The authors would like to thank all members of the laboratories of Dr. Barletta and Dr. Powers for helpful comments while developing the protocol. We thank Wendy Austin for helpful discussions and proofreading of the manuscript. The work described in this manuscript was funded by seed pilot grants to each investigator listed above from the University of Nebraska-Lincoln Redox Biology Center (parent grant #NCRR 2P20RR 017675, D. Becker, P.I.). In addition, we thank Dr. Ofelia Chacon for providing funds from her R21 grant (1R21AI087561-01A1) for research supplies and Mr. Halouska's partial salary support to standardize NMR techniques included in this publication.

1. Protocol Text
This protocol highlights the adaption of NMR methodology to M. tuberculosis (Class III agent). Therefore, Biosafety Level 3 (BSL3) practices need to be followed when conducting M. tuberculosis research in an annually certified laboratory. Exposure to laboratory-generated aerosols is the most important hazard encountered by personnel working with these microorganisms. The following procedures are conducted at our institution and variations may exist based on the Institutional Biosafety Committee's recommendations. Common personnel protective equipment will consist of a Tyvek suit, bouffant cap, booties, N95 respirator, eye protection, sleeves, and a double pair of nitrile gloves. Work involving M. tuberculosis cultures and/or manipulations with open M. tuberculosis containers is performed in the type A2 or B2 biological safety cabinet. Plastic-covered absorbent paper is placed on the working surface. All materials/supplies to be disposed or removed from the facility must be placed in two biohazard bags and decontaminated by autoclaving. Work surfaces and equipment utilized within the cabinet (FastPrep-24 lysis homogenizer, spectrophotometer, bucket with ice, etc.) must be disinfected after each work session with 1% Amphyl (tuberculocidal, bactericidal, fungicidal, and virucidal agent). M. tuberculosis cultures must be placed under double containment for transportation to larger equipment located outside of the biological safety cabinet such as freezers, incubators, centrifuges, and refrigerators. Centrifugation is conducted with enclosed safety cups and O-ring screw top tubes. For further analysis outside the BSL3 laboratory, cell-free extracts are filtered through a 0.2 &mu;m filter or microorganisms heat killed at 95 &deg;C for 15 min.2 Samples are plated to verify the absence of colony-forming units prior to removal from containment.
<ol>
 <li>Transfer 110 ml of Middlebrook 7H9 complete media containing polysorbate 80 (Tween 80 to prevent clumping) broth (MADC-TW) or a suitable medium to a 250 ml Erlenmeyer flask. Triplicate cultures are routinely grown per condition when using 13C-metabolites, and ten identical cultures are grown for 1D 1H NMR profiling. For two conditions (e.g., with and without addition of drug), double volume of culture per replicate can be grown and split into two identical cultures. All reagent and solution recipes are provided at the end of the protocol.</li>
 <li>Inoculate broth with 0.150 ml of M. tuberculosis 50% glycerol stock (allow to thaw on ice). See Note 1 below.</li>
 <li>Let the culture grow at 37 &deg;C shaking at 100 rpm for approximately 6 days (OD600 0.6-0.8). See Note 2 below.</li>
 <li>If no antibiotics, alternative additions or other treatments are needed, the cultures are ready to be harvested. If treatments are used, continue directly to Step 5. Remove a 0.5 ml sample from each flask, transfer to a microcentrifuge tube and place at 4 &deg;C for culture titration and quality control: contamination tests, phenotypic analysis, PCR tests. See Note 3 below. At this point, proceed to Step 6.</li>
 <li>Remove flasks from shaker and perform the desired treatment (e.g., add drug or metabolite). Place flasks back in the shaker and incubate for an additional period (e.g., 6-18 hr). At the end of this time, take another 1.0 ml aliquot and determine OD600. Remove a 0.5 ml sample from each flask, transfer to a microcentrifuge tube and place at 4 &deg;C for culture titration and quality control.</li>
