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.