Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

This protocol allows us to easily concentrate and identify volatile metabolites and actively produced volatiles from microbial organisms in a variety of biological samples. VASE is a more user-friendly method of concentrating low-abundance volatiles. All you need is a sample under near-vacuum, and let physics do the rest.

Access to volatile analysis of clinical samples has important implications for metabolic biomarker discovery in a number of different disease states. For example, the pathogen driving and infection or the success of antibacterial therapy could be detected with volatile analysis of saliva, sputum, or breath samples. To prepare fecal samples, add one milliliter of deionized water to 100 milligrams of feces in a 1.5-milliliter microcentrifuge tube and vortex for three minutes.

Keep the samples on ice when not in use. Add 485 milliliters of BHI medium with 20-millimolar 13C glucose or BHI with 30%deuterium to 15 microliters of fecal and water mixture, ensuring that the final volume of the sample is 500 microliters. Prepare samples in technical triplicates.

To prepare sewage samples, add 500 microliters of sewage to 500 microliters of BHI medium with 13C glucose or 30%deuterium for a total volume of one milliliter. Prepare samples in triplicates and keep them on ice. To prepare saliva samples, add 50 microliters of saliva to 500 microliters of BHI medium with 13C glucose or 30%deuterium for a total volume of 550 microliters.

Prepare samples in triplicates and keep them on ice. To prepare sputum samples, add 15 microliters of sputum into a vial. Prepare samples in triplicate and keep them on ice.

Place empty VOA vials on the cold plate and place the cold plate on ice in the biosafety hood. Turn on the 5600 SPEU and adjust it to the required temperature. Label 20-milliliter VOA vials according to samples, replicates, and HSP IDs using a water resistant marker that resists water in case condensation forms on the outsides of the vial while on ice.

Inside the biosafety hood, unscrew the white cap on the vial, quickly pipette sample into the vial, and assemble the lid liner, black cap, and HSP. Place the vial containing the sample and HSP back on the cold plate. Once all samples have been prepared in the glass vials, turn on the vacuum pump, place the vials under vacuum, and remove the vacuum source.

Double check the pressure after placing all samples under vacuum using the pressure gauge. If a vial is leaking, ensure that the cap is screwed on tightly and that the white O-rings of the HSP and lid liners are correctly in place. Place vials in the SPEU for the optimized time and temperature with agitation at 200 RPM.

Extract cultures for one hour at 70 degrees Celsius and extract stable isotype probing experiments with fecal, sewage, saliva, and sputum samples for 18 hours at 37 degrees Celsius. Place the cold plate at minus 80 degrees Celsius for use after the extraction period is complete. When the extraction is complete, place samples on the cold plate for 15 minutes to draw out water vapor from the HSP and vial headspace, then transfer the HSPs to their sleeves.

Set up the sequence of samples on the Entech software. Open the program and select 5800 and Sequence in the options to the right of the Instrument drop-down menu. Save the sequence table, select Run on the left-hand side, then Start with Blank in Desorber if the blank HSP isn’t the desorber.

Note that HSPs will be handled by the SPR for each sample in the sequence. Let the SPR warm up. Allow the SPR to run all samples automatically.

The sequence on the GC-MS side will automatically record the data in separate files. Add a peak to the processing method by selecting Calibrate followed by Edit Compound, Name, and Insert Compound under External Standard Compound. Add the name of the compounds, retention time, and quant signal target ion.

Add the three largest peaks, which include compounds with a greater than 75%probability, ensuring that the alignment of each identifying ion of the compound lies within the center of the peak. Save it by selecting OK, followed by Method and Save. Once the process method is set up, proceed to Quantitate and Calculate, then View and QEDIT Quant Result to quantitate the data.

Here, vacuum-assisted sorbent extraction was followed by thermal desorption on a GC-MS to survey the volatile profiles of bacterial mono-and co-cultures, and identify actively produced volatiles with stable isotope probing from human feces, saliva, sewage, and sputum samples. The mono-and co-cultures consisted of the bacterial species staphylococcus aureus, pseudomonas aeruginosa, and acinetobacter baumannii. 43 annotated volatile molecules were detected from the mono-and co-cultures at 24-and 48-hour time points.

There was more incorporation of 13C into fully-labeled volatile molecules in comparison to the deuterium. 13C was incorporated into 2-butanone, 3-hydroxy, 2, 3-butanedione diacetyl, acetic acid, and phenol for all fecal, sewage, and saliva samples. Acetone, butanoic acid, and propanoic acid were detected as labeled in saliva and sewage.

whereas dimethyl trisulfide and disulfide dimethyl were enriched in both fecal and saliva samples. The volatiles 1-propanol, 2-butanone, benzophenone, ethanol, and methylthiol acetate were enriched only in sewage and 2, 3-pentanedione in saliva. Deuterium was incorporated into the volatiles acetic acid, benzaldehyde, 4-methyl-dimethyl trisulfide, and phenol from either saliva or sewage samples.

Acetic acid, dimethyl trisulfide, acetone, and propanol 2-methyl were more abundant in the cultured sputum samples than uncultured sputum samples. A permutated multi-variate analysis of variants, a PERMANOVA, was performed on a Bray-Curtis distance matrix of the volatile abundances from the cystic fibrosis sputum samples. We found that the subject who donated the sample explains 51%of the variation in 13C-labeled cultured sputum and 33%of the variation in uncultured sputum.

Here, the success of stable isotope labeling with 13C glucose in volatiles from cultured sputum samples collected from seven people with cystic fibrosis is shown. Volatiles that have higher 13C incorporation for the majority of samples are shown in panel 5A, those with lower-percent 13C incorporation in the majority of sputum samples are in panel 5B, and molecules with lower 13C percent conversion in a minority of the sputum samples are shown in panel 5C. Always double check your vial pressures after about a minute.

A broken vacuum defeats the sensitivity and speed of the VASE method. Following this procedure, if stable isotope probing was performed, the DNA can be extracted from the remaining material to identify microbial organisms or species that may have contributed to the production of the volatile molecules. This method is also useful in detecting volatiles from any sample type without isotope probing.

With advantages of sensitivity and low sample volume, many applications can benefit from this technique.