This work was funded by Science Foundation Ireland (SFI 08/RFP/BMT1689), the Health Research Board in Ireland [HRA-POR/2012/4 and HRA-POR-2015-1145] and Royal City of Dublin Hospital Trust.

The experiments outlined in this protocol were carried out using the attenuated H37Ra strain of Mtb, which can be handled in a Containment Level 2 laboratory. All manipulations of live mycobacteria were carried out in Class II biological safety cabinet (BSC). Experimental procedures were designed to minimize the generation of aerosols. Eukaryotic cell culture (THP-1 cells) was also carried out in a Class II BSC. The laboratory carried out a risk assessment and ensured that all procedures were carried out in line with institutional and national biological safety regulations. The human monocytic THP-1 cell line was used to perform the method as described (step 1). Cells are differentiated into macrophages following stimulation with phorbol 12-myristate 13-acetate (PMA) before infection with mycobacteria.
1. Cell culture
<ol>
	<li>Propagate H37Ra seed stock to log phase in Middlebrook 7H9 (MB) broth supplemented with albumin-dextrose-catalase (ADC) enrichment (10%) and 0.05% polysorbate 80. Store H37Ra stock in 1 mL aliquots in a -80 &#176;C freezer for up to 1 year.</li>
	<li>Thaw a 1 mL vial of Mtb-H37Ra and transfer it to a T25 flask with a filter cap containing 9 mL of MB supplemented broth approximately 1 week before the planned experiment. Incubate at 37 &#176;C for 5-7 days in a static incubator.</li>
	<li>Grow THP-1 cells in RPMI-1640 supplemented with non-heat killed 10% fetal calf serum (complete (c)RPMI) in a T75 flask in a CO2 humidified incubator at 5% CO2/37 &#176;C and subculture twice per week to maintain a density of less than 1 x 106 cells/mL.</li>
	<li>Differentiate THP-1 cells into macrophages 3 days before infection by gently pipetting cells several times using a serological pipette in T75 flasks to disperse any clumps and place them in a 50 mL conical tube.</li>
	<li>Centrifuge cells at 300 x g for 10 min at room temperature, decant off the supernatant, and gently resuspend the pellet in 2 mL of RPMI. Perform cell count to estimate cells/mL.</li>
	<li>Seed 2 mL of THP-1 macrophages in 12-well tissue culture plates at a density of 100,000 cells/mL in cRPMI with 100 ng/mL PMA for 72 h. Remove PMA-containing medium from cells and replenish with fresh cRPMI before Mtb infection.</li>
	<li>Set up individual plates for each time point required.</li>
	<li>Seed cells at the same density (100,000 cells/mL) in 2-well glass chamber slides to determine the multiplicity of infection (MOI).</li>
	<li>Place in a 5% CO2 humidified incubator for 3 days at 37&#8239;&#176;C.</li>
</ol>
2. Quantification of Mtb uptake
<ol>
	<li>Determination of Mtb uptake by macrophages (MOI)
	<ol>
		<li>Set up the Class II biological safety cabinet (BSC) on the day of infection and work on two layers of tissue paper to catch any spills. Set up waste discard containers according to local regulations.</li>
		<li>Remove 6-8 mL of mycobacterial culture from the T25 flask and transfer it into a 15 mL polypropylene tube. 
		NOTE: Smaller volume tubes can be used for smaller experiments.</li>
		<li>Centrifuge the tube in a benchtop centrifuge at room temperature for 10 min at 2890 x g.</li>
		<li>Carefully remove the tube from the centrifuge and transfer it to the biological safety cabinet. Wait 1 min to allow the bacteria to settle.
		<ol>
			<li>Pour off the supernatant into the disinfectant discard container, recap tube, and resuspend the bacteria in the remaining medium by tapping the side of the tube. Wait 1 min to allow the bacteria to settle.</li>
		</ol>
		</li>
		<li>Add 2 mL of pre-warmed cRPMI, mix gently, and transfer to a 50 mL conical tube.</li>
		<li>Resuspend the mycobacteria very carefully using a 25 G needle and 5 mL syringe. To resuspend, draw up the mycobacteria suspension into the syringe and eject very gently down the sidewall of the tube to minimize aerosol production. Repeat 6-8 times. 
		NOTE: Exercise utmost caution as this is a high-density culture of mycobacteria. To avoid the risk of needle stick injury, use blunt needles where possible, and Luer lock syringes.</li>
		<li>Dispose of the needle and syringe in a sharps container in the BSC.</li>
