Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry
October 29th, 2020
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.
Understanding the mode of action of novel antibacterial compounds is an important but difficult task. This method provides insights into such mechanisms and it's easy to implement. This technique allows a real-time observation of antibacterial effects in a noninvasive manner enabling further sample analysis.
Although practicing with microbiological techniques is a prerequisite, the experiment is easy to perform. Taking your time with data analysis is very important when performing microcalorimetry for the first time. Begin by using a spectrophotometer at a wavelength of 600 nanometers to measure the optical density of the overnight culture.
Dilute the culture to a 5 times 10 to the 5th colony forming units per milliliter of fresh MHB medium concentration and add 150 microliters of cells to each 1.5 milliliter tube of antibiotic at the concentration of interest. Then mix the compound with the cells by vortexing. To prepare the inserts, add 120 microliters of each bacteria antibiotic mixture into individual plastic inserts in the appropriate wells of a 48-well plate and use tweezers to place all of the titanium vials into the holders.
Gently transfer the inserts into the titanium vials in the holder plate and loosely place titanium lids onto all of the vials. When all of the inserts have been transferred, place the holder onto the designated area on the sample station and use a torque wrench set to 40 centinewton meters to tighten all of the lids. In the system software, start a new experiment and retract the sample insertion arm from the instrument.
Place the cup holder on the bridge with column eight facing the sample insertion opening and gently push the cup holder into the instrument at position one. Wait 10 minutes for the system to stabilize before labeling the experimental wells. Next, push the sample insertion arm until the cup holder is at position two.
After allowing the system to stabilize for 20 minutes, push the sample insertion arm to position three and retract the sample insertion arm until it is at the running position. Highlight all of the wells in the software and select reaction start, then run the experiment until the heat emission reads are stably back at zero. At the end of the analysis, select stop.
The software will then ask if you are sure. Select yes and save the experiment for data analysis. Then insert the sample insertion arm completely into the instrument and engage the magnets to retrieve the cup holder.
To analyze the data, open the software and select open experiment. In the popup window, select the experiment of interest and click open. Click select all and define baseline to normalize the data in each position.
In the popup window, select a time period of greater than 30 minutes within the lag phase. After selection, the baseline will appear in green in the thermogram. Close the define baseline section window, then click save and close the software.
Next, open the web-based SymCel calorimetry analysis application. Click browse to upload the file of interest and select the experiment. The metabolic parameters will be automatically calculated for the 32 samples.
To fit the heat flow data to Gompertz and/or Richards Growth models, click growth function. Growth models will be displayed in the cumulative section for comparison to the raw data in the flow section. To download the calculated parameters, click download measures, select the file location and click save.
The file will be exported to a spreadsheet for further analysis. Here, the thermograms obtained by exposing A.baumannii DSM-30008 to ciprofloxacin in serial dilution are displayed. Concentrations between 0.005 and 0.1 micromolar have a minimal effect on A.baumannii growth and metabolism.
Treating the cells with 0.5 micromolar ciprofloxacin, however, leads to a significant shift in the lag phase duration and to a lower maximum heat flow. These two changes together affect the time to peak, resulting in an increase of approximately six hours. In this figure, the cumulative released heat is plotted against time with the effect of each concentration reflected by an incline of slope.
Quantification of the thermogram incline allows calculation of the maximum metabolic rate of A.baumannii in the presence of ciprofloxacin with a concomitant decrease in the metabolic rate observed in cells treated with 0.5 micromolar ciprofloxacin. Rifampicin treatment has a dramatic effect on the thermograms of A.baumannii DSM-30008. A significant reduction of heat emission correlating with a decrease in metabolic activity is also observed.
Note the increase in time to peak for all of the tested concentrations and the decrease in the metabolic rate caused by the decrease in slope for all of the concentrations that are usually ascribed to a bactericidal effect. To ensure optimal results, use reverse pipetting and prevent additional fluid at the sides of the insert when transferring the sample, also make sure that the lids are properly closed.
