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

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.