		<li>Transfer the suspension to a 2 mL microfuge tube (with screw-on cap) and centrifuge at room temperature for 3 min at 100 x g to pellet any remaining clumps. Return the tube to the safety cabinet and wait 1 min to allow the bacteria to settle.</li>
		<li>Transfer the top 1-1.5 mL of the supernatant to a new tube. Discard the original tube in the waste bucket containing disinfectant. Mix well and add various amounts of the mycobacterial suspension (e.g., 5, 25, 50, 150 &#181;L) to the 2-well glass chamber slides and incubate for 3 h in a CO2 incubator at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Staining for acid fast bacteria (AFB) 
	NOTE: After 3 h incubation, the macrophages are washed and fixed with paraformaldehyde to inactivate mycobacteria. The slides are then stained using a Modified Auramine O kit (see Table of Materials) to estimate mycobacteria phagocytosed per cell. Due to their waxy cell wall, mycobacteria retain the Auramine dye after an acid-alcohol wash. The macrophage nuclei are then counter-stained with Hoechst. This method allows for the number of phagocytosed bacteria per cell to be counted and is used to determine the multiplicity of infection (MOI) of the macrophages.
	<ol>
		<li>Remove the medium from the glass chamber slide after pipetting up and down three times to dislodge bacteria that have not been phagocytosed.</li>
		<li>Wash once with 2 mL of room temperature PBS.</li>
		<li>Store stocks of 4% paraformaldehyde, dissolved in PBS in aliquots at -20 &#176;C for up to 6 months. Thaw an aliquot of 4% paraformaldehyde immediately before use. Dilute to 2% paraformaldehyde with PBS and add 2 mL per well.</li>
		<li>Incubate for 10 min at room temperature. The glass chamber slide can be removed from the safety cabinet at this stage for staining.</li>
		<li>Wash the slide under a gentle stream of tap water.</li>
		<li>Dispense enough Auramine onto the slide to cover the cells using a plastic transfer pipette and incubate for 1 min at room temperature in the dark (cover with aluminum foil).</li>
		<li>Wash excess dye off the slide with tap water and add the decolorizer/quencher for 1 min in the dark.</li>
		<li>Wash off the excess with tap water and incubate for 15 min at room temperature with Hoechst 33342 (10 &#181;g/mL in PBS) in the dark.</li>
		<li>Wash off the Hoechst stain with tap water, remove the chambers, drain excess water from the slide, add a drop of antifade and coverslip, and air dry.</li>
		<li>Examine the slide under the fluorescent microscope using the 100x oil objective. Mycobacteria will fluoresce green under the FITC filter. The nuclei fluoresce blue under the DAPI filter (Figure 1C).</li>
		<li>Determine the MOI by counting the number of mycobacteria phagocytosed per cell and the percentage of cells infected.</li>
		<li>Calculate the volume of mycobacterial suspension needed to achieve the required MOI based on the surface area of a well in the plate; for example, the surface area of the glass chamber slides used in this experiment is 4 cm2. Low MOI (approximately 1-2 bacilli/cell) is desirable for experiments conducted over several days (e.g., 5 days).</li>
	</ol>
	</li>
	<li>Infection of macrophages
	<ol>
		<li>Mix the mycobacteria suspension well and add the amount needed to the cells on 12-well plates once the volume required to achieve the desired MOI has been determined.</li>
		<li>Incubate at 37 &#176;C for 3 h to allow mycobacteria to be phagocytosed.</li>
		<li>Remove extracellular bacteria by washing the wells with either warm RPMI or HBSS several times.</li>
		<li>Lyse the macrophages in one well (3 h sample) to determine the percentage time to positivity (TTP) of the initial inoculum (3 h sample) as outlined in step 3 below.</li>
		<li>Add fresh cRPMI and the required drug doses or vehicle control to the remaining wells, incubate the plates in the CO2 incubator at 37 &#176;C for the time necessary (dependent on the experimental design but usually at several intervals between 1 to 8 days).</li>
	</ol>
	</li>
</ol>
3. Harvesting samples for the liquid culture detection system
