an assay to evaluate drug efficacy against a mycobacterium tuberculosis infection model

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

For drug testing, prepare two anti-tuberculosis drugs in triplicate on a new 96-well plate as follows. First, add 125 microliters of 7H9-C medium to wells B2 through G10. Next, prepare the two drugs at double the highest desired final concentration in 1 milliliter of 7H9-C.
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 B11 through G11 into B10 through G10. 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 4. After mixing column 4, discard 125 microliters into a waste container. Columns 2 and 3 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 B2 through G11. 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 7H9-C medium to wells B2 through G11 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 0.8 milligrams per milliliter in water. Filter through a 0.22-micrometer pore-size PVDF membrane for sterilization. Prepare a working solution in resazurin by mixing the stock solution, water, and Tween-80 in a 2 to 1 to 1 ratio. Final concentrations are 0.4 milligrams per milliliter resazurin in 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 B2 through G11 of the drug-treated plate. Place the plate on the reader and start the program.

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Nanyang Technological University

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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 (green fluorescent protein) expressing plasmid pMN437 is used throughout this protocol. It is possible to substitute the Mtb-GFP strain with non-gfp expressing wild-type strain to enable easier accessibility of the strain for researchers. However, gfp expression is desirable to enable visual confirmation of phagocytosis and the 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 (Oleic Albumin Dextrose Catalase), 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 10 mL of 7H9-C in a 30 mL square bottom PETG flask.</li>
	<li>Incubate at 37 &#176;C on a 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 (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).</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)&#8203;
	<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&#8203;
	<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 
	&#8203;NOTE: See Figure 1 for the 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 the 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 with 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 the 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 the 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 the remaining media (~50 &#181;L) by tilting the plate and inserting the pipette tip into 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 a 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 polyvinylidene fluoride (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. The final concentrations are 0.4 mg/mL resazurin and 5% Tween-80.</li>
	<li>Using a plate reader, set up 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 the 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>

<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 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 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 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</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>

Source: Schaaf, K., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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.

<img alt="Figure 1" src="/files/ftp_upload/21926/21926_Figure1.jpg" /> 
Figure 1: Drug plate layout. (A) Example template of the drug challenge plate used in step 4. This allows for the parallel testing of two drugs in triplicate wells at 8 defined concentrations. As a representative experiment, we used rifampicin (RIF) and moxifloxacin (MOXI) starting at 2 &#181;M and 2.5 &#181;M, respectively. (B) An alternative template for throughput screening is also provided to show the possibility of testing of 58 compounds at a single concentration.
<img alt="Figure 2" src="/files/ftp_upload/21926/21926_Figure2.jpg" /> 
Figure 2: Generation of&#160;Mtb/macrophage aggregate structures in 96-well plate format.&#160;(A) Representative bright field and GFP merged images of&#160;Mtb-macrophage aggregates on Day 9 post-infection. Images were captured with a 4X objective using an automated cell imaging program that documents the entire well by stitching a montage of 3 by 3 individually captured images. (B) An individual non-stitched image from the visual field is shown in (A). (C) A representative merged bright field and GFP image of an&#160;Mtb/macrophage aggregate structure captured with a 10X objective.

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

Take a multiwell plate containing an established Mycobacterium tuberculosis or Mtb infection model.
The wells contain cell aggregates comprising a core of densely packed Mtb and Mtb-infected necrotic macrophages, surrounded by uninfected macrophages &#8212; mimicking in vivo infection.
Take a serial dilution of the antibiotics rifampicin and moxifloxacin.
Discard media from the infection plate, transfer antibiotic dilutions to the wells, and incubate.
The antibiotics diffuse through the cell aggregates and enter the bacterial cells.
Rifampicin binds bacterial RNA polymerase, inhibiting RNA synthesis and causing bacterial death.
Moxifloxacin binds bacterial DNA topoisomerase, inhibiting replication and arresting bacterial growth. The binding also induces stress responses, including the accumulation of reactive oxygen species, killing the bacteria.
Post-incubation, add resazurin &#8212; a redox indicator. Inside living and metabolically active bacteria, the oxidoreductase enzyme reduces blue resazurin to pink fluorescent resorufin.
Measure the fluorescence to detect viable bacteria.
Bacterial survival decreases with increasing antibiotic concentration, exhibiting their efficacy against Mtb.

31 Views. Un test per valutare l'efficacia di un farmaco contro un modello di infezione da Mycobacterium tuberculosis

Video: Un test per valutare l'efficacia di un farmaco contro un modello di infezione da Mycobacterium tuberculosis - Experiment

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Identificazione di High-throughput di combinazioni di farmaci sinergici con il metodo2 di sovrapposizione

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

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Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potenziamento dell'efficacia dell'anticorpo antitumorale di farmaci antineoplastici: rilevazione dell'anticorpo-farmaco sinergismo usando l'equazione di indice di combinazione

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

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An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

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

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