Overview
This article describes a method for elucidating the mode of action of novel antibacterial compounds using isothermal microcalorimetry. The technique allows for real-time, noninvasive observation of antibacterial effects, providing insights into drug-microbe interactions.
Key Study Components
Area of Science
- Microbiology
- Pharmacology
- Biochemistry
Background
- Understanding antibacterial mechanisms is crucial in drug discovery.
- Microcalorimetry offers a unique approach to study these interactions.
- Real-time data collection enhances the analysis of drug effects.
- Practicing microbiological techniques is essential for accurate results.
Purpose of Study
- To provide a method for profiling antibacterial compounds.
- To observe the effects of antibiotics on bacterial metabolism.
- To facilitate further analysis of drug-microbe interactions.
Methods Used
- Isothermal microcalorimetry with calScreener.
- Preparation of bacterial cultures and antibiotic mixtures.
- Data analysis using specialized software.
- Thermogram analysis to assess metabolic rates.
Main Results
- Ciprofloxacin significantly alters the growth and metabolism of A.baumannii.
- Heat emission data correlates with metabolic activity changes.
- Rifampicin shows a dramatic reduction in metabolic rates.
- Quantification of thermograms aids in understanding bactericidal effects.
Conclusions
- The method provides valuable insights into antibiotic action.
- Microcalorimetry is effective for real-time monitoring of bacterial responses.
- Proper technique and data analysis are crucial for reliable results.
Frequently Asked Questions
Executive Industry Relevance
Rapid elucidation of antibacterial mode-of-action is a critical inflection point in antibiotic discovery, directly impacting target validation and portfolio triage. Isothermal microcalorimetry enables real-time, quantitative profiling of metabolic responses, distinguishing bactericidal from bacteriostatic effects without user intervention. This approach supports predictive confidence and accelerates the advancement of novel natural product leads against resistant pathogens.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct interrogation of antibacterial mechanism through metabolic fingerprinting.
- Supports functional target validation by differentiating bactericidal and bacteriostatic responses.
- Facilitates biological de-risking and prioritization of novel compounds from natural sources.
Screening & Assay Development
- Provides standardized, quantitative thermogram outputs for robust assay development.
- Delivers reproducible metabolic readouts suitable for high-throughput screening adaptation.
- Enables rapid comparison of compound effects across bacterial species and antibiotic classes.
Translational & Preclinical Research
- Aligns metabolic response profiles with translational biomarker development for preclinical models.
- Supports continuity from early discovery through preclinical validation by enabling in-depth fingerprint analyses.
- Reduces mechanistic ambiguity, informing risk-adjusted advancement decisions.
Pipeline & Workflow Integration
Isothermal microcalorimetry integrates into the discovery-to-preclinical continuum, bridging early target validation, screening, and translational research for antibacterial candidates.
- Discovery Biology: Quantitative thermogram analysis supports hypothesis testing and mechanistic de-risking.
- Screening: Standardized metabolic outputs enable assay reproducibility and screening readiness.
- Analytics: Automated calculation of metabolic parameters and growth model fitting facilitate cross-condition comparisons.
- Translational Research: Reference thermograms and metabolic fingerprints support biomarker alignment and preclinical continuity.
- Enterprise Reuse: Growing reference data pools enhance interpretability and enable platform reuse across antibacterial portfolios.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in antibacterial profiling.
- Operational Value: Delivers standardized, scalable, and reproducible metabolic measurements.
- Strategic Value: Accelerates go/no-go decisions and improves capital efficiency in early-stage antibiotic discovery.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of novel antibacterial leads.
Implementation Considerations
- Requires expertise in microbiological techniques and metabolic data analysis.
- Needs access to isothermal microcalorimetry instrumentation and compatible analytical software.
- Demands cross-team standardization of sample preparation and data interpretation protocols.
- Adaptation of measurement conditions may be necessary for different bacterial species.
- Interpretation benefits from access to growing pools of reference thermograms for benchmarking.
