We thank Dr. Frank Wolschendorf for access to the Cytation 3 automated imaging plate reader. This work was funded in part by NIH grant R01-AI104499 to OK. Parts of the work were performed in the UAB CFAR facilities and by the UAB CFAR Flow Cytometry Core/Joint UAB Flow Cytometry Core, which are funded by NIH/NIAID P30 AI027767 and by NIH 5P30 AR048311.

NOTE: As M. tuberculosis mc26206 is an avirulent strain17,18, all work in this protocol can be performed in a Biosafety Level 2 facility (BSL-2).
1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc26206 (Mtb-GFP)
NOTE: The M. tuberculosis H37Rv derived auxotroph strain mc26206 (&#916;panCD, &#916;leuCD) transformed with the gfp expressing plasmid pMN437 is used throughout this protocol16. 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 confirmation of phagocytosis and formation of Mtb/macrophage aggregates. For long term storage, Mtb-GFP were frozen at -80 &#176;C in complete 7H9 media (described in step 1.1) supplemented with 20% glycerol.
<ol>
	<li>Prepare complete Mtb-GFP media (7H9-C) by supplementing 7H9 medium with 10% Middlebrook OADC, 0.02% tyloxapol, 24 &#181;g/mL D-pantothenic acid, 50 &#181;g/mL L-leucine and 50 &#181;g/mL Hygromycin B.</li>
	<li>Thaw one vial of Mtb-GFP and add to 10 mL of 7H9-C in a 30 mL square bottom PETG flask.</li>
	<li>Incubate at 37 &#176;C on rotary shaker (50 rpm). Take optical density measurements (OD600) every few days until OD600 reaches 1.0 (approximately 5-8 days).</li>
	<li>Dilute and passage culture as needed to maintain an OD600 between 0.2-1.0.</li>
	<li>For infection, use an established culture of Mtb-GFP within the logarithmic growth phase (OD600 of 0.6-1). To avoid clumpy cultures, sonicate the Mtb culture flask in a water bath (130 W; 3 x 5 s pulses) once or twice a week</li>
</ol>
2. Culture Conditions for THP-1 Cells
<ol>
	<li>Prepare complete THP-1 cell media (RPMI-C) by supplementing RPMI 1640 medium with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 10 mM HEPES.</li>
	<li>Incubate cells in a T-75 flask at 37 &#176;C in a humidified atmosphere of 5% CO2.</li>
	<li>Count cells using a hemocytometer or flow cytometry every other day and maintain THP-1 cells at a density between 0.1 to 0.6 million per mL.</li>
</ol>
3. Infection Protocol to Generate Mtb/Macrophage Aggregate Structures
<ol>
	<li>THP-1 cell preparation (per 96-well plate)
	<ol>
		<li>Centrifuge 7 x 106 THP-1 cells at 250 x g for 5 min. Resuspend in 7 mL RPMI-C.</li>
	</ol>
	</li>
	<li>Mtb-GFP preparation
	<ol>
		<li>Centrifuge 2.8 x 108 Mtb-GFP at 3,200 x g for 5 min in a swinging-bucket centrifuge using a conversion of OD600 1.0 = 3 x 108 bacteria/mL.</li>
		<li>Wash once with RPMI-C and centrifuge as in step 3.2.1.</li>
		<li>Resuspend in 7 mL RPMI-C and vortex for 10 s.</li>
	</ol>
	</li>
	<li>Infection 
	NOTE: See Figure 1 for template.
	<ol>
		<li>Prepare a 96-well plate by adding 200 &#181;L of sterile water in rows A and H and columns 1 and 12 for a water rim to prevent evaporation of culture medium.</li>
		<li>Add 200 &#181;L RPMI-C to column 2 (B2 to G2) for the background control (Blank).</li>
		<li>To infect, add Mtb-GFP suspension (step 3.2) to THP-1 cell suspension (step 3.1) and mix well by pipetting. The final THP-1 cell density is 5 x 105 per mL and the corresponding multiplicity of infection is 40.</li>
		<li>Pour the THP-1/Mtb-GFP suspension into a 25 mL reservoir.</li>
		<li>Add 200 &#181;L of THP-1/Mtb-GFP suspension to all remaining 96-wells (B3 through G11) using a multi-channel pipette. 
		NOTE: If working with multiple 96-well plates, regularly resuspend the remaining THP-1/Mtb suspension in the reservoir to ensure an even mixture is added to each well.</li>
		<li>Incubate at 37 &#176;C with 5% CO2 for 7-10 days.</li>
		<li>Change media every 2 days by slowly removing 100 &#181;L spent media from the top of each well and gently adding 100 &#181;L pre-warmed RPMI-C using a multi-channel pipet. Do not resuspend wells and disturb the Mtb/macrophage aggregates on the bottom of the wells.</li>
