Detection of the Dipicolinic Acid Biomarker in Bacillus Spores Using Curie-Point Pyrolysis Mass Spectrometry and Fourier Transform Infrared Spectroscopy

摘要

Thirty-six strains of aerobic endospore-forming bacteria confirmed by polyphasic taxonomic methods to belong to Bacillus amyloliquefaciens,Bacillus cereus,Bacillus licheniformis,Bacillus megaterium,Bacillus subtilis(us sphaericus, and Brevi laterosporus were grown axenically on nutrient agar, and vegetative and sporulated biomasses were analyzed by Curie-point pyrolysis mass spectrometry(PyMS)and diffuse reflectance-absorbance Fourier-transform infrared spectroscopy(FT-IR).Chemometric methods based on rule induction and genetic programming were used to determine the physiological state(vegetative cells or spores) correctly,and these methods produced mathematical rules which could be simply interpreted in biochemincal terms.For PyMS it was found that m/z 105 was characteristic and is a pyridine ketonium ion(C_6H_3ON~+)obtained from the pyrolsis of dipicolinic acid (pyridine-2,6-dicarboxylic acid;DPA),a substance found in spores but not in vegetative cells;this was confirmed using pyrolysis-gans 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 data sets recorded hundreds of spectral variables from whole cells simultaneously,a simple biomarker can be used for the rapid unequivocal detections of spores of these organisms.