The authors would like to thank the team of Symcel for fruitful discussions and we would like to acknowledge the great support from Ganna Oliynyk and Wilhelm Paulander. We would also like to thank Daniel Kohnh&#228;user for providing the natural product and Stefanie Schmidt for skillful technical support.

NOTE: The instrument temperature must be set according to the bacterium used at least a day in advance to ensure stability of the system. Here, Acinetobacter baumannii DSM30008 samples are run at 30 &#176;C.
1. Culture preparation
<ol>
	<li>Streak out the strain under investigation (here: A. baumannii) on a CASO agar plate and incubate overnight in a static incubator at 30 &#176;C.</li>
	<li>Prepare an overnight culture by inoculating a single colony in MHB (Mueller-Hinton broth) and incubate on a shaking incubator at 180 rpm at 30 &#176;C.</li>
</ol>
2. Sample preparation
<ol>
	<li>Use 1.5 mL tubes to prepare the concentration range of selected drug or compound (e.g., by adding 1.5 &#181;L of 100x stock solutions in DMSO; see step 2.3).</li>
	<li>Dilute the overnight culture in MHB medium.
	<ol>
		<li>Measure the optical density of the overnight culture using a spectrophotometer at a wavelength of 600 nm.</li>
		<li>Dilute the culture to obtain 5 x 105 colony-forming units (CFU)/mL in fresh MHB medium. An OD600 of one equals approximately 5 x 108 CFU/mL (e.g., Escherichia coli, Staphylococcus aureus, A. baumannii). 
		NOTE: It is important to calibrate the conversion factor OD600 to CFU/mL for each individual bacterial strain under the applied culturing conditions.</li>
	</ol>
	</li>
	<li>Add 150 &#181;L of the cells to the test tubes prepared in step 2.1, ensuring correct final concentration of tested drug or compound and correct final cell concentration.</li>
	<li>Mix the compound with the cells by vortexing. 
	NOTE: The sample plate (see Table of Materials) has six rows, each containing eight wells, a total of 48-wells; Row A and Row F are the thermodynamic reference. Therefore, no samples can be loaded into those wells, corresponding media is loaded into those wells. 32 test samples can be measured per run, using wells in rows B-E. Individual test sample should be run at minimum in duplicate (here: all samples were run as triplicates). Growth controls should be included.</li>
</ol>
3. Insert preparation
<ol>
	<li>Transfer 120 &#181;L from the mixture prepared in step 2.4 to the plastic inserts. 
	NOTE: Use reverse pipetting in step 3.1 to prevent liquid from spraying on the sides of the plastic inserts, which can lead to interference with correct signal readouts.</li>
	<li>Place all titanium vials into the holders (see Table of Materials) with tweezers.</li>
	<li>Gently transfer inserts into the titanium vials in the holder plate.</li>
	<li>Loosely place titanium lids on all titanium vials.</li>
</ol>
4. Insert loading
<ol>
	<li>Transfer the holder with titanium cups onto the sample station and place on designated area.</li>
	<li>Use the torque wrench, set to 40 cNm force, to tighten all the lids.</li>
</ol>
5. Running of samples
<ol>
	<li>In calView software, start a new experiment (Supplemental Figure 1).</li>
	<li>Retract the sample insertion arm from the instrument.</li>
