Name: an automated culture system for use in preclinical testing of host-directed therapies for tuberculosis
Uploaded: 2021-08-16T15:00:00
Description: Watch this Scientific Journal Video about An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis at JoVE.com

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 ins...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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) &#8805;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&#160; 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 &#181;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&#8217;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&#947;, recombinant human</td>
			<td>R&#38;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...

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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 ...

The main advantage of this protocol is that it is less labor-intensive and time-consuming than the traditional method of counting colony forming units to estimate the intracellular growth of Mycobacterium tuberculosis. This technique makes it easier for researchers to rapidly screen FDA-approved drugs with the aim of repurposing them as host directed therapies to treat tuberculosis. The method is very simple and straightforward.Basic knowledge of mycobacterial and EO carrier T-cell culture techniques would be enough to follow the protocol step by step. To begin, transfer six to eight milliliters of attenuated mycobacterial culture H37Ra from the T25 flask into a 15 milliliter polypropylene tube. Centrifuge the tube in a benchtop centrifuge.After carefully removing the tube, transfer the tube to the biological safety cabinet and wait one minute for the bacteria to settle. Pour the supernatant into the disinfectant discard container. Then, recap the tube and suspend the bacteria in the remaining medium by tapping the side of the tube gently and allowing the bacteria to settle for one minute.Add and mix two milliliters of pre-warmed complete RPMI and transfer to a 50 milliliter conical tube. To resuspend the mycobacteria, draw up the suspension in a five milliliter syringe equipped with a 25 gauge needle. Then, eject very gently down the side wall of the tube to minimize the aerosol production, and repeat the process six to eight times.Dispose of the needle and syringe in a sharps container in the biosafety cabinet. Transfer the suspension to a two milliliter microcentrifuge tube and centrifuge to pellet down any remaining clumps. Return the tube to the safety cabinet and allow the bacteria to settle for one minute.Transfer the upper 1 to 1.5 milliliters of the supernatant to a new tube. Discard the original tube in the waste bucket containing the disinfectant. Mix well and add various amounts of the mycobacterial suspension to the two well glass chamber slides, and incubate the slides for three hours at 37 degrees Celsius.After pipetting up and down three times to dislodge non-phagocytosed bacteria, remove the medium from the glass chamber slide. Wash once with two milliliters of PBS. Thaw the aliquots of 4%PFA in PBS stored at minus 20 degrees Celsius.Dilute the aliquot to 2%PFA and add two milliliters to each well. After incubating for 10 minutes at room temperature, remove the slide from the safety cabinet for staining. Discard PFA in the PFA chemical waste container and wash the slide under a gentle stream of tap water.Using a plastic transfer pipette, dispense enough auramine to cover the cells on the slide, and incubate the slide for one minute at room temperature in the dark. Wash excess dye off the slide with tap water and add the quencher for one minute in the dark. After washing off the excess quencher, incubate for 15 minutes at room temperature with Hoechst stain in the dark.Then wash off Hoechst stain. And after draining excess water, add a drop of antifade and a cover slip. Examine the air-dried slide under the fluorescent microscope using the 100x oil objective.Mycobacteria will fluoresce green under Fitz filter and nuclei will fluoresce blue under DAPI filter. Count the number of mycobacteria phagocytosed per cell and the percentage of infected cells to determine MOI. Calculate the volume of mycobacterial suspension needed to achieve the required MOI based on the surface area of a well in the plate.After mixing the mycobacteria suspension, add the calculated volume to the cells on 12 well plates to achieve the desired MOI. Incubate the plate at 37 degrees Celsius for three hours to allow mycobacteria to be phagocytosed. Then remove the extracellular bacteria by washing the wells with warm RPMI several times.Add 500 microliters of lysis buffer for 10 minutes to lyse the macrophages in one well of the three-hour sample, and collect the lysate to determine the baseline infection rate. Then add fresh complete RPMI and required drug doses, or vehicle control, to the remaining wells. Incubate the plates in the carbon dioxide incubator at 37 degrees Celsius for one to eight days, depending on the experiment design.To determine the percentage time to positivity, or TTP, of the initial inoculum of the three-hour sample, warm Middlebrook broth and instrument culture bottles to room temperature. Transfer the medium from the 12-well plate to the corresponding labeled conical tubes and add 500 microliters of sterile lysis buffer to each well. After 10 minutes, gently scrape the cells from the well with a sterile scraper and combine them with the medium in the appropriate conical tube.Once the well is washed with 0.5 milliliters of sterile PBS and combined with the medium and lysate, break the clumps by gently passing each sample through a 25 gauge needle syringe six to eight times. Dilute the sample in MB broth by adding 900 microliters of MB broth to 100 microliters of sample, and repeat the harvest process at the required time points during incubation. Prepare the instrument cluster bottles by sterilizing the rubber cap with 70%alcohol.Transfer enough nutrient supplements for all the samples into a conical tube, and use a needle and syringe to inject 0.5 milliliters of nutrient supplement into the instrument culture bottle. Then, pipette 500 microliters of the one-in-10 diluted sample in a one milliliter v-bottom tube. Use a needle and syringe to inject the 500 microliters of sample into the assigned instrument culture bottles.After carefully transporting the bottles from the biosafety cabinet to the instrument for loading, press the loading"button on the automated microbial detection system. Scan the barcodes on instrument culture bottles and place the bottles into the detection system incubator at 37 degrees Celsius for up to 42 days. Read and record the time taken to reach positivity from the instrument screen and calculate percentage TTP.A positive TTP indicates mycobacterial growth. The automated liquid culture instrument monitored carbon dioxide levels every 10 minutes and calculated TTP, which is the number of days from inoculation until cultures are flagged as positive. An inverse relationship between TTP and log(10)values of CFU in the initial inoculum was illustrated.When intracellular growth of Mycobacterium tuberculosis within macrophages determined by enumerating CFU was compared to the automated culture method described, a significant correlation between results was obtained. Mycobacterium tuberculosis growth was graphically presented by comparing the TTB values for up to eight days after infection of macrophages to the initial TTP value. A similar trend between CFU and liquid culture demonstrated significant inhibition of Mycobacterium tuberculosis growth in macrophages in the presence of all-trans retinoic acid, or AtRA, in solution, or the equivalent dose of AtRA encapsulated in PLGA microparticles.A dose-response experiment was carried out in infected THP-1 cells to determine the efficacy of the antibiotic Linezolid alone, or the combination of linezolid and interferon gamma. No significant interaction was observed between the drugs. AFP staining allows us to use a low MOI to minimize cell death when infecting macrophages, and to ensure that the same MOI is used regardless of the treatment used.Following this protocol, lead anti-TB drugs can be identified and studied further to investigate their in vivo efficacy.

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