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

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....

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