	<li>Place the cup holder on the &#8220;bridge&#8221;, column 8 facing the sample insertion opening and gently push the cup holder into the instrument at the designated &#8220;Position 1&#8221;. Wait 10 minutes for the system to stabilize. Label the experimental wells.</li>
	<li>Push the sample insertion arm until the cup holder is at the designated &#8220;Position 2&#8221;. Wait 20 minutes for the system to stabilize.</li>
	<li>Push the sample insertion arm into &#8220;Position 3&#8221; and retract the sample insertion arm until it is at the &#8220;Running position&#8221;. Highlight all the wells in the software and select Reaction Start (Supplemental Figure 4).</li>
	<li>Run the experiment until the heat emission reads are stably back at zero. 
	NOTE: Be sure that all wells behave similar within these steps. Supplemental Figure 5 illustrates what should be observed if the wells are loaded correctly. If loaded incorrectly, repeat steps 5.2-5.4.</li>
</ol>
6. Remove the cup holder
<ol>
	<li>In the software, select Stop (Supplemental Figure 6). The software will then ask if &#8220;you are sure&#8221;, select Yes (Supplemental Figure 7) and save the experiment on a drive or desktop for data analysis (Supplemental Figure 8).</li>
	<li>Insert the sample insertion arm completely into the instrument and engage the magnets to retrieve the cup holder.</li>
	<li>Loosen the lids, remove the inserts and place vials and lids into glass holders and place for 4 hours at 180 &#176;C followed by placing the vials and lids into a desiccator to ensure lids and vials are dry.</li>
</ol>
7. Analyzing data
<ol>
	<li>Open the software (Supplemental Figure 9), select Open experiment in the left upper corner (Supplemental Figure 10). In the popup window select the experiment of interest and press Open (Supplemental Figure 11). The application will open the experiment in default wells view (Supplemental Figure 12).</li>
	<li>Press Select all or Ctrl+A (Supplemental Figure 13).</li>
	<li>Press Define Baseline, this parameter normalizes the data in each position (Supplemental Figure 14). In the popup window select a time period of &#62;30 min located in the lag phase (the heat flow has to be low, between zero and ten &#181;W; Supplemental Figure 15). After selection of base line time period the chosen baseline will appear in green in the thermogram. Close the Define Baseline Section window.</li>
	<li>Press Save or Ctrl+S and close the software (Supplemental Figure 16).</li>
	<li>Open web-based Symcel Calorimetry analysis application (https://symcel.shinyapps.io/symcel_calorimetricgrowth/).</li>
	<li>To upload the file to the Calorimetry analysis application, press Browse (Supplemental Figure 17), select the experiment and press Open (Supplemental Figure 18). The metabolic parameters will be calculated automatically for the 32 samples in the web application (Supplemental Figure 19).</li>
	<li>In order to fit the heat Flow data to Gompertz and/or Richard&#180;s growth models click Growth Function. Growth models fit will be displayed in the section &#8220;Cumulative&#8221;, also compared to the raw data in the section &#8220;Flow&#8221; (Supplemental Figure 20).</li>
	<li>To download all calculated parameters, press Download Measures. Select the file location and press Save (Supplemental Figure 21). The file will be exported to a spreadsheet for further calculations.</li>
</ol>

