Decontamination of Bacillus anthracis Spores at Subzero Temperatures by Complete Submersion

Abstract

Introduction: Bacillus anthracis, the etiological agent of anthrax, produces long-lived spores, which are resistant to heat, cold, pH, desiccation, and chemical agents. The spores maintain their ability to produce viable bacteria even after decades, and when inhaled can cause fatal disease in over half of the clinical cases. Owing to these characteristics, anthrax has been repeatedly selected for both bioweapon and bioterrorism use. In the event of a bioterrorism attack, surfaces in the vicinity of the attack will be contaminated, and recovering from such an event requires rapid and effective decontamination. Previous decontamination method development has focused mainly on temperatures >0°C, and have shown poor efficacy at subzero temperatures. Methods: In this study, we demonstrate the use of calcium chloride (CaCl₂) as a freezing point depression agent for pH-adjusted sodium hypochlorite (NaOCl) for the effective and rapid decontamination of B. anthracis Sterne strain spores at subzero temperatures. Results: We show the complete decontamination of 10⁶ B. anthracis Sterne strain spores at temperatures as low as -20°C within 2.5 min by submersion in solution containing 25% (w/v) CaCl₂, 0.50% NaOCl, and 0.40% (v/v) acetic acid. We also demonstrate significant reduction in number of spores at -28°C. Conclusions: The results show promise for rapidly decontaminating equipment and materials used in the response to bioterrorism events using readily available consumer chemicals. Future study should examine the efficacy of these results on complex surfaces.

Keywords: anthrax; bioterrorism; calcium chloride; decontamination; subzero.

Figure 1.

Average percentage recovery of Bacillus anthracis Sterne strain spores after application of decontamination solutions containing 25% (w/v) CaCl₂ and NaOCl concentrations (v/v) of 0.05%, 0.10%, 0.20%, and 0.50% for 10 min. Three experimental replicates were conducted for each of the NaOCl concentrations: blue circles indicate individual replicate values, while the red line indicates the arithmetic mean of the individual replicates. CaCl₂, calcium chloride; NaOCl, sodium hypochlorite.

Figure 2.

Total recovery of Bacillus anthracis Sterne strain spores after 20 min at −20°C from solutions containing 25% (w/v) CaCl₂, 0.50% NaOCl, and the following CH₃COOH concentrations (v/v): (1) 0.40% (N = 15), (2) 0.31% (N = 15), (3) 0.02% (N = 10), and (4) 0.00% (N = 10), and the neutralizer only, no decontamination control (N = 5).

Figure 3.

Average percentage recovery of Bacillus anthracis Sterne strain spores after application of decontamination Solution 1 [25% (w/v) CaCl₂, 0.50% NaOCl, 0.40% (v/v) CH₃COOH] at temperatures of −5°C, −15°C, −20°C, and −28°C, for 5, 10, 15, and 20 min; in addition 2.5 min at −20°C was tested. Each combination of time and temperature was replicated experimentally at least five times: blue circles indicate individual replicate values, while the red line indicates the arithmetic mean of the individual replicates. No spores were recovered at any time points across any temperature, except for −28°C, which exhibited significant reduction of spores at all time points (2.5 min p = 3.43 × 10^–11; 5 min p = 1.17 × 10^–5; 10 min p = 8.85 × 10^–24; 15 min p = 9.80 × 10^–12; 20 min p = 1.21 × 10^–5).

Figure 4.

The visible damage to steel disks caused by application of decontamination Solution 1 (25% (w/v) CaCl₂, 0.50% NaOCl, 0.40% (v/v) CH₃COOH) for 0, 0.17, 0.5, 1, 1.5, 3, and 24 h.

Figure 5.

The efficacy of decontamination Solution 1 [25% (w/v) CaCl₂, 0.50% NaOCl, 0.40% (v/v) CH₃COOH] conducted on 7 consecutive days, showing the average percentage recovery of Bacillus anthracis Sterne strain spores. Five experimental replicates were conducted for each day using the same solution preparation: blue circles indicate individual replicate values, while the red line indicates the arithmetic mean of the individual replicates.