An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model

Abstract

Source: Schaaf, K., et al. A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis. J. Vis. Exp. (2017)

This video demonstrates an assay to evaluate the efficacy of drugs against Mycobacterium tuberculosis (Mtb) infection. An in vitro infection model containing Mtb/macrophage aggregates is generated that mimics in vivo infection. Upon introducing suitable antibiotics, their efficacy is tested via resuzarin assay to monitor the survival of bacteria with increasing drug concentrations.

Protocol

1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc²6206 (Mtb-GFP)

NOTE: The M. tuberculosis H37Rv derived auxotroph strain mc²6206 (ΔpanCD, ΔleuCD) transformed with the gfp (green fluorescent protein) expressing plasmid pMN437 is used throughout this protocol. It is possible to substitute the Mtb-GFP strain with non-gfp expressing wild-type strain to enable easier accessibility of the strain for researchers. However, gfp expression is desirable to enable visual confir....

Results

Figure 1: Drug plate layout. (A) Example template of the drug challenge plate used in step 4. This allows for the parallel testing of two drugs in triplicate wells at 8 defined concentrations. As a representative experiment, we used rifampicin (RIF).......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
7H9	BD Difco	271310	Follow manufacturer's recommendations
Middlebrook OADC	BD Biosciences	212351
Tyloxapol	Sigma	T8761	Prepare 20% stock solution in H2O; filter sterilize
D-Pantothenic acid hemicalcium salt	Sigma	P5710	Prepare 24 mg/ml stock solution in H2O; filter sterilize
L-leucine	MP Biomedicals	194694	Prepare 50 mg/ml stock solution in H2O; filter sterilize
Hygromycin B	EMD Millipore	400051	Prepare 200 mg/ml stock solution in H2O
Nalgene Square PETG media bottle	Thermo Fisher	2019-0030
RPMI 1640 media	Hyclone	SH30027.01
Fetal Bovine Serum	Atlanta Biologicals	S12450H
L-glutamine	Corning	MT25005CI
HEPES	Hyclone	SH30237.01
Cytation 3 plate reader	Biotek		Interchangable with any fluorescent plate reader and microscope
Gen5 Software	Biotek		Recording and analysis of rezasurin coversion
Rifampicin	Fisher Scientific	BP2679250	Prepare 10 mg/ml stock solution in H2O
Moxifloxacin Hydrochloride	Acros Organics	457960010	Prepare 10 mg/ml stock solution in H2O
Resazurin Sodium Salt	Sigma	R7017	Prepare 800 μg/mL stock solution in H2O; filter sterilize
Tween-80	Fisher Scientific	T164500	Prepare 20% stock solution in H2O; filter sterilize