Name: capturing actively produced microbial volatile organic compounds from human-associated samples with vacuum-assisted sorbent extraction
Uploaded: 2022-06-01T12:30:00
Description: 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

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 ...

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