Detection of Drug Susceptibility and Drug Interactions in Mycobacterium tuberculosis using Resazurin Microtiter and Checkerboard Assay

Summary

Here, we present a protocol to visualize and standardize resazurin microtiter assay (REMA) and its checkerboard variation for evaluating the drug susceptibility and interaction profiles of Mycobacterium tuberculosis. This method provides insights, in vitro, for optimizing drug combinations to be used in treatment regimens and combating multidrug-resistant tuberculosis.

Abstract

The emergence of multidrug-resistant tuberculosis (MDR-TB) necessitates rapid and accurate drug susceptibility testing (DST) methods to guide effective treatment. This study introduces a methodology combining the resazurin microtiter assay (REMA) and checkerboard assay to determine minimum inhibitory concentration (MIC) and evaluate drug-drug interactions of anti-tuberculosis drugs against Mycobacterium tuberculosis. The REMA, adapted to a 96-well format, leverages the reduction of resazurin dye by metabolically active M. tuberculosis as a visual indicator of drug susceptibility. Varying concentrations of anti-TB drugs are tested against M. tuberculosis isolates, and color changes are observed to determine the MIC. Subsequently, a checkerboard assay is employed to assess potential synergistic, additive, or antagonistic effects between drug combinations. This simple and inexpensive method yields results within seven days, offering a significant advantage over traditional DST methods. This method provides valuable insights into the DST of M. tuberculosis isolates and facilitates the identification of promising drug combinations for improved treatment outcomes against MDR-TB.

Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a formidable global health challenge, exacerbated by the emergence of drug-resistant strains. According to the World Health Organization (WHO), in 2023, there were an estimated 10.6 million new TB cases and 1.3 million deaths, highlighting the pressing need for more effective diagnostic and therapeutic strategies¹. Multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) pose major challenges to disease control due to the complexity, toxicity, and prolonged duration of current treatment regimens^2,3.

The recent introduction of novel anti-TB agents, such as bedaquiline and delamanid, targeting unique biochemical pathways in M. tuberculosis, represents a breakthrough in TB management^4,5. These drugs have been approved primarily for use in fluoroquinolone- and rifampicin-resistant TB cases as part of WHO-endorsed longer and shorter MDR-TB regimens^6,7. However, the determination of optimal drug combinations and dosage regimens, particularly when these agents are used in conjunction with established therapies, remains a critical area of investigation^8,9. Current methods for drug susceptibility testing (DST), while reliable, are often time-intensive, resource-intensive, and impractical for evaluating complex drug combinations in a clinical or research setting¹⁰. This underscores the need for simple, scalable phenotypic assays that can evaluate both drug efficacy and interaction profiles in M. tuberculosis.

Among such phenotypic approaches, the resazurin microtiter assay (REMA) has emerged as a widely accepted and conventional method for assessing drug susceptibility. The assay is based on the reduction of the blue resazurin dye to pink resorufin by metabolically active bacteria, serving as a visual indicator of cell viability. Although popularized by Palomino et al. in 2002¹¹, the principle of using resazurin to assess bacterial growth dates back to the work of Pital et al.¹². Over the past two decades, the REMA method has been extensively validated and incorporated into standard operating procedures for mycobacterial research and diagnostic testing^13,14,15.

Complementing the REMA, the checkerboard assay, as introduced by Caleffi-Ferracioli et al., extends the utility of REMA by enabling two-drug interaction studies in a microdilution format using the same resazurin-based readout¹⁶. While promising in principle, this method remains primarily a research tool useful for screening potential synergistic or antagonistic drug combinations during early preclinical evaluation rather than a routine diagnostic assay.

This manuscript does not present REMA or its checkerboard variant as novel methods. Rather, the goal of this study is to provide a visual representation of a useful and validated methodology for MIC determination and drug interaction testing, which has been described and successfully applied in earlier studies. We provide a step-by-step protocol with detailed guidance on inoculum preparation, drug titration, plate layout, MIC interpretation, and checkerboard interaction analysis. This manuscript aims to support broader, more consistent use of REMA and checkerboard assays in TB drug development and experimental pharmacology.

