Animal studies were performed in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee of Texas A&#38;M University. Review and approval from a biohazard safety officer or committee at your institution&#160;may be required.&#160;
Caution: All the procedures require BSL3 containment. The personnel are required to wear personal protective equipment at all times. All manipulations are performed inside the Biosafety Cabinet (BSC) and all sharps are disposed of in sharps containers. Work surfaces and BSC are cleaned with buffered phenol and 70% ethanol before initiating work and after work.&#160;Procedures would normally be done at biosafety level 3 with Mycobacterium tuberculosis, but for illustration and filming purposes, the authors demonstrate these procedures at biosafety level 2 with less virulent bacteria.
1. Strains and culture conditions
Note: M. tuberculosis strain CDC1551 is used in this study, but any M. tuberculosis strain can be used in the same manner.
<ol>
	<li>Grow the bacteria in M-OADC-TW (7H9 broth supplemented with 0.5% glycerol, 10% oleic acid dextrose complex without catalase, and 0.05% Tween-80) medium standing at 37 &#176;C to an OD600 of 0.5 (~ 0.2 x 107 CFU).</li>
	<li>Dilute the culture in M-OADC-TW (series of 1:10 dilutions of the bacteria). Plate the dilutions of the bacteria (105, 106, 107, 108) in triplicate onto selective 7H11 plates to allow determination of CFU.</li>
	<li>Incubate the plates for four weeks at 37 &#176;C or until colonies can be accurately counted.</li>
	<li>Centrifuge the bacterial inoculum at 8,534 x g for 5 min to obtain a pellet. Wash the pellet once with 10 mL of saline (0.9% NaCl) and re-suspend the pellet in 15 mL of saline (0.9% NaCl).</li>
</ol>
2. Aerosol infection of mice using a Madison Chamber
<ol>
	<li>Allow mice to acclimate to the new surroundings for a week.</li>
	<li>Weigh the mice prior to loading them into the chamber.</li>
	<li>Plug three cords into the power strip: the main chamber power, the vacuum pump, and the air compressor, in that order.</li>
	<li>Carefully unscrew the glass jar. Bring the glass jar to the biosafety cabinet and add the challenge inoculum suspended in 15 mL of saline (0.9% NaCl) to achieve ~ 104&#160;- 106 CFU of bacteria in the lungs.</li>
	<li>Close the lid of the glass jar in the biosafety cabinet. Attach the jar to the nebulizer unit and adjust the vertical stainless steel tube so that the lower (intake) end is about a quarter of an inch below the level of the fluid in the jar.</li>
	<li>Load all the animals needed for the experiment (the chamber can hold up to 90 mice) into the chamber and close all the latches on the door.</li>
	<li>Check the main (room) air flow meter. Ensure that the center of the float (ball) runs about 50 L/min as measured on the scale on the left.</li>
	<li>Press Start button on the control panel of the chamber. Set the airflow rate through the compressor air flow meter (the smaller meter in the left) as 4 L/min on the scale. Check visually to ensure that the challenge inoculum is being nebulized.</li>
	<li>After 15 min, when the red light on the front of the control panel appears and an audible signal indicates the end of the run, press Reset button on the lower right corner of the control panel to reset the timers.</li>
	<li>Hold down the small red button on the door of the chamber to release the vacuum.</li>