 <li>Place cultures on ice for 5 min. After this step, leave cells on ice throughout the entire remaining protocol. Harvest cultures by centrifugation at 2,000 x g and 4 &deg;C for 15 min in 50 ml tubes using a benchtop centrifuge. Each culture will require four tubes with 25 ml each (a total of 100 ml is required to obtain adequate signal-to-noise for 2D 1H-13C NMR experiments, where only 50 ml per culture is required for 1D 1H NMR experiments).</li>
 <li>Wash each cell pellet two times with ice-cold ddH2O (approximately 15 ml the first time and 10 ml the second time) by the centrifugation parameters described above. For the second wash, combine the 10 ml aliquots into the same tube before spinning. Resuspend the cell pellet in a final volume of 1.0 ml ddH2O (need to adjust for cell pellet volume). If a single cell suspension free of clumping is desired at this step, cells can be briefly sonicated and/or passed through a 27-gauge needle three times. Alternatively, the cell pellet can be frozen at -80 &deg;C and stored until further processing. In the latter case, frozen pellets are thawed on ice prior to resuspension. Resuspend the cell pellets in a final volume of 1.0 ml ddH2O as stated above.</li>
 <li>Transfer the 1.0 ml cell suspension to a 2.0 ml screw cap tube containing Lysing Matrix B (0.1 mm silica spheres). Place the FastPrep-24 lysis homogenizer inside the biosafety cabinet. Put the samples into the sample holder, securing the retention spoke plate on top of the tube. Process the samples for 60 sec in the homogenizer at a speed setting of 6 meters/sec.</li>
 <li>Spin the samples in a microcentrifuge at 15,000 x g and 4 &deg;C for 10 min to pellet cell debris and intact cells.</li>
 <li>Remove supernatant and pass sample through a syringe filter (0.2 &mu;m) into a sterile tube. Plate 0.1 ml of sample (or a representative fraction of the sample, as for example 10%) on MADC agar to verify that there are no viable cells. Investigators may also conduct more than one filtration step and/or verify the presence of viable cells in the extracts using live-dead staining procedures so as to prevent or identify any potential biosafety concerns. Freeze samples in an ethanol-dry ice bath to store at -80 &deg;C until they are ready to be lyophilized and processed in an NMR facility.</li>
 <li>After 2 months, check plates to verify the absence of colony forming units. If no CFU's are found, samples may be taken out of the BSL3 laboratory. Further analysis is carried out in a standard NMR facility, which normally serves multi-users and operates under no specific containment requirements.</li>
 <li>Lyophilize the samples to dryness, then resuspend in 0.7 ml of NMR buffer and transfer to a microcentrifuge tube. Centrifuge for 3 min at 13,000 x g. Remove 0.6 ml and transfer to a 5 mm NMR tube. Alternatively, lyophilized samples could be mailed to an external facility as non-biohazardous regular samples.</li>
 <li>NMR data is collected immediately. Despite all precautions, active enzymes may still be present and the sample is typically not stable for long periods of time. Changes in the NMR spectrum are noticeable when the samples are left out at room temperature or at 4 &deg;C for more than 1 week. Also, NMR data collection is alternated between untreated and drug treated cultures, where samples are randomly selected from each category. This prevents unnecessary bias because a particular category had a longer delay before the NMR spectra were collected. A bias in the data would occur if the NMR spectra for all the untreated samples were collected first, and then followed by the drug treated samples. If the samples are unable to be immediately analyzed by NMR, the samples should be stored in 1 ml Eppendorf tubes at -80 &deg;C.</li>
 <li>After inserting the samples into a BACS-120 sample changer, instrument calibration, which involves a few routine steps such as lock, shim, tune, and optimization of the 90 &deg; pulse length, is required to maximize the quality of the results. A single sample is used to calibrate the 90 &deg; pulse length and tune the instrument for the remaining samples. Locking, shimming, and NMR data collection for every sample is automated using ICONNMR and gradshim.</li>