Protocol

NOTE: Chemicals and materials utilized in the example experiments are available in the Table of Materials file.

1. Preparation of 7H9-S Media

1. For 100 mL of 7H9-S media, weigh 0.47 g of 7H9 powder and 0.1 g of casitone, then dissolve in 90 mL of distilled water; stir until fully dissolved.
2. Sterilize the broth in a 250 mL flask using an autoclave at 121 °C for 15 min.
3. Once autoclaved and cooled, add 10 mL of Oleic Albumin Dextrose Catalase (OADC) supplement and 0.5 mL of glycerol.
4. Incubate overnight at 37 °C to check for sterility.
   NOTE: The sterility of the prepared 7H9-S medium is verified by incubating it overnight at 37 °C. The medium should remain clear the next day, with no turbidity observed.
5. Store the 7H9-S medium at 4 °C, away from direct light.

2. M.tuberculosis  Inoculum preparation

1. Preparation of inoculum from M.tuberculosis culture grown in Lowenstein Jensen (LJ) solid medium
   1. Using a sterile inoculation loop, collect a full loop of M. tuberculosis culture from a freshly grown (21-28 days old) LJ medium.
      NOTE: Carefully avoid scraping too deep into the medium, which may disturb older colonies.
   2. Place the collected bacteria into a sterile bijou bottle containing sterile glass beads and 2.5 mL of 7H9-S broth. Ensure that the glass beads help in the mechanical disruption of clumps during vortexing.
      NOTE: Fresh cultures between 21-28 days are essential to ensure reliable susceptibility test results. Avoid using older cultures, as they may lead to inconsistent outcomes.
   3. Secure the bijou bottle and vortex vigorously for at least 1 min. Ensure continuous mixing to break up bacterial clumps, resulting in a relatively turbid suspension. The goal is a homogenous distribution of bacteria within the medium.
   4. Transfer the culture suspension into a fresh bijou bottle and allow it to stand undisturbed for 15 min. This step allows the larger bacterial clumps to settle at the bottom.
   5. Using the McFarland 1.0 Standard as a reference, check the turbidity of the culture suspension and adjust with 7H9-S broth until the desired turbidity is achieved.
      NOTE: A McFarland Standard, typically 1.0, is used as the reference for turbidity equivalent to approximately 1.97 × 10⁶ CFU/mL¹⁷. The standard is prepared by combining 0.1 mL of 1% barium chloride and 9.9 1 mL of 1% sulfuric acid, resulting in a fine precipitate of barium sulfate, which provides the turbidity. The bacterial suspension is visually compared against the McFarland Standard by holding both tubes against a white background with black lines. The turbidity of the bacterial suspension is adjusted to match the standard.
2. Preparation of inoculum from M.tuberculosis culture grown in 7H9 liquid medium
   1. Using a sterile pipette, transfer 2.5 mL of M. tuberculosis culture (2-3 weeks old) to a fresh bijou bottle with glass beads.
   2. Vortex the mixture for at least 1 min to ensure a homogenous suspension. The vortexing action helps to break any clumps.
   3. Transfer the 2.5 mL of culture suspension to a fresh bijou bottle and leave it for 15 min to allow clumps to settle at the bottom.
   4. Compare the turbidity against the McFarland 1.0 Standard and adjust using 7H9-S broth to achieve the required concentration.
3. Dilution of inoculum
   1. Prepare a 1:10 dilution: To dilute the inoculum, add 1 mL of the bacterial suspension to 9 mL of 7H9-S broth in a sterile container (This will give approximately 1.97 × 10⁵ CFU/mL).