		<li>Visually examine the wells by fluorescence microscopy (4-10X objective) daily, taking note of the size of Mtb/macrophage aggregates. By day 7-10, Mtb/macrophage aggregates should be sufficiently large (refer to Figure 2 for reference) to proceed for drug efficacy testing (Section 4).</li>
		<li>If desired, capture images of the 96-wells using an automated cell imaging system fitted with bright field and GFP filter sets to document Mtb/macrophage aggregate formation. 
		NOTE: If users do not have access to an automated imaging system, images of representative wells can be taken using any appropriate microscope with GFP and bright field capabilities.</li>
	</ol>
	</li>
</ol>
4. Growth Inhibition Assay to Assess Drug Efficacy Against Mtb Derived from Mtb/Macrophage Aggregates
<ol>
	<li>Drug preparation (triplicate conditions for two drugs) 
	NOTE: See Figure 1A for template.
	<ol>
		<li>In a separate 96-well plate, add 125 &#181;L of 7H9-C media to B2 through to G10.</li>
		<li>Prepare two drugs at double the highest desired final concentration in 1 mL of 7H9-C to account for the dilution in step 4.2.4.</li>
		<li>Add 250 &#181;L of each drug to wells B11-C11-D11 and E11-F11-G11, respectively for triplicate treatments.</li>
		<li>Using a multi-channel pipet, serially dilute the test drugs two-fold by moving 125 &#181;L from B11-G11 into B10-G10. Mix by pipetting 5 times at each step.</li>
		<li>Continue to move 125 &#181;L from column to column (right to left) across the plate, and stop after column 4.</li>
		<li>After mixing column 4, discard 125 &#181;L into a waste container. Columns 2 and 3 should not contain any drugs to allow for use as a background (Blank) and positive growth controls. 
		NOTE: Alternatively, follow template in Figure 1B for testing drug libraries. Each 96-well plate can accommodate 58 drugs at a single concentration. Prepare this drug plate using double the desired final concentration since it will be diluted in half in step 4.2.4.</li>
	</ol>
	</li>
	<li>Drug treatment:
	<ol>
		<li>Retrieve the 96-well plate containing the Mtb-infected macrophages (Mtb/macrophage aggregates).</li>
		<li>Carefully decant all the relevant wells (B2 through G11) with a multi-channel pipet. Perform this in a two-step manner: first remove 150 &#181;L without tilting the plate, and then remove remaining media (~50 &#181;L) by tilting the plate and inserting the pipette tip to the bottom edge of the well. As Mtb/macrophage aggregates are adherent to the bottom of the well, no material should be lost.</li>
		<li>Gently add 100 &#181;L of 7H9-C media to all relevant wells (B2 through G11) of the plate containing the Mtb/macrophage aggregates.</li>
		<li>Using a multi-channel pipet, transfer 100 &#181;L from the drug containing 96-well plate (step 4.1) to the corresponding wells of the infection plate.</li>
		<li>Place in sealed bag and incubate for 3 days at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
5. Quantification of Drug Efficacy Using the Resazurin Microtiter Assay
<ol>
	<li>Prepare resazurin stock solution at a final concentration of 0.8 mg/mL in H2O. Filter through 0.22 &#181;m pore size PVDF membrane for sterilization.</li>
	<li>Prepare resazurin working solution by mixing resazurin stock solution, H2O and Tween-80 (20% solution in H2O) in a 2:1:1 ratio. Final concentrations are 0.4 mg/mL resazurin and 5% Tween-80.</li>
	<li>Using a plate reader, setup a program to read fluorescence at 530 nm excitation and 590 nm emission every 30 min for 24 h at 37 &#176;C. Pre-warm plate reader to 37 &#176;C.</li>
	<li>Add 20 &#181;L of resazurin working solution to all relevant wells (B2 through G11) of the drug treated plate (step 4.11) using a multi-channel pipet.</li>
	<li>Place plate on the plate reader and start the program set up in step 5.3. 
	NOTE: To facilitate the processing of high volumes of assay plates, it is sufficient to develop the assay in a 37 &#176;C incubator and perform single fluorescence reads every 24 h. This is also applicable to users who do not have access to a plate reader with kinetic and incubation capabilities.</li>
</ol>