	<li>Open the chamber door and remove the animals. Place the mice back into their cages.</li>
	<li>Remove the glass nebulizer jar and place in a sealed, leak-proof transport container. Place the container inside the bio-safety cabinet and discard the challenge suspension into a designated waste container.</li>
	<li>Place the used nebulizer jar inside a biohazard bag and seal the bag for transport to the autoclave.</li>
	<li>At the end of the infection procedure, spray the inside of the chamber with buffered phenol and 70% ethanol and allow the chamber to sit for 10 min.</li>
	<li>Then, wipe down all accessible interior surfaces very thoroughly. Dispose of all contaminated paper towels, etc. in the biohazard trash. Autoclave the trash, waste container and used nebulizer jars.</li>
	<li>Place the animals back in the containment room until the imaging time-point.</li>
	<li>On the day of imaging, transfer the animals in a secondary container to the imaging room.</li>
</ol>
3. Animal anesthesia
<ol>
	<li>Anesthetize the mice with isoflurane using a customized gas anesthesia system.</li>
	<li>Weigh each of the two charcoal filter canister located on top of the anesthesia unit.</li>
	<li>Replace with a new canister if the weight is 50 g above the initial weight.</li>
	<li>Check the vaporizer unit to ensure sufficient isoflurane for the procedure.</li>
	<li>Place the nose cone holder inside the imaging chamber. Place the number of nose cones required for the procedure and seal the remaining openings with the nose cone blockers.</li>
	<li>Turn on the oxygen supply from the high-pressure cylinder and set it at 55 psi.</li>
	<li>Turn on the evacuation pump located in front of the anesthesia unit and set it to 8 L/min.</li>
	<li>Turn on the oxygen toggle located in front of the anesthesia unit.</li>
	<li>Turn on the gas flow to the anesthesia induction chamber to set the flow of gas at 1.5 L/min. Turn the gas flow off.</li>
	<li>Turn on the gas flow to the imaging chamber to set the flow of gas at 0.25 L/min. Turn the gas flow off.</li>
	<li>Turn on the isoflurane vaporizer and set it at 2 - 2.5%. Adjust the isoflurane level according to the number and weight of animals being used for the experiment by rotating the dial on the isoflurane vaporizer (2 - 2.5% for a mouse weighing ~20 g; 4% for a guinea pig weighing 300 g).</li>
	<li>Place the mice in the anesthesia chamber and close the lid. Turn on the gas flow to the anesthesia induction chamber.</li>
	<li>Leave the mice in the anesthesia induction chamber for 5 - 10 min until completely anesthetized.</li>
	<li>Once the mice are anesthetized, apply an optical ointment to the eyes to protect them while imaging.</li>
	<li>Place the mice in ventral or sternal recumbency&#160;such that their noses are placed in the nose cone to facilitate anesthesia of mice during the imaging procedure.</li>
</ol>
4. Reporter enzyme fluorescence (REF) imaging
<ol>
	<li>Inject the substrate (20 &#181;M, 2.5 &#181;L/g of weight) by intraperitoneal injection into the infected as well as the control mice.&#160;The mice are under anesthesia inside the imaging chamber for less than 1 min.&#160;</li>
	<li>Start the imaging system.</li>
	<li>Initialize the system by clicking on Initialize. Wait until the temperature bar turns to green. 