3. Preparation of antibiotics and storage

1. Preparation of stock and working solutions: Prepare the drugs used in the example experiment, along with their stock and working solution preparation methods, as summarized in Table 1.
   1. Isoniazid (INH or I): To prepare a 1 mg/mL stock solution of isoniazid, dissolve 2 mg of isoniazid powder in 2.0 mL of distilled water. Sterilize the solution using a 0.22 µm syringe filter.
   2. Moxifloxacin (MOX or M): To prepare a 1 mg/mL stock solution of Moxifloxacin, dissolve 2 mg of moxifloxacin powder in 2.0 mL of distilled water. Sterilize the solution using a 0.22 µm syringe filter.
   3. Bedaquiline (BDQ or J): Bedaquiline is poorly soluble in water. To prepare a 1 mg/mL stock solution, dissolve 2 mg of bedaquiline powder in 2.0 mL of DMSO. Sterilize the solution using a 0.22 µm syringe filter.
   4. Delamanid (DEL or D): Similar to bedaquiline, delamanid has poor water solubility. To prepare a 1 mg/mL stock solution, dissolve 2 mg of delamanid powder in 2.0 mL of DMSO. Sterilize the solution using a 0.22 µm syringe filter.
2. Storage: Aliquot 100 µL of each drug stock solution into sterile cryovials and store them at -20 °C for up to 3 months. Once thawed, discard any remaining solution. Do not refreeze.

4. Preparation of Resazurin solution and storage

1. Dissolve resazurin to a final concentration of 0.01% or 0.02% in distilled water. Filter the solution through a 0.2 µm filter for sterilization.
2. Store the sterilized resazurin solution at 4 °C for up to 1-2 weeks, protected from light to prevent degradation.

5. Preparation of the REMA plate

NOTE: Refer to Figure 1 for a visual guide on the REMA 96-well microtiter plate setup. The 96-well plate provides sufficient space to run drug assays in duplicate for each drug in six two-fold dilutions.

1. Add 7H9-S broth: Using a multichannel pipette, add 100 µL of 7H9-S broth to columns 2-11, rows B-G. Ensure the distribution is even across all wells.
2. Add drug solutions: Add 100 µL of the working INH solution to wells B2 and B6. Similarly, add the MOX, DEL, and BDQ working solutions to their respective wells (B3 and B7, B4 and B8, B5 and B9).
3. Dilute the drug concentrations: Using a multichannel pipette, perform two-fold serial dilutions from rows B to G (columns 2-9), ensuring to discard the final 100 µL after mixing in row G.
4. Growth control wells: Add 100 µL of 7H9-S broth to wells B10 and C10 for the growth control.
5. Negative and sterility controls: Add 200 µL of 7H9-S broth to wells B11 and C11 to serve as negative and sterility controls.
6. Prevent evaporation: Add 200 µL of sterile distilled water to all outer wells to minimize evaporation during incubation.
7. Inoculate plates: Inoculate each well with 100 µL of the 1:10 diluted culture suspension, except for the negative control well. Ensure uniform inoculation across all wells.
8. Seal and incubate: Seal the plates in plastic bags to maintain sterility and incubate at 37 °C for 7 days.

6. REMA checkerboard titration assay

NOTE: Refer to Figure 2A, B for a visual guide on the REMA checkerboard titration assay template for drug combinations (e.g., BDQ and DEL).

1. Dilute drugs vertically and horizontally: Dilute the first drug (BDQ) vertically (rows B-H) and the second drug (DEL) horizontally (columns 2-8) to generate various two-drug combinations.
   NOTE: Prepare the serially diluted working solution in a sterile vial (from 4 µg/mL- 0.06 µg/mL) for a drug that is diluted horizontally.
2. Add BDQ working solution for vertical dilution: Add 100 µL of the working BDQ solution to well B2 and B8.
3. Add 7H9-S broth: Using a multichannel pipette, add 50 µL of 7H9-S broth to columns C2-H8. Ensure the distribution is even across all wells.
4. Dilute the drug concentrations: Using a multichannel pipette, perform two-fold dilutions from rows B-H (columns 2-8), ensuring to discard the final 50 µL after mixing in row H.
5. Add DEL working solution for horizontal dilution: Add serially diluted working solutions of the drugs from 4 µg/mL to 0.06 µg/mL to the corresponding wells.
6. Growth control wells: Add 100 µL of 7H9-S broth to wells B9 and C9 for the growth control.
7. Negative and sterility controls: Add 200 µL of 7H9-S broth to wells B10 and C10 to serve as negative and sterility controls.
8. Inoculate plates: Inoculate each well with 100 µL of the 1:10 diluted culture suspension, except for the negative control well. Ensure uniform inoculation across all wells.
9. Seal and incubate: Seal the plates in plastic bags to maintain sterility and incubate at 37 °C for 7 days.

