2.3. Time Kill Curves

Time kill curves (TKC) were determined following the CLSI guidelines [22]. Two trials were performed at low and high inoculum.

Low inoculum assay: for each isolate of S. aureus overnight cultures were diluted in each growth medium and incubated for 120 min at 37 °C to obtain final inoculum close to 10⁵ CFU/mL. Subsequently, they were transferred to test tubes at 37 ºC with antimicrobial dilutions at 0 (control), 0.125, 0.25, 0.5, 1, 2, 4, 8 and 16 multiples of the MIC value previously obtained, and aliquots of 25 μL were sampled at 0, 1, 2, 4, 8, 12 and 24 h from each test tube.

High inoculum assay: the same methodology describe at low inoculum was carried out but using a final bacterial density close to 10⁸ CFU/mL. Antimicrobial dilutions at 0 (control), 0.5, 1, 2, 4, 8, 16, 32 and 64 multiples of MIC were used and aliquots of 25 μL were sampled at 0, 1, 2, 4, 8, 12 and 24 h from each test tube.

In both assays, bacterial counts were determined by serial dilution and culture on CAMHB agar plates at 37 °C. The lower limit of detection was 40 CFU/mL. Moreover, each assay was performed in duplicate on two different days and the averaged value (geometric mean) was calculated from each sampling time.

In this research, three growth mediums and two inoculum sizes were used and 6 data sets were obtained at low and high inoculum in CAMHB, serum and milk with 108 TKC per data set.

A more detailed description of TKC determination and tabulation of data sets can be found in the Supplementary Material section.