Supercritical carbon dioxide and hydrogen peroxide cause mild changes in spore structures associated with high killing rate of Bacillus anthracis

The present work examines chemical and structural response in B. anthracis spores killed by a mixture of supercritical carbon dioxide (SCCO2) and hydrogen peroxide (H2O2). Deactivation of 6-log of B. anthracis spores by SCCO2 + H2O2 was demonstrated, but changes in structure were observed in only a small portion of spores. Results from phase contrast microscopy proved that this treatment is mild and does not trigger germination-like changes. TEM imaging revealed mild damage in a portion of spores while the majority remained intact. Dipicolinic acid (DPA) analysis showed that < 10% of the DPA was released from the spore core into the external milieu, further demonstrating only modest damage to the spores. Confocal fluorescent microscopy, assessing uptake of DNA-binding dyes, directly demonstrated compromise of the permeability barrier. However, the magnitude of uptake was small compared to spores that had been autoclaved. This work suggests that SCCO2 + H2O2 is quite mild compared to other sterilization methods, which has major implications in its application. These results provide some insight on the possible interactions between spores and the SCCO2 + H2O2 sterilization process.