Volatile Emissions from Mycobacterium avium subsp. paratuberculosis Mirror Bacterial Growth and Enable Distinction of Different Strains

Abstract

Control of paratuberculosis in livestock is hampered by the low sensitivity of established direct and indirect diagnostic methods. Like other bacteria, Mycobacterium avium subsp. paratuberculosis (MAP) emits volatile organic compounds (VOCs). Differences of VOC patterns in breath and feces of infected and not infected animals were described in first pilot experiments but detailed information on potential marker substances is missing. This study was intended to look for characteristic volatile substances in the headspace of cultures of different MAP strains and to find out how the emission of VOCs was affected by density of bacterial growth. One laboratory adapted and four field strains, three of MAP C-type and one MAP S-type were cultivated on Herrold’s egg yolk medium in dilutions of 10-0, 10-2, 10-4 and 10-6. Volatile substances were pre-concentrated from the headspace over the MAP cultures by means of Solid Phase Micro Extraction (SPME), thermally desorbed from the SPME fibers and separated and identified by means of GC-MS. Out of the large number of compounds found in the headspace over MAP cultures, 34 volatile marker substances could be identified as potential biomarkers for growth and metabolic activity. All five MAP strains could clearly be distinguished from blank culture media by means of emission patterns based on these 34 substances. In addition, patterns of volatiles emitted by the reference strain were significantly different from the field strains. Headspace concentrations of 2-ethylfuran, 2-methylfuran, 3-methylfuran, 2-pentylfuran, ethyl acetate, 1-methyl-1-H-pyrrole and dimethyldisulfide varied with density of bacterial growth. Analysis of VOCs emitted from mycobacterial cultures can be used to identify bacterial growth and, in addition, to differentiate between different bacterial strains. VOC emission patterns may be used to approximate bacterial growth density. In a perspective volatile marker substances could be used to diagnose MAP infections in animals and to identify different bacterial strains and origins.

Diversity of food-borne Bacillus volatile compounds and influence on fungal growth

“…; Trefz et al . ) but also on the nature of the pathogenic fungi against which the VOCs were tested. This points out to the fact that the observed variations in fungal responses may reflect differences in the site of action or in the ability of fungi to detoxify the metabolites.…”

Physiological variability in volatile organic compounds (VOCs) in exhaled breath and released from faeces due to nutrition and somatic growth in a standardized caprine animal model

“…VOC analyses were performed using GC-MS. An Agilent 7890 A gas chromatograph connected to an Agilent 5975 C inert XL mass selective detector (MSD) was used to separate and detect VOCs desorbed from SPME fibres, as described in Trefz et al (2013a). VOCs desorbed from the NTDs were separated by a gas chromatograph (Agilent 7890 A) and detected by a mass selective detector (Agilent 5975 C inert XL MSD), as previously described in Trefz et al (2013b).…”

“…In order to obtain unequivocal identification and quantification, the selected marker substances were verified by means of pure reference substances. For the calibration and determination of limit of detection (LOD, signal-to-noise ratio 3:1) and limit of quantification (LOQ, signal-to-noise ratio 10:1), different concentration levels of the reference substances were measured as previously described for SPME (Trefz et al 2013a) and NTME (Trefz et al 2012). Humidified standards were used for the quantification of NTME-GC-MS data.…”

Volatile Organic Compound and Metabolite Signatures as Pathogen Identifiers and Biomarkers of Infectious Disease

“…logarithmic versus stationary phase), sample storage conditions (e.g. short-term versus long-term), and the type of culture media used [34,62,63]. To confound analysis further, patient samples are far less well-defined than laboratory cultures of reference strains, and therefore vary greatly in terms of growth phase, host response, viscosity, confounding co-morbidities, and medications (including antibiotics) [43,61,64].…”