NOTE: On the day of infection, extracellular mycobacteria are removed by washing, and intracellular mycobacteria are harvested by lysis of one well of macrophages (3 h sample) to determine the initial amount phagocytosed as a baseline control for infection. At subsequent times both the medium, cell lysate, and washes are combined to measure total mycobacterial growth. Extracellular and intracellular growth can also be assessed separately if desired.
<ol>
	<li>Harvesting 3 h sample to determine TTP
	<ol>
		<li>Wash off extracellular mycobacteria from all the wells after the initial 3 h of infection as outlined in step 2.3.3. Add 1 mL of fresh media to the 3 h control well to equalize the lysate volume with those of later time points. 
		&#8203;NOTE: See step 3.2.7 if extracellular mycobacteria are to be excluded from the analysis.</li>
	</ol>
	</li>
	<li>Sample collection
	<ol>
		<li>Warm MB broth and instrument culture bottles to bring them to room temperature.</li>
		<li>Transfer the medium from the 12-well plate to the corresponding labeled conical tubes.</li>
		<li>Add 500 &#181;L of sterile lysis buffer (0.1% Triton x-100 in PBS filtered through a sterile 0.2 &#181;m filter) to each well for 10 min.</li>
		<li>Gently scrape the cells from the well with a sterile scraper and combine with the medium in the appropriate conical tube.</li>
		<li>Wash the well with 0.5 mL of sterile PBS and transfer to the appropriate tube.</li>
		<li>Gently pass each sample through a needle and syringe (25 G) 6-8 times to break up the clumps. Dilute samples 1:10 in MB broth; 100 &#181;L sample + 900 &#181;L MB medium.</li>
		<li>At the required times/days (usually between 1 to 8 days), harvest the remaining samples by following steps 3.2.1-3.2.6 above. 
		&#8203;NOTE: Investigators may prefer to exclude extracellular mycobacteria from their analysis, in which case, in step 3.2.2 above, the medium from each well is discarded, and macrophages are washed several times before adding lysis buffer.</li>
	</ol>
	</li>
	<li>Inoculating and loading instrument culture bottles 
	NOTE: Details of the liquid culture instrument and related consumables are provided in the Table of Materials.
	<ol>
		<li>Sterilize the rubber cap of the instrument culture bottle with tissue paper soaked in 70% alcohol and allow it to air dry. 
		NOTE: This step needs to be carried out in the BSC.</li>
		<li>Prepare bottles by transferring enough Nutrient supplements for all the samples into a conical tube (0.5 mL/bottle). Use a needle and syringe to inject 0.5 mL of Nutrient supplement into the instrument culture bottle.</li>
		<li>Pipette 500 &#181;L of the diluted sample (1:10) into a 1 mL V-bottomed tube.</li>
		<li>Use a needle and syringe to inject the 500 &#181;L of sample into the assigned instrument culture bottle.</li>
		<li>Sterilize the rubber cap of the instrument culture bottle with tissue paper soaked in 70% alcohol and allow it to air dry. Wipe the bottles with tissue paper soaked in 70% alcohol before removal from the BSC.</li>
		<li>Carefully transport bottles from the biosafety cabinet to the instrument for loading.</li>
		<li>Press the loading button on the automated microbial detection system.</li>
		<li>Scan the barcodes on instrument culture bottles and place the bottles into the detection system incubator at 37 &#176;C for up to 42 days. Read and record the time taken to reach positivity from the instrument screen. 
		NOTE: The barcode allows the instrument to identify the bottle and link reflectance readings with a particular bottle.</li>
		<li>Calculate percentage time to positivity (TTP) by comparing the TTP of the initial intracellular inoculum (Day 0) to that of macrophages cultured for the indicated times. A positive change in percentage TTP means mycobacterial growth13. 
		For example, for day 3: 
		Percentage change in time to positivity = <img alt="Equation 1" src="/files/ftp_upload/62838/62838eq01.jpg" /></li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The authors have used the liquid culture method described in this protocol to monitor Mtb growth in monocyte-derived macrophages and alveolar macrophages and THP-1 cells differentiated with PMA11,16,17. This technique can also be modified for use with non-adherent cells12. More recently, the instrument was also used in preclinical studies to evaluate inhaled all-trans retinoic acid (AtRA) as an HDT for TB...

