Combining spore germination and heat inactivation to decontaminate materials contaminated with Bacillus anthracis spores.

AIMS: To add a spore germination step in order to reduce decontamination temperature and time requirements compared to the current hot, humid air decontamination parameters, which are 75-80°C, ≥72 h, 70-90% RH, down to ≤60°C and ≤24 h total decontamination time. METHODS AND RESULTS: Bacillus anthracis spore germination with l-alanine+inosine+calcium dipicolinate (CaDPA) was quantified at 0-40°C, several time points and spore concentrations of 5-9 log10 per ml. Germination efficiency at 0-40°C was >99% at <8 log10 spores per ml. The temperature optimum was 20°C. Germination efficiency was significantly higher but slower at 0°C compared to ≥30°C at ≥8 log10 spores per ml. A single germinant application followed by 60°C, 1-h treatment consistently inactivated >2 log10 (>99%) of spores. However, a repeat application of germinant was needed to achieve the objective of ≥6 log10 spore inactivation out of a 7 log10 challenge (≥99·9999%) for ≤24 h total decontamination time for nylon and aircraft performance coating. CONCLUSIONS: l-alanine+inosine+CaDPA stimulated germination across wide temperature and spore concentration ranges. SIGNIFICANCE AND IMPACT OF THE STUDY: Germination expands the scope of spore decontamination to include materials from any industry sector that can be sprayed with an aqueous germinant solution.

Hot, humid air decontamination of a C-130 aircraft contaminated with spores of two acrystalliferous Bacillus thuringiensis strains, surrogates for Bacillus anthracis.

AIM: To develop test methods and evaluate survival of Bacillus thuringiensis kurstaki cry(-) HD-1 and B. thuringiensis Al Hakam spores after exposure to hot, humid air inside of a C-130 aircraft. METHODS AND RESULTS: Bacillus thuringiensis spores were either pre-inoculated on 1 × 2 or 2 × 2 cm substrates or aerosolized inside the cargo hold of a C-130 and allowed to dry. Dirty, complex surfaces (10 × 10 cm) swabbed after spore dispersal showed a deposition of 8-10 log10 m(-2) through the entire cargo hold. After hot, humid air decontamination at 75-80°C, 70-90% relative humidity for 7 days, 87 of 98 test swabs covering 0·98 m(2) , showed complete spore inactivation. There was a total of 1·67 log10 live CFU detected in 11 of the test swabs. Spore inactivation in the 98 test swabs was measured at 7·06 log10 m(-2) . CONCLUSIONS: Laboratory test methods for hot, humid air decontamination were scaled for a large-scale aircraft field test. The C-130 field test demonstrated that hot, humid air can be successfully used to decontaminate an aircraft. SIGNIFICANCE AND IMPACT OF THE STUDY: Transition of a new technology from research and development to acquisition at a Technology Readiness Level 7 is unprecedented.

Test methods and response surface models for hot, humid air decontamination of materials contaminated with dirty spores of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam.

AIMS: To develop test methods and evaluate survival of Bacillus anthracis ∆Sterne or Bacillus thuringiensis Al Hakam on materials contaminated with dirty spore preparations after exposure to hot, humid air using response surface modelling. METHODS AND RESULTS: Spores (>7 log10 ) were mixed with humic acid + spent sporulation medium (organic debris) or kaolin (dirt debris). Spore samples were then dried on five different test materials (wiring insulation, aircraft performance coating, anti-skid, polypropylene, and nylon). Inoculated materials were tested with 19 test combinations of temperature (55, 65, 75°C), relative humidity (70, 80, 90%) and time (1, 2, 3 days). The slowest spore inactivation kinetics was on nylon webbing and/or after addition of organic debris. CONCLUSIONS: Hot, humid air effectively decontaminates materials contaminated with dirty Bacillus spore preparations; debris and material interactions create complex decontamination kinetic patterns; and B. thuringiensis Al Hakam is a realistic surrogate for B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: Response surface models of hot, humid air decontamination were developed which may be used to select decontamination parameters for contamination scenarios including aircraft.

Decontamination of materials contaminated with Francisella philomiragia or MS2 bacteriophage using PES-Solid, a solid source of peracetic acid.

