Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

Manual checkerboard array synergy testing is a labor-intensive and error-prone method. In contrast, our automated inkjet printer-based checkerboard method dramatically improves speed, accuracy, and throughput. The automated checkerboard method makes it possible to screen large numbers of drugs for synergistic combinations, while the time-kill method can confirm and further explore the activity of these combinations.

Novel antibiotic discovery is not keeping pace with the spread of resistance, but the investigation of synergistic combinations offers a possibility for salvaging existing drugs to treat resistant bacteria. Although we demonstrate this technique with antibacterials, it could also be used with other classes of antimicrobials or even for different types of combination drug or compound testing. In this video, we demonstrate an automated checkerboard array method and a manual time-kill method.

We suggest performing checkerboard array studies first to identify promising combinations, then investigating these further with time-kill studies. To begin, make antimicrobial stock solutions of colistin and minocycline, and then perform quality control on the stock solutions as described in the accompanying text protocol. Next, add about one milliliter of 0.9%sodium chloride, saline, to 12-millimeter-by-75-millimeter round bottom glass culture tubes.

Select one or two colonies from an overnight plate of bacteria, and place them into the culture tubes. Vortex the tubes gently to suspend the bacteria in the solution. Check the concentration of bacteria using a McFarland reader.

Adjust the concentration by adding more saline or more bacteria to achieve a 0.5 McFarland turbidity reading. Then, dilute the bacteria one to 300 by adding 100 microliters of the suspension to 30 milliliters of cation-adjusted Mueller-Hinton broth in a 50-milliliter conical tube to reach a final cell density of five times 10 to the fifth colony-forming units per milliliter. Now, prepare the antimicrobials for printing by following along in the accompanying text protocol.

Save the protocol, and then click on the Run button at the top left, followed by the Start button. Load a 384-well plate into the plate holder, and press Loaded under the Load Plate 1 Synergy prompt. Then, place a T8+cassette into the cassette slot, and press Loaded under the Load a T8+Cassette prompt.

When prompted, add antibiotic stock solution to the indicated reservoirs on the cassette. After each solution is added, press the Filled button. Once the D300 Dispenser has added antibiotic stock in appropriate volumes to each well and the Run Completed box appears, click Close, remove the plate, and turn off the D300.

Next, pour the previously prepared bacterial suspension into a sterile reagent reservoir. Use a multichannel pipette to add 50 microliters of the suspension to all wells in the checkerboard array. Then, add 50 microliters of cation-adjusted Mueller-Hinton broth without bacteria to an empty well.

This will be the negative control well to confirm sterility of the media. Incubate in a 35-degree Celsius ambient air incubator for 16 to 20 hours. To analyze the test, follow along in the accompanying text protocol.

For the time-kill synergy test, again prepare the antimicrobial stock solutions as described in the text protocol. Then, make a 0.5 McFarland suspension of the test organism in sterile saline. Add 100 microliters of the 0.5 McFarland suspension to five milliliters of CAMHB in a 25-by-150-millimeter glass round bottom culture tube with stainless steel closure, and vortex gently to mix.

Using a sterile inoculating loop, isolation streak a drop of the diluted suspension onto a blood agar plate, and incubate the plate at 35 degrees Celsius in ambient air to confirm inoculum purity. Replace the closure on the tube, and incubate it in a test tube rack on a shaker at 35 degrees Celsius in ambient air for at least 3 hours, until logarithmic-phase growth is reached. While the initial culture is incubating, add 10 milliliters of cation-adjusted Mueller-Hinton broth to five autoclaved glass culture tubes.

For a time-kill synergy study, at least one drug should be at a concentration that does not affect the growth curve individually. This can be determined by evaluating the effects of individual drug concentrations prior to the synergy study. To the first tube, add 10 microliters of one-milligram-per-milliliter colistin stock to obtain a final colistin concentration of one microgram per milliliter, as this is a concentration that is ineffective against the strain used in this example.

For the second tube, add 10 microliters of one-milligram-per-milliliter minocycline stock to obtain a final concentration of one microgram per milliliter. This concentration is also ineffective against the strain being used in this example. To the third tube, add 10 microliters of one-milligram-per-milliliter minocycline stock and 10 microliters of one-milligram-per-milliliter colistin stock.

This is the same quantity of colistin and minocycline used in tubes one and two. In the fourth and fifth tubes, add no antibiotics. This will be the growth control and the negative control tubes, respectively.

