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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American Thoracic Society and The Centers for Disease Control and Prevention.</a> Am J Respir Crit Care Med. 149 (5), 1359-1374 (1994).</li><li>Koul, A., Arnoult, E., Lounis, N., Guillemont, J., Andries, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+new+drug+discovery+for+tuberculosis.">The challenge of new drug discovery for tuberculosis.</a> Nature. 469 (7331), 483-490 (2011).</li><li>Palomino, J. C., Martin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TMC207+becomes+bedaquiline,+a+new+anti-TB+drug.">TMC207 becomes bedaquiline, a new anti-TB drug.</a> Future Microbiol. 8 (9), 1071-1080 (2013).</li><li>Zuniga, E. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+anti-tuberculosis+drug+efficacy:+rethinking+bacterial+populations+and+how+we+model+them.">Understanding anti-tuberculosis drug efficacy: rethinking bacterial populations and how we model them.</a> Int J Infect Dis. 32, 76-80 (2015).</li><li>Ekins, S., 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=Looking+back+to+the+future:+predicting+in+vivo+efficacy+of+small+molecules+versus+Mycobacterium+tuberculosis.">Looking back to the future: predicting in vivo efficacy of small molecules versus Mycobacterium tuberculosis.</a> J Chem Inf Model. 54 (4), 1070-1082 (2014).</li><li>Christophe, T., 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=High+content+screening+identifies+decaprenyl-phosphoribose+2'+epimerase+as+a+target+for+intracellular+antimycobacterial+inhibitors.">High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors.</a> PLoS Pathog. 5 (10), e1000645 (2009).</li><li>Hartkoorn, R. C., 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=Differential+drug+susceptibility+of+intracellular+and+extracellular+tuberculosis,+and+the+impact+of+P-glycoprotein.">Differential drug susceptibility of intracellular and extracellular tuberculosis, and the impact of P-glycoprotein.</a> Tuberculosis (Edinb). 87 (3), 248-255 (2007).</li><li>Queval, C. J., 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=A+microscopic+phenotypic+assay+for+the+quantification+of+intracellular+mycobacteria+adapted+for+high-throughput/high-content+screening.">A microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening.</a> J Vis Exp. (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. K., Karakousis, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+the+duration+of+tuberculosis+treatment+be+shortened+with+higher+dosages+of+rifampicin?.">Can the duration of tuberculosis treatment be shortened with higher dosages of rifampicin?.</a> Front Microbiol. 6, 1117 (2015).</li><li>van Ingen, J., 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=Why+Do+We+Use+600+mg+of+Rifampicin+in+Tuberculosis+Treatment?.">Why Do We Use 600 mg of Rifampicin in Tuberculosis Treatment?.</a> Clin Infect Dis. 52 (9), e194-e199 (2011).</li><li>Donald, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+concentrations+of+antituberculosis+agents+in+adults+and+children.">Cerebrospinal fluid concentrations of antituberculosis agents in adults and children.</a> Tuberculosis (Edinb). 90 (5), 279-292 (2010).</li><li>Schaaf, K., 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=A+Macrophage+Infection+Model+to+Predict+Drug+Efficacy+Against+Mycobacterium+Tuberculosis.">A Macrophage Infection Model to Predict Drug Efficacy Against Mycobacterium Tuberculosis.</a> Assay Drug Dev Technol. 14 (6), 345-354 (2016).</li><li>Sampson, S. 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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A., 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=Assessment+of+immunity+to+mycobacterial+infection+with+luciferase+reporter+constructs.">Assessment of immunity to mycobacterial infection with luciferase reporter constructs.</a> Infect Immun. 67 (9), 4586-4593 (1999).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7H9</td>
			<td>BD Difco</td>
			<td>271310</td>
			<td>Follow manufacturer&#39;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 H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>D-Pantothenic acid hemicalcium salt</td>
			<td>Sigma</td>
			<td>P5710</td>
			<td>Prepare 24 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>MP Biomedicals</td>
			<td>194694</td>
			<td>Prepare 50 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>EMD Millipore</td>
			<td>400051</td>
			<td>Prepare 200 mg/ml&#160; stock solution in H2O</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&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP2679250</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Moxifloxacin Hydrochloride</td>
			<td>Acros Organics</td>
			<td>457960010</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Resazurin Sodium Salt</td>
			<td>Sigma</td>
			<td>R7017</td>
			<td>Prepare 800 &#956;g/mL stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher Scientific</td>
			<td>T164500</td>
			<td>Prepare 20% stock solution in H2O; 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...

Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

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

7.7K 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 ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

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 In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...