Anal. Chem. 2000, 72, 119-127 Detection of the Dipicolinic Acid Biomarker in Bacillus Spores Using Curie-Point Pyrolysis Mass Spectrometry and Fourier Transform Infrared Spectroscopy Royston Goodacre,*,² Beverley Shann,² Richard J. Gilbert,² EÄ adaoin M. Timmins,² Aoife C. McGovern,² Bjùrn K. Alsberg,²,³ Douglas B. Kell,² and Niall A. Logan§ Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion, SY23 3DD, Wales, U.K., Department of Computer Sciences, University of Wales, Aberystwyth, Ceredigion, SY23 3DB, Wales, U.K., and School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow, G4 0BA, Scotland, U.K. Thirty-six strains of aerobic endospore-forming bacteria biochemical changes in the developing spore. It becomes encased confirmed by polyphasic taxonomic methods to belong to in two novel layers, a peptidoglycan layer (the spore cortex) and Bacillus amyloliquefaciens, Bacillus cereus, Bacillus a number of layers of spore coats that contain proteins unique to licheniformis, Bacillus megaterium, Bacillus subtilis spores.1 The spore also accumulates a substantial deposit (5- (including Bacillus niger and Bacillus globigii), Bacil- 14% of dry weight) of pyridine-2,6-dicarboxylic acid (dipicolinic lus sphaericus, and Brevi laterosporus were grown acid; DPA), which is unique to spores, as well as a large amount axenically on nutrient agar, and vegetative and sporulated of divalent cations.2 biomasses were analyzed by Curie-point pyrolysis mass Members of the genus Bacillus are widely distributed in soil, spectrometry (PyMS) and diffuse reflectance-absorbance water, and air, and because their spores are so resistant their Fourier-transform infrared spectroscopy (FT-IR). Chemo- control is of considerable importance in the food processing metric methods based on rule induction and genetic industry and in the preparation of sterile products.3 In addition, programming were used to determine the physiological the rapid identification of Bacillus anthracis spores is of importance state (vegetative cells or spores) correctly, and these because of its potential use as a biological warfare agent.4 methods produced mathematical rules which could be Therefore, there is a need for a generic characterization system simply interpreted in biochemical terms. For PyMS it was that can be used to carry out the large-scale and rapid detection found that m/z 105 was characteristic and is a pyridine of bacterial spores. ketonium ion (C H ON+) obtained from the pyrolysis of 6 3 Although spores can be observed by simple microscopy, this dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA), a approach does not lead itself to automation, and hence, analytical substance found in spores but not in vegetative cells; this techniques are being employed to detect bacterial spores much was confirmed using pyrolysis-gas chromatography/mass more rapidly and with more specificity. The theoretical fluores- spectrometry. In addition, a pyridine ring vibration at 5 1447-1439 cm-1 from DPA was found to be highly cence of dipicolinic acid and its anion have been studied, while characteristic of spores in FT-IR analysis. Thus, although terbium dipicolinate photoluminescence has been used to detect 6 the original data sets recorded hundreds of spectral the presence of sporulating bacteria. The vibrational spectroscopic 7 variables from whole cells simultaneously, a simple bio- methods of UV resonance Raman spectroscopy and Fourier 8 marker can be used for the rapid and unequivocal transform infrared spectroscopy have also been used to differenti- detection of spores of these organisms. (1) Buchanan, C. E.; Henriques, A. O.; Piggot, P. J. In Bacterial Cell Wall; Hakenbeck, J.-M. A. R., Ed.; Elsevier Science Publishers: New York, 1994; The genus Bacillus consists of Gram-positive bacteria, which pp 167-186. can respond to slowed growth or starvation by initiating the (2) Murrell, W. G. In The Bacterial Spore; Gould, G. W., Hurst, A., Eds.; - process of sporulation. These organisms as well as exhibiting Academic Press: London, 1969; pp 215 273. (3) Doyle, M. P.; Beuchat, L. R.; Montville, T. J. Food Microbiology: Funda- morphological variation of vegetative cells can undergo differentia- mentals and Frontiers; Amercian Society of Microbiology Press: Washington, tion to distinct resting bodies or spores, and after a period of time, DC, 1997; p 768. these spores can germinate to produce a single vegetative cell. (4) Barnaby, W. The Plague Makers: The Secret World of Biolgoical Warfare; Vision Paperbacks: London, 1997. As sporulation proceeds, there are striking morphological and (5) Hameka, H. F.; Jensen, J. O.; Jensen, J. L.; Merrow, C. N.; Vlahacos, C. P. J. Mol. Struct. (THEOCHEM) 1996, 365, 131-141. * Corresponding author: (telephone) +44 (0)1970 621947; (telefax) +44 (6) Pellegrino, P. M.; Fell, N. F.; Rosen, D. L.; Gillespie, J. B. Anal. Chem. 1998, (0)1970 621947; (e-mail) [email protected]. 