	NOTE:&#160;The imaging chamber consists of a heated platform to maintain body temperature of the animal while imaging.</li>
	<li>For imaging acquisition setting, select Fluorescent | Trans-illumination for whole animal or Epi-illumination for lung tissues, Structure and Overlay in Acquisition Control Panel.</li>
	<li>Set Field of view to B for single mouse and lamp level to high.</li>
	<li>Set exposure time to auto, medium binning, 2 - 3 for the&#160;f/stop, and excitation filter at 745 nm and emission filters from 780 nm to 840 nm.</li>
	<li>For sequence setup, click on Sequence Setup, select 9 -&#160;12 Trans-illumination points in the lung area.</li>
	<li>Click on Acquire for image acquisition.</li>
	<li>Place the mouse back into the cage post-imaging.</li>
	<li>Monitor the mice until fully recovered or sacrifice for quantifying CFU if imaging at a time-point of the experiment. Perform a modified Karnofsky score to monitor the well-being of mice post-imaging.&#160;</li>
</ol>
5. Analysis of REF imaging
<ol>
	<li>Open the imaging software.</li>
	<li>Load image files by clicking Browse icon.</li>
	<li>For spectral unmixing, click on Spectral Unmixing in the tool palette and click the spectra required to unmix. Click on start unmixing.</li>
	<li>For optical surface reconstruction, click on Surface Topography.</li>
	<li>Select orientation to dorsal, subject to fur mouse and, click Generate surface.</li>
	<li>Draw Crop image and set Threshold image for a region of interest. Click Done.</li>
	<li>For organ registration, go to the registration tab in 3D Optical Tools dropdown menu.</li>
	<li>Register organs of interest in an organ atlas.</li>
	<li>Adjust organ size or location to the generated surface topography using transform tool on/off located on the panel-Registration Tools.</li>
	<li>Go to Fluorescent tomography (FLIT) 3D reconstruction, and select analyze tab. Select the images to be included for analysis, image type to Normalized Transmission Fluorescence (NTF) efficiency, click Start.</li>
	<li>Check on select all and reconstruct in Data Preview tab.</li>
	<li>For fluorescence quantification, go to Region of Interest (ROI) Tools, add ROI cube to the organ of interest and adjust cube size to cover the organ of interest. Click on measure 3D ROIs.</li>
	<li>In the ROI measurements window, go to 3D ROI measurements, select data type to source voxels and measurements unit to pmolM-1cm-1.</li>
	<li>Save and/or export the result of FLIT 3D reconstruction analysis into a figure or data file.</li>
</ol>
6. Quantification of bacteria by CFU
<ol>
	<li>Euthanize the mice by intraperitoneal injection of 0.1 mL pentobarbital sodium (390 mg/mL).</li>
	<li>Check for pedal reflex by squeezing the pads of the feet of mice to ensure there is no reflex reaction.</li>
	<li>Explant the lung tissue from the mice using sterile forceps and scissors. Homogenize the lung tissue in 1 mL of 1x phosphate-buffered saline (PBS).</li>
	<li>Make 10-fold serial dilutions of the lung homogenate in sterile 1x PBS.</li>
	<li>For plating, spot three aliquots of 20 &#181;L each of the respective dilution on the agar plate.</li>
	<li>Incubate the plates in an incubator at 37 &#176;C and monitor for bacterial growth.</li>
	<li>Count the colonies in each spot after the incubation time and express as CFU/ mL by correcting for volume and dilution using the following equation: 
	CFU/ mL = (C/V) x M 
	Where C = colony counts per spot, V = volume of sample inoculated on each plate (mL), and M = multiplication factor (reciprocal of the dilution used). 
	NOTE: If 11 colonies are counted in one spot for a sample volume of 0.002 mL at a sample dilution of 10-4, using the equation, the colony count will be calculated as 
	(11/0.02)&#160;x 104 = 5.5 x 106 CFU/ mL</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2,7-disubstituted+cephalosporin+derivatives+as+beta-lactamase+substrates+and+methods+for+their+use+for+the+diagnosis+of+tuberculosis.">2,7-disubstituted cephalosporin derivatives as beta-lactamase substrates and methods for their use for the diagnosis of tuberculosis.</a> US patent. , (2015).</li><li>Shao, Q., Zheng, Y., Dong, X., Tang, K., Yan, X., Xing, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Covalent+Reporter+of+&#946;-Lactamase+Activity+for+Fluorescent+Imaging+and+Rapid+Screening+of+Antibiotic-Resistant+Bacteria.">A Covalent Reporter of &#946;-Lactamase Activity for Fluorescent Imaging and Rapid Screening of Antibiotic-Resistant Bacteria.</a> Chem Eur J. 19 (33), 10903-10910 (2013).</li><li>Li, L., Li, Z., Shi, W., Li, X., Ma, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+Selective+Near-Infrared+Fluorescent+Off-On+Probe+and+Its+Application+to+Imaging+Different+Levels+of+&#946;-Lactamase+in+Staphylococcus+aureus.">Sensitive and Selective Near-Infrared Fluorescent Off-On Probe and Its Application to Imaging Different Levels of &#946;-Lactamase in Staphylococcus aureus.