 <li>A 1D 1H NMR spectrum is collected using the Bruker zgesgp pulse sequence with water suppression using excitation sculpting. A total of 32k data points with a sweep width of 5482.5Hz, 128 scans, and 16 dummy scans are used. A 2D 1H-13C HSQC spectrum is collected using the Bruker hsqcetgp pulse sequence. A total of 2048 data points with a sweep width of 5000.0 Hz is collected along the direct 1H dimension, and 64 data points with a sweep width of 18864.9 Hz along the indirect 13C dimension. The spectrum is collected with 128 scans and 16 dummy scans to obtain good signal to noise.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/3673/3673fig1.jpg" /> 
Figure 1. A flow diagram of the experimental procedures is depicted.
NMR buffer
Stock solution of 50 mM potassium phosphate buffer with 50 &mu;M TMSP:
<ul>
 <li>2.17 g K2HPO4 (potassium phosphate dibasic)</li>
 <li>1.70 g KH2PO4 (potassium phosphate monobasic)</li>
 <li>7.86 mg Sodium-3-Trimethylsilylpropionate (TMSP-2,2,3,3-D4 (D,98%) )</li>
 <li>Dissolve in 500 ml of "100%" D2O</li>
 <li>The final solution should be at a pH 7.2 (uncorrected)</li>
</ul>
MADC-TW or MOADC-TW (1 L)
<ul>
 <li>4.7 g Middlebrook 7H9 broth base</li>
 <li>900 ml ddH2O</li>
 <li>2 ml glycerol</li>
 <li>Mix the components and autoclave for 25 min. Cool liquid to touch and add the following solutions:</li>
 <li>100 ml ADC when preparing MADC or 100 ml OADC when preparing MOADC</li>
 <li>2.5 ml 20% Tween 80 (alternatively, 1 ml of non-metabolizable 20% Tyloxapol)</li>
 <li>10 ml 1% cycloheximide</li>
</ul>
ADC (1 L)
<ul>
 <li>20 g D(+)-Glucose</li>
 <li>50 g BSA Fraction V</li>
 <li>8.5 g NaCl</li>
 <li>800 ml ddH2O</li>
 <li>Dissolve together, adjust volume to 1 L and sterilize with 0.2 &mu;m filter. Store at 4 &deg;C.</li>
</ul>
Alternatively, to prepare OADC (1L), add all components listed above plus 50 ml of 1% oleic acid (recipe below makes a 250 ml batch). Dissolve together, adjust volume to 1 L and sterilize with 0.2 &mu;m filter. Bottle needs to be wrapped in aluminum foil and store at 4 &deg;C.
<ul>
 <li>2.5 g Oleic Acid</li>
 <li>250 ml 0.2M NaOH</li>
 <li>Thaw oleic acid (solidifies upon refrigeration) by heating at 55 &deg;C for 10 min and add to NaOH solution and heat with stirring for 60 min. Store in sterile glass bottles that are wrapped in aluminum foil. Seal bottles with parafilm and store at 4 &deg;C.</li>
</ul>
If preferred, you can purchase commercial BD BBL Middlebrook ADC or OADC with catalase Enrichment.
1% Cycloheximide (100ml)
<ul>
 <li>1 g cycloheximide</li>
 <li>100 ml ddH2O</li>
</ul>
Dissolve and sterilize with 0.2 &mu;m filter. Store at 4 &deg;C. Caution: cycloheximide is toxic so handle with extreme care.
20% (v/v) Tween 80 (100ml)
<ul>
 <li>20 ml Tween 80</li>
 <li>80 ml ddH2O</li>
</ul>
Alternatively, weigh 20 g of Tween 80 (approximately 18.9 ml; density 1.06 g/ml), to prepare 20% w/v solution. Heat solution to 55 &deg;C for 30 min to dissolve, and mix completely. Sterilize liquid with a 0.2 &mu;m filter. Store the final solution at room temperature.
20% (v/v) Tyloxapol (100ml)
<ul>
 <li>20 ml Tyloxapol</li>
 <li>80 ml ddH2O</li>
</ul>
Alternatively, weigh 20 g of Tyloxapol (approximately 18.2 ml; density 1.1 g/ml), to prepare 20% w/v solution. Heat solution to 55 &deg;C for 30 min to dissolve, and mix completely. Sterilize liquid with a 0.2 &mu;m filter. Store the final solution at room temperature.
Note 1: Glycerol stocks of M. tuberculosis are prepared using the following protocol.
Stocking of M. tuberculosis
<ul>
 <li>Grow M. tuberculosis in 50 ml MADC to saturation (OD600 = 1.5 to 2.0) at 37 &deg;C under shaking conditions (100 rpm). Depending on the strain, this will take 7-14 days.</li>
 <li>Spin samples at 2,000 x g at 4 &deg;C for 15 min to pellet culture. Remove supernatant.</li>
 <li>Resuspend in 6 ml of sterile 50% glycerol.</li>
 <li>Aliquot 1.5 ml into 4 Corning cryogenic vials and label appropriately.</li>
 <li>Immediately flash freeze vials in ethanol/dry ice bath and store at -80 &deg;C.</li>
</ul>
