This work was supported by BC Lung Association and Mitacs.

1. Bacterial Strain and Growth Medium
<ol>
	<li>Make albumin dextrose and salt stock solution (ADS) by solubilizing 25 g of bovine serum albumin, 10.0 g of dextrose, and 4.05 g of sodium chloride in 460 mL of deionized water. Filter-sterilize the ADS and store at 4 &#176;C.</li>
	<li>Make 7H9 broth by adding 4.7 g of 7H9 powder and 2 mL of glycerol to 900 mL of purified water. Autoclave the 7H9 broth at 121 &#176;C for 10 min and allow it to cool to room temperature before proceeding. Make 7H9ADST by adding 100 mL of ADS and 0.5 mL of Tween80 to 900 mL of 7H9 broth. Store at 4 &#176;C.</li>
	<li>Weigh 50 mg of kanamycin sulfate and dissolve in 1 mL of deionized water; the final concentration is 50 mg/mL. Filter-sterilize and store at -20 &#176;C. Add 0.5 mL of kanamycin stock solution per 1 L of 7H9ADST. 
	NOTE: This medium should be made fresh, so scale the volumes appropriately according to the culture size.</li>
	<li>Grow M. tuberculosis in 7H9ADST supplemented with kanamycin in standing culture. Shake the culture daily and dilute it before the OD600 reaches 1.0 to avoid clumping. 
	&#8203;NOTE: The M. tuberculosis strain used for the development of this method was H37Rv transformed with pJAK2.A plasmid12. pJAK2.A is an integrative plasmid based on the pMV361 vector, which allows high-level expression of the firefly luciferase gene from the hsp60 promoter and can be selected using kanamycin.</li>
</ol>
2. THP-1 Medium and Maintenance
<ol>
	<li>Add 50 mL of heat-inactivated fetal bovine serum (FBS) and 5 mL of 200 mM L-glutamine to 500 mL of RPMI 1640 to make RPMI incomplete medium (approximately 10% FBS and 2 mM glutamine).</li>
	<li>Maintain an THP-1 cell culture according to standard protocol13. Briefly, grow THP-1 cells in RPMI incomplete medium while maintaining a cell density of 0.2 to 1 million per mL of medium between passages.</li>
</ol>
3. High-throughput Intracellular Screening Using Luciferase-expressing M. tuberculosis H37Rv
<ol>
	<li>Measure the optical density of an actively growing bacterial suspension in a spectrophotometer at a wavelength of 600 nm. Calculate the bacterial density using the conversion factor of 0.1 OD600 = 3 x 107 bacteria per mL.</li>
	<li>Pipette out sufficient bacteria for a multiplicity of infection (MOI) of 10:1 into a new centrifuge tube. Pellet at 3,000 x g for 10 min and aspirate the liquid. Add 50 &#181;L of human serum to 450 &#181;L of RPMI1640. Scale the volume to appropriate values for the experiment.</li>
	<li>To opsonize the bacteria, resuspend the pellet at a density of 1 x 108 bacteria per 500 &#181;L of RPMI1640 containing 10% human serum. Allow the mixture to incubate at 37 &#176;C for 30 min. Determine the THP-1 cell culture density by counting with a hemocytometer and an inverted microscope.</li>
	<li>Pellet the cells in sterile centrifuge tubes at 100 x g and 37 &#176;C for 10 min. Aspirate the supernatant and resuspend the cells in RPMI incomplete at a density of 1 million cells per mL. Add phorbol-12-myristate-13-acetate (PMA) to a 40 ng/mL final concentration. 
	NOTE: This will be referred to as the differentiation mix.</li>
	<li>Combine opsonized M. tuberculosis with THP-1 differentiation mix at a MOI of 10:1 and aliquot the final mix at 100 &#181;L per well in a 96-well flat-bottom white plate. Regularly stir the mixture to ensure uniformity. Allow the differentiation and infection to proceed overnight at 37 &#176;C in a humidified incubator containing 5% CO2.</li>
	<li>Wash the wells twice with 100 &#181;L of RPMI each. Add compounds diluted to the desired concentrations in RPMI incomplete and incubate for 3 days.</li>
	<li>Aspirate the medium from the wells. Add 50 &#181;L of luciferase assay reagent to each well. Seal the plates with transparent adhesive plate sealers. Allow 5 min of incubation at 22 &#176;C and then obtain a readout in a luminometer at 1 s per well.</li>
</ol>
4. Cytotoxicity Analysis Using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay14
<ol>
	<li>Differentiate THP-1 cells in RPMI incomplete supplemented with 40 ng/mL of PMA in clear 96-well plates. Maintain a cell density of 1 million per mL and aliquot 100 &#181;L per well. Allow differentiation to proceed overnight at 37 &#176;C in a humidified incubator containing 5% CO2 .</li>
	<li>Aspirate the medium from the wells and wash them twice with RPMI 1640. Add compounds diluted in RPMI incomplete to the wells. Incubate for 3 days.</li>
	<li>Dissolve 0.5 g of MTT in 100 mL of phosphate-buffered saline (PBS) to make a stock solution of 5 mg/mL. Sterile filter and store at -20 &#176;C, away from light; it is best to make this solution fresh.</li>
	<li>2.5 h before the end of the 3-day incubation period, add 25 &#181;L of MTT solution to each well and complete the incubation period.</li>
	<li>Prepare 50% N,N-dimethyl formamide (DMF) by mixing 250 mL of DMF with 250 mL of deionized water.</li>
	<li>Prepare MTT extraction buffer as follows: Weigh 100 g of SDS in a 500-mL bottle and add 300 mL of 50% DMF. Apply low heat to allow the SDS to dissolve. Add 10 mL of pure acetic acid and 12.5 mL of 1 M HCl. Fill up to the 500-mL mark with 50% DMF; the final composition of the extraction buffer is 50% DMF, 20% SDS, 2.5% acetic acid, and 2.5% 1 M hydrochloric acid.</li>
	<li>At the end of the treatment period, add 100 &#181;L of extraction buffer (warmed to 45 &#176;C to dissolve any crystals) to each well. Allow the mixture to incubate overnight at 37 &#176;C in a humidified incubator containing 5% CO2. Read the absorbance at 570 nm. 
	&#8203;NOTE: The cytotoxicity assay is best performed in parallel with an intracellular screen using same-batch and age of THP-1 cells.</li>
</ol>
5. In-broth Activity Analysis Using a Resazurin Assay3
<ol>
	<li>Grow M. tuberculosis in 7H9ADST to the mid-log phase (~ 0.5 - 0.8 OD600). Dilute the culture with the 7H9ADST to 0.01 OD600. Dilute the compounds in 7H9ADST to 2x the testing concentrations and aliquot 100 &#181;L of each diluted compound into each well.</li>
	<li>Transfer 100 &#181;L of the diluted bacterial suspension into each well. Allow the plates to incubate at 37 &#176;C in a humidified incubator for 5 days. Dissolve 10 mg of resazurin in 100 mL of deionized water and sterile filter.</li>
	<li>Add 30 &#181;L of resazurin solution and monitor the color change after 48 h; bacterial growth is indicated by a color conversion from blue to pink. 
	NOTE: A quantitative analysis can also be performed by measuring either the fluorescence at 590 nm with excitation at 530 - 560 nm or the absorbance at 570 nm and 600 nm15.</li>
</ol>

