Dose-response assessment of <i>Bacillus anthracis</i> surrogate spore inactivation on different surfaces and environmental conditions using pH-adjusted peracetic acid

ABSTRACT A deliberate release of environmentally persistent Bacillus anthracis spores in an urban setting could cause severe public health, economic, and environmental disruption. Rapid, scalable decontamination methods are essential for effective recovery and minimizing long-term impacts. This requires understanding how decontaminant dose, surface type, and environmental conditions influence spore inactivation. The present study evaluates bespoke peracetic acid (PAA) formulations for inactivating Bacillus thuringiensis HD-1 cry- spores, a surrogate for B. anthracis . Urban surfaces nebulized with B. thuringiensis spores (7 Log 10 CFU·cm⁻²) were treated with PAA (3.7% wt/wt) or PAA plus rheology modifier (RM, 4.9% wt/wt), both adjusted to pH 6 for material compatibility, and applied at ~500–2,000 L·ha⁻¹. RM was added to retain the decontaminant for longer on the surface of porous materials. Samples were incubated for 2 h under “mild” (7°C, 94% relative humidity [RH]) or “warm” (28°C, 32% RH) conditions. PAA applied at 76 kg·ha⁻¹ (delivered using 2,046 ± 43 L·ha⁻¹ [0.2 L·m⁻²]) achieved ≥5 log 10 reduction within 2 h on porous (wood) and non-porous (steel) surfaces. At the statistically same application of 2,040 ± 42 L·ha⁻¹ ( P = 0.69), PAA + RM showed slightly lower efficacy (≥4.6 Log 10 ) despite its higher dose (98 kg·ha⁻¹). Also, a general trend of greater sporicidal efficacy was observed under warm conditions for a given surface, dose, and formulation. These data suggest a role of diffusion-dominated kinetics influencing sporicidal efficacy, where temperature and formulation viscosity can affect the level of spore inactivation. Suspension tests indicated that the virulent B. anthracis Ames strain was more susceptible to PAA than the surrogate. Finally, dose-response comparisons showed that PAA achieved sporicidal performance statistically equivalent to formaldehyde. IMPORTANCE The UK Biological Security Strategy (Cabinet Office, 2023) recognizes the need for an effective response through a pre-developed, well-validated technical strategy to remediate a scene or area that has been contaminated by hazardous biological material. Although standardized American Society for Testing and Materials (ASTM) and British Standards adopted from European Norms (BS EN) methods may help initial decontaminant selection, significant information remains for remediation planners on how applied doses of decontaminants (volume and concentration) interact with different surface types and environmental conditions to influence spore reductions. By linking these parameters, this work provides critical information toward developing actionable guidance for effective and efficient incident-scale remediation. These findings are pivotal for remediation planners, who must determine the precise decontaminant quantities required, account for associated on-site storage needs, and ensure correct equipment specifications for optimized application. Additionally, although formaldehyde solution is well-proven for B. anthracis inactivation in the open environment, our dose-response comparison with PAA-based decontaminant provides evidence toward establishing the latter as a viable, non-carcinogenic, and safer alternative for large-scale environmental decontamination. Further exploitability of the B. thuringiensis -based PAA dose-response data was demonstrated by validation with virulent strains of B. anthracis , where it was found that surrogate-based decontamination protocols are likely to be effective against virulent B. anthracis .