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

Abstract

Aim: To establish a basis for rapid remediation of large areas contaminated with Bacillus anthracis spores.

Methods and results: 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 2 h exposure. Pre-germinating spores (500 mmol l^-1 l-alanine and 25 mmol l^-1 inosine, pH 7) 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 2 h 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).

Conclusions: 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.

Significance and impact of the study: 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.

Keywords: Bacillus anthracis; anthrax; biocides; decontamination; dose-response; formaldehyde; germination; spores.

FIGURE 1

Photographs of the experimental setup for the manually triggered laboratory‐scale spray applicator and a steel surface sprayed with water to show droplet distribution. The graph shows a linear regression model with ±95% CI bands plotted against increasing spray volume and time when applied to a wood surface.

FIGURE 2

(a) Photograph showing configuration of the nebulizer system to generate and deposit aerosolised Bacillus thuringiensis cry‐ spores or dye onto surfaces; (b) layout of stainless steel surfaces (each 16 cm²) within the deposition field under the funnel canopy for dye droplet deposition measurements; (c) UV–visible calibration curve relating absorbance at 634 nm with Green ‘S’ concentration (μl ml⁻¹).

FIGURE 3

(a) Recovery of Bacillus thuringiensis cry‐ spores from steel (n = 21), cement (n = 15) and wood (n = 21) surfaces following nebulization (where * indicates p ≤ 0.05 using one‐way ANOVA with multiple comparisons). The dotted‐line denotes the theoretical spore deposition calculated from nebulized dye (5.03‐log10 CFU per cm²). (b) Frequency of droplet size distribution by %volume, from nebulizing water or B. thuringiensis cry‐ spores in water (2.8 × 10¹⁰ CFU per ml) using the Omcrom NE‐C28‐E system. The experimental set‐up for these droplet measurements is shown in the photograph: A SprayTec laser droplet analyser was used, where the distance between the nozzle tip and the center of the lazer beam was 45 mm and the sampling time was 90 s. (c) Representative photomicrographs showing spore inoculum (10⁸ CFU per ml) nebulized onto glass taken at ×200 and ×400 magnification.

FIGURE 4

(a) Bacillus thuringiensis cry‐ spore log10 reduction measured on steel (initial loading: 6.3 ± 0.2 log10 CFU per cm²), wood (initial loading: 5.9 ± 0.2 log10 CFU per cm²) and cement surfaces (5.8 ± 0.2 log10 CFU per cm²) following 2 h treatment to increasing formaldehyde doses (kg ha⁻¹) applied using the LSSA; 3D scatter plots showing the relationship between formaldehyde concentration (g l⁻¹), application volume (l ha⁻¹) and spore killing (log10 reduction) on steel (b), wood (c) and cement (d) surfaces.

FIGURE 5

Heat‐shock or non‐heat‐shock counts of Bacillus thruingiensis cry‐ spores recovered from steel and wood surfaces, with or without, germinant treatment for 2 h (*p ≤ 0.05 one‐way ANOVA with multiple comparisons).

FIGURE 6

Comparison of Bacillus thuringiensis cry‐ spore log10 reductions on steel with and without pre‐germination.

FIGURE 7

(a) Pre‐heat shock spore log10 reduction and (b) post‐heat shock spore log10 reduction of Bacillus thuringiensis cry‐ and seven Bacillus anthracis strains; Sterne; LSU149; LSU463; Ames, Vollum; LSU465 and LSU 442 following incubation for 24 h with phosphate buffered alanine and inosine germinant at 21°C (where *p ≤ 0.05, **p ≤ 0.01 and ****p ≤ 0.0001 using one‐way ANOVA with multiple comparison).