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

Abstract

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 (including Bacillus niger and Bacillus globigii), Bacillus 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 biochemical terms. For PyMS it was found that m/z 105 was characteristic and is a pyridine ketonium ion (C6H3ON+) obtained from the pyrolysis of dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA), a substance found in spores but not in vegetative cells; this was confirmed 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 data sets recorded hundreds of spectral variables from whole cells simultaneously, a simple biomarker can be used for the rapid and unequivocal detection of spores of these organisms.

Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification

“…The variance corrected average spectra were then used for assessing changes in the spectral information. Cluster analysis was employed and was carried out as detailed elsewhere [37,38]. The initial stage involved the reduction of the dimensionality of the MS data by principal components analysis (PCA).…”

“…The application of PCA and analysis of the resultant information by hierarchical cluster analysis is one approach to obtain global information and thereby observe the relative similarity between the spectra, and has been shown to be useful in the analysis of MS and vibrational spectroscopic data [37,38]. Accordingly, PCA was performed on the variance corrected average spectra, and the first few PC scores (typically five) that explained at least 95% of the total variance in the two data sets were used to construct dendrograms for the two cone potentials ( Figure 7).…”

Biphenyl Phytoalexin in Sorbus pohuashanensis Suspension Cell Induced by Yeast Extract

“…FCW ( Figure 1a) grew rapidly during days 1-5, with growth rate slowing between days 5-9, and reached a plateau between days 9-14; while DCW grew quickly during days 1-5, and slowed over the final 9 days (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). DCW reached its peak (0.89 g per flask) on the 5th day after subculture, as shown in Figure 1b, then continued to decrease during the culture, and this was due to the decrease of nutrient intake in cell proliferation caused by nutrient consumption in the later period of the culture.…”

“…PCA involves a mathematical procedure for reducing the dimensionality of multidimensional matrices while retaining a large amount of the original information in the data set [14]. Quintuplicate measurements from the same sample were found to be highly reproducible, as the scores of replicate measurements were more or less superimposed.…”

Measurement of dietary exposure: a challenging problem which may be overcome thanks to metabolomics?

“…For example, FT-IR [22] and NMR [37,61] generate global chemical fingerprints with little need for specialised sample preparation [23,24]. However, these methods are less sensitive than FIE-MS [3] and, generally, require a further level of directed analysis to link any differences in wavenumber (FT-IR) or chemical shifts (NMR) to specific chemistry [23].…”