Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

Summary

This protocol describes how to study all possible combinations that can be obtained between four drugs in one single experiment. This method is based on the standard 96-well plate micro dilution assay and the calculation of fractional inhibitory concentrations (FICs) to evaluate the results.

Abstract

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The Quadruple checkerboard, also called the Q-checkerboard, aims at maximizing the number of possible combinations that can be obtained between four drugs in one experiment to minimize the time and work needed to accomplish the same results with other protocols. This protocol is based on the simple micro dilution technique where the drugs are diluted and combined together in several 96-well plates.

In the first set of 96-well plates, Muller-Hinton broth is added followed by the first required drug (e.g., Cefotaxime here) to serially dilute it. After the first step is done, another set of 96-well plates is used to dilute the second drug (e.g., Amikaci), which will be transferred by removing a specific volume of drug 2 and put in the corresponding wells in the first set of 96-well plates that contains drug one. The third step is done by adding the required concentrations of the third drug (e.g., Levofloxacin), to the appropriate plates in the initial set containing combination of drug 1 and 2. The fourth step is done by adding the required concentrations of the fourth drug (e.g., Trimethoprim-sulfamethoxazol) into the appropriate plates in the first set. Then, E. coli ESBL bacterial inoculum will be prepared and added.

This method is important to evaluate all the possible combinations and has a wider range of possibilities to be tested furthermore for in vivo testing. Despite being a tiring technique requiring a lot of focus, the results are remarkable and time saving where a lot of combinations can be tested in a single experiment.

Introduction

With the increase in resistance due to the overuse and misuse of antibiotics^1,2, the need to develop new drugs and agents to treat bacterial infections has become crucial. New approaches such as developing new drugs are very important to overcome the resistance crisis. However, the pharmaceutical industry is not interested in developing new antimicrobial agents. Moreover, if new drugs are developed, bacteria will keep on evolving and developing resistance against these new drugs^3,4. Thus, the problem of resistance will not be solved, making the need for another approach a must that should be considered and studied to overcome bacterial resistance.

Drug combination is a very important concept for treating bacterial infections mainly those that are caused by multidrug resistant pathogens^5,6. It decreases the course of treatment, decreases the dose given; thus, decreasing the toxicity of the given drug, helps in decreasing the rate of resistance development and, in a way, sensitizes the bacteria to the given drugs as described in the concept of collateral sensitivity^5,7,8,9.

Resistance development to one drug requires a single mutation; however, resistance development to a combination of drugs targeting multiple pathways requires several independent mutations that are slowed down by this combination. An example to decreased resistance while using combination therapy is the decreased rate of resistance to Rifampin in Mycobacterium Tuberculosis¹⁰. Another example is a study done by Gribble et al. that showed the rate of emergence of resistant strains in patients taking Piperacillin alone to be higher than in those taking a combination of carboxypenicillin and aminoglycoside¹⁰. Studies have shown that resistance development to aminoglycosides in evolving bacteria made these strains sensitive to various other drugs⁵. The combination between the beta-lactam class drug amoxicillin and the lactamase inhibitor clavulanic acid showed success in treating resistant bacterial strains⁸.

Decreasing the time of treatment is a good advantage resulting from drug combinations. For example, a therapy of combined penicillin or ceftriaxone with gentamicin for 2 weeks will give the same efficacy given by penicillin or ceftriaxone alone when given for 4 weeks¹¹. Combining drugs allows for the usage of lower dosages of drugs that are not effective when given alone such as the Sub-MICs. The example of sulfonamides can be given where the use of triple-sulfonamides minimizes, at lower doses, the toxicity produced which is crystal formation or crystalluria when using insoluble sulfonamides at full doses¹².

Thus, decreasing the dosage given and the time of treatment will eventually decrease the toxicity of the drugs on the body. The idea of developing methods to assess the interaction between combined drugs is very important. In one study, the results showed that combination therapy is more effective for the treatment of resistant species of Acinetobacter and P. aeruginosa⁸.

Giving drugs in combination
There are different methods by which we can study drug combinations, such as the checkerboard method, the time-kill curve method, and the E-test method¹³. The checkerboard method can study all the possible combinations between the two drugs in question in one experiment itself. In addition, it was developed to study a combination of three drugs¹⁴. Now, we extend this to study a combination of four drugs mainly for the treatment of multidrug-resistant pathogens.

The time-kill curve assay is usually performed to test for the bactericidal effect of a certain drug. It was also used to test for the effect of drug combinations where several drugs are combined at specific concentrations. This protocol requires the preparation of several sterile tubes or cups where in each cup we add the broth, combination of drugs, and the required bacterial strain. After incubation and recording of the optical density at several time points, the results are compared with the normal growth rate of the used strain to see whether the growth rate increased, decreased, or did not change¹³.

