We thank Heather Maughan and Linda M. Kalikin for careful editing of this manuscript. This work was supported by NIH NHLBI (grant 5R01HL136647-04).

1. Headspace Sorbent Pen (HSP) and sample analysis considerations
NOTE: The HSP containing the sorbent Tenax TA was selected to capture a broad range of volatiles. Tenax has a lower affinity for water compared to other sorbents, which enables it to trap more VOCs from higher-moisture samples. Tenax also has a low level of impurities and can be conditioned for re-use. Sorbent selection was also made in consideration with the column installed in the GC-MS (see the Table of Materials</stron...

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To identify volatile production in in vitro cultures and human-associated samples, volatile analysis of mono- and co-cultures of P. aeruginosa, S. aureus, and A. baumanii and stable isotope probing of different biological samples were performed. In the analysis for the mono- and co-cultures, volatiles were detected by performing a short extraction for 1 h at 70 &#176;C. The volatile analysis of mono- and co-cultures allowed the survey of the compounds produced both by individual species and dur...

Volatile organic compounds (VOCs) have great promise for bacterial detection and identification because they are emitted from all organisms, and different microbes have unique VOC signatures. Volatile molecules have been utilized as a non-invasive measurement for detecting various respiratory infections including chronic obstructive pulmonary disease1, tuberculosis2 in urine3, and ventilator-associated pneumonia4, in addition to distinguishing subjects with cystic fibrosis (CF) from healthy control subjects5,6. Volatile signatures have even been used to distinguish specific pathogen infections in CF (Staphylococcus aureus7, Pseudomonas aeruginosa8,9, and S. aureus vs. P. aeruginosa10). However, with the complexity of such biological samples, it is often difficult to pinpoint the source of specific VOCs.
One strategy for disentangling the volatile profiles from multiple infecting microbes is to perform headspace analysis of microorganisms in both mono- and co-culture11. Headspace analysis examines the analytes emitted into the &#34;headspace&#34; above a sample rather than those embedded in the sample itself. Microbial metabolites have often been characterized in mono-cultures because of the difficulty in determining the origin of microbial metabolites in complex clinical samples. By profiling volatiles from bacterial mono-cultures, the types of volatiles a microbe produces in vitro may represent a baseline of its volatile repertoire. Combining bacterial cultures, e.g., creating co-cultures, and profiling the volatile molecules produced may reveal the interactions or cross-feeding between the bacteria12.
Another strategy for identifying the microbial origin of volatile molecules is to provide a nutrient source that is labeled with a stable isotope. Stable isotopes are naturally occurring, non-radioactive forms of atoms with a different number of neutrons. In a strategy that has been utilized since the early 1930s to trace active metabolism in animals13, the microorganism feeds off of the labeled nutrient source and incorporates the stable isotope into its metabolic pathways. More recently, a stable isotope in the form of heavy water (D2O) has been used to identify metabolically active S. aureus in a clinical CF sputum sample14. In another example, 13C-labeled glucose has been used to demonstrate the cross-feeding of metabolites between CF clinical isolates of P. aeruginosa and Rothia mucilaginosa12 .
With the advancement of mass spectrometry techniques, methods of detecting volatile cues have moved from qualitative observations to more quantitative measurements. By using gas chromatography mass spectrometry (GC-MS), processing of biological samples has become within reach for most laboratory or clinical settings. Many methods to survey volatile molecules have been used to profile samples such as food, bacterial cultures, and other biological samples, and air and water to detect contamination. However, several common methods of volatile sampling with high-throughput require solvent and are not performed with the advantages provided by vacuum extraction. In addition, larger volumes or quantities (greater than 0.5 mL) of sampled materials are often required for analysis15,16,17,18,19, although this is substrate-specific and requires optimization for each sample type and method.