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The authors declare no competing financial interests for this work.

The goal of this study was to create a simple and cost-effective HTS method using a human intracellular infection model for M. tuberculosis. Tuberculosis is a human disease characterized by the infection of alveolar macrophages by M. tuberculosis. Due to biosafety issues, research involving biological models of both the bacterium and the host cells has been used in the past. However, it has been shown that the usage of surrogate bacteria and non-human models are poor predictors of hit-to-lead success in...

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. Drug-sensitive TB is a treatable disease that requires multiple antibiotics for a period of 6 months. Despite being a treatable disease, TB mortality was estimated to be 1.5 million in 20151. In the past 10 years, there have been increasing concerns over the prevalence of drug-resistant M. tuberculosis. Multidrug-resistant TB (MDR-TB) is defined as TB that is resistant to at least Isoniazid (INH) and Rifampicin (RMP), and most MDR-TB strains are also resistant to select second-line TB drugs, thus limiting treatment options. The effects of drug resistance create a greater challenge for treating patients co-infected with Human Immunodeficiency Virus (HIV); 400,000 patients worldwide died of HIV-associated TB in 20151. Disappointingly, the United States Food and Drug Administration has approved only one new TB drug against MDR-TB, bedaquiline, in the past 40 years2. Advances in finding better and shorter TB therapies are urgently needed in order to stay ahead in the fight against TB and MDR-TB.
Traditionally, TB drug screens are performed under in vitro growth conditions in growth medium, whereby compounds are added to a growing culture and their effectiveness in eradicating the microorganisms are determined by counting colony forming units (CFU) on solid medium. As CFU counts are labor intensive, time consuming, and costly, various indirect methods have been developed to alleviate this problem. Such methods include the Alamar Blue viability assay3, the determination of fluorescence4 from green fluorescent protein (GFP) or luminescence5 from luciferase-expressing bacteria, and the estimation of total adenosine triphosphate (ATP)6,7.
Typical TB is characterized by an M. tuberculosis infection of the lung, where the bacteria reside and replicate inside the phagosomes of alveolar macrophages8. The simple in-broth phenotypic screen may suit extracellular pathogens; however, in the historical perspective, hit compounds against M. tuberculosis identified using this method often fail to live up to expectations during downstream validation steps in infection models. We propose that TB drug is best performed in an intracellular host cell infection model. Nevertheless, intracellular models possess many technological and biological barriers to high-throughput screening (HTS) development. A big hurdle is the complexity of the infection process, exemplified by numerous steps and the elaborate removal of extracellular bacteria by in-between washing. A second major hurdle is the lengthy time requirements, as growth detection, normally done by CFU counting on culture plates, is a process that takes over 3 weeks to complete. One solution to replace CFU counts has been provided by automated fluorescent microscopy in combination with fluorescent bacteria. However, this solution requires an initial equipment investment that is out of reach for many research labs. A simple, low-cost, and disease-relevant HTS method would greatly enhance the drug discovery process.
In this study, we report a new, modular HTS system that is aimed at providing a rapid, and highly scalable, yet economical, assay suitable for determining the activity of compounds against intracellular M. tuberculosis. This system is composed of three modules: (i) intracellular, (ii) cytotoxicity, and (iii) in-broth assays. The combined final result provides a comprehensive description of the compound properties, with additional information as to the potential mode of action. This screening system has been used in several projects with various compound libraries that target mode of action, including the analysis of drug synergy9, the stimulation of autophagy10, and the inhibition of M. tuberculosis-secreted virulence factor (unpublished). Compounds of unknown mode of action have also been studied11. A modified version of this method was also adopted by our industrial partner as the primary screening method to identify new compounds against intracellular M. tuberculosis11.