E-test method is usually done to test for the minimal inhibitory concentration (MIC) where a strip containing a gradient concentration of the drug in question is put on an inoculated plate. It was also used to test the combination between two drugs where two strips are added to the plate in a perpendicular manner intersecting at their MICs¹³.

According to literature, there is no gold standard to define and study synergy; thus, it is difficult to assess which one of the methods used to study combination is better and which one produces better and more reliable results mainly¹³. However, Time-kill assay is labor intensive, time consuming, and expensive^15,16, while the E-test method is developed to study a combination between two drugs only. Checkerboard can study all the possible combinations between the two drugs tested and this is why this technique has been chosen to be developed.

Protocol

1. Preparation steps

1. Prepare Muller-Hinton broth (MHB) by adding 25 g of MH broth to 1 L of distilled water and mix. Autoclave at 121 °C for 2.5 h. Then, store the autoclaved media at room temperature or in the fridge.
2. Subculture the bacteria in question (E. coli ESBL) on the agar media using the four-quadrant streaking method and incubate overnight at 37 °C.
   1. Using a sterile loop, take one colony and spread it in the first half of the MacConkey agar plate by doing close parallel streaks.
   2. Using the loop, spread the bacteria in the second quadrant by streaking it from the first quadrant.
   3. From the second quadrant, extend the streaks in the third quadrant using close parallel streaks.
   4. Spread the bacteria in the center of the fourth quadrant by streaking it from the third quadrant.

2. Panel preparation

1. Place four 96-well plates next to each other to form a square. Using a tape, tape their bottom together.
2. Repeat this step to obtain four panels each containing 4 plates and name them A1, A2, A3, and A4.
3. Add 50 µL of MH broth to the wells between column 2 and column 11 in the 16 96-well plates of the four panels.
4. Add 200 µL of MH broth to the well H12 serving as the negative control well in the 16 96-well plates of the four panels.
5. Add 150 µL of MH broth to the wells A1 and H1 serving as positive control wells in the 16 96-well plates of the four panels.

3. Drug 1, Cefotaxime, serial dilution

1. To a conical tube, add 15 mL of sterile dH₂O.
   1. Calculate the volume to be removed from the stock solution of the drug following the formula C₁V₁ = C₂V₂. Thus, V₁ = (C₂ x 15 mL) / C₁.
      NOTE: In our case, the Cefotaxime stock solution is 10⁵ μg/mL and C₂ is 256 μg/mL; thus, V₁ = 38.4 µL.
2. Remove the calculated volume from the 15 mL of sterile dH₂O, and then add the drug.
3. Pipette 50 µL of the prepared drug solution into each well in column 11 and column 12 except H12.
4. Start the serial dilution by removing 50 µL from column 11 and putting it into the corresponding wells in column 10, and then from 10 until reaching column 2 where the 50 µL taken from column 2 will be discarded.
5. Repeat steps 3.4 and 3.5 for all the 16 96-well plates of the four panels.

4. Drug 2, Amkacin, serial dilution

1. To a conical tube, add 10 mL of sterile dH₂O.
2. Calculate the volume to be removed from the stock solution of the drug following the formula C₁V₁ = C₂V₂. Thus, V₁ = (C₂ x 10 mL) / C₁.
   NOTE: In our case, the Amikacin stock solution is 10³ μg/mL and C₂ is 64 μg/mL; thus, V₁ = 64 µL.
3. Remove the calculated volume from the 10 mL of sterile dH₂O, and then add the drug.
4. Take eight separate 96-well plates.
5. To each plate, add 100 µL of MHB to the wells between rows G and B.
6. Add 100 µL of the previously prepared drug 2 solution to the wells of row G.
7. Dilute serially from row G to row B by taking 100 µL from each well and finally discard the 100 µL from the wells of row B.
8. Repeat steps 4.5, 4.6, and 4.7 to prepare eight plates.

5. Transfer of drug 2 to the four panels

1. Pipette 50 µL of drug 2 from the wells between rows G and B into the corresponding wells in each plate in the four panels. One prepared 96-well plate contains 100 µL of drug 2 that is enough for two plates in one panel.