AIM: The aim of the study was to develop test methods and evaluate survival of Francisella philomiragia cells and MS2 bacteriophage after exposure to PES-Solid (a solid source of peracetic acid) formulations with or without surfactants. METHODS AND RESULTS: Francisella philomiragia cells (≥7·6 log10 CFU) or MS2 bacteriophage (≥6·8 log10 PFU) were deposited on seven different test materials and treated with three different PES-Solid formulations, three different preneutralized samples and filter controls at room temperature for 15 min. There were 0-1·3 log10 CFU (<20 cells) of cell survival, or 0-1·7 log10 (<51 PFU) of bacteriophage survival in all 21 test combinations (organism, formulation and substrate) containing reactive PES-Solid. In addition, the microemulsion (Dahlgren Surfactant System) showed ≤2 log10 (100 cells) of viable F. philomiragia cells, indicating the microemulsion achieved <2 log10 CFU on its own. CONCLUSIONS: Three PES-Solid formulations and one microemulsion system (DSS) inactivated F. philomiragia cells and/or MS2 bacteriophage that were deposited on seven different materials. SIGNIFICANCE AND IMPACT OF THE STUDY: A test method was developed to show that reactive PES-Solid formulations and a microemulsion system (DSS) inactivated >6 log10 CFU/PFU F. philomiragia cells and/or MS2 bacteriophage on different materials.

Decontamination of materials contaminated with Bacillus anthracis and Bacillus thuringiensis Al Hakam spores using PES-Solid, a solid source of peracetic acid.

AIMS: To develop test methods and evaluate survival of Bacillus anthracis Ames, B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores after exposure to PES-Solid (a solid source of peracetic acid), including PES-Solid formulations with bacteriostatic surfactants. METHODS AND RESULTS: Spores (≥ 7 logs) were dried on seven different test materials and treated with three different PES-Solid formulations (or preneutralized controls) at room temperature for 15 min. There was either no spore survival or less than 1 log (<10 spores) of spore survival in 56 of 63 test combinations (strain, formulation and substrate). Less than 2.7 logs (<180 spores) survived in the remaining seven test combinations. The highest spore survival rates were seen on water-dispersible chemical agent resistant coating (CARC-W) and Naval ship topcoat (NTC). Electron microscopy and Coulter analysis showed that all spore structures were intact after spore inactivation with PES-Solid. CONCLUSIONS: Three PES-Solid formulations inactivated Bacillus spores that were dried on seven different materials. SIGNIFICANCE AND IMPACT OF THE STUDY: A test method was developed to show that PES-Solid formulations effectively inactivate Bacillus spores on different materials.

Test method development to evaluate hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores.

AIMS: To develop test methods and evaluate the survival of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air. METHODS AND RESULTS: Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH-adjusted bleach decontamination. CONCLUSIONS: Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores with similar kinetics. SIGNIFICANCE AND IMPACT OF THE STUDY: Hot, humid air is a potential alternative to conventional chemical decontamination.

Effect of animal sera on Bacillus anthracis Sterne spore germination and vegetative cell growth.

AIMS: The aims of this work were to investigate the effects of sera on B. anthracis Sterne germination and growth. Sera examined included human, monkey and rabbit sera, as well as sera from eight other species. METHODS AND RESULTS: Standard dilution plate assay (with and without heat kill) was used as a measure of germination, and spectroscopy was used to measure growth. In addition, a Coulter Counter particle counter was used to monitor germination and growth based on bacterial size. Spores germinated best in foetal bovine and monkey sera, moderately with human sera and showed limited germination in the presence of rabbit or rat sera. Vegetative bacteria grew best in foetal bovine sera and moderately in rabbit sera. Human and monkey sera supported little growth of vegetative bacteria. CONCLUSION: The data suggested sera can have a significant impact on germination and growth of Sterne bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: These data should be considered when conducting in vitro cell culture studies and may aid in interpreting in vivo infection studies.

Decontamination of a hard surface contaminated with Bacillus anthracisΔSterne and B. anthracis Ames spores using electrochemically generated liquid-phase chlorine dioxide (eClO2).

AIMS: To evaluate the inactivation of Bacillus anthracisΔSterne and Ames spores using electrochemically generated liquid-phase chlorine dioxide (eClO(2)) and compare two sporulation and decontamination methods with regard to cost, safety and technical constraints. METHODS AND RESULTS: Spores were prepared via agar and broth methods and subsequently inoculated and dried onto clean, autoclave-sterilized glass coupons. Bacillus anthracis spore inactivation efficacy was evaluated using the modified three-step method (AOAC 2008.05) and a single-tube extraction method. Spores (7·0 ± 0·5 logs) were inactivated within 1 min at room temperature using freshly prepared eClO(2). Bacillus anthracisΔSterne spores decreased in size after eClO(2) treatment as measured using a Beckman Coulter Multisizer. CONCLUSIONS: eClO(2) saturation of a hard surface was an effective B. anthracis sporicide. Broth sporulation and the single-tube extraction method required less time and fewer steps, yielded a higher percentage of phase-bright spores and showed higher spore recovery efficiency compared with AOAC 2008.05, making it more amenable to biosafety level 3 (BSL3) testing of virulent spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Two test methods demonstrated the sporicidal efficacy of eClO(2). A new single-tube extraction test protocol for decontaminants was introduced.