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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Research

JoVE Journal

Immunology and Infection

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 ...

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

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

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

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

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

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

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

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

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

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

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

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