Name: metabolic profiling to determine bactericidal or bacteriostatic effects of new natural products using isothermal microcalorimetry
Uploaded: 2020-10-29T13:30:00
Duration: 7 min 28 s
Description: Watch this Scientific Journal Video about Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry at JoVE.com

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>Acinetobacter baumannii </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 &#181;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 CaCl2, 0.8 mM MgCl2</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>LABSOLUTE</td>
			<td>7696404</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 100 - 1000 &#181;L</td>
			<td>Brand</td>
			<td>705880</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 2 - 20 &#181;L</td>
			<td>Brand</td>
			<td>705872</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 20 - 200 &#181;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 &#181;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 &#181;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 ...

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