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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: July 24th, 2021

DOI:

10.3791/62311

1Faculty of Medicine and Medical Sciences, University of Balamand, 2Department of Clinical Microbiology, Michigan Health Clinics, 3College of Medicine, Central Michigan University

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.

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.

With the increase in resistance due to the overuse and misuse of antibiotics1,2, 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 drugs3,4. Thus, the problem of resistance will not be solved, making the need fo....

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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 clos.......

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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.......

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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 .......

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None.

....

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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

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