New and simplified method for drug combination studies by checkerboard assay

Abstract

To evaluate the effect of two combined antimicrobial drugs, one method currently in use is the checkerboard assay in a 96-well microplate, which gives a good in vivo estimation of the drug-drug combination effect. Appropriate and consolidated methods are described in numerous scientific publications which are, however, in turn, laborious and time-spending, specifically for the setting of the 96-well microplate preparation. Each drug of every combination must be prepared and dispensed individually in several steps, often limiting its use in terms of consumed materials and working time. In our method, the strengths of the previous consolidated techniques are kept, although the toughness and the execution time are drastically reduced. No special laboratory apparatuses are needed. All the procedures of our method can be referred to the CLSI or EUCAST guideline. The method provides few main steps, which can be summarised in:•Preparation of the microorganism inoculum and three concentrations of antimicrobial drugs.•Easy dispensing of all reagents into the microplates with a multichannel pipette.•Evaluation of the microorganism optical density (OD) by a microplate reader, and calculation of growth percentage for each of the 77 combinations.

Keywords: Antagonism; Antimicrobials; CLSI; Drug-drug interaction; EUCAST; MIC; Minimal inhibitory concentration; Synergy.

Graphical abstract

Fig. 1

Disposition of drug A in the 96-well microplate.

Fig. 2

Dilution of drug A in the 96-well microplate by multichannel pipette.

Fig. 3

Disposition of drug B in the 96-well microplate and dilution by multichannel pipette.

Fig. 4

The final setting of 96-well microplate.