Note 2: Inoculating strain H37Rv (-80 &deg;C stock up to 1.5 years old) into 70 ml of MADC media yields an OD600 of about 0.6 after 5 days of growth at 37 &deg;C under shaking conditions (100 rpm) in an Innova 40 Shaker.
Note 3: To test culture contamination, investigators could inoculate a culture aliquot onto standard rich medium and verify absence of growth after overnight incubation. Routinely, cultures are plated on MADC agar to observe colony morphology and examine by phase-contrast microscopy. If desired, cultures can be verified by PCR using IS6110 primers as described.3
2. Representative Results
A sample that is well prepared will yield an NMR spectrum similar to the one depicted in Figure 2. This spectrum is a representation of the M. tuberculosis wild type metabolic pool. The identified metabolites are directly or indirectly associated with the D-alanine pathway. Also, the magnitude of peak intensities is proportional to metabolite concentrations present in the cell extract. Therefore changes in peak intensities between untreated cultures, drug treatment, and mutant strains can indicate perturbations in metabolites and metabolic pathways. The M. tuberculosis 1D 1H NMR spectra were collected on a Bruker Avance 500 MHz spectrometer equipped with a triple-resonance, Z-axis gradient cryoprobe. The spectrum contains ca. 400 peaks, from which it was possible to identify and quantify 40-50 metabolites, including amino acids, nucleotide precursors, and glycolytic and citric acid intermediates. A BACS-120 sample changer with Bruker Icon software was used to automate the NMR data collection. 1D 1H NMR spectra were collected using excitation sculpting4 to efficiently remove the solvent and maintain a flat baseline, eliminating any need for baseline collection that may induce artifacts in subsequent principal component analysis (PCA) or orthogonal partial least squares discriminant analysis (OPLS-DA). A 1D 1H NMR spectrum is collected at 25 &deg;C with a spectrum width of 5482.5 Hz and 32K data points. A total of 16 dummy scans and 128 scans were used to obtain the spectrum. The ACD Labs 1D NMR processor software was used to semi-automatically process all the 1D 1H NMR spectra. The spectra were Fourier transformed, phased, and referenced to the TMSP peak (0.0 ppm). The NMRpipe software package was used to individually process the 2D 1H-13C NMR spectra and analyzed with NMRDraw. The Bruker FID data file was first converted to a file format recognizable by NMRpipe and then the spectrum was Fourier transformed, phase corrected, and zero filled. The observed NMR peaks in the 1D 1H NMR spectrum and 2D 1H-13C NMR spectrum are assigned to specific metabolites using 1H and 13C chemical shift tolerances of 0.05 and 0.50 ppm, respectively, and the Madison Metabolomics Consortium Database (MMCD),5the BioMagResBank,6 and the Human Metabolome Database.7 Specifically, the 1D and 2D NMR spectra are manually peak-picked, where the peak list of NMR chemical shifts is then uploaded to the Human Metabolome Database. The metabolites identified by the Human Metabolome Database are assigned to the NMR spectrum based on both maximizing the number of matching NMR resonances and belonging to a metabolic network. Each compound or metabolite typically has multiple C-H pairs and correspondingly multiple NMR resonances. Thus, the more of these NMR resonances that are observed in the experimental NMR spectrum, the more likely the metabolite is present. Similarly, identifying multiple metabolites associate with the same pathway increases the likelihood of correct assignments. The presence of metabolites and metabolic pathways are verified with the Kyoto Encyclopedia of Genes and Genomes (KEGG)8 and the MetaCyc databases.9 Mycobacterium smegmatis is a useful model system for M. tuberculosis and other mycobacterial pathogens. As described elsewhere, samples of M. smegmatis could also be disrupted by sonication.10
<img alt="Figure 2" src="/files/ftp_upload/3673/3673fig2.jpg" /> 
Figure 2. 1D 1H NMR spectrum of Mycobacterium tuberculosis cell extract. NMR peaks associated with representative metabolites are labeled. Abbreviations are the following: AMP, adenosine mono phosphate; &alpha;-Kg, &alpha;-ketoglutarate; Glu, Glutamate; and Ala, alanine.

