Checkerboard MIC and Effect Model analysis
According to Clinical and Laboratory Standards Institute guidelines (Clinical and Laboratory Standards Institute, 2018),(Clinical and Laboratory Standards Institute, 2021), checkerboard MICs of aztreonam/nacubactam were determined by broth microdilution method using cation-adjusted Mueller-Hinton broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). A 2-fold step dilution series of each drug was added to the wells of a 96-well plate at a volume of 1:1. Bacteria were added to the plate to a final concentration being approximately 2-8×105 CFU mL-1. After incubating at 37 °C for 20 h, the lowest concentration of aztreonam with each nacubactam concentration, at which no bacterial growth was observed, was defined as MIC.
The results obtained by checkerboard assay for each strain were plotted and analysed using the Inhibitory Effect SigmoidI max Model [equation (1)] to draw an approximate curve representing the dose-response relationship of the aztreonam MIC to nacubactam concentration.
log2 (MIC) = log2 (MIC0) - (I max × CNACɤ )×(CNACɤ+ IC50ɤ )-1Eq.(1)
where MIC0 is the MIC of aztreonam in the absence of nacubactam, CNAC is the concentration of nacubactam (mg L-1), I max is the maximum inhibitory effect, IC50 is the concentration of nacubactam at which 50% of the maximum inhibition, ɤ is the sigmoid coefficient.