</a> Anal Chem. 86 (12), 6115-6120 (2014).</li><li>Ashizawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+lung+cancer+and+tuberculoma+in+the+same+lesion:+demonstration+by+high+resolution+and+contrast-enhanced+dynamic+CT.">Coexistence of lung cancer and tuberculoma in the same lesion: demonstration by high resolution and contrast-enhanced dynamic CT.</a> BRIT J RADIOL. 77 (923), 959-962 (2004).</li><li>Figueroa, C. J., Riedel, E., Glickman, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+radiographic+differentiation+of+lung+nodules+caused+by+mycobacteria+and+lung+cancer:+a+case-control+study.">Clinical and radiographic differentiation of lung nodules caused by mycobacteria and lung cancer: a case-control study.</a> BMC Infect Dis. 15 (482), (2015).</li></ol>

When using imaging techniques, such as REF, there are key strategies that allow generation of robust and consistent data. Optical imaging produces scattered light in tissues which can impact the depth of penetration, since it is difficult to capture light emitted in all directions. Use of a near infra-red fluorophore (NIR) substrate for REF imaging having an excitation and emission wavelength in the Querange of 700 - 900 nm facilitates minimal absorption of the fluorescent signal by mammalian tissues. The custom designed substrate was constructed by linking a NIR fluorophore, IRDye 800Cw to a quencher, IRDye QC-1, by a lactam ring allowing fluorescence resonance energy transfer-based quenching. IRDye has excellent tissue penetration and light scattering characteristics and does not have any evident harmful effect on mammals36, being cleared from the blood and organs by 24 h. Fluorescence signal significantly increases beginning 4 h after substrate administration, reaching maximal levels 6 h after administration.
The bacterial infectious dose, mode of administration of infectious dose and the substrate as well as time-points of imaging post infection by performing pilot studies should be standardized before embarking on large, complex, experiments. Pilot studies can greatly reduce time and cost when imaging a large number of animals because a standard procedure can be optimized prior to doing the key experiment. Bacterial loads should be determined in the organs/tissues of interest following whole body imaging using trans-illumination and ex vivo lung imaging using epi-illumination to validate the source of the signal and determine the quality of correlation with bacterial numbers present8. Pilot studies will provide insight into the threshold of detection, dynamic range of the technique as well as determination of the optimal experimental conditions for imaging.
The primary advantages of using REF imaging as compared to other fluorescent and bioluminescent strategies are its high sensitivity and ability to image natural M. tuberculosis strains. REF imaging utilizes the catalytically robust enzyme BlaC that is conserved in all M. tuberculosis clinical isolates and tuberculosis-complex strains. The great sensitivity of REF imaging is due to the rapid catalytic rate of BlaC in combination with retention of the cleaved fluorescent product by the host cell. Signal continuously increases as long as the substrate is available resulting in nearly limitless build-up of the signal within the infected cells and tissues. This increased sensitivity of REF imaging as compared to alternative approaches of imaging allows specific detection of M. tuberculosis both in vitro and in vivo8,23,24,29.
REF imaging can be used for bacterial detection without genetic modifications enabling its direct application to any infection model, either laboratory animals8,37 or human clinical materials29,38. REF can be used to detect and image a wide array of pathogens39,40, since fluorogenic substrates can be developed for numerous enzymatic targets other than BlaC such as proteases, kinases, ureases, and &#946;-galactosidases. However, careful thought should be given to the target in order to ensure it displays optimal characteristics for imaging. BlaC represents a good model enzyme for characteristics that will ensure successful application of this strategy. REF imaging provides an immediate read-out on bacterial load present in lungs during infections, which greatly speeds progress in study of tuberculosis pathogenesis, since determination of bacterial numbers normally requires three to six weeks, but even in more rapid-growing organisms this approach will save a great deal of time. REF could also be used to discriminate carcinomas from tuberculosis, a key problem in diagnosis of nodular lesions in patients41,42. REF serves as a novel tool to accelerate translational tuberculosis imaging and can even be applied to humans, potentially allowing rapid prediction of therapeutic outcomes.