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The project was performed in collaboration with Symcel, developer of the calScreener technology. The authors have nothing to disclose.

Isothermal microcalorimetry measures energy emitted from a specimen over time and this energy release is a result of all biological, physical and (bio-)chemical processes. The measured heat flow can be exploited to evaluate or determine antibacterial effects of substances as it enables continuous real-time monitoring of metabolic activity.
In order to obtain reliable data for analysis, correct starting colony-forming units (CFU) need to be determined individually for each species or strain used. In case the CFU count is too low, this leads to a prolonged lag phase as it takes longer for the system to reach a critical amount of biomass producing enough heat to be detected. In case the CFU count is too high the lag phase will be very short, and the amount of heat produced can cause heat transfer to neighboring sensor cells (reference and experimental wells) and cause distortions of the thermograms. High numbers of CFU will also lead to a faster oxygen depletion and switch to anaerobic conditions. It also has to be taken into account that during the first 30 minutes of the experiment, when the system is equilibrating, data collection is not possible and the actual recording of effects is delayed. In addition, incorrect CFU determination leads to false MIC determination, ultimately affecting the experiment and the changes observed25. Another critical point is the correct determination of the baseline. Usually, the baseline signal is selected during the lag phase when the heat flow signal is zero and ideally, the time range for baseline definition is &#62;30 min. However, this is not always possible as the time that the bacteria requires to reach the heat signal detection limit differs between strains and species. Some species or strains require more than 30 minutes to reach the heat signal detection limit while other strains or species reach it within the first 30 minutes. In that case, it is possible to select the baseline signal at the end of the experiment when all the heat signals have dropped back to zero and remain stable. Alternatively, baselines from other experiments with the same bacterial strain can be used, which is however not recommended.
The system allows for some flexibility in terms of design and optimization of experiments and troubleshooting. Volumes used are in the range of 100-300 &#181;L when using plastic inserts and 100-600 &#181;L when using titanium cups without the plastic inserts. The recommended working volume by the manufacturer is 120 &#181;L. The use of different volumes in setting up a new experimental series, and while finding optimal conditions for measurements, has an impact mainly on two parameters. By using lower volumes, the amount of required test compound can be decreased, which is particularly important for compounds, which are available only in small amounts. In addition, the used volume directly impacts oxygen availability during the measurement with lower volumes increasing the amount of available oxygen required for bacterial growth. Oxygen depletion is one of the main factors contributing to the maximum possible duration of the experiment. Importantly, it is possible to use solid media, not only liquid media. This is especially important for slow-growing microorganisms as growth on the interface between solid and gas phase enables better oxygen access9.
IMC is a useful analytic tool to discover unknown processes with applications in physics, chemistry and biology. The method measures the heat exchange within a closed system and analysis of the recorded heat exchange provides additional information that cannot always be obtained with standard methods. In microbiology and antibiotics research, one of the biggest advantages of IMC is its ability to distinguish between live, dead and persister or dormant cells, which is not possible using standard turbidity methods11. In addition, IMC is highly sensitive and it can detect heat emission from as few as 104-105 cells9. Another advantage is that the experimental setup is fast and easy, and it allows for continuous, real-time tracking with minimal to no user interference. Further, IMC is non-destructive, which enables further analysis of samples. Data analysis allows for decoupling of biomass formation until late exponential or early stationary phase and metabolic activity in stationary phase.
Besides the novel and exciting application features mentioned above, there are also drawbacks to this method. The major limitation is that IMC instruments measure the total heat produced and released within a specific system, which also include non-specific signals. This highlights the importance of experimental planning with appropriate controls to be able to assess the heat signal changes that are measured by recording heat flow9,20.
In our hands, IMC is an important tool to study antibacterial effects of new natural products and to determine effective concentration ranges. Apart from differentiating bacteriostatic and bactericidal effects, it could be used in future as part of target identification studies and MoA determination. This can be done by comparing thermograms of different antibiotic classes to thermograms of new active compounds as displayed here. However, it still has to be investigated whether certain quantifiable parameters that can be extracted from the measured data are sufficient for such comparisons or if it will be necessary to work on algorithms giving fingerprints based on full thermograms. Another possible application in the field of antibiotic research is the comparison of wildtype to resistant clones, coupled with whole genome sequencing, which can help elucidating the mode-of-resistance (MoR) of new antibacterials. Because of the static nature of the method, investigation of efficacy of new agents on either biofilm formation or on already established biofilms could lead to better understanding of the biological process and the effect of selected agents on microbes in different stages of dormancy. IMC records total energy released in the form of heat making it a suitable method to investigate also sub-inhibitory effects of active substances that might be coupled to transcriptomics. This method can also be used in clinical settings to detect contamination of samples or for the determination of antibiograms helping to rapidly decide on definite treatment of the patients11.

