By carefully examining those compounds detected by TD-GC-MS in at least two of the M. bovis BCG cultures,
but which were absent in the LJ medium controls, seven potential markers of M. bovis BCG were identified and are given in Table 1. In addition, SIFT-MS analysis of the culture headspace indicated that hydrogen sulphide, H2S, was produced by M. bovis BCG but was absent in the medium controls. The headspace of the BCG cultures also contained significantly more acetaldehyde and methanol than was present in the headspace of the controls. Ammonia (NH3) was significantly depleted in the culture headspace compared with the medium, indicating utilization by the mycobacteria. Headspace from LJ cultures of BCG and M. smegmatis was tested and the resultant chromatograms selleck screening library compared to LJ slopes that were not inoculated. Of the seven VOC previously identified by TD-GC-MS, one was observed to be present exclusively
in the headspace of cultures. The remaining six VOC did not have retention times that coincided Roxadustat purchase with peaks that varied according to growth, either they were not present in sufficient quantity or retention times coincided those of other interfering compounds in culture headspace. The observed peak ran concurrently with reference samples of phenylethyl alcohol (PEA) (Sigma-Aldrich, Gillingham, UK) on both columns. A retention time of 7.06 s was recorded when using the zNose 7100 (Fig. 1) and 3.50 s with the zNose 4200 (Fig. 3). PEA production clonidine was dependent on growth of the bacteria
and when testing with the ZNose was observed when sufficient bacteria were present to be seen by eye. For M. bovis BCG, PEA was observed in culture headspace a minimum of 5 days following inoculation with a 10 μL loop of culture. The time taken for PEA to appear in the headspace of M. smegmatis culture was between 1and 2 days. Peaks increased in size as the culture within the bottle increased and decreased when cultures reached confluence. For M. smegmatis, the peaks were observed for a period of < 1 week, whereas for BCG, peaks were observed for up to 5 weeks (Fig. 2). Growth of bacteria and production of PEA was encouraged if caps on the culture bottles were loosened during the incubation to allow exchange of gases (data not shown). The PEA peak was not observed when LJ was inoculated with heat killed bacteria. Following inoculation of LJ slopes containing compounds inhibitory to the growth of mycobacteria belonging to the M. tuberculosis complex, such as 0.5 mg mL−1p-nitro benzoic acid, PEA production was absent for BCG but present for M. smegmatis (Fig. 3). PEA was not observed when LJ slopes were inoculated with Escherichia coli DH5 or when mycobacteria were grown on Middlebrook 7H9 agar slopes (data not shown).