Mycobacterium tuberculosis (Mtb), the causative agent of TB, was the most significant infectious disease killer globally in 20191. To evade host defenses, Mtb subverts the mycobactericidal activity of innate immune cells such as macrophages and dendritic cells (DCs), allowing it to persist intracellularly and replicate2. The lack of an effective vaccine to prevent adult pulmonary TB and the increasing emergence of drug-resistant strains highlight the urgent need for new therapies.
Adjunctive host-directed therapies (HDT) could shorten treatment time and help overcome resistance3. Preclinical assessment of HDT candidates in vitro to determine mycobactericidal activity within macrophages often relies on the quantification of Mtb growth by colony-forming units (CFU) on solid agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. Commercially available automated, broth-based microbial detection systems are more commonly used in clinical microbiology laboratories for detection and drug susceptibility testing of Mtb and other mycobacterial species in clinical specimens4. These instruments measure growth indirectly based on the bacterial metabolic activity leading to physical changes in the culture media (change in CO2 or O2 levels or pressure) monitored over time5. The readout is time to positivity (TPP), which has previously been shown to correlate with Mtb CFU in sputum specimens of TB patients in response to treatment6,7 and in lysates of infected murine lung and spleen8. In addition, liquid culture detection systems have been used to measure the effect of conventional pathogen-directed therapies on the growth of mycobacteria in axenic culture and cultured macrophages9,10. The instrument has also been used to investigate the innate ability of dendritic cells and of alveolar macrophages to control intracellular growth of Mtb11,12. This experimental protocol demonstrates that a liquid culture diagnostic system can be adapted to perform preclinical screening of HDT for TB in cultured macrophages. Compared to CFU enumeration, the main advantage of this technique is that it considerably reduces the experimental labor and time required to quantify intracellular mycobacterial growth/survival. This technique relies on access to an automated culture instrument that can be used to assess intracellular mycobacterial survival in immune cells treated with a broad range of pharmacological reagents targeting cellular functions to boost host immunity.