6. Drug 3, Levofloxacin, addition

1. To four different conical tubes, add 14 mL of sterile dH₂O.
2. Calculate the volume to be removed from the stock solution of the drug following the formula C₁V₁ = C₂V₂. Thus, V₁ = (C₂ x 14 mL) / C₁.
   NOTE: In our case, Levofloxacin is prepared in four different concentrations from a stock solution of 5 x 10³ μg/mL.
   C₁ = 2 μg/mL, V₁ = 5.6 µL
   C₂ = 4 μg/mL, V₁ = 11.2 µL
   C₃ = 8 μg/mL, V₁ = 22.4 µL
   C₄ = 16 μg/mL, V₁ = 44.8 µL
3. Remove the calculated volume from the 14 mL of sterile dH₂O from each tube, and then add the drug.
4. After preparing the required concentrations of the third drug, take 50 µL and add it to the corresponding wells between rows B and G and columns 2 and 12 in the corresponding plate in each panel where C1 corresponds to the four P1 plates in the four panels, C2 corresponds to the four P2 plates in the four panels, C3 corresponds to the four P3 plates in the four panels, and C4 corresponds to the four P4 plates in the four panels.

7. Drug 4, Trimethoprim-sulfamethoxazole, addition

1. To a conical tube, add 14 mL of sterile dH₂O.
2. Calculate the volume to be removed from the stock solution of the drug following the formula C₁V₁ = C₂V₂. Thus, V₁ = (C₂ x 14 mL) / C₁.
   NOTE: In our case, trimethoprim-sulfamethoxazole is prepared in four different concentrations from a stock solution of 48 x 10³ μg/mL.
   C₁ = 512 μg/mL, V₁ = 149.33 µL
   C₂ = 1024 μg/mL, V₁ = 298.66 µL
   C₃ = 2048 μg/mL, V₁ = 597.33 µL
   C₄ = 4096 μg/mL, V₁ = 1 194.66 µL
3. Remove the calculated volume from the 14 mL of sterile dH₂O of each tube, and then add the drug.
4. After preparing the required concentrations of the fourth drug, take 50 µL and add it to the corresponding wells between rows B and G and columns 2 and 12 in the four plates in the corresponding panel where C1 corresponds to panel 1, C2 corresponds to panel 2, C3 corresponds to panel 3, and C4 corresponds to panel 4.

8. Preparation and addition of bacterial inoculum E. coli ESBL

1. Using a sterile loop, transfer one colony of the bacterial isolate E. coli ESBL previously cultured on a plate into 2 mL of sterile MHB and vortex.
2. Check for the turbidity where it should be 0.5 McFarland using a densitometer.
3. Add 80 mL of sterile MH broth to a sterile urine cup.
4. Add bacterial inoculum from the 0.5 McFarland inoculum to the urine cup following C₁V₁ = C₂V₂, where V₁ = (10⁶ x 80 mL) / 10⁸ = 800 µL.
5. Pipette 50 µL of the inoculum solution 10⁶ CFU/mL into each well except H12, which is the sterility control well.
6. Incubate the panels at 37 °C overnight.
7. After incubation, add 50 µL of Iodotetrazolium to record the growth in the wells.

9. Protocol for the FIC template (Supplemental File)

1. Write the highest concentration of drug 1 (Cefotaxime) in the yellow cell in panel A.
2. Write the highest concentration of drug 2 (Amikacin) in the yellow cell in panel B.
3. Write the highest concentration of drug 3 (Levofloxacin) in the yellow cell in panel C.
4. Write the highest concentration of drug 4 (Trimethoprim-sulfamethoxazole) in panel D.
5. Trace the Redline (Growth/no Growth interface) by highlighting the wells having growth in red.
6. Write the MIC of drug 1 in the table of drug 1 (ATB1).
7. Write the MIC of drug 2 in the table of drug 2 (ATB2).
8. Write the MIC of drug 3 in the table of drug 3 (ATB3).
9. Write the MIC of drug 4 in the table of drug 4 (ATB4).
10. To calculate FIC for ATB1
    1. Determine the wells on the Growth/no Growth interface.
    2. In table ATB1, double click on the cell next to FIC1 and drag the yellow cell on the left of panel A to the first selected well.
    3. Repeat step 9.10.2 for every selected well where well 1 corresponds to FIC1, well 2 corresponds to FIC2, and so on.
11. To calculate the FIC for ATB2
    1. In table ATB2, double click on the cell next to FIC1 and drag the yellow cell on the left of panel B to the first pre-selected well.
    2. Repeat step 9.11.1 for every pre-selected well.
12. To calculate the FIC for ATB3
    1. In table ATB3, double click on the cell next to FIC1 and drag the yellow cell on the left of panel C to the first pre-selected well.
    2. Repeat step 9.12.1 for every pre-selected well.
13. To calculate the FIC for ATB4
    1. In table ATB4, double click on the cell next to FIC1 and drag the yellow cell on the left of panel D to the first pre-selected well.
    2. Repeat step 9.13.1 for every pre-selected well.
14. In the table labeled ATB1+2+3+4, sum the FIC1 of each ATB automatically. The same will occur for the other FICs (FIC2, FIC3, and so on).
    1. In the cell containing FIC and highlighted in yellow, double click on it and select the summed ΣFICs from table ATB1+2+3+4.
15. Repeat these steps for each of the nine sheets representing the nine plates.
    ​NOTE: In the sheet FIC all, the table will show the final summed FIC with the interpretation of the obtained value.