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S., Early, J., Parish, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+for+early-stage+tuberculosis+drug+discovery.">The future for early-stage tuberculosis drug discovery.</a> Future Microbiol. 10 (2), 217-229 (2015).</li><li>Evangelopoulos, D., Fonseca, d. a., D, J., Waddell, S. 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(83), e51114 (2014).</li><li>Sorrentino, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+intracellular+screen+for+new+compounds+able+to+inhibit+Mycobacterium+tuberculosis+growth+in+human+macrophages.">Development of an intracellular screen for new compounds able to inhibit Mycobacterium tuberculosis growth in human macrophages.</a> Antimicrob Agents Chemother. 60 (1), (2015).</li><li>Sarathy, J., Dartois, V., Dick, T., Gengenbacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+drug+uptake+in+phenotypically+resistant+nutrient-starved+nonreplicating+Mycobacterium+tuberculosis.">Reduced drug uptake in phenotypically resistant nutrient-starved nonreplicating Mycobacterium tuberculosis.</a> Antimicrob Agents Chemother. 57 (4), 1648-1653 (2013).</li><li>Dutta, N. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+elicited+by+a+double+leucine+and+pantothenate+auxotroph+of+Mycobacterium+tuberculosis+in+guinea+pigs.">Protection elicited by a double leucine and pantothenate auxotroph of Mycobacterium tuberculosis in guinea pigs.</a> Infect Immun. 72 (5), 3031-3037 (2004).</li><li>Jain, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+transduction+designed+for+precise+high-throughput+unmarked+deletions+in+Mycobacterium+tuberculosis.">Specialized transduction designed for precise high-throughput unmarked deletions in Mycobacterium tuberculosis.</a> MBio. 5 (3), e01245-e01214 (2014).</li><li>Davis, J. 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The authors have nothing to disclose.

Here, we have described in detail an alternative Mtb infection model suitable for drug efficacy testing. This model takes into account two key factors that should be given more consideration during the early TB drug development process: the presence of physiologically relevant barriers to drug penetration and metabolic changes of Mtb during infection. While we have previously shown the benefits of our infection model and proposed the possibility of scaling up the infection for throughput compatibility<s...

Tuberculosis (TB) remains a serious global health threat despite the availability of anti-TB chemotherapy regimens for over 40 years1. This is due in part to the requirement for long treatment periods of over 6 months using multiple drug combinations, which leads to patient non-compliance2. The emergence of drug-resistant TB in recent years has further compounded problems in a field where successful development of clinically approved drugs is virtually non-existent3. Indeed, despite exhaustive anti-TB drug development, only a single drug has been FDA approved for clinical use in the past 40 years4. Thus, new generations of anti-TB drugs are urgently needed to address this problem.
A key problem in TB drug discovery is the lack of successful transfer from compounds with in vitro activity to efficacy in the clinical setting5,6,7. Initially, target based approaches were used to screen for anti-Mtb drugs5, which failed to translate into whole bacterial cells. Even when Mtb cells are used, it is often performed using broth grown cultures, which do not accurately predict drug efficacy in vivo8,9. These problems have been recognized and drug screening assays against macrophages containing Mtb or latent Mtb have been successfully established8,10,11,12. However, even these more advanced assays do not give sufficient consideration to the penetration barriers that drugs encounter in the non-vascularized pulmonary lesions, and in the necrotic foci at the site of infection. Indeed, even for the first-line TB drug rifampicin, sub-optimal dosing has been questioned due to inadequate in vivo tissue and cerebral spinal fluid (CSF) penetration13,14,15 as well as decreased efficacy against intracellular Mtb8,9. As such, new models and assays that would take into account these parameters during the early lead development process would undoubtedly improve TB drug discovery efforts.
To address this need, we recently established an inexpensive, rapid, and BSL-2 compatible alternative infection model for Mtb drug efficacy testing16. This infection model produced densely packed Mtb within large macrophage aggregate structures, which recapitulated physiologically relevant cellular penetration barriers and generated macrophage-passaged Mtb with an altered physiological status resembling latent Mtb. Mtb derived from this infection model was combined with the resazurin microtiter assay (REMA) to evaluate drug efficacy, which produced results consistent with other intracellular infection models and correlated well with the reported ability of common TB drugs to achieve high CSF concentrations relative to serum concentrations16.
Here we describe in detail the generation of Mtb/macrophage aggregate structures to produce macrophage-passaged Mtb suitable for drug susceptibility testing using REMA. In particular, we show how this infection system could be adapted to a 96-well format for compatibility with throughput screening of candidate anti-TB drugs.

