4.4. Antimicrobial Susceptibility Testing

The MIC of the antibiotics was determined using the round-bottomed 96-well microtiter plates (Greiner Bio-one, Monroe, NC, USA) following the broth microdilution procedure described by Wiegand and colleagues [40]. Briefly, 50 µL of sterile distilled water was aliquoted into the wells 2 to 10, which served as the antibiotics’ diluent. Subsequently, 100 µL of the highest concentration of the antibiotics to be investigated was dispensed into well 1. It was serially diluted by transferring 50 µL of the antibiotics from well 1 through well 10 and finally discarded after dilution in the last well allowing for the geometric serial dilution of the antibiotics across the rows. Each well containing the antibiotic solution was inoculated with 50 µL of the test organism earlier standardised. Well 11 served as the growth control (GC), containing only the inoculum, while well 12 served as the sterility control (SC), only containing the assayed antibiotics.

The microtiter plates were covered and incubated at 35 ± 1 °C for 16–20 ± 2 h. The results were read after the addition of the 30 µL resazurin dye (w/v, 0.015%) (Glentham Life Sciences, Corsham, UK) or the 2,3,5, triphenyl tetrazolium chloride (Merck, Darmstadt, Germany), depending on the availability of the dyes, with a further 2 h incubation period for the observation of a colour change. The well with the lowest concentration of the antibiotics that completely inhibited the growth of the bacteria, as indicated by no observable colour change, was read as the MIC value, which was interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI breakpoints [41,42]. The tests were performed in triplicates.