New and simplified method for drug combination studies by checkerboard assay

1Specifications Table

2When two or more drugs are tested together simultaneously, they can give different phenotypic effects: synergism, additivity, antagonism or indifference. To evaluate the effect of the combination between two antimicrobial drugs, the microbiologist uses the agar dilution method and the disk diffusion method. An additional 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,. The latter uses a liquid medium and a two-dimensional, two-agents microdilution checkerboard to evaluate combinations of antimicrobial agents against organisms,. In the checkerboard assay, two antimicrobics are tested in double serial dilutions, and the concentration of each drug is tested both alone and in combination. Thus, it is possible to determine the effect of the individual drug, but above all, the effect produced by their combination. The nature of the interaction between the two antimicrobials is then determined either algebraically or geometrically. The most used, appropriate and consolidated method is described in the Clinical Microbiology Procedures Handbook 3rd Edition, which is, 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, we use the strengths of the previously cited protocol but reducing its laboriousness and execution time, using only three concentrations of antimicrobial drugs, and dispensing the regents with a multichannel pipette, unlike a single channel pipette. Furthermore, no special laboratory equipment is needed.

3All the procedures of our method refer to the CLSI or EUCAST guideline in some cases with some modifications. Though most drug interaction methods are not used in the clinical laboratory workflow, the numerous tests developed for this purpose are very useful in the practice of research laboratories.

4Additionally, it is possible not only to test antimicrobial molecules with each other but also in combination with compounds that, normally, by themselves, do not have antimicrobial properties.

5With the crisis generated in recent years, most antibiotics or antifungal drugs fail because of increasing antimicrobial resistance. The use of molecules in combination could be the key to the development of new formulations that do not necessarily require the discovery of new molecules.

6Isolated bacterial colonies.Two antimicrobics or compounds from stock solutions or powder.Mueller-Hinton broth (MHB).Cation-adjusted Mueller-Hinton broth (CAMHB).Double concentrated Cation-adjusted Mueller-Hinton broth (2XCAMHB).Diluents for the antimicrobial agent stock solutions.Sterile distilled reagent-grade water.Sterile 0.9% NaCl.V-shaped Sterile pipetting reservoir or sterile 90 mmor 100 mm diameter Petri dish.Electronic portable pipette controller or manual pipette controller.Sterile serological pipette: 2, 5, 10, 25, 50 mL.Manual or electronic single channel pipettes delivery tips (1000, 200 and 10 µL).Manual or electronic multichannel pipette (8 or 12 channel) deliver 200 µL tips.Sterile pipet tips: 1000, 200 and 10 µL.Sterile, clear round bottom, not treated and individually wrapped 96-well microplates with lid, or clear flat bottom, not treated and individually wrapped 96-well microplates with lid.50, 15 and 1.5 mL sterile disposable plastic vials.100 and 250 mL microbiological glass flask.Lids for glass flasks or bacteriological hydrophobic cotton.Glass beaker.

7Isolated bacterial colonies.

8Two antimicrobics or compounds from stock solutions or powder.

9Mueller-Hinton broth (MHB).

10Cation-adjusted Mueller-Hinton broth (CAMHB).

11Double concentrated Cation-adjusted Mueller-Hinton broth (2XCAMHB).

12Diluents for the antimicrobial agent stock solutions.

13Sterile distilled reagent-grade water.

14Sterile 0.9% NaCl.

15V-shaped Sterile pipetting reservoir or sterile 90 mmor 100 mm diameter Petri dish.

16Electronic portable pipette controller or manual pipette controller.

17Sterile serological pipette: 2, 5, 10, 25, 50 mL.

18Manual or electronic single channel pipettes delivery tips (1000, 200 and 10 µL).

19Manual or electronic multichannel pipette (8 or 12 channel) deliver 200 µL tips.

20Sterile pipet tips: 1000, 200 and 10 µL.

21Sterile, clear round bottom, not treated and individually wrapped 96-well microplates with lid, or clear flat bottom, not treated and individually wrapped 96-well microplates with lid.

2250, 15 and 1.5 mL sterile disposable plastic vials.

23100 and 250 mL microbiological glass flask.

24Lids for glass flasks or bacteriological hydrophobic cotton.

25Glass beaker.

26Autoclave.Vortex mixer.Magnetic stirrer.35±2°C ambient air orbital shaker-incubator.35±2°C ambient air incubator.96-well microplate reader equipped with a 600 nm optical filter or close to 600 nm.

27Autoclave.

28Vortex mixer.

29Magnetic stirrer.

3035±2°C ambient air orbital shaker-incubator.

3135±2°C ambient air incubator.

