Name: a high-throughput compatible assay to evaluate drug efficacy against macrophage passaged mycobacterium tuberculosis
Uploaded: 2017-03-24T15:00:00
Description: Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

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;leu...

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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 ...

The overall goal of this procedure is to reproducibly generate mycobacterium tuberculosis infected macrophage aggregate structures in 96 well plate format for drug susceptibility testing using a viability dye. This method can improve the early drug development process for anti tuberculosis compounds, which is hindered by the unproductive translation of candidate compounds into the clinical setting. The main advantage of this simple, inexpensive, BSL two compatible infection model is that it recapitulates physiologically relevant cellular penetration barriers, reminiscent of tuberculosis granulomas.This produces drug susceptibility results more predictive of in vivo efficacy. Begin this procedure by preparing green fluorescent protein expressing M.tuberculosis MC squared 6206, hereafter referred to as MTBGFP, for infection. Keep in mind that this is avirulent strain, so all work in the protocol described here can be performed in a biosafety level two facility.For each 96 well plate to be set up centrifuge at 2.8 times 10 to the eighth MTBGFP at 3, 200 times G for five minutes in a swinging bucket centrifuge. After the spin, aspirate the supernatant and add five milliliters of RPMIC to wash the bacteria. Pipette up and down to resuspend the cell.Then centrifuge again. After the second spin pour off the supernatant and resuspend the bacterial cells in seven milliliters of RPMIC, then vortex for 10 seconds. Next, for each 96 well plate to be set up, centrifuge seven times 10 to the sixth THP1 human monocytic cells at 250 times G for five minutes.After the centrifugation pour off the supernatant and resuspend the THP1 cells in seven milliliters of RPMIC. Next prepare a 96 well plate for infection by adding 200 microliters of sterile water to the wells in rows A and H and columns one and 12. This water rim will prevent the evaporation of culture medium.Add 200 microliters of RPMIC to column two, B two to G two, for the background control or blank. To infect the THP1 monocyte use a pipette to transfer the entire prepared MTBGFP bacterial suspension to the THP1 cell suspension and mix will by pipetting up and down. The final THP1 cell density is five times 10 to the fifth per milliliter and the corresponding well duplicity of infection is 40.Next, pour the THP1 MTBGFP suspension into a 25 milliliter reservoir, and then using a multichannel pipette, add 200 microliters of THP1 MTBGFP suspension all of the remaining 96 wells, E three through G 11. Incubate the plate at 37 degrees Celsius, with 5%CO2 for seven to 10 days. Every two days throughout the incubation, use a multichannel pipette to change the medium by slowly removing 100 microliters of spent medium from the top of each well and gently adding 100 microliters of pre warmed RPMIC.When changing the medium it is important to take care not to disturb the MTB macrophage aggregates on the bottom of the wells. Each day visually examine the wells using a fluorescence microscope, equipped with bright field and GFP filter sets with a four to 10 X objective. Note the size of MTB macrophage aggregates and capture images if desired.By day seven to 10 MTB macrophage aggregates should be sufficiently large to proceed for drug efficacy testing, as described later in the video. For drug testing, prepare two anti tuberculosis drugs in triplicate on a new 96 well plate as follows. First, add 125 microliters of 7H9C medium to wells B two through G 10.Next prepare the two drugs at double the highest desired final concentration in one milliliter of 7H9C. Using a pipette add 250 microliters of each drug to wells B, C, and D 11 and then E, F, and G 11 respectively for triplicate treatments. Next, using a multichannel pipette serially dilute the test drugs two fold by moving 125 microliters from B 11 through G 11 into B 10 through G 10.Mix by pipetting five times at each step. Continue to move 125 microliters from column to column right to left across the plate, and stop after column four. After mixing column four discard 125 microliters into a waste container.Columns two and three should not contain any drugs. This will allow them to be used as a background, and for positive growth controls. Next retrieve the 96 well plate containing the MTB infected macrophages from the incubator.Without tilting the plate, use a multichannel pipette to remove 150 microliters of medium from wells B two through G 11. Then tilt the plate as shown here, and insert the pipette tips into the bottom edge of the wells and remove the remaining medium, about 50 microliters. As MTB macrophage aggregates adhere to the bottom of the well, no material should be lost.However, removal of the medium should be as gentle as possible and care must be taken to avoid resuspension during this process. Gently add 100 microliters of 7H9C medium to wells B two through G 11 of the plate, which contain the MTB macrophage aggregates. Using a multichannel pipette transfer 100 microliters of the drug containing 96 well plate to the corresponding wells of the infection plate.Then place the plate in a sealed bag and incubate it for three days at 37 degrees Celsius. The resazurin assay relies on the oxidative species produced by metabolically active MTB to convert the blue resazurin to fluorescent pink resorufin. The change in color and fluorescence can be used as a surrogate marker to determine the amount of bacterial growth.Prepare a stock solution of resazurin at a final concentration of 8 milligrams per milliliter in water. Filter through a 22 micrometer pore size PVDF membrane for sterilization. Prepare a working solution in resazurin by mixing the stock solution, water, and Tween-80 in a two to one to one ratio.Final concentrations are 4 milligrams per milliliter resazurin, and 5%Tween-80. Using a plate reader, set up a program to read the fluorescence at 530 nanometer excitation and 590 nanometer emission for 24 hours every 30 minutes at 37 degrees Celsius. Pre warm the plate reader to 37 degrees Celsius.Using a multichannel pipette, add 20 microliters of resazurin working solution to wells B two through G 11 of the drug treated plate. Place the plate on the reader and start the program. To confirm the robustness of adapting this infection model to 96 well plate format the susceptibility of MTB rifampicin RIF in moxifloxacin moxy was assessed as described in this video.As shown here, MTB macrophage aggregate structures were successfully generated in a 96 well plate format thereby enabling through put compatibility. To quantitatively measure the conversion of resazurin as an indicator of bacterial growth fluorescence of individual wells was monitored kinetically for 24 hours. The presence of viable MTB cells is determined by the conversion of the blue resazurin dye to its pink reduced form, which is quantitatively reflected by the relative fluorescence z nets at the time point of saturation.These representative mini graphs show the resulting fluorescence units shown on the Y axis versus time shown on the X axis. To generate drug susceptibility killing curves rifampicin and moxifloxacin treated wells were normalized to the no drug control maximal MTB growth as 100%survival. Background control blank signals were subtracted from every sample well.The percent survival was plotted for each individual concentration of drug treatment to generate a killing curve. These data show that the minimal inhibitory concentration defines the lowest concentration of the antibiotic at which 90%growth inhibition is observed is greater than two micrograms per milliliter, for both rifampicin and moxifloxacin against MTB derived from our infection model. As such, the minimal inhibitory concentration of these two drugs against MTB using our infection model and assay is more reflective of the activity of these drugs in vivo.After watching this video, you should have a good understanding of how to reliably and reproducibly generate MTB infected macrophage aggregate structures for drug susceptibility testing using the resazurin assay. Following this procedure modifying the drug template from two drugs as shown to a 58 drug library panel is easily performed to enable high through put drug screening.

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

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 ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

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 ...