The aim of this method is to apply isothermal microcalorimetry (IMC) as a medium throughput assay in mode-of-action (MoA) profiling of new antibacterial compounds. This method reveals data regarding the activity of compounds on bacterial species and provides information on the bactericidal or bacteriostatic nature of the compound itself.
The rise of emerging antimicrobial resistance (AMR) is a global problem and it leads to less effective treatments of common infections with known antibiotics1. However, the search for new compounds and drugs that can replace or work in combination with known antibiotics are ongoing and this is built on diverse approaches. Natural products are a key player in current drug discovery campaigns and particularly in anti-infective drug discovery2. However, identifying new lead structures for antibiotic development is a lengthy and financially demanding process3. Thus, the early steps of discovery are extremely important in order to filter on most promising scaffolds already at an early stage. Initial steps in natural product drug discovery include obtaining a compound&#8217;s structure, determining the activity in vitro along with MoA and target identification. Most successful compounds being eligible for further development should display a favorable spectrum of activity (i.e., broad-spectrum activity in the case of antibacterials) and a novel MoA by which pre-existing AMR can be overcome. Promising scaffolds are then typically screened in secondary assays, which include in vivo bioavailability, toxicity, and metabolism4. Besides the financial concerns, natural product drug discovery faces further challenges concerning the costs and technical difficulties related to compound isolation and purification, which, in turn, can make it difficult to obtain multi-milligram or even gram amounts in the early stages of the discovery process5,6. Therefore, it is of utmost importance in natural product research to be able to perform state-of-the-art primary screening with minimal compound amounts in order to take a well-informed decision about further investments to make a novel natural product accessible for pre-clinical development. With the use of IMC for antibacterial profiling, the amount of compound needed is significantly reduced in comparison to standard methods. The technique also provides more in-depth information regarding the interaction of new drugs with the microbial community7.
IMC is a well-established method for measuring total energy as a result of all biological, physical and biochemical processes and reactions in a biological system. Bacterial energy release is proportional to the total metabolic reactions8. Within a closed system, such as the used microcalorimeter, the heat levels can be measured in the microwatt range to study the metabolic kinetics of bacteria9,10,11,12. The heat (energy) that is released by bacteria is linked to cellular functions that underlie their metabolism and that are not necessarily proportional to the cellular biomass.
Initially, the applicability of isothermal calorimetry for microbiological assays has been limited due to its low throughput and high testing volumes. However, the used microcalorimeter is unique as it combines the advantages of isothermal calorimetry with increased throughput and lower compound requirements, which makes it a valuable tool for drug discovery applications10. Furthermore, the instrument provides further advantages over alternative methods for measuring bacterial growth kinetics, such as the standard turbidity method, which is based on the measurement of optical density at 600 nm (OD&#60;600). Measuring OD600 is based on the assumption that increased optical density is equal to microbial growth, thereby neglecting the presence of non-viable cells. This method has also been criticized as it excludes small colony variants and persister cells11. In contrast, IMC allows the real-time observation of any type of viable cells. If cells are dormant, they still exhibit metabolic activity and they can thus be detected by IMC, whereas such phenomena are not detectable by the standard turbidity method11. Other advantages of IMC include a shorter antimicrobial susceptibility testing time, measuring drug interactions in a complex community and standard analysis methods without destroying the sample7.
The IMC technology has been implemented in a wide range of studies, ranging from microbiology to thermogenesis and cancer biology13,14,15,16,17. The microbial applications include the determination of minimum inhibitory concentrations (MIC) of compounds against various bacterial strains. Several studies have been done and it has been concluded that MIC data from isothermal calorimetry for the majority of bacterial species can be obtained faster and results are similar compared to other (standard) methods for MIC determination12,18,19. Further applications of IMC include observing the interaction of drugs and combination of drug treatments with complex bacterial communities such as biofilms11. A study focusing on MoA profiling showed that the microcalorimeter can detect a difference in first- and second-generation cephalosporins, while different antibiotics with the same MoA exhibit a similar heat flow curve compared to each other18.
Here, we describe the use of IMC for MoA profiling of new natural products using the new isothermal microcalorimetry instrument. The method is used to determine effective antibiotic concentrations and to describe characteristics of antibiotics in terms of bactericidal or bacteriostatic mechanisms. The method can be broadly implemented in MoA profiling of compounds and it might replace or at least complement standard microbiological methods. Future studies will include in-depth fingerprint analyses that will enable the differentiation of antibiotic classes based on target mechanisms.