<table><tbody><tr><td>RPMI 1640</td><td>Sigma-Aldrich</td><td>R5886</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific</td><td>12483020</td><td></td></tr><tr><td>Middlebrook 7H9</td><td>Becton, Dickinson and Company</td><td>271210</td><td></td></tr><tr><td>Tween80</td><td>Fisher Scientific</td><td>T164</td><td></td></tr><tr><td>Albumin, Bovine pH7</td><td>Affymetrix</td><td>10857</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>BP350</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>kanamycin sulfate</td><td>Fisher Scientific</td><td>BP906</td><td></td></tr><tr><td>PMA</td><td>Sigma-Aldrich</td><td>P8139</td><td></td></tr><tr><td>MTT</td><td>Sigma-Aldrich</td><td>M2128</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide (DMF)</td><td>Fisher Scientific</td><td>D131</td><td></td></tr><tr><td>1 M Hydrocholoric acid (HCl)</td><td>Fisher Scientific</td><td>351279212</td><td></td></tr><tr><td>Acetic acid</td><td>Fisher Scientific</td><td>351269</td><td></td></tr><tr><td>SDS</td><td>Fisher Scientific</td><td>BP166</td><td></td></tr><tr><td>Resazurin</td><td>Alfa Aesar</td><td>B21187</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229</td><td></td></tr><tr><td>THP-1</td><td>American Type Culture Collection</td><td>TIB-202</td><td></td></tr><tr><td>&lt;em&gt;M. tuberculosis&lt;/em&gt; H37Rv</td><td></td><td></td><td></td></tr><tr><td>96-well flat bottom white plate</td><td>Corning</td><td>3917</td><td></td></tr><tr><td>95-well flat bottom clear plate</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Transparent plate sealer</td><td>Thermo Fisher Scientific</td><td>AB-0580</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>Biomate 3</td><td></td></tr><tr><td>Microplate spectrophotometer</td><td>Biotek</td><td>Epoch</td><td></td></tr><tr><td>luminometer</td><td>Applied Biosystems</td><td>Tropix TR717</td><td></td></tr></tbody></table>

system for efficacy and cytotoxicity screening of inhibitors targeting intracellular mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

High-throughput intracellular screening using M. tuberculosis expressing the luciferase gene
Figure 2A and Table 1 contain the raw data collected by the luminometer, expressed in relative luminescent units (RLU), showing the effect of an increasing concentration of the TB drug rifampicin on M. tuberculosis inside THP-1 cells. Figure 2A is a scatter plot of the raw...

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System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

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Universidad de Cartagena UDEC

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Immunology and Infection

8.5K Views.  University of British Columbia. We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Video: System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

High-Throughput Screening Assay for Mycobacterium abscessus Using Dual Reporters

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

Author Spotlight: Scalable Drug Screening Protocol for Efficient Discovery of M. abscessus Treatments

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Accelerating Tuberculosis Determination Using the Micro-CFU Method

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

Author Spotlight: Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

Medicine

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

Source: Schaaf, K., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55453">A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis.</a> 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.

1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc26206 (Mtb-GFP)
NOTE: The M. tuberculosis H37Rv derived auxotroph strain ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

Evaluating the Effectiveness of a Test Compound against Mycobacteria in Broth

Source: Zheng, X., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55273">System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis.</a> J. Vis. Exp. (2017)
This video demonstrates an in-broth assay to determine antibiotic efficacy against Mycobacterium tuberculosis. The bacterial culture is incubated with different concentrations of a bactericidal antibiotic. The bactericidal efficacy is measured by assessing the presence of viable bacteria using a resazurin assay.

1. Bacterial Strain and Growth Medium

	Make albumin dextrose and salt stock solution (ADS) by solubilizing 25 g of bovine serum albumin, 10.0 g of ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Summary

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Chapters in this video

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

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

Evaluating the Effectiveness of a Test Compound against Mycobacteria in Broth