7. Incubation and interpretation of results

1. Resazurin staining: After 7 days of incubation, add 30 µL of resazurin (0.01%-0.02%) to each well. Seal and incubate the plate overnight for color development.
2. Interpret color change.
   1. Examine the wells visually under consistent white light or daylight-balanced illumination.
      NOTE: The growth control (no drug) should turn blue to pink, indicating active bacterial metabolism. A blue color indicates complete inhibition of growth. Pink denotes active growth, while purple (a mixed hue) represents partial inhibition.
   2. Compare the intensity of the color to both the negative control (media only) and the positive control (bacterial growth without drug) for accurate interpretation. To minimize observer bias, use dual independent readings or plate photography.
      NOTE: Visual judgment can vary with ambient lighting conditions, so consistent illumination is essential.
3. Minimal inhibitory concentration (MIC)
   1. Define MIC as the lowest concentration of drug at which the well remains completely blue, indicating ≥99% inhibition of metabolic activity. Consider wells showing purple or pink color indicative of partial or complete growth.
      NOTE: The MIC determination by REMA approximates the principles of the classical proportion method, in which the MIC is considered the lowest concentration that inhibits ≥99% of the inoculum relative to the drug-free control. While REMA does not use CFU counts, the visual endpoint represents a phenotypic correlate of this proportion-based threshold. This interpretation aligns with previously established colorimetric protocols^11,14.

8. Calculation of fractional inhibitory concentration (FIC) Index

NOTE: The fractional inhibitory concentration (FIC) index helps determine the interaction between two drugs (synergy, antagonism, or indifference). It is calculated based on the MIC values obtained from the checkerboard assay.

1. Identify the MIC for each drug alone: From the REMA assay, determine the MIC for each drug when used individually.
2. Identify the MIC for each drug in combination: From the checkerboard titration assay, identify the MIC of each drug when used in combination with the second drug.
3. Calculate the FIC for each drug.
   NOTE: Refer to Table 2 for the formulas used to calculate the FIC and FIC index. Table 3 shows consolidated MIC based on REMA assay. Representative MIC and FIC index values are shown in Table 4.
   1. Interpret the FIC Index as follows:
      FIC ≤ 0.5: Indicate synergy - the drugs work together to inhibit growth more effectively than either drug alone.
      FIC > 0.5 to < 4.0: Indicate additivity - the combined effect is equal to the sum of the individual effects.
      ​FIC ≥ 4.0: Indicate antagonism - the drugs interfere with each other's activity.
4. Example: Calculate the FIC Index for BDQ and DEL in H37Rv
   1. Determine the MIC of BDQ for H37Rv (e.g., 0.125 µg/mL). In combination with DEL, record the MIC of BDQ as 0.03 µg/mL.
   2. Determine the MIC of DEL for H37Rv (e.g., 0.063 µg/mL). In combination with BDQ, record the MIC of DEL as 0.125 µg/mL.
   3. Calculate FIC for BDQ:
      FIC(BDQ) = MIC of BDQ in combination/MIC of BDQ alone = 0.03 / 0.125 = 0.24
   4. Calculate FIC for DEL:FIC(DEL) = MIC of DEL in combination / MIC of DEL alone = 0.125 / 0.063 = 1.98
   5. Add both FICs to obtain the FIC Index:FIC Index = FIC(BDQ) + FIC(DEL) = 0.24 + 1.98 = 2.26
   6. Interpret the result.
      NOTE: An FIC index of 2.26 falls within the additive interaction range (>0.5 to <4.0), indicating that the combination of BDQ and DEL has an additive effect on M. tuberculosis growth inhibition.