<table><tbody><tr><td>&lt;em&gt;Acinetobacter baumannii &lt;/em&gt;</td><td>DSMZ</td><td>DSM-30008</td><td>reference strain used in this study</td></tr><tr><td>calPlate</td><td>Symcel</td><td>1220093</td><td>48-well plate for titanium cups to be inserted</td></tr><tr><td>calScreener</td><td>Symcel</td><td>1200001</td><td>isothermal microcalorimetry instrument</td></tr><tr><td>calView software</td><td>Symcel</td><td></td><td>collection and analysis software</td></tr><tr><td>calWell</td><td>Symcel</td><td>1901004</td><td>micro-bio grade (non-active) 48-well plate with plastic inserts which are inserted into the titanium cups</td></tr><tr><td>CASO agar</td><td>Carl Roth</td><td>X937.1</td><td>isolation and cultivation of microorganisms</td></tr><tr><td>Chloroamphenicol</td><td>Sigma-Aldrich</td><td>C0378</td><td>antibiotic</td></tr><tr><td>Ciprofloxacin</td><td>Sigma-Aldrich</td><td>17850</td><td>antibiotic</td></tr><tr><td>Cuvettes</td><td>Brand</td><td>759015</td><td>1.5 mL cuvettes</td></tr><tr><td>Disposable inoculation loops</td><td>Sarsted</td><td>86.1562.050</td><td>10 &amp;micro;L inoculation loops</td></tr><tr><td>Dimethylsulfoxid (DMSO)</td><td>Thermo Fisher Scientific</td><td>85190</td><td></td></tr><tr><td>Eppendorf tubes</td><td>Eppendorf</td><td>30120086</td><td>1.5 mL eppendorf safe-lock tubes</td></tr><tr><td>Ethanol</td><td>Thermo Fisher Scientific</td><td>10428671</td><td></td></tr><tr><td>Falcon Tubes</td><td>Sarsted</td><td>62.554.502</td><td>15 mL falcon tubes</td></tr><tr><td>Hydrochloride (HCL)</td><td>Thermo Fisher Scientific</td><td>10316380</td><td></td></tr><tr><td>Methanol</td><td>Thermo Fisher Scientific</td><td>A412-500</td><td></td></tr><tr><td>Mueller Hinton Broth</td><td>Sigma-Aldrich</td><td>70192</td><td>liquid medium for antibiotic susceptibility studies</td></tr><tr><td>Mueller Hinton Broth II cation adjusted media</td><td>Sigma-Aldrich</td><td>90922</td><td>Mueller Hinton Broth cation-adjusted to 1.25 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.8 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Petri dishes</td><td>LABSOLUTE</td><td>7696404</td><td></td></tr><tr><td>Pipette 100 - 1000 &amp;micro;L</td><td>Brand</td><td>705880</td><td></td></tr><tr><td>Pipette 2 - 20 &amp;micro;L</td><td>Brand</td><td>705872</td><td></td></tr><tr><td>Pipette 20 - 200 &amp;micro;L</td><td>Brand</td><td>705878</td><td></td></tr><tr><td>Pipette tips 100 - 1000 L</td><td>Brand</td><td>732032</td><td></td></tr><tr><td>Pipette tips 2 - 200 &amp;micro;L</td><td>Brand</td><td>732028</td><td></td></tr><tr><td>Polymyxin B sulfate</td><td>Sigma-Aldrich</td><td>P0972</td><td>antibiotic</td></tr><tr><td>Rifampicin</td><td>Sigma-Aldrich</td><td>R3501</td><td>antibiotic</td></tr><tr><td>Serological pipette</td><td>Thermo Fisher Scientific</td><td>170356N</td><td>10 mL Nunc serological pipette</td></tr><tr><td>Spectrophotometer</td><td>Eppendorf AG</td><td>6135 000.017</td><td></td></tr><tr><td>Sterile filters</td><td>Minisart</td><td>16534----------K</td><td>0.2 &amp;micro;m pore size sterile filters</td></tr><tr><td>Syringe 50 mL</td><td>NORM-JECT</td><td>22778</td><td></td></tr><tr><td>Tetracycline</td><td>Sigma-Aldrich</td><td>87128</td><td>antibiotic</td></tr><tr><td>Titanium cups</td><td>Symcel</td><td>1220089</td><td>inserted in 48-well titanium calPlate</td></tr><tr><td>Titanium lids</td><td>Symcel</td><td>1220091</td><td>screwed and tightend to the titanium cups</td></tr><tr><td>Trimethroprim</td><td>Sigma-Aldrich</td><td>T7883</td><td>antibiotic</td></tr><tr><td>Tweezers</td><td>Symcel</td><td>1900602</td><td></td></tr></tbody></table>

metabolic profiling to determine bactericidal or bacteriostatic effects of new natural products using isothermal microcalorimetry

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an&#160;innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.

Watch this Scientific Journal Video about Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry at JoVE.com

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as ...

metabolic-profiling-to-determine-bactericidal-or-bacteriostatic

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.6K Views.  Saarland University. Inzicht in de werkingswijze van nieuwe antibacteriële verbindingen is een belangrijke maar moeilijke taak. Deze methode biedt inzicht in dergelijke mechanismen en is eenvoudig te implementeren. Deze techniek maakt een real-time observatie van antibacteriële effecten op een niet-invasieve manier mogelijk verdere monsteranalyse.Hoewel oefenen met microbiologische technieken een voorwaarde is, is het experiment eenvoudig uit te voeren. Het nemen van de tijd met data-analyse is erg belan...

Video: Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

Biochemistry

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Immunology and Infection

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) ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

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 ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Using TLC-Direct Bioautography Assay to Identify Natural Products for Biocontrol

Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

Author Spotlight: Discovering New Biopesticides from Bioactive Soil Microbe-Derived Natural Products

We describe the use of thin-layer chromatography direct bioautography assay and liquid chromatography-mass spectrometry to identify microbial natural products that display antagonism against fungal pathogens using the pathogen Sclerotinia sclerotiorum and biopesticidal Bacillus isolates as a model system.

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

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