<table><tbody><tr><td>7H9</td><td>BD Difco</td><td>271310</td><td>Follow manufacturer&#039;s recommendations</td></tr><tr><td>Middlebrook OADC</td><td>BD Biosciences</td><td>212351</td><td></td></tr><tr><td>Tyloxapol</td><td>Sigma</td><td>T8761</td><td>Prepare 20% stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>D-Pantothenic acid hemicalcium salt</td><td>Sigma</td><td>P5710</td><td>Prepare 24 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>L-leucine</td><td>MP Biomedicals</td><td>194694</td><td>Prepare 50 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>Hygromycin B</td><td>EMD Millipore</td><td>400051</td><td>Prepare 200 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Nalgene Square PETG media bottle</td><td>Thermo Fisher</td><td>2019-0030</td><td></td></tr><tr><td>RPMI 1640 media</td><td>Hyclone</td><td>SH30027.01</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S12450H</td><td></td></tr><tr><td>L-glutamine</td><td>Corning</td><td>MT25005CI</td><td></td></tr><tr><td>HEPES</td><td>Hyclone</td><td>SH30237.01</td><td></td></tr><tr><td>Cytation 3 plate reader</td><td>Biotek</td><td></td><td>Interchangable with any fluorescent plate reader and microscope</td></tr><tr><td>Gen5 Software</td><td>Biotek</td><td></td><td>Recording and analysis of rezasurin coversion</td></tr><tr><td>Rifampicin&amp;nbsp;</td><td>Fisher Scientific</td><td>BP2679250</td><td>Prepare 10 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Moxifloxacin Hydrochloride</td><td>Acros Organics</td><td>457960010</td><td>Prepare 10 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Resazurin Sodium Salt</td><td>Sigma</td><td>R7017</td><td>Prepare 800 &amp;mu;g/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>Tween-80</td><td>Fisher Scientific</td><td>T164500</td><td>Prepare 20% stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr></tbody></table>

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.

To confirm the robustness of adapting this infection model to 96-well plate format, we here examined the drug susceptibility of Mtb derived from our 96-well adapted infection model to rifampicin (RIF) and moxifloxacin (MOXI) according to the template given in Figure 1A. We demonstrate that the generation of Mtb/macrophage aggregate structures key to this assay can be reliably produced in a 96-well plate format (Figure 2), thereby enablin...

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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.

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 ...

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Immunology and Infection

7.8K Views.  University of Alabama at Birmingham. 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. 

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

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

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 ...

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 ...

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.

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.

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

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. ...

Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic strains and genetically engineered cells. Here, an alternative method to detect biochemical interaction between the macrophage microbial sensor SLAMF1 and Mycobacterium tuberculosis is described. Two technical approaches employing flow cytometry and fluorescence microscopy were developed. Total cell protein extracts from human macrophages were generated, then incubated with whole cells of M. tuberculosis (WCMtb)&#160;or M. tuberculosis antigens (Mtb Ags) overnight at 4 &#176;C and finally cross-linked using formaldehyde/glycine/ethylene glycol bis (succinimidyl succinate) treatment. SLAMF1 interaction with WCMtb by flow cytometry was detected with a PE-specific anti-SLAMF1 antibody. The existence of interaction by fluorescence microscopy was performed by attaching Rhodamine-PE stained Mtb Ags to poly-D-lysine coated slides, which were incubated with the total protein extract from monocyte-derived macrophages. After cross-linking treatment, SLAMF1 was visualized using primary (anti-SLAMF1) and secondary (Alexa Fluor 488) antibodies. The assays provided a strong biochemical tool to measure pathogen-immunoreceptor interactions, overcoming the difficulties associated with transgenic cell lines and protein gene expression modulation experiments.

This study provides a protocol for evaluating the interaction of Mycobacterium tuberculosis with the SLAMF1 microbial sensor. The assays were conducted on human monocyte-derived macrophages using flow cytometry and fluorescence microscopy. The described tools are relevant for studying interactions between pathogens and immunoreceptors.

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic ...

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://app.jove.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://app.jove.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 ...

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

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