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Crespo E.,Radboud University Nijmegen | De Ronde H.,Royal Tropical Institute KIT | Kuijper S.,Vant Hoff Institute for Molecular science HIMS | Pol A.,Radboud University Nijmegen | And 4 more authors.
Rapid Communications in Mass Spectrometry | Year: 2012

RATIONALE Several mycobacterial species can produce serious infections in humans, and the treatment required depends on the infecting species. Fast identification, ideally with minimal manipulation of the infecting species, is therefore critical; here, we propose a method potentially allowing cultures to be identified by headspace analysis and use it to screen for differences between mycobacterial species based on the volatiles released during growth. METHODS Short-chain volatile organic compound emissions from two non-tuberculosis slow growing mycobacterial species, Mycobacterium avium and Mycobacterium kansasii, and a non-pathogenic fast growing species, Mycobacterium smegmatis, in Middlebrook M7H9 culturing media were followed online with a proton transfer reaction quadrupole mass spectrometer. RESULTS Measurable differences between the headspace of the two slow growing mycobacteria M. kansasii and M. avium were found, as well as differences with respect to the faster growing mycobacteria M. smegmatis. Three compounds, attributed to sulfur-containing volatiles - dimethyl sulfide, propanethiol and dimethyl disulfide - were found to be specific to M. avium. CONCLUSIONS Clear differences were detected in the low molecular weight volatile emissions compounds of the mycobacterial species under study, without the need for sample manipulation. Further studies with other mycobacterial species will reveal if the differences observed are specific to the species studied here. Furthermore, the use of an ion trap as a mass analyzer with the same ionization technique, allowing molecular detection over a wider molecular range, could allow the detection of additional biomarkers thus capturing a wider molecular range. Copyright © 2012 John Wiley & Sons, Ltd.

Crespo E.,Radboud University Nijmegen | Cristescu S.M.,Radboud University Nijmegen | de Ronde H.,Royal Tropical Institute KIT | Kuijper S.,Vant Hoff Institute for Molecular science HIMS | And 3 more authors.
Journal of Microbiological Methods | Year: 2011

The metabolic activity of plants, animals or microbes can be monitored by gas headspace analysis. This can be achieved using Proton Transfer Reaction Mass Spectrometry (PTR-MS), a highly sensitive detection method for trace gas analysis. PTR-MS is rapid and can detect metabolic responses on-line as they occur. Here, we study the headspace of actively growing cultures of paired ciprofloxacin sensitive and resistant bacterial strains (Mycobacterium smegmatis in Middlebrook M7H9 liquid media) after the addition of the antibiotics ciprofloxacin and gentamicin in real time. Following the emission patterns of the mycobacteria over time allowed volatile markers specific for the bacterial response to each antibiotic to be detected. A proportion of the measured responses were very rapid, occurring within three hours after the addition of the compounds and varied between isolates with different resistance phenotypes. Specifically, we observed a two fold increase of m73 (unidentified C4 compound) within 10. h after the addition of ciprofloxacin and a threefold increase of m45 (acetaldehyde) within 4. h after the addition of gentamicin as compared to values before the addition. Monitoring the emission of specific volatiles into the culture headspace thus has the potential for rapid drug susceptibility testing. Moreover, these and other differences in the measured responses to the two tested compounds provide evidence that monitoring multiple compounds may also give an indication of the mechanism of action of the compound added. © 2011 Elsevier B.V.

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