Effects of Gamma Irradiation on the Molecular and Physical Detection Properties of Bacillus Spores

Abstract

Gamma irradiation is a technique for inactivation of biological warfare agents (BWAs). Detection of these irradiated agents by sensor technologies may be affected,; altering sensitivity compared to that of non-irradiated organisms. Here for the first time, we aim to determine if spore morphology, or other physical or chemical properties beyond viability and PCR, is altered by the gamma irradiation process. This study analyzed common detection methods to determine the effects the irradiation process has on Bacillus thuringiensis subspecies kurstaki (Btk) spores compared to nonirradiated spores. Liquid concentrations of 108, 106, and 104 CFU ml-1 of Btk spores and one dry powder sample of 109 CFU g-1 were exposed to varying levels of gamma irradiation. Cell viability studies showed complete inactivation for all concentrations at doses of 5.43 kGy and higher. PCR analysis indicated no loss in sensitivity with increasing doses for both wet and dry spore samples. Visual inspection of the spores through scanning electron microscopy showed a change in morphology as the dosage of irradiation increased. With The inability to distinguish whole spores from cell debris occurred in at the 10.86 kGy samples level. Similarly, fluorescence readings remained stable among all but one dose with only the 10.86 kGy sample showing an increase in fluorescence. For PCR based detection methods, no bias towards irradiated versus non-irradiated spore samples exist while optical detection technologies are likely affected by the physical changes to spore morphology and rupturing. Results from this study demonstrate that gamma irradiation of Bacillus spores causes damage to the organism which may make them unrecognizable alter how they to are perceived by detection technologies. When using irradiated analyte to evaluate performance of detection technologies, the results may be impacted by the irradiation process.