The overall objective of this proposal is to evaluate and characterize variable frequency microwave (VFM) technology for use in dry bacterial spore sterilization with the ultimate goal of producing a device capable of eliminating microbial/viral/biological toxin based bioterrorism agents from mail. The final VFM device will be designed to integrate with current automated mail handling systems. Phase I of this proposal will specifically address bacterial endospores (spores) because their current public health significance (i.e. anthrax attacks through USPS) and extreme environmental stability makes spores a societally important and technically challenging target. In addition, considerable scientific literature exists regarding molecular mechanisms of spore killing. Four specific aims will be addressed in Phase I to gain a greater understanding of variable frequency microwave spore sterilization. These specific aims are (1) understand the kinetic parameters of spore sterilization using VFM technology as compared to those for dry heat sterilization; (2) explore a range of microwave frequencies between 2 and 20 GHz to examine the role applied frequency on degree of spore sterilization; (3) to gain an understanding of whether mechanisms other than spore heating are important in VFM sterilization; and (4) examine spore mutants deficient in small acid soluble proteins (SASPs), the recA gene involved in DNA repair and various spore coat/assembly genes (singly and in combinations) to elucidate VFM spore killing mechanisms from a molecular level viewpoint. Surrogate spores to B. anthracis will be used in this Phase I study. B. subtilis spores will be used due to their commercial availability as bio-indicators for dry heat sterilization and the mechanisms of spore resistance are best understood for this organism. Spores will be tested for VFM sterilization in concentrations between 10e6 and 10e12 spores in simulated mail packages. Spore kill kinetics (D and Z values) will be evaluated using standard statistical methods based on log surviving spores versus time of VFM application at a given treatment temperature. Kill kinetics will be studied at five temperatures between 150-190øC for specific aim 1. Specific aims 2, 3 and 4 will be evaluated at a single "optimal" temperature from aim 1. These studies will be validated with additional species including live veterinary vaccine strains of B. anthracis (Sterne 34F2 for example) as part of Phase II process studies