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+liquid+chromatography-mass+spectrometry+(LC-MS)+in+metabolic+profiling+and+metabolomic+studies+for+biomarker+discovery.">The future of liquid chromatography-mass spectrometry (LC-MS) in metabolic profiling and metabolomic studies for biomarker discovery.</a> Biomark Med. 1, 159-185 (2007).</li><li>Ludwig, C., Viant, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+J-resolved+NMR+spectroscopy:+review+of+a+key+methodology+in+the+metabolomics+toolbox.">Two-dimensional J-resolved NMR spectroscopy: review of a key methodology in the metabolomics toolbox.</a> Phytochem. Anal. 21, 22-32 (2010).</li><li>Simpson, R. J., Inglis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proteins and Proteomics: A Laboratory Manual. , 425-595 (2003).</li><li>Beste, D. J., McFadden, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=System-level+strategies+for+studying+the+metabolism+of+Mycobacterium+tuberculosis.">System-level strategies for studying the metabolism of Mycobacterium tuberculosis.</a> Mol. Biosyst. 6, 2363-2372 (2010).</li><li>Rhee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+carbon+metabolism+in+Mycobacterium+tuberculosis:+an+unexpected+frontier.">Central carbon metabolism in Mycobacterium tuberculosis: an unexpected frontier.</a> Trends Microbiol. , (2011).</li><li>Who. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+Organization.">Health Organization.</a> Anti-tuberculosis Drug Resistance in the World: Report No. 4. , (2008).</li><li>Jassal, M., Bishai, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensively+drug-resistant+tuberculosis.">Extensively drug-resistant tuberculosis.</a> Lancet Infect Dis. 9, 19-30 (2009).</li></ol>

