Rapid Detection and Discrimination of Bacillus Species Using Immunomagnetic Separation Combined with Surface-enhanced Raman Spectroscopy

Introduction: Bacillus species are Gram-positive, spore-forming bacteria which include B. anthracis, a Class A bioterrorism agent according to the Centers for Disease Control and Prevention and the Department of Homeland Security. Because of the potential high risk of anthrax spores, it is important to be able to quickly and accurately detect anthrax spores in environmental and food samples.

Purpose: This research aimed to discriminate between three Bacillus species, B. anthracis, B. thuringiensis, and B. mycoides using SERS and establish an IMS-SERS procedure that can detect B. anthracis spores in milk within 20 minutes.

Methods: In this study, Surface-Enhanced Raman Spectroscopy (SERS) was first utilized in order to discriminate between Bacillus species. In order to detect B. anthracis in food systems, immunomagetic separation (IMS) was used to capture out of solution. This is then treated with dodecylamine, which digests the sporal coat. The resulting solution is analyzed for the spore biomarker dipicolinic acid (DPA).  

Results: The results show that Bacillus species and their cell states (live cell, dead cell, spore) could be differentiated when SERS spectra were analyzed using principal component analysis and hierarchical cluster analysis. The limit of detection was 2x10⁷ spores/ml due to the difficulty to optically locate the spores under a Raman microscope. In order to achieve a lower limit of detection, the spores were captured by IMS. This method was shown to have a ~50% recovery of B. anthracis spores from water and milk within 15 min.  The spores captured by IMS were treated with dodecylamine, which allowed a lower limit of detection at 2x10³spores/ml within 20 min.

Significance: Based on published toxicological data, detection at this limit is sufficient. This method could be extended to detect other spore-forming bacteria and in a variety of matrices.