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

Summary

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.

Abstract

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

Introduction

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 subjects^5,6. Vola....

Protocol

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).

1. Generate negative controls by extracting media and/or sample blanks with the same conditions used for sample extraction.
2. ....

Results

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 .......

Discussion

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 °C. The volatile analysis of mono- and co-cultures allowed the survey of the compounds produced both by individual species and dur.......

Materials

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