No conflicts of interest declared.

A significant number of studies have analyzed the transcriptomic and proteomic profiles of M. tuberculosis under a variety of in vitro and in vivo conditions.11-16 Ultimately, changes in gene expression and enzyme activity lead to variations in the concentrations of small molecular weight molecules. The complete description of these compounds constitutes the metabolome. Thus, the effects of drugs and varying growth conditions on metabolic pathways may be followed by metabolomic analy...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the Reagent/Equipment</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>ADC Enrichment</td>
 <td>BD BBL Middlebrook</td>
 <td>212352</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BACS-120 Sample Changer</td>
 <td>Bruker</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bruker Avance NMR</td>
 <td>Bruker</td>
 <td>&nbsp;</td>
 <td>500 MHz</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Fisher Scientific</td>
 <td>BP1600-100</td>
 <td>Fraction V</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Allegra X-15R</td>
 <td>Benchtop</td>
 </tr>
 <tr>
 <td>Centrifuge Tubes</td>
 <td>Corning</td>
 <td>430291</td>
 <td>50 ml sterile polypropylene</td>
 </tr>
 <tr>
 <td>Cryogenic Vials</td>
 <td>Corning</td>
 <td>430488</td>
 <td>2.0 ml sterile polypropylene</td>
 </tr>
 <tr>
 <td>Cycloheximide</td>
 <td>A.G. Scientific</td>
 <td>C-1189</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td>D(+) - Glucose</td>
 <td>ACROS</td>
 <td>41095-0010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Deuterium Oxide</td>
 <td>Sigma Aldrich</td>
 <td>617385</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Erlenmeyer Flask</td>
 <td>VWR</td>
 <td>89095-266</td>
 <td>Sterile, flat base, polycarbonate, 0.22 &mu;m PTFE membrane vented cap</td>
 </tr>
 <tr>
 <td>Flash Freeze Flask</td>
 <td>VWR</td>
 <td>82018-226</td>
 <td>750 ml</td>
 </tr>
 <tr>
 <td>Freeze Dryer</td>
 <td>VWR</td>
 <td>82019-038</td>
 <td>4.5 L Benchtop</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>GibcoBRL</td>
 <td>15514-029</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Incubator</td>
 <td>New Brunswick</td>
 <td>Innova 40</td>
 <td>Benchtop shaker</td>
 </tr>
 <tr>
 <td>Lysing Matrix B</td>
 <td>MP Biomedicals</td>
 <td>6911-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lysis Machine</td>
 <td>MP Biomedicals</td>
 <td>FastPrep-24</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td>Eppendorf</td>
 <td>5415D</td>
 <td>Benchtop</td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td>Beckman Coulter</td>
 <td>Microfuge 22R</td>
 <td>Benchtop</td>
 </tr>
 <tr>
 <td>Middlebrook 7H9 Broth</td>
 <td>Difco</td>
 <td>271310</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NMR tubes</td>
 <td>Norell</td>
 <td>ST500-7</td>
 <td>5mM</td>
 </tr>
 <tr>
 <td>OADC Enrichment</td>
 <td>BD BBL Middlebrook</td>
 <td>212351</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Oleic Acid</td>
 <td>Sigma</td>
 <td>O1008</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium Phosphate Dibasic</td>
 <td>VWR</td>
 <td>BDH0266</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium Phosphate Monobasic</td>
 <td>VWR</td>
 <td>BDH0268</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rotor - Microfuge 22R</td>
 <td>Beckman Coulter</td>
 <td>F241.5P</td>
 <td>Sealed and polypropylene</td>
 </tr>
 <tr>
 <td>Rotor - Allegra X-15R</td>
 <td>Beckman Coulter</td>
 <td>SX4750</td>
 <td>With bio-certified covers</td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Fisher Scientific</td>
 <td>S271-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium-3-trimethylsilylpropionate-2,2,3,3-D4</td>
 <td>Cambridge Isotope</td>
 <td>DLM-48</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spectrophotometer</td>
 <td>Beckman Coulter</td>
 <td>DU-530</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spectrophotometer Cuvettes</td>
 <td>LifeLINE</td>
 <td>LS-2410</td>
 <td>1.5 ml polystyrene, 2 clear sides</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Becton Dickinson</td>
 <td>309585</td>
 <td>Sterile, 3 ml Luer-Lok</td>
 </tr>
 <tr>
 <td>Syringe Filter</td>
 <td>Nalgene</td>
 <td>190-2520</td>
 <td>0.2 &mu;m sterile cellulose acetate</td>
 </tr>
 <tr>
 <td>Tween 80</td>
 <td>Fisher Scientific</td>
 <td>BP338-500</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies at JoVE.com

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.

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) ...

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15.2K Views.  University of Nebraska-Lincoln. 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.

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

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). ...

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 ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

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 Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

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 ...

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 ...