<table><tbody><tr><td>IX51 Fluorescent Microscope</td><td>Olympus, Japan</td><td>N/A</td><td>AFB detection and imaging</td></tr><tr><td>2 mL microtube, flat bottom, screw cap, sterile</td><td>Sarstedt, North Carolina, USA</td><td>72.694.006</td><td>Mtb infection of macropahges</td></tr><tr><td>5 mL syringe, Luer lock</td><td>BD Biosciences, San Jose, CA, USA</td><td>SZR-150-031K</td><td>Mtb infection of macropahges/CFU</td></tr><tr><td>50 mL tube, sterile</td><td>Sarstedt, North Carolina, USA</td><td>62.547.254</td><td>Mtb infection of macropahges</td></tr><tr><td>all-trans-Retinoic Acid (ATRA) &amp;ge;98% (HPLC)</td><td>Sigma Aldrich, Missouri, USA</td><td>R2625</td><td>Host directed therapy candidate</td></tr><tr><td>BacT/ALERT 3D Microbial Detection System</td><td>Biomerieux ( Hampshire, UK)</td><td>247001</td><td>Broth-based colormetric detection system</td></tr><tr><td>BACT/ALERT MP&amp;nbsp; BACT/ALERT MP Nutrient Supplement</td><td>Biomerieux ( Hampshire, UK)</td><td>414997</td><td>Broth-based colormetric detection assay</td></tr><tr><td>BACT/ALERT MP culture bottles</td><td>Biomerieux ( Hampshire, UK)</td><td>419744</td><td>Broth-based colormetric detection assay</td></tr><tr><td>BD BBL Middlebrook ADC Enrichment, 20 mL</td><td>BD Biosciences, San Jose, CA, USA</td><td>M0553</td><td>Mycobacterium liquid culture</td></tr><tr><td>BD BBL Middlebrook OADC Enrichment, 20mL</td><td>BD Biosciences, San Jose, CA, USA</td><td>M0678</td><td>Colony Forming Units</td></tr><tr><td>Cell scraper, 25 cm</td><td>Sarstedt, North Carolina, USA</td><td>83.1830</td><td>Harvest of lmacrophage lysates</td></tr><tr><td>Corning Syringe Filter, 0.2 &amp;micro;m</td><td>Corning Incorporated, Germany</td><td>431219</td><td>Sterilization of lysis buffer</td></tr><tr><td>Cover glass (borosilicate), 24 x 50 mm, #1.5 thickness</td><td>VWR International Limited</td><td>631 - 0147</td><td></td></tr><tr><td>Cycloheximide, from microbial</td><td>Sigma Aldrich, Missouri, USA</td><td>C7698</td><td>Colony Forming Units</td></tr><tr><td>Dako Fluorescent Mounting Medium</td><td>Agilent Technologies Ireland Limited</td><td>S3023</td><td>Antifade mounting medium</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma Aldrich, Missouri, USA</td><td>D8537</td><td>Mtb infection of macropahges</td></tr><tr><td>Fetal Bovine Serum, Gibco</td><td>Thermo Fisher, Massachusetts, USA</td><td>10270106</td><td>Macrophage cell culture</td></tr><tr><td>Glycerol, Difco</td><td>BD Biosciences, San Jose, CA, USA</td><td>228220</td><td>Colony Forming Units</td></tr><tr><td>Hoescht 33342 (bisBenzimide H 33342 trihydrochloride)</td><td>Sigma Aldrich, Missouri, USA</td><td>B2261</td><td>Nuclear stain</td></tr><tr><td>IFN&amp;gamma;, recombinant human</td><td>R&amp;amp;D Systems Inc, Minnesota, USA</td><td>285-IF</td><td>Host directed therapy candidate</td></tr><tr><td>Labtek 2-well chamber slide, sterile, Nunc</td><td>Thermo Fisher, Massachusetts, USA</td><td>TKT-210-150R</td><td>Mtb infection of macropahges</td></tr><tr><td>L-Asparagine, anhydrous</td><td>Sigma Aldrich, Missouri, USA</td><td>A4159</td><td>Colony Forming Units</td></tr><tr><td>Linezolid</td><td>Sigma Aldrich, Missouri, USA</td><td>PZ0014</td><td>Antibiotic</td></tr><tr><td>Microlance Hypodermic Needle 25 G</td><td>BD Biosciences, San Jose, CA, USA</td><td>300400</td><td>Mtb infection of macropahges/CFU</td></tr><tr><td>Middlebrook 7H10 Agar Base</td><td>BD Biosciences, San Jose, CA, USA</td><td>M0303</td><td>Colony Forming Units</td></tr><tr><td>Middlebrook 7H9 Broth Base</td><td>BD Biosciences, San Jose, CA, USA</td><td>M0178</td><td>Mycobacterium liquid culture</td></tr><tr><td>Modified Auramine O Stain and Decolourizer</td><td>Scientific Device Laboratory, IL, USA</td><td>345-250</td><td>AFB stain</td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich, Missouri, USA</td><td>158127</td><td>Mtb infection of macropahges</td></tr><tr><td>Petri dishes, 92 x 16mm (20/bag)</td><td>Sarstedt, North Carolina, USA</td><td>82.1473.001</td><td>Colony Forming Units</td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma Aldrich, Missouri, USA</td><td>P8139</td><td>Macrophage cell culture</td></tr><tr><td>Polysorbate 80, Difco</td><td>BD Biosciences, San Jose, CA, USA</td><td>231181</td><td>Mycobacterium liquid culture</td></tr><tr><td>RPMI-1640, Gibco</td><td>Thermo Fisher, Massachusetts, USA</td><td>52400025</td><td>Macrophage cell culture</td></tr><tr><td>Sterile Cell Spreader, L-Shaped</td><td>Fisherbrand, Thermo Fisher, MA, USA</td><td>RB-44103</td><td>Colony Forming Units</td></tr><tr><td>T25 TC flask, angled neck, filter cap, sterile, Nunc</td><td>Thermo Fisher, Massachusetts, USA</td><td>156367</td><td>Mycobacterium liquid culture</td></tr><tr><td>THP-1 cell line</td><td>ATCC, Virginia, USA</td><td>ATCC TIB-202</td><td>Macrophage cell culture</td></tr></tbody></table>