The slow growth rate of M. tuberculosis is a major roadblock in rapid diagnosis of tuberculosis1,2,3. While culture based diagnosis takes weeks to produce results, acid-fast smear has diagnostic limitations4 in children5 and patients co-infected with human immunodeficiency virus6,7. Optical imaging technologies have recently been recognized as an alternative to traditional diagnostic methods for tuberculosis8,9. Fluorescence and bioluminescence can be used to optically image M. tuberculosis in live animals in real-time10,11,12,13,14,15,16,17,18,19. Optical imaging has the benefit of a rapid and specific assessment of an infection with M. tuberculosis20,21,22.
We outline details for optical imaging of M. tuberculosis in live mice using REF. This method is very specific and sensitive23,24 and, similar to other optical methods, is less expensive than other methods of tuberculosis (TB) imaging25, including computed tomography (CT)26, magnetic resonance imaging (MRI)27, and F-fluorodeoxyglucose positron emission tomography/CT (F-FDG PET/CT)28. REF utilizes custom fluorescent or bioluminescent substrates that upon cleavage by a bacterial enzyme, produce a fluorescent product8,29. Hence, it has the advantage of not requiring a recombinant mycobacterial reporter strain30,31. The FRET substrate described is comprised of a fluorochrome and a quencher connected by a &#946;-lactam ring that is hydrolyzed by BlaC (&#946;-lactamase), naturally constitutively expressed by tuberculosis-complex Mycobacterium8,32. The bacteria directly generate signal due to REF catalytic activity that allows amplification by many orders of magnitude and sensitive detection of M. tuberculosis.
The REF substrate used in this study has excellent tissue penetration in live animals and reduced background due to its long wavelength. With this long-wavelength substrate it is possible to achieve a threshold of detection for M. tuberculosis of nearly 100 colony forming units (CFU) in vitro and &#60;1000 CFU in the lungs of mice in vivo (whole animal)8,33. REF can be used as a diagnostic tool for sputum, clinical materials and even directly in patients with micro endoscopic systems16,32,33,34 due its high sensitivity and specificity. REF can be applied to any tuberculosis clinical strain, because it uses a naturally produced bacterial enzyme, BlaC, for detection present in all strains. These characteristics make REF imaging a valuable tool in pre-clinical tuberculosis research in general to facilitate therapeutic and vaccine evaluation as well as analysis of pathogenesis, but may also ultimately be applied to diagnosis in tuberculosis patients.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td style="width:163px;">VETONE</td>
			<td align="right" style="width:119px;">501027</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:216px;">CNIR800</td>
			<td style="width:163px;">Custom synthesized</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Fatal Plus solution</td>
			<td style="width:163px;">Vortech Pharmaceutical Ls, Ltd</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">7H9 Middlebrook broth</td>
			<td style="width:163px;">BD</td>
			<td align="right" style="width:119px;">271310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">OADC Middlebrook enrichment</td>
			<td style="width:163px;">BD</td>
			<td align="right" style="width:119px;">212351</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sporcidin</td>
			<td style="width:163px;"></td>
			<td style="width:119px;">RE-1284F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">7H11 Middlebrook Agar</td>
			<td style="width:163px;">BD</td>
			<td align="right" style="width:119px;">212203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Madison Chamber</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IVIS Spectrum</td>
			<td style="width:163px;">Perkin Elmer</td>
			<td align="right">124262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">XGI-8-gas Anesthesia System</td>
			<td style="width:163px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Living Imaging software</td>
			<td style="width:163px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transparent nose cones</td>
			<td style="width:163px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">M. tuberculosis strain CDC1551</td>
			<td style="width:163px;">ATCC</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Female BALB/C mice, 5-7 weeks</td>
			<td style="width:163px;">Jackson Laboratory</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging mycobacterium tuberculosis in mice with reporter enzyme fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

