An Automated Miniaturized Method to Perform and Analyze Antimicrobial Drug Synergy Assays

Abstract

In the light of emerging antibiotic resistance mechanisms found in bacteria throughout the world, discovery of drugs that potentiate the effect of currently available antibiotics remains an important aspect of pharmaceutical research in the 21st century. Well-established clinical tests exist to determine synergy in vitro, but these are only optimal for low-throughput experimentation while leaving analysis of results and interpretation of high-throughput microscale assays poorly standardized. Here, we describe a miniaturized broth microdilution checkerboard assay and data analysis method in 384-well plate format that conforms to the Clinical Laboratory and Standards Institute (CLSI) methods. This method has been automated and developed to rapidly determine the synergism of current antibiotics with various beta-lactamase inhibitors emerging from our antimicrobial research efforts. This technique increases test throughput and integrity of results, and saves test compound and labor. We facilitated the interpretation of results with an automated analysis tool allowing us to rapidly qualify inter- and intraplate robustness, determine efficacy of multiple antibiotics at the same time, and standardize the results of synergy interpretation. This procedure should enhance high-throughput antimicrobial drug discovery and supersedes former techniques.

Fig. 1.

Flow diagram of a 384-wpf synergy assay. Separate plates are used to dilute the test inhibitor/s (assay plate) and antibiotics (antibiotic plate), which are then combined using a 384-well transfer device into the assay plate. Test inhibitors (BLIs) are not added to the first two columns of assay plate, just antibiotic and bacteria, allowing for the MIC of antibiotic alone to be measured while also leaving several wells available with no drug serving as positive controls. Bacteria (30 μL) are added to columns 1–23. In column 24, 30 μL of the highest concentration of antibiotic but, no bacteria, is added serving as the negative growth control. A color diagram is included along with reference legend to help identify wells of interest. ABX, antibiotic; BLIs, beta-lactamase inhibitors; MIC, minimum inhibitory concentration; wpf, well plate format.

Fig. 2.

The Synergy RunTool displaying separate areas of interpretation. (A) The RunTool first imports raw data and displays it overlaid with regions color coded for compound and control allocation. (B) Data are then normalized and MICs determined per the methods described implementing user-defined controls. Bacterial growth versus no growth is automatically assigned the heat map colors depicted biased upon the automatically interpreted MIC values. (C) The same application calculates the ΣFIC using the formula described in the manuscript. The value calculated in each well is used to determine whether the compound is synergistic with the antibiotic assigning a shade of green if it is. A value of ≤0.5 means the compound synergizes with the antibiotic. (D) 384-wpf synergy testing yields two sets of data per plate pertaining to the left and right side of the plate. Results are shown in a color-coded table that quickly allows determination of Test Inhibitor MIC (alone), ABX MIC (alone), synergy shown as the fold potentiation, Min [Test Inhibitor] Restore = the μM concentration required to obtain its designated fold or full restoration of ABX efficacy, and the ΣFIC. Again, a synergistic result is shaded green. In the “Left” result, full restoration of ABX efficacy was achieved. (E) The statistics generated using the Synergy RunTool and also corresponding to the data generated as part of the illustration above. Separate statistics are calculated for each side of the plate. BLI Left is the compound tested in columns 1–12 of the assay plate and BLI Right is the bacteria tested in columns 13–23 of the assay plate. The chart shows values for the maximum BLI (inhibitor) concentration tested, maximum antibiotic (ABX) concentration, and the ABX MIC. The Synergy RunTool also calculates the [BLI]_MIN needed to reduce ABX MIC by 2-, 4-, 8-, or 16-fold and the Z′ which determines assay quality as previously discussed. The MIC cutoff is shown and is determined as the sum of 3 × SD plus the average of the no bacteria control, as previously described in the text. All of these data are readily archived in the Scripps database for the ease of access by medicinal chemists. ΣFIC, Total Fractional Inhibitory Concentration.

Fig. 3.

(A) The statistics generated using the Synergy RunTool corresponding to the data generated as part of the discovery of probe ML302 (CID53362017). The bacteria tested was clinical isolate YMC07/8/B3323, an Acinetobacter spp. expressing VIM2. Fold reduction of the MIC of imipenem by ML302 is shown against this strain as 2×, 4×, 8×, and 16× fold. (B) These data were then archived in the Scripps database and also published to the PubChem web site (https://pubchem.ncbi.nlm.nih.gov/bioassay/624096#section=BioAssay-Target), which is available to the public. Note the maximum potentiation is listed for this and all other compounds published to the link above.