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. 2015 Aug 12;6:663. doi: 10.3389/fmicb.2015.00663

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At least a portion of this work is authored by Drs. Christopher J. Doona, Florence E. Feeherry, Kenneth Kustin, Gene G. Olinger and Alexander J. Malkin on behalf of the U.S. Government and, as regards Drs. Doona, Feeherry, Kustin, Olinger and Malkin and the U.S. Government, is not subject to copyright protection in the United States. Foreign and other copyrights may apply.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Increasing RH at a fixed dose of (A) 500 ppm ClO₂ for 4 h (∼2000 ppm-h) and (B) 1000 ppm ClO₂ for 4 h (∼4000 ppm-h) results in a higher percentage of log 6 B. atrophaeus spore strips being killed. (A) B. atrophaeus D_EtO = 3.3 (n = 120), = B. atrophaeus D_EtO = 3.8 (n = 160); (B) B. atrophaeus D_EtO = 3.3 (n = 140), and B. atrophaeus D_EtO = 3.8 (n = 140). Data shown is for spore strips packaged in Tyvek. For the D_EtO = 3.3 strips exposed to ∼2000 ppm-h ClO₂, ≥80% kill is achieved at 70 %RH and above, but when we increase the dose to ∼4000 ppm-h the same level of kill is achieved at %RH of 60. The relationship between low D_EtO value and increased susceptibility to ClO₂ is evident, though, in this example, we see that at 90 %RH all four sample sets are behaving roughly the same. (C) B. atrophaeus D_EtO = 3.3, log 6 spore strips packaged in img medical-grade glassine, and img Tyvek are not equally susceptible to a set ClO₂ dose at 79 %RH (NE = no effect). Spore strips were exposed to 250 ppm ClO₂ for 4 h (dose ≈1000 ppm-h), where n = 20 each for glassine and Tyvek; 2000 ppm-h, where n = 40 (glassine) and n = 20 (Tyvek); and 4000 ppm-h, where n = 40 (glassine) and n = 20 (Tyvek) at constant temperature and %RH. (D) We did not achieve 100% kill for glassine-packaged log 6 spore strips until we subjected a lot of B. atrophaeus with D_EtO value of 3.0 (n = 20) to 2000 ppm for 10 h (∼20,000 ppm-h) at 79 %RH. This high dose killed 80 and 85% of B. atrophaeus D_EtO = 3.3 and 3.8 (n = 20 each) spore strips, respectively. (E) For all three species and B. atrophaeus with two different D_EtO values (3.3, with n = 80 for each Tyvek and no packaging; and 3.8, with n = 189 for each Tyvek and no packaging), probabilities were predicted for a spore surviving after treatment, with RH = 0.79 and N = 10⁶. Note that for B. thuringiensis (n = 98 each for Tyvek and no packaging), measurements were only made at N = 1.2 × 10⁶. For both sets of B. atrophaeus, measurements were made at N = 1.3 × 10⁶, so in these cases this plot involves extrapolation in N. B. thuringiensis, the closest phylogenetic relative to Bacillus anthracis used in this study, is more likely to survive at any given ClO₂ dose than either of the B. atrophaeus strains, though it’s resistance is similar to the strain of G. stearothermophilus (n = 80 each for Tyvek and no packaging) assayed.