Next, prepare 96 deep-well polypropylene plates with two-milliliter wells for serial dilutions by adding 900 microliters of sterile saline to rows B through H of columns one to four with a multichannel pipette. When the culture is in logarithmic growth phase, remove the culture tube from the shaker, and vortex it gently. Transfer one milliliter of the suspension to a 12-by-75-millimeter glass culture tube, and check the density with a McFarland reader.

If the density is less than 1.0 McFarland, return the tube to the shaker and incubate longer. If it is greater than 1.0 McFarland, add cation-adjusted Mueller-Hinton broth to the tube, vortex gently, and re-sample until the suspension is at 1.0 McFarland. It's important that the culture is in the logarithmic growth phase when preparing the starting inoculum.

You can construct a growth curve to determine when an organism reaches this phase. Then, add 100 microliters of the 1.0 McFarland suspension to tubes one through four, and vortex them gently. Immediately after mixing and at one, two, four, six, and 24 hours, remove a 150-microliter aliquot from each culture tube by tilting the tube so that only the sterile pipette tip enters the tube and not the unsterile pipettor shaft during aliquot withdrawal.

Add aliquots, respectively, to consecutive wells in the first row of the previously prepared 96 deep-well plate. Return tubes to a test tube rack on a shaker in a 35-degree Celsius ambient air incubator immediately after removing aliquots at each time point. Using a multichannel pipette, remove 100 microliters from row A of the plate, and add it to row B, containing 900 microliters of saline, creating a one-to-10 dilution.

Pipette the solution up and down four to five times to mix, and then discard the tips. First, label Mueller-Hinton agar plates with the antibiotic conditions and dilution to be plated. Then, plate the diluted samples for colony counts using the drop plate method, using a multichannel pipette with extra-long tips.

Remove 10 microliters from each well in column one, and dispense carefully in a row onto the appropriately labeled plate. Continue until all the rows are dispensed onto plates. In addition, at the 24-hour time point, place a 10-microliter drop taken directly from the negative control tube onto the spot dedicated for this in order to test for sterility.

Then, invert all plates, and incubate them overnight at 35 degrees Celsius in ambient air. Then, count the number of colonies on each area, and calculate the cell density. Mark colonies with a fine-tip permanent marker on the reverse of the plate to avoid double-counting or missing colonies.

For each dilution series, identify drops with three to 30 colonies. Count the colonies in these drops, and record the count along with the dilution factor. Shown here is a grid from a checkerboard array synergy experiment in which minocycline in concentrations of zero to 32 micrograms per milliliter was combined with colistin at concentrations of zero to 16 micrograms per milliliter and tested against a strain of E.coli.

Wells with OD600 values below 07 represent no growth and are shaded red, while wells with OD600 values above or equal to 07 represent growth and are shaded green. For each drug, the minimum inhibitory concentration is the lowest concentration of drug that inhibits bacterial growth. For minocycline, this is 32 micrograms per milliliter, and for colistin, it is eight micrograms per milliliter.

The shading is retained here, but values within the wells in which growth is inhibited are replaced by fractional inhibitory concentration, or FIC, index values. In each well, the FIC index of each drug is calculated by dividing the concentration of antibiotic in that well by the drug's minimum inhibitory concentration. Wells with an FIC index of less than or equal to 0.5, which is considered the cutoff for synergy, are indicated with a broken-line border, and the well with the lowest FIC index is bolded.

Because the minimum FIC value is in the synergistic range, the combination is considered synergistic. An example of a case that does not demonstrate synergy is shown here, as the minimum FIC index at which growth is inhibited is one, which is greater than 0.5. Finally, in this example, several skipped wells occurred, which represents bacterial growth that is inhibited, despite the presence of bacterial growth in adjacent wells with higher concentrations of antibiotic.

If more than one skipped well occurred in a checkerboard array, the results should be discarded and the assay repeated. An example of a graph demonstrating results of a time-kill synergy assay is presented here. Please see the accompanying text protocol for details on interpretation of the time-kill synergy assay.

Further information about the activity of synergistic combinations can be obtained through evaluations of pharmacokinetics and pharmacodynamics and through in vivo studies with animal models. Always follow appropriate safety procedures, including use of personal protective equipment, when working with bacteria. If you generate aerosols or use high-risk pathogens, perform all work in a biosafety cabinet.