3296-well microplate reader equipped with a 600 nm optical filter or close to 600 nm.

33Day 1

34Weigh an appropriate amount of powdered culture medium.Using a magnetic stirrer, dissolve the powder into deionised water until the required volume is reached (See recipe reported on the package insert or in the datasheet) and then sterilise by autoclaving at 121°C for 15 min.

35Weigh an appropriate amount of powdered culture medium.

36Using a magnetic stirrer, dissolve the powder into deionised water until the required volume is reached (See recipe reported on the package insert or in the datasheet) and then sterilise by autoclaving at 121°C for 15 min.

37The culture media required are:10 mL of MHB.50–100 mL of 2 × CAMHB.

3810 mL of MHB.

3950–100 mL of 2 × CAMHB.

40Using a magnetic stirrer, dissolve 0.9 g of NaCl into 100 mL of deionised water and then sterilise by autoclaving at 121°C for 15 min.

41Using a magnetic stirrer, dissolve 0.9 g of NaCl into 100 mL of deionised water and then sterilise by autoclaving at 121°C for 15 min.

42Weigh an appropriate amount of powder of drug A and drug B and dissolve each one into its appropriate solvent as requested by CLSI in the chapter: “Solvents and Diluents for Preparing Stock Solutions of Antimicrobial Agents” or as recommended by the EUCAST guidelines. If the antibiotic or compound is not listed in the guidelines, use the solvent that keeps the compound more stable (This information is often available in the datasheet).

43Weigh an appropriate amount of powder of drug A and drug B and dissolve each one into its appropriate solvent as requested by CLSI in the chapter: “Solvents and Diluents for Preparing Stock Solutions of Antimicrobial Agents” or as recommended by the EUCAST guidelines. If the antibiotic or compound is not listed in the guidelines, use the solvent that keeps the compound more stable (This information is often available in the datasheet).

44Withdraw with a sterile loop an isolated colony from a nutrition agar plate.Inoculated the colony into a 10 mL of MHB.Grow the bacteria at 35±2°C in an orbital shaker incubator at 200 rpm under aerobic conditions for 18 h ±2.

45Withdraw with a sterile loop an isolated colony from a nutrition agar plate.

46Inoculated the colony into a 10 mL of MHB.

47Grow the bacteria at 35±2°C in an orbital shaker incubator at 200 rpm under aerobic conditions for 18 h ±2.

48Note: After 18 h ±2 of incubation, under the condition indicated, the culture will reach the concentration of ∼ 109 CFU/mL.

49Day 2

50Place 100 μL of the 2 × CAMHB medium in a V-shaped reservoir or sterile Petri dish and dispense into each well of the microplate, by multichannel pipette, proceeding by column from left to right or vice versa.

51Place 100 μL of the 2 × CAMHB medium in a V-shaped reservoir or sterile Petri dish and dispense into each well of the microplate, by multichannel pipette, proceeding by column from left to right or vice versa.

52Prepare 1.5 mL of stock solution (SA) in 2 × CAMHB medium to obtain a solution 4-fold more concentrated than the highest concentration of the drug A to be tested.Prepare 500 μL of 2 × stock solution (2 × SA) in 2 × CAMHB medium to obtain a solution 8-fold more concentrated than the highest concentration of the drug A to be tested.Dispense 100 μL of SA into row A from columns 1–11. (Fig. 1)Dispense 100 μL of 2 × SA into well A12. (Fig. 1)

53Prepare 1.5 mL of stock solution (SA) in 2 × CAMHB medium to obtain a solution 4-fold more concentrated than the highest concentration of the drug A to be tested.

54Prepare 500 μL of 2 × stock solution (2 × SA) in 2 × CAMHB medium to obtain a solution 8-fold more concentrated than the highest concentration of the drug A to be tested.

55Dispense 100 μL of SA into row A from columns 1–11. (Fig. 1)

56Dispense 100 μL of 2 × SA into well A12. (Fig. 1)

57Note: The purpose of adding the Stock solution 2 × SA is to keep constant the concentration of drug A during the next serial dilution (point 9).

58Dilute the drug A from row A to G using a multichannel pipette set to 100 μL and discard the remaining volume from row G.

59Dilute the drug A from row A to G using a multichannel pipette set to 100 μL and discard the remaining volume from row G.

60Note: With an 8-channel pipette, dilute in two steps using 6 tips. The first from the 1–6 and then from 7 to 12. When starting again with the second step, change the tips ( Fig. 2 ).

61Prepare 1.0 mL of stock solution (SB) in 2 × CAMHB medium to obtain a solution 4-fold more concentrated than the highest concentration of drug B to be tested.Dispense 100 μL of SB into each column 12 wells. (Fig. 3).

