In 2015, a private company notified the CDC that inactivated Bacillus anthracis (Ba) spores in their possession were viable. Upon an exhaustive review of the situation it was determined that the material in question originated from a Department of Defense (DoD) Lab and was further distributed to other areas around the country and world. Since that time a major focus by the Center for Disease Control (CDC) and DoD has been to re-assess and better characterize all inactivation procedures currently being performed across all capable laboratories. ECBC has been a crucial part of this process and has been tasked to provide the Chemical Biological Defense Program with a methodology to properly inactivate BA spores while maintaining molecular and immunological targets for assay development, performance verification, and proficiency testing. To that end, along with more robust inactivation methods needing to be developed, analysis techniques to rapidly detect and determine viable and non-viable biological material is a priority. Through the work being led by Dr. Sandy Gibbons at ECBC in coordination of the Bacillus anthracis Gamma Inactivation Study, the ECBC Spectroscopy Branch was able to secure a small sample set of viable and non-viable gamma irradiated Ba sterne spores currently being investigated. Utilizing Raman Chemical Imaging (RCIM) we were able to ascertain several key spectral differences between the viable Ba sterne samples and gamma irradiated spore samples. Figure 1 shows a spectral area focused on calcium dipicolinate (CaDP) which is a common marker for presence of certain types of bacterial spores. The CaDP vibrational mode observed at 1018 cm-1 in the viable sample is clearly diminished in the gamma irradiated sample leaving only the 1000 cm-1 ring breathing mode of phenylalanine present. This demonstrates that spectral differences are present and can assist with spectral discrimination between the processes. This work presents a study of Raman spectral features from specific biological sources and identifies those vibrational chemical components that give rise to the spectral features allowing for viable/non-viable discrimination. The ultimate goal of this work is to determine if a spectral analysis method be can developed to rapidly verify the difference between viable and non-viable bacteria including determination of the inactivation method.
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