Detection of the dipicolinic acid biomarker in Bacillus spores using Curie-point pyrolysis mass spectrometry and fourier transform infrared spectroscopy

Thirty-six strains of aerobic endospore-forming bacteria confirmed by polyp hasic taxonomic methods to belong to Bacillus amyloliquefaciens, Bacillus c ereus, Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis (incl uding Bacillus niger and Bacillus globigii), Bacillus sphaericus,and Brevi laterosporus were grown axenically on nutrient agar, and vegetative and spo rulated biomasses were analyzed by Curie-point pyrolysis mass spectrometry (PyMS) and diffuse reflectance-absorbance Fourier-transform infrared spectr oscopy (FT-IK), Chemometric methods based on rule induction and genetic pro gramming were used to determine the physiological state (vegetative cells o r spores) correctly, and these methods produced mathematical rules which co uld be simply interpreted in biochemical terms. For PyMS it was found that m/z 105 was characteristic and is a pyridine ketoniun ion (C6H3ON+) obtaine d from the pyrolysis of dipicolinic acid (pyridine-2,6-dicarboxylic acid; D PA), a substance found in spores but not in vegetative cells; this was conf irmed using pyrolysis-gas chromatography/mass spectrometry, In addition, a pyridine ring vibration at 1447-1439 cm(-1) from DPA was found to be highly characteristic of spores in FT-IR analysis. Thus, although the original da ta sets recorded hundreds of spectral variables from whole cells simultaneo usly, a simple biomarker can be used for the rapid and unequivocal detectio n of spores of these organisms.