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

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.

Evidence for cytoskeletal changes secondary to plasma membrane functional alterations in the in vitro cell response to Clostridium perfringens epsilon-toxin.

To investigate the mode of action of Clostridium perfringens epsilon-toxin, MDCK cells were treated with purified toxin and incubated at 37 degrees C for up to 24h. Exposure to epsilon-toxin caused a time-dependent decrease in cell-cell and cell-substrate interactions. After 30min of treatment retraction of the cell body and the emission of filopodia were detectable in a number of cells. Longer exposure resulted in cell rounding and cell blebbing which reached a maximum after 5h of toxin treatment. A parallel modification in the cytoskeleton was also detected. Actin marginalization and the entanglement of microtubules and intermediate filaments were observed by fluorescence microscopy after 30min of toxin exposure. Functional alterations of the plasma membrane of MDCK cells were assessed by flow cytometry. After 10 or 30min of intoxication an increase in cell volume was detected, indicating an alteration in plasma membrane permeability. These findings provide evidence for cytoskeletal changes and plasma membrane functional alterations in the in vitro cell response to C. perfringens epsilon-toxin.

Production of a non-toxic site-directed mutant of Clostridium perfringens epsilon-toxin which induces protective immunity in mice.

A panel of ten site-directed mutants of Clostridium perfringens epsilon-toxin was generated. All of the mutated proteins expressed in Escherichia coli were recognized in immunoblots by a neutralizing mAb raised against wild-type native epsilon-toxin. The cytotoxicity of the site-directed mutated toxins was assayed in vitro against MDCK cells. One mutation resulting in loss of activity in the assay was identified. This non-toxic protein was derived by substituting a proline for the histidine at residue 106 of the toxin. Immunization of mice with the non-toxic mutated epsilon-toxin resulted in the induction of a specific antibody response and immunized mice were protected against 1000 LD50 doses of wild-type recombinant epsilon-toxin.