62Prepare 1.0 mL of stock solution (SB) in 2 × CAMHB medium to obtain a solution 4-fold more concentrated than the highest concentration of drug B to be tested.

63Dispense 100 μL of SB into each column 12 wells. (Fig. 3).

64Dilute the drug B Column 12–2 using a multichannel pipette set to 100 μL and discard the remaining volume taken from column 2. (Fig. 3).

65Dilute the drug B Column 12–2 using a multichannel pipette set to 100 μL and discard the remaining volume taken from column 2. (Fig. 3).

66Prepare from 10 to 40 mL of bacterial inoculum 106 CFU/mL in 0,9% NaCl, from the culture prepared the day before (see chapter: Preparation of microbial inoculum).Place 100 μL of inoculum in a V-shaped reservoir or sterile Petri dish and dispense into each well of the microplate using a multichannel pipette.

67Prepare from 10 to 40 mL of bacterial inoculum 106 CFU/mL in 0,9% NaCl, from the culture prepared the day before (see chapter: Preparation of microbial inoculum).

68Place 100 μL of inoculum in a V-shaped reservoir or sterile Petri dish and dispense into each well of the microplate using a multichannel pipette.

69Note:

70Before diluting the bacterial culture at step 10, mix by vortex for at least 20 s. The final inoculum concentration in the microplate will be 5 × 105 CFU/mL.

71A mirror plate, without bacteria and with the same reagents, should be implemented to obtain no-growth control. Moreover, the mirror plate is useful as O.D. background in the data analysis (i.e., when compound precipitates may appear during the incubation).

72Place the microplate with its cover into a static incubator at 35±2°C for 18±2 h.

73Place the microplate with its cover into a static incubator at 35±2°C for 18±2 h.

74Note: If there should be an excessive variation in volume during incubation, it is possible to seal the microplate by sealing film. Also, a tray with water can be added to the incubator to prevent the dry environment favouring evaporation from the plate.

75Day 3

76Mix the contents of the wells using a multichannel pipette.Place the microplate in a microplate reader.Read the optical density (OD) at 600 nm or around 600 nm (e.g. 595 or 605).

77Mix the contents of the wells using a multichannel pipette.

78Place the microplate in a microplate reader.

79Read the optical density (OD) at 600 nm or around 600 nm (e.g. 595 or 605).

80From the optical density, it is possible to calculate the percentage of growth for each well.

81Based on the distribution of the reagents that have been made, the plate is organised as follows (Fig. 4):In column 1 from A to G wells, there are the double serial dilutions of drug A. A1 is the well where the drug A has the highest concentration (HA).In the raw H from columns 12–2, there are the double serial dilutions of drug B. H12 is the well where the drug B has the highest concentration (HB).The well H1 represent the bacterial growth control without drugs (100% growth).All the other wells of the microplate represent all the 77 possible combinations formed by the combination of the serial dilutions of each antibiotic.

82In column 1 from A to G wells, there are the double serial dilutions of drug A. A1 is the well where the drug A has the highest concentration (HA).

83In the raw H from columns 12–2, there are the double serial dilutions of drug B. H12 is the well where the drug B has the highest concentration (HB).

84The well H1 represent the bacterial growth control without drugs (100% growth).

85All the other wells of the microplate represent all the 77 possible combinations formed by the combination of the serial dilutions of each antibiotic.

86The percentage of growth in each well was calculated as:

87Note: ODbackground is the value of OD where no growth is appreciable or a value previously determined using the same method, where the reagents without bacteria were placed.

88From the data, it is also possible to calculate the Minimal Inhibitory Concentration (MIC) for each combination. MIC is defined as the lowest drug concentration, which reduces bacterial growth by more than 80%.

89Several models can be used to analyse the data obtained from the new method of checkerboard assay and assess the nature of in vitro interaction.

90Some applied examples can be extrapolated from the following publication: Segatore, B. et al. (2012) Phytomedicine, 19 (3-4), pp. 341-347. Celenza, G. et al. (2012) Phytomedicine, 19 (7), pp. 596-602. Bellio, P. et al. (2015) Phytomedicine, 22 (2), pp. 223-230.

91Segatore, B. et al. (2012) Phytomedicine, 19 (3-4), pp. 341-347.

92Celenza, G. et al. (2012) Phytomedicine, 19 (7), pp. 596-602.

93Bellio, P. et al. (2015) Phytomedicine, 22 (2), pp. 223-230.

94In these studies, data were analysed by nonparametric models based on the Loewe additivity model and the Bliss Independence theory.

95The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.