Dose-response analysis of Bacillus thuringiensis HD-1 cry- spore reduction on surfaces using formaldehyde with pre-germination.

Abstract

To establish a basis for rapid remediation of large areas contaminated with Bacillus anthracis spores. Representative surfaces of wood, steel and cement were coated by nebulization with B. thuringiensis HD-1 cry- (a simulant for B. anthracis) at 5.9± 0.2, 6.3± 0.2 and 5.8± 0.2 log10 CFU per cm2 , respectively. These were sprayed with formaldehyde, either with or without pre-germination. Low volume (equivalent to ≤2500 L ha-1 ) applications of formaldehyde at 30 g l-1 to steel or cement surfaces resulted in ≥4 or ≤2 log10 CFU per cm2 reductions respectively, after 2h exposure. Pre-germinating spores (500 mmol l-1 l-alanine and 25 mmol l-1 inosine, pH7) followed by formaldehyde application showed higher levels of spore inactivation than formaldehyde alone with gains of up to 3.4 log10 CFU per cm2 for a given dose. No loss in B. thuringiensis cry- viability was measured after the 2h germination period, however, a pre-heat shock log10 reduction was seen for B. anthracis strains: LSU149 (1.7 log10), Vollum and LSU465 (both 0.9 log10), LSU442 (0.2 log10), Sterne (0.8 log10) and Ames (0.6 log10). A methodology was developed to produce representative spore contamination of surfaces along with a laboratory-based technique to measure the efficacy of decontamination. Dose-response analysis was used to optimize decontamination. Pre-germinating spores was found to increase effectiveness of decontamination but requires careful consideration of total volume used (germinant and decontaminant) by surface type. To be practically achievable, decontamination of a wide area contaminated with B. anthracis spores must be effective, timely and minimize the amount of materials required. This study uses systematic dose-response methodology to demonstrate that such an approach is feasible.