70, 1755-1760. ² Institute of Biological Sciences, University of Wales. (7) Ghiamati, E.; Manoharan, R.; Nelson, W. H.; Sperry, J. F. Appl. Spectrosc. ³ Department of Computer Sciences, University of Wales. 1992, 46, 357-364. § Glasgow Caledonian University. (8) Helm, D.; Naumann, D. FEMS Microbiol. Lett. 1995, 126,75-80. 10.1021/ac990661i CCC: $19.00 © 2000 American Chemical Society Analytical Chemistry, Vol. 72, No. 1, January 1, 2000 119 Published on Web 11/19/1999 ate between spores and vegetative bacteria. Flow cytometry9 has scatterplots and dendrograms. More recently, various related but also been explored for the rapid detection of spores. Finally, a much more powerful approaches, most often referred to within number of workers have investigated methods based on mass the framework of chemometrics, have been applied to the spectrometric analyses. However, bacterial spores are nonvolatile ªsupervisedº analysis of hyperspectral data;18 arguably the most and so to introduce these and their component chemical species significant of these is the application of intelligent systems based to a mass spectrometer they first have to be pyrolyzed. The volatile on artificial neural networks (ANNs).19,20 fragments (or pyrolysate) from bacterial spores have then been Although ANNs are excellent at identifying unknown bacterial measured following electron ionization using a quadrupole mass isolates to the correct genera and species, the information in terms analyzer,10 a tandem mass spectrometer,11 or gas chromatogra- of which masses in the mass spectrum or wavenumbers in the phy/ion mobility spectrometry;12 however, none of these has yet infrared spectrum are important is not readily available. The use gained acceptance as the analytical method of choice for spore of ANNs therefore is often perceived as a ªblack boxº approach detection in a military context. to modeling spectra and so has limited use for the deconvolution The above studies used only a handful of Bacillus species, and of hyperspectral data in (bio)chemical terms. Therefore, in this some merely a single strain. Most studies for detecting B. study a number of rule induction methods21 and methods involving anthracis spores that have been deliberately released for military evolutionary computation22,23 were employed to aid in the decon- or terrorist actions have concentrated on Bacillus subtilis, and in volution of these hyperspectra. particular its pigmented variant ªBacillus globigiiº(B. subtilis var. niger), which is perhaps surprising since phylogenetic analysis EXPERIMENTAL SECTION of their 16S rRNA sequences shows that B. subtilis and B. globigii Bacteria and Cultivation. Identities of the 36 Bacillus species are distinct from B. anthracis,13 which itself is phylogenetically studied were confirmed by use of a polyphasic approach using indistinguishable from Bacillus cereus. Therefore the present study conventional biochemical (API tests) and nucleic acid technolo- investigates a wide variety of genetically distinct bacteria (36 gies.24 Full details are shown in Table 1. representatives of seven mesophilic Bacillus species; B. cereus was Vegetative cells were collected by incubating the 36 bacteria represented by five strains and was used to draw analogy to B. on Lab M blood agar base plates (without blood) at 37 °C for 10 anthracis; the latter was omitted from this study on grounds of h. Spores were prepared by incubation on Lab M blood agar base -1 25 safety). plates + 5 mg/L MnSO4 at 30 °C for 7 days. After incubation, The rapid, fully automated, analytical methods that were the biomass was collected in physiological saline (0.9% NaCl). employed in this study included Curie-point pyrolysis mass These bacterial slurries contained ∼109 cells/mL and were stored spectrometry (PyMS), and diffuse reflectance-absorbance Fourier at -20 °C until analysis. To assess the level of sporulation, all 72 transform infrared (FT-IR) spectroscopy, in the mid-infrared range. cultures were examined microscopically. The vegetative cells PyMS and FT-IR are physicochemical methods that measure contained no spores, while the sporulated biomass comprised predominantly the bond strengths of molecules and the vibrations >80% spores; no attempt was made to remove any cell debris or of bonds within functional groups, respectively.14,15 Therefore they vegetative cells from the spore preparations. give quantitative information about the total biochemical composi- Pyrolysis Mass Spectrometry. Five-microliter aliquots of the tion of a sample. However, the interpretation of these multi-