Results

Determination of MIC of anti-TB drugs by REMA assay
The activities of Bedaquiline, Delamanid, Moxifloxacin, and Isoniazid were evaluated using REMA against drug-sensitive H37RV and drug-resistant clinical isolate of M. tuberculosis. Bedaquiline, Delamanid, and Isoniazid were used in the concentration range of (1 µg/mL to 0.03 µg/mL) and Moxifloxacin was in the range of (2 µg/mL to 0.06 µg/mL). The bacterial suspensions of drug-resistant clinical isolates of M. tuberculosis and H37Rv...

Discussion

The global threat posed by drug-resistant tuberculosis (DR-TB) has become a pressing issue^1,19. This has created an urgent need for developing improved, rapid methods for drug susceptibility testing (DST), particularly for second-line and newer anti-TB drugs. Such methods are essential to enhance treatment strategies, manage anti-TB drugs effectively, and reduce the risks of disease progression and further resistance²⁰. A method that evalu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
250 mL square bottle with cap	Schott Duran	218203655	Media preparation
7H9 broth	Becton Dickinson	271310	Growth media for M. tuberculosis (Room temparature)
96-well plate with lid	Corning	3596	96-well Clear Flat Bottom Polystyrene TC-treated Microplates, Individually Wrapped, with Lid, Sterile (Room temparature)
ART-Tips (1000 μL)	TARSONS	528106	Hydrophobic polyethylene filters with  Polypropylene Racked Filter Tip-Sterile (Room temparature)
ART-Tips (200 μL)	TARSONS	528104	Hydrophobic polyethylene filters with  Polypropylene Racked Filter Tip-Sterile (Room temparature)
Barium chloride dihydrate	Merck	217565	McFarland Standard preparation (Room temparature)
Bedaquiline (BDQ)	Jansen and Jansen	Gift from Jansen and Jansen	Anti-TB drug (4 °C)
Casitone	Becton Dickinson	225930	Supplement for bacterial culture (Room temparature)
Delamanid (DEL)	Otsuka Pharmaceuticals	Gift from Otsuka Pharmaceuticals	Anti-TB drug (4 °C)
Dimethyl sulfoxide	Sigma-Aldrich	67-68-5	Drug stock preparation (Room temparature)
Filter 0.2 μm	Millipore	SLGVR33RS	0.22  μm PVDF ,disposable , sterile syringe filter (Room temparature)
Glycerol	Merck	356350	Added to culture media (Room temparature)
Inoculating Loop	Sigma-Aldrich	I8263	For culture suspension preparation (Room temparature)
Isoniazid (INH)	Sigma-Aldrich	I3377-5G	Anti-TB drug (4 °C)
LJ medium Slant	BD Mycobactosel	221414	Growth media for M. tuberculosis (4 °C)
McCartney Bijou Narrow Mouth bottle, 14 mL	Supertek	12.124.0014	For culture suspension preparation (Room temparature)
McCartney Bijou Narrow Mouth bottle, 7 mL	Supertek	12.124.0007	For culture suspension preparation (Room temparature)
Micropipettes (1000 μL)	Gilson	F144059M	For precise liquid handling (Room temparature)
Micropipettes (200 μL)	Gilson	FA10005M	For precise liquid handling (Room temparature)
Moxifloxacin (MOX)	Sigma-Aldrich	SML1581-50MG	Anti-TB drug (4 °C)
Multichannel pipette(20–200 μL)	VWR	849070024	For simultaneous pipetting across wells
OADC	Becton Dickinson	211886	Supplement for bacterial culture (4 °C)
Pipette fillers	Merck	Z333980	For precise liquid handling (Room temparature)
Resazurin sodium salt	Sigma-Aldrich	62758-13-8	Redox indicator for REMA assays (Room temparature)
Serological pipettes,10 mL	Corning	CLS4100	Sterile, polystyrene disposable pipettes were used (Room temparature)
Solid-glass beads, diam. 6 mm	Sigma-Aldrich	Z265950	For culture suspension preparation (Room temparature)
Sulfuric acid 95–97%	Merck	100731	McFarland Standard preparation (Room temparature)
Syringes for filter	Dispo Van	100032	For use with syringe filters
Vortex Mixer	REMI	CM-101 PLUS	For culture suspension preparation
Zipper-top poly bag	Sigma-Aldrich	Z162949