Differentiation of Bacillusendospore species from fatty acid methyl ester biomarkers

Abstract

A simple method to detect and differentiate Bacillus anthracis (BA), Bacillus thuringiensis (BT), Bacillus atrophaeus (BG), and Bacillus cereus (BC) endospores using biomarker compounds, including dipicolinic acid methyl ester (DPAME) and fatty acid methyl esters (FAMEs), has been developed. The method is based on thermochemolysis methylation (TCM) of the endospores and gas chromatography-mass spectrometry (GC-MS) of the reaction products. A suspension of the sample mixed with sulfuric acid (H₂SO₄) and tetramethylammonium hydroxide (TMAH) in methanol (MeOH) at room temperature is sampled using a coiled wire filament (CWF) device, which consists of a tiny platinum helical wire coil attached to a retractable plunger that moves the coil in and out of a syringe needle housing. Sampling is accomplished by dipping the CWF in an endospore sample suspension, evaporating the suspension liquid, and then introducing the CWF into the injection port. While DPAME can be used for the general detection of endospores, specific saturated and unsaturated C₁₅, C₁₆, and C₁₇ fatty acid methyl esters provide additional information for differentiating various Bacillus species grown at different temperatures and in different media. DPAME could be detected in samples containing as few as 6000 endospores, and the GC-MS peak area percent reproducibility for FAMEs varied from 3 to 13% (RSD). Better than 97% correct predictability of Bacillus species identity was obtained from a blind experiment consisting of 145 samples.