REF imaging of mice infected with M. tuberculosis along with uninfected control mouse are shown in Figure 1A. The infected mice produced a significantly (P = 0.0057) higher fluorescence signal from the lungs at 6 weeks post-infection compared to control upon substrate administration. A typical time course to study the therapeutic efficacy against M. tuberculosis using REF could be imaging at week 1, 2, 4, 6, 15, 24 post-infection. Fluorescence intensity is quantified using epi-illumination for lung tissues (Figure 1B). The consistently increasing signal from week 2 to week 6 in lungs of infected mice suggests that REF is successfully able to detect M. tuberculosis in vivo. A drop in the background signal of control mice at a later time point (Figure 1B) could be attributed to the increase in body mass and volume of mice over a period of 6 weeks thereby reducing the excitation wavelength penetration. 3D FLIT reconstruction of fluorescence sources in mice infected with M. tuberculosis is represented in Figure 2A. The image sequences are acquired at multiple trans-illumination points in mouse using the same excitation and series of emission filters. These image sequences are then used for 3D FLIT reconstruction for fluorescent source distribution within the animal subject. Figure 2A-D demonstrates the different directions (coronal, sagittal and transaxial) of mouse tomography with lung organ registration. NTF efficiency maps for checking the reconstruction quality are represented in Figure 2E. NTF allows subtracting the background light leakage from trans-illumination images through an extra image captured with neutral density filter. The image taken with the specific excitation filter (Figure 2E-measured) is normalized to the transmission image measured with the same emission filter and an open excitation filter (Figure 2E- simulated) to give the signal produced by the substrate alone. The similar measured and simulated NTF efficiency profile in both the horizontal (Figure 2F) and vertical (Figure 2G) profiles with nearly 0% percentage error (Figure 2E-% error) provides evidence of a good quality 3D reconstruction with reduced artifacts and improved signal localization and sensitivity.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56801/56801fig1.jpg" /> 
Figure 1. Imaging of M. tuberculosis with REF. (A) in vivo imaging of mice uninfected and infected with M. tuberculosis at 2, 4, and 6 weeks post-infection. The color bar represents the intensity of the fluorescent signal in photons per second per cm2 from low (yellow) to high (red). (B) Quantification of fluorescence intensity from mice infected with M. tuberculosis. Fluorescence values for both control (black) and infected (red) are represented along with the standard error for each time point. The significance of results was determined by Students t-test, p values of &#60;0.05 were considered significant. <a href="https://cloudflare.jove.com/files/ftp_upload/56801/56801fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56801/56801fig2.jpg" /> 
Figure 2. 3D FLIT reconstruction of fluorescence sources in mice infected with M. tuberculosis. Mouse tomography represented in different directions; A) coronal, B) sagittal, and C) transaxial with D) lung organ registration. The 3D region of interest (ROI) is represented as a red cube in the lung for source measurements. (E) NTF efficiency maps of the measured and simulated for checking the reconstruction quality. The measured and simulated NTF efficiency profile was compared, providing good quality of 3D reconstruction (similar measured and simulated NTF efficiency). F) Horizontal and G) vertical signal profiles representing measured (blue) and simulated (red) NTF efficiency curve. The horizontal and vertical red bars indicate the source position. The color bar represents the intensity of the fluorescent signal in photons per second per cm2 from low (blue) to high (red). <a href="https://cloudflare.jove.com/files/ftp_upload/56801/56801fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence at JoVE.com

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

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JoVE Journal

Immunology and Infection

7.3K Views.  Texas A&M Health Science Center. We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Video: Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

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 ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

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 ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...