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

Abstract

The present work examines chemical and structural response in B. anthracis spores killed by a mixture of supercritical carbon dioxide (SCCO(2)) and hydrogen peroxide (H(2)O(2)). Deactivation of 6-log of B. anthracis spores by SCCO(2)+H(2)O(2) 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 SCCO(2)+H(2)O(2) 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 SCCO(2)+H(2)O(2) sterilization process.

Figure 1

Schematic of supercritical CO₂ apparatus

Figure 2

Phase contrast images of untreated, pure CO₂ processed, and CO₂+H₂O₂ processed spores

Figure 3

TEM images of B. anthracis spores

Figure 4

DPA release from B. anthracis spores. (a) Controls and spores treated with CO₂+H₂O₂. (b) Controls and spores treated with pure CO₂.

Figure 5

Fluorescent images of BacLight stained B. anthracis spores

Figure 5

Fluorescent images of BacLight stained B. anthracis spores

Figure 6

Percentages of different staining patterns of B. anthracis spores