Rapid Identification of Bacillus anthracis Spores in Suspicious Powder Samples by Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)

Abstract

cRapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 ؋ 10 6 spores, equivalent to a 55-g sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min.

Bacillus cereus Sensu Lato Genomes: Basis for Identifying Anthrax Disease Strain Sources

“…Together with another report (8) of a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) proteomic analysis of proteins directly from anthrax spores (which would allow for identification with 30-min analysis), the data here presumably provide background strain source identification for the small annual number of natural animal (some in Texas) and a few human anthrax disease cases and future bioterrorism threats. Rapid recognition allows for the successful decontamination of sources and antibiotic treatment of patients, as required by the military and police.…”

MALDI Profiling and Applications in Medicine

“…The MALDI protein profi ling approach has also been utilised to characterise rarer pathogenic bacteria and it was noted that of the various organisms studied fewer bacteria needed a second confi rmation analysis when using MALDI compared to conventional approaches (50 compared to 620) [ 73 ]. Besides its application in identifying clinical bacterial infections, the MALDI protein profi ling approach has also been reported in being able to confi rm the identifi cation of Bacillus anthracis spores in suspicious powder in less than half an hour [ 17 ]. Due to the promise suggested by such laboratory investigations inter-laboratory comparisons have been performed in order to show the reproducibility of the technique between centres, to compare competing MALDI mass spectrometers and commercial databases available and to compare the technique to existing methods of identifi cation [ 23 , 36 , 42 , 48 , 53 , 66 , 93 ].…”

MALDI-TOF Mass Spectrometry Applied to Classification and Identification of Bacteria

“…Protocol 3 was found to be appropriate for inactivation of viable cells but not of resistant spores. In order to remove spores from the extracts, the inclusion of centrifugal filtration steps (pore size 0.1 mm) was suggested (Drevinek et al, 2012;Dybwad, van der Laaken, Blatny, & Paauw, 2013). A procedure starting with suspending the biomass in 80% (v/v) TFA was suggested as an alternative to Protocol 3 for reliable inactivation of spores and highly pathogenic bacteria by Sauer (2009) andLasch et al (2008).…”