdilution and resazurin colorimetric assay were proposed by Silveira, et al. and Teh, et al. [65,66]. Broth microdilution was carried out with the same procedure as above. After overnight incubation, 0.01% resazurin (7-hydroxy-3H-phenoxazine-3-one 10-oxide; Sigma-Aldrich) was added to all wells and incubated at 37 °C for another 4 h. The color change was observed. The lowest concentration before the color change was determined as the MIC, the blue color represented no growth of bacteria and the pink one meant bacterial survival. The tests were performed in at least two independent experiments in triplicates.