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).

The automated liquid culture instrument used in this study monitors CO2 levels every 10 min. A color change in the sensor at the bottom of the instrument bottle is measured colorimetrically and expressed as reflectance units. The instrument software then applies detection algorithms to calculate time to positivity (TTP), i.e., the number of days from inoculation until cultures are flagged as positive (Figure 1A). An inverse relationship between TTP and log10CFU (determi...

Watch this Scientific Journal Video about An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis at JoVE.com

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

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 ...

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Research

JoVE Journal

Immunology and Infection

1.7K Views.  The University of Dublin. Het grote voordeel van dit protocol is dat het minder arbeidsintensief en tijdrovend is dan de traditionele methode om kolonievormende eenheden te tellen om de intracellulaire groei van Mycobacterium tuberculosis te schatten. Deze techniek maakt het voor onderzoekers gemakkelijker om snel door de FDA goedgekeurde geneesmiddelen te screenen met als doel ze opnieuw te gebruiken als gastheergerichte therapieën om tuberculose te behandelen. De methode is heel eenvoudig en ongecompliceerd....

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

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in &gt; 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

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 ...

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 ...

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

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

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

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

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

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. ...

Preparing Mtb Secretome to Study Host-Pathogen Interactions

Preparation of Mycobacterium tuberculosis Culture Filtrate to Understand TB Pathogenesis

Mycobacterium tuberculosis (Mtb) secretes many proteins into the host to modulate various cellular processes, such as host signaling, immune response, apoptosis, etc., to enhance survival. While the function of a few secretory factors has been elucidated, many are yet to be characterized. Hence, investigating the Mtb secretory protein is an essential area of research. Mtb secretes proteins into the extracytoplasmic space with the help of three different secretion systems: general, twin-arginine, and specialized Type VII secretion system. One has to prepare the secretome to identify novel secretory molecules and functionally characterize the known factors. Here, we outline the method for preparing the secretome and sample preparation for mass spectrometry. Secretome preparation requires the utmost care to avoid contamination of cytosolic proteins, which can be monitored by probing with antibodies against well-characterized membrane, secretory, and cytosolic proteins. The culture filtrate thus prepared can be used for different purposes, which include investigating host immunomodulation properties.

Preparation of Mycobacterium Tuberculosis Culture Filtrate to Understand TB Pathogenesis

Here, we demonstrate the preparation of Mtb culture filtrate (CF), which is the bacterial growth media that consists of proteins secreted by the bacteria. Mtb secretes proteins to modulate host signaling and immune response, which are crucial for pathogenesis. The CFs prepared are analyzed by mass spectrometry to identify all the secretory factors.

Mycobacterium tuberculosis (Mtb) secretes many proteins into the host to modulate various cellular processes, such as host signaling, immune response, ...

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

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.

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

Samenvatting

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