Here, vacuum-assisted sorbent extraction (VASE) followed by thermal desorption on a GC-MS was employed 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 (Figure 1). With limited sample quantities, VOCs were extracted from as little as 15 &#181;L of sputum. Isotope probing experiments with human samples required adding a stable isotope source, such as 13C glucose, and media to cultivate the growth of the microbial community. The active production of volatiles was identified as a heavier molecule by GC-MS. Extraction of volatile molecules under a static vacuum enabled the detection of volatile molecules with increased sensitivity20,21,22.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>13C glucose</td>
			<td>Sigma-Aldrich</td>
			<td>389374-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Stg Diaph Pump</td>
			<td>Entech Instruments</td>
			<td>01-10-20030</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL VOA vials</td>
			<td>Fisher Scientific</td>
			<td>5719110</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm Black Caps with hole, no septum</td>
			<td>Entech Instruments</td>
			<td>01-39-76044B</td>
			<td>holds lid liner in place on vial</td>
		</tr>
		<tr>
			<td>24 mm vial liner for sorbent pens</td>
			<td>Entech Instruments</td>
			<td>SP-L024S</td>
			<td>allows pens to make a vacuum seal at top of vial</td>
		</tr>
		<tr>
			<td>5600 Sorbent pen extraction unit (SPEU)</td>
			<td>Entech Instruments</td>
			<td>5600-SPES</td>
			<td>5600 Sorbent Pen Extraction Unit -120 VAC</td>
		</tr>
		<tr>
			<td>96-well assay plates</td>
			<td>Genesee</td>
			<td>25-224</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI) media</td>
			<td>Sigma-Aldrich</td>
			<td>53286-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemStation Stofware</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DB-624 column</td>
			<td>Agilent</td>
			<td>122-1364E</td>
			<td>60 m, 0.25 mm ID, 1.40 micron film thickness, in GC-MS</td>
		</tr>
		<tr>
			<td>Deuterium oxide</td>
			<td>Sigma-Aldrich</td>
			<td>151882-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexsi sofware</td>
			<td>Dexsi (open source)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GC-MS (7890A GC and 5975C inert XL MSD with Triple-Axis Detector)</td>
			<td>Agilent</td>
			<td></td>
			<td>7890A GC and 5975C inert XL MSD with triple-axis detector</td>
		</tr>
		<tr>
			<td>Headspace Bundle HS-B01, 120VA</td>
			<td>Entech Instruments</td>
			<td>SP-HS-B01</td>
			<td>Items for running headspace extraction included in bundle</td>
		</tr>
		<tr>
			<td>Headspace sorbent pen (HSP) - blank</td>
			<td>Entech Instruments</td>
			<td>SP-HS-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Headspace sorbent pen (HSP) Tenax TA (35/60 Mesh)</td>
			<td>Entech Instruments</td>
			<td>SP-HS-T3560</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (2 mL)</td>
			<td>VWR</td>
			<td>53550-792</td>
			<td></td>
		</tr>
		<tr>
			<td>O-rings</td>
			<td>Entech Instruments</td>
			<td>SP-OR-L024</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Preparation Rail</td>
			<td>Entech Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbent pen thermal conditioner</td>
			<td>Entech Instruments</td>
			<td>3801-SPTC</td>
			<td></td>
		</tr>
		<tr>
			<td>Todd Hewitt (TH) media</td>
			<td>Sigma</td>
			<td>T1438-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

capturing actively produced microbial volatile organic compounds from human-associated samples with vacuum-assisted sorbent extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

Mono- and co-cultures of S. aureus, P. aeruginosa, and A. baumannii 
The mono- and co-cultures consisted of the bacterial species S. aureus, P. aeruginosa, and A. baumannii. These are common opportunistic pathogens found in human wounds and chronic infections. To identify the volatile molecules present in the mono- and co-cultures, a short 1-h extraction was performed at ...

Watch this Scientific Journal Video about Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction at JoVE.com

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

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

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.

capturing-actively-produced-microbial-volatile-organic-compounds-from

Research

JoVE Journal

Biochemistry

3.9K visualizações.  University of California Irvine. Este protocolo nos permite concentrar e identificar facilmente metabólitos voláteis e produzir ativamente voláteis de organismos microbianos em uma variedade de amostras biológicas. O VASE é um método mais fácil de usar para concentrar voláteis de baixa abundância. Tudo que você precisa é de uma amostra sob quase vácuo, e deixe a física fazer o resto.O acesso à análise volátil de amostras clínicas tem implicações importantes para a descoberta de biomarcadores metabó...