Improved Discrimination of Bacillus anthracis from Closely Related Species in the Bacillus cereus Sensu Lato Group Based on Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Discrimination of highly pathogenic bacteria, such asBacillus anthracis , from closely related species based on molecular biological methods is challenging. We applied matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) to a collection ofB. anthracis strains and close relatives in order to significantly improve the statistical confidence of identification results for this group of bacteria. Protein mass spectra of 189 verified and diverseBacillus strains of theBacillus cereus sensu lato group were generated using MALDI-TOF MS and subsequently analyzed with supervised and unsupervised statistical methods, such as shrinkage discriminant analysis (SDA) and principal-component analysis (PCA). We aimed at identifying specific biomarkers in the protein spectra ofB. anthracis not present in closely relatedBacillus species. We could identify 7, 10, 18, and 14B. anthracis -specific biomarker candidates that were absent inB. cereus ,B. mycoides ,B. thuringiensis , andB. weihenstephanensis strains, respectively. Main spectra (MSP) of a defined collection ofBacillus strains were compiled using the Bruker Biotyper software and added to an in-house reference library. Reevaluation of this library with 15 hitherto untested strains ofB. anthracis andB. cereus yielded improved score values. TheB. anthracis strains were identified with score values between 2.33 and 2.55 using the in-house database, while the same strains were identified with scores between 1.94 and 2.37 using the commercial database, and no false-positive identifications occurred using the in-house database.