Emax plus

The experiment was conducted in 96-well plates as described previously [25 (link)]. One agent underwent a 2-fold serial dilution along the x-axis, while the other was similarly diluted along the y-axis, resulting in a matrix where each well held both agents at varying concentrations. Bacterial cultures, cultivated overnight, were diluted in saline to achieve a 0.5 McFarland turbidity. This was then further diluted 1:50 in CAMHB, and each well was inoculated, reaching an approximate concentration of 5 × 10⁵ CFU/mL. Wells containing only CAMHB, with or without bacteria, served as the positive and negative controls, respectively. The plates were incubated at 37 °C for 18 h and checked for visible turbidity, verified with an EMax Plus microplate reader (Molecular Devices, USA) at 590 nm. The fractional inhibitory concentration (FIC) for each agent was determined by dividing the MIC of the compound with colistin by its MIC when alone. Similarly, the FIC for colistin was determined by dividing its MIC with each compound by its standalone MIC. The FIC index was derived by adding both FIC values together. FIC indices were classified as synergistic (≤0.5), indifferent (0.5 < x ≤ 4), or antagonistic (>4).

Half of the maximal inhibitory concentration (IC₅₀) value was performed as previously described [60 (link)]. In brief, cells were seeded in 96 well plates and cultured for 24 h. After treatment, cells were treated at various concentrations of synthetic (±)-kusunokinin and neratinib for 72 h. Cell viability was determined using 3-(4,5-dimethyl thiazol-2 yl)-2,5-diphenyltetrazolium bromide (tetrazolium salt MTT, Cat No.: M6494, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The absorbance was measured at 570 and 650 nM using a microplate reader (Emax^® Plus, Molecular Devices, San Jose, CA, USA). Each treatment was performed in triplicate.

The adjuvant properties of the guanidinylated polymyxins were evaluated using the checkerboard assay as previously described [10 (link)]. The bacterial solution was prepared as described in the antimicrobial susceptibility assay. The antibiotic of interest and the adjuvant were serially diluted two-fold along the x- and y-axis on a 96-well plate, respectively, resulting in varying concentrations of both agents in each well. Equal volumes of bacterial solution were added to the designated wells. The well consisting of bacterial solution served as the positive control, while the wells containing only media served as the negative control. The plate was then incubated at 37 °C for 18 h. After incubation, an Emax Plus microplate reader (Molecular Devices, Union City, CA, USA) was used to measure the OD at a wavelength of 590 nm to confirm the turbidity. The fractional inhibitory concentration (FIC) index was then determined to establish the relationship between the antibiotic and adjuvant. The FIC index corresponds to the sum of the FICs of the antibiotic and adjuvant. The FIC of each agent is calculated by dividing its MIC when it is used in combination by its MIC when used alone. FIC indices ≤0.5, 0.5 < x ≤ 4 and >4 indicate synergy, additivity and antagonism, respectively.