10. MIC determination using the microdilution assay for the four drugs

1. Label four different rows with the abbreviation of each tested drug. For example, CTX for Cefotaxime, AMK for Amikacin, LEVO for Levofloxacin, and SXT for Trimethoprim-sulfamethoxazole.
2. Pipette 200 µL of sterile Muller Hinton broth into well number 1 and well number 12 in each used row. Well number 12 will serve as the negative control well.
3. Pipette 100 µL of sterile Muller Hinton broth to the wells 2 till 11 in each used row.
4. Calculate the volume of each drug to be added using the formula C₁V₁ = C₂V₂. Thus, V₁ = (C₂ x 4 x V₂) / C₁. V₂ is the final volume in the wells which is 200 µL, C₁ is the concentration of the stock solution, and C₂ is the initial concentration that we need to have in the first well.
   NOTE: Here, we use the following:
   Cefotaxime: C₁ = 10⁵ μg/mL, where we dilute it to 10⁴ μg/mL, C₂ = 256 μg/mL; thus, V₁ = 20.48 µL
   Amikacin: C₁ = 10³ μg/mL, C₂ = 64 μg/mL; thus, V₁ = 51.2 µL
   Levofloxacin: C₁ = 5 x 10³ μg/mL, where we dilute it to 5 x 10² μg/mL, C₂ = 16 μg/mL; thus, V₁ = 25.6 µL
   Trimethoprim-Sulfamethoxazole: C₁ = 48 x 10³ μg/mL, C₂ = 4096 μg/mL; thus, V₁ = 68.26 µL
5. Pipette the required volume for each drug in the first well of the corresponding row after removing the same volume from the 200 µL broth to obtain a total volume of 200 µL after the addition of the drug.
6. Serially dilute by removing 100 µL from well 1 into well 2 and so on until reaching well 10 where the 100 µL removed from well 10 will be discarded. Note that well 11 serves as the positive control well.
7. Pipette 100 µL of 10⁶ prepared bacterial inoculum into each well in each used row except for well number 12 serving as the negative control.
8. Incubate at 37 °C overnight.

Results

Figure 2A represents the results obtained by combining Cefotaxime and Amikacin with specific concentrations of Levofloxacin and Trimethoprim-sulfamethoxazole. We can see in the left part of the figure the four plates that are schematically presented with the concentrations of the drugs in the right part of the figure. The arrows represent the wells on the Growth/no Growth interface. The colored wells are the wells that contain growth. We notice that the fourth plate does not contain growth i...

Discussion

The Quadruple Checkerboard method resembles the checkerboard and the three dimensional checkerboard in its protocol. However, certain crucial steps should be taken into consideration to avoid errors during the experiment.

Make sure to test for the MIC of each drug against the tested isolate before starting the protocol to know what are the concentrations that are needed to start the dilutions with for drug 1 and drug 2 that need to be serially diluted in the plates. Concerning drug 3 and ...

Materials

Name	Company	Catalog Number	Comments
1000 µL tips	Citotest	4330000402
200 µL tips	Citotest	4330-0013-17
50 mL centrifuge tube	corning	430828	For drug 3 and 4 preparation
5 mL polysterene round-bottom Tube	Falcon	352058	For 0.5 MacFarland bacterial inoculum preparation
90mm petri dishes	JRZ Plastilab		As bed for the solutions to be added using the multichannel pipette
96-well plates	corning	3596	For serial diltuion and combining drugs
Bactrim 200, 40 mg (Trimethoprim-sulamethoxazole	By CRNEXI SAS Fontenay-sous-Bois, France	10177403	Drug 4
Ceforane, 1 g (Cefotaxime)	PHARCO Pharmaceuticals	24750/2006	Drug 1
Densitometer
E. Coli ESBL strain	Retreived as a medical strain from the Saint-George Hospital Lebanon		Bacterial strain
Mac Conkey + crystal violet agar	BIO-RAD	64169508	For making agar plates used for subculturing
Miacin 500 mg/2 mL (Amikacin)	HIKMA Pharmaceuticals	2BXMIA56N-AEF	Drug 2
Muller-Hinton Broth	BIO-RAD	69444	For making bacterial media
Multichannel Pipette	Thermo Scientific	GJ54761	For serial dilution and addition of media, bacteria and drugs
Paper Tape
Single Channel pipettes	Thermo Scientific	OH19855 HH40868	For the addition of media, bacteria and drugs
Tavanic, 500 mg (Levofloxacin)	sanofi aventis	221937/2009	Drug 3