Bovine tuberculosis (bTB) is a zoonotic disease of international public health, trade, agricultural, and wildlife management significance1,2. The disease is caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex3,4,5. It is estimated that in 2014, 9.6 million new cases human tuberculosis (hTB) and 1.5 million associated deaths occurred worldwide6, with the majority caused by M. tuberculosis; however, zoonotic tuberculosis is often under-reported. Muller et al. 2013 reported that approximately 10–37% of hTB cases may be caused by M. bovis infection, especially in developing countries where the prevalence of livestock bTB may reach 10–14%7,8,9,10,11,12.

A primary hurdle for mycobacterial disease diagnosis is the long incubation time required to culture and confirm the presence of mycobacteria in biological samples. Culture may take approximately eight weeks before final results can be confirmed. Several in vitro blood based tests (i.e., interferon-ɣ release assay) have been developed to confirm that an individual has been exposed to mycobacteria. However, these tests suffer from cross-reactivity with closely related non-tuberculous mycobacteria resulting in false positive test results. Development of technologies to reduce the time between the start of culture, detecting growth, and positively identifying the mycobacterial agent will improve both the diagnosis and appropriate treatment of infected humans, and eradication of bTB from wildlife and livestock.

In recent years increased attention has been given to the concept of using microbial volatile organic compound (VOC) emissions as “signature markers” (a.k.a. biomarkers) for faster, more economical, and noninvasive disease diagnosis in humans and animals. These VOC emissions may be collected from breath, blood, skin, urine, feces and other bodily secretions. Studies have identified potential VOC biomarkers related to multiple diseases such as cholera, cancer, diabetes, uremia, schizophrenia, asthma, liver disease, chronic lung disease, Pseudomoniasis, tuberculosis, and others13,14,15,16. Several methods of collecting and analyzing VOCs for potential diagnosis of M. tuberculosis and M. bovis have been described. Closed loop stripping analysis (CLSA) - gas chromatography- mass spectrometry (GC-MS) was used to detect VOCs from multiple strains of M. tuberculosis from cultures17, and select ion flow tube (SIFT)-MS has been used to measure VOCs present in the headspace of M. bovis BCG cultures18 and to measure VOCs in the breath of children with cystic fibrosis14, NH 3 levels in human breath for Helicobacter pylori screening, and detect acetonitrile levels in smokers’ breath19.

Electronic ‘e-nose’ technology has been used for detection of VOCs present in sputum samples collected from humans infected with M. tuberculosis14, and VOCs from the headspace of cultures of M. bovis BCG and M. smegmatis18. ‘E-nose’ has also been reported capable of differentiating between M. tuberculosis, three other bacteria, and a control19, monitoring smokers’ habits by measuring breath CO, detecting H. pylori presence in association with chronic gastritis, and detecting N 2 O produced by respiratory inflammation16. Thermal desorption (TD)-GC-MS has been used to determine VOCs present in the headspace of M. bovis BCG cultures18, and cattle breath20; and solid-phase microextration (SPME)-GC-MS was used to determine biomarkers M. tuberculosis and M. bovis in cultures and M. tuberculosis in human breath21,22. Other methods that have been used to collect VOCs associated with diseases that could be utilized in the future for TB detection include ion mobility spectrometry (IMS)14,23, proton transfer reaction (PTR)-MS14,15,16, and laser spectroscopy24. Select ion flow tube-MS, ‘e-noses’, IMS, PTR-MS have the advantage of being fast and potentially mobile. The downside of these methods includes decreased sensitivity, and the inability to chemically identify or profile VOCs. GC-MS-based methods may be slower, more expensive, and the instrumentation is not typically mobile; however, they have the advantage of being able to reproducibly identify and profile known and unknown microbial VOCs at low concentration ranges14,16.

Solid-phase microextration is an attractive technology for collecting microbial VOCs due to its simplicity, ease of use, and ability to sample and pre-concentrate a wide range of potential target compounds. Dynamic headspace extraction of VOCs can increase mass transfer to the SPME fiber compared to static headspace extraction, thus, reducing sampling times while improving mass loading of the fiber25,26. In contrast, both CLSA and TD are capable of pre-concentrating VOCs but require extra equipment and are more labor intensive than SPME. Syhre et al.21 collected VOCs from seven mycobacterial and 16 other respiratory pathogen cultures using three different SPME fiber types; 100 µm polydimethylsiloxane (PDMS), 2 cm 50/30 µm divinylbenzene (DVB)/Carboxen/PDMS, and 70 µm Carbowax/DVB. The SPME fiber coated with 2 cm 50/30 µm DVB/Carboxen/PDMS was found to recover higher concentrations of all target VOCs. Other studies have utilized the 50/30 µm DVB/Carboxen/PDMS-type and Carboxen/PDMS-type SPME fiber coatings for microbial VOC collection in human and cattle breath samples22,27,28, in bovine nasals excretions29, and in bovine fecal excretions from cattle vaccinated with M. bovis challenge30. SPME has been also used for the in-vivo and in-vitro collection of rumen gases31,32, VOCs emitted from wildlife marking fluids33,34, decaying carcasses35.

The objectives of this research were to (1) design, build, and test a lab-scale dynamic VOC sampling platform specifically capable of simultaneous biosecure SPME collection of headspace VOCs emitted from controlled bacterial cultures and a media control; (2) apply this screening method to M. avium paratuberculosis (MAP); the vaccine strain of M. bovis Bacillus Calmette-Guérin (BCG); and M. kansasii cultures to demonstrate a proof-of-concept detection method that is faster than standard culture methods.

Our first working hypothesis was that by sampling the recirculating culture headspace air, we would be able to detect trace-levels of microbial VOCs early in the incubation process with minimal background interference. Our second hypothesis was that SPME sampling of microbial VOCs followed by GC-MS analysis would be suitable to detect differences in microbial VOCs emitted by different cultured strains of mycobacteria.

If successful, this proof-of-concept identification of VOC biomarkers would allow differentiation between the microbial agents prior to the eight weeks often required for cultured mycobacterial strain identification. The knowledge gained from this work could be directly applied for diagnosis of hTB and bTB, for detection and identification of VOC biomarkers produced by culture of other pathogenic bacteria, and as a reference library for pathogen-produced VOCs present in other samples such as breath, feces, urine, blood, and other biofluids.