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.

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.

Analysis of broth-cultured Bacillus atrophaeus and Bacillus cereus spores.

AIMS: To compare physical properties of spores that were produced in broth sporulation media at greater than 10(8) spores ml(-1). METHODS AND RESULTS: Bacillus atrophaeus reproducibly sporulated in nutrient broth (NB) and sporulation salts. Microscopy measurements showed that the spores were 0.68 +/- 0.11 microm wide and 1.21 +/- 0.18 microm long. Coulter Multisizer (CM3) measurements revealed the spore volumes and volume-equivalent spherical diameters, which were 0.48 +/- 0.38 microm(3) and 0.97 +/- 0.07 microm, respectively. Bacillus cereus reproducibly sporulated in NB, sporulation salts, 200 mmol l(-1) glutamate and antifoam. Spores were 0.95 +/- 0.11 microm wide and 1.31 +/- 0.17 microm long. Spore volumes were 0.78 +/- 0.61 microm(3) and volume-equivalent spherical diameters were 1.14 +/- 0.11 microm. Bacillus atrophaeus spores were hydrophilic and B. cereus spores were hydrophobic. However, spore hydrophobicity was significantly altered after treatment with pH-adjusted bleach. CONCLUSIONS: The utility of a CM3 for both quantifying Bacillus spores and measuring spore sizes was demonstrated, although the volume between spore exosporium and spore coat was not measured. This study showed fundamental differences between spores from a Bacillus subtilis- and B. cereus-group species. SIGNIFICANCE AND IMPACT OF THE STUDY: This is useful for developing standard methods for broth spore production and physical characterization of both living and decontaminated spores.

Differential susceptibility of macrophage cell lines to Bacillus anthracis-Vollum 1B.

Bacillus anthracis (BA) is a spore forming bacterium and the causative agent of anthrax disease. Macrophages (Mphis) play a central role in anthrax disease. An important step in disease progression is the ability of BA to secrete lethal toxin (LeTx) that kills Mphis. LeTx is a heterodimer composed of protective antigen (PA) and lethal factor (LF). Researchers have shown that Mphi cell lines demonstrate differential susceptibility to purified LeTx; for example RAW264.7 and J774A.1 Mphis are sensitive to LeTx whereas IC-21 Mphis are resistant. Research has also suggested that exogenous factors, including other BA proteins, can influence the activity of LeTx. For this reason, the objective of the current work was to examine if RAW264.7, J774A.1, and IC-21 Mphis demonstrated differential susceptibility when cultured with a LeTx-producing strain of BA. Here, we co-cultured Mphis with LeTx+ Vollum 1B (V1B) spores for >15 h and assayed for Mphi cell death by morphology, trypan blue (TB) staining, neutral red (NR) activity, and lactate dehydrogenase (LDH) activity in the culture media. Following the addition of V1B spores, necrosis (approximately 50% mortality) was observed in RAW264.7 and J774A.1 Mphis at 7.5 and 10 h, respectively. By 15 h, both RAW264.7 and J774A.1 Mphis demonstrated 100% mortality. In contrast, IC-21 Mphis, under identical culture conditions, remained viable (98%) and activated throughout the course of the experiment (>24 h). The mechanism of RAW264.7 cell death appeared to involve LeTx because the V1B-induced cytotoxicity was dose-dependently reversed by the addition of anti-PA antibody to the culture media. These observations suggest there is differential susceptibility of Mphi cell lines to the LeTx+ V1B strain of BA. Further development of this in vitro model may be useful to further characterize the interactions between Mphis and BA spores.

Gene Expression Analysis during Conidial Germ Tube and Appressorium Development in Colletotrichum trifolii.

Preinfection development in Colletotrichum spp. exhibits three morphologies (conidia, germ tubes, and appressoria) and is directed by a complex interplay of environmental signals. Germ tube morphogenesis for Colletotrichum trifolii and the related fungus Colletotrichum gloeosporioides f. sp. aeschynomene was shown to be partially dependent on a balance between self-germination inhibitors and environmental nutrients or cutin. The degree of responsiveness to these environmental signals was strikingly different between the two fungal species. A solid contact surface stimulated germ tube morphogenesis and was the only apparent requirement for appressorium morphogenesis in both fungi. A population of C. trifolii conidia was incubated on a solid surface in the presence of cutin to stimulate nearly synchronous preinfection morphogenesis for gene expression analysis. RNA analysis of signal-transducing genes from C. trifolii, including genes for a serine-threonine kinase (TB3), calmodulin, and protein kinase C, showed that maximum transcription of all three genes occurred in conidia prior to or during germ tube morphogenesis. Transcription of melanin biosynthetic genes THR1 and SCD1 (Y. Kubo, Y. Takano, and I. Furusawa, Colletotrichum Newsl. II:5-10, 1996; N. S. Perpetua, Y. Kubo, N. Yasuda, Y. Takano, and I. Furusawa, Mol. Plant-Microbe Interact. 9:323-329, 1996) was highest prior to and during appressorium morphogenesis.

A kinase-encoding gene from Colletotrichum trifolii complements a colonial growth mutant of Neurospora crassa.

Colletotrichum trifolii is a fungal pathogen which is responsible for anthracnose disease of alfalfa. To initiate research on molecular communication in this fungus, a kinase-encoding gene (TB3) and the corresponding cDNA were cloned and sequenced. The deduced amino acid sequence of TB3 closely resembles that of a Neurospora crassa serine/threonine protein kinase, COT1, required for hyphal elongation and branching. The C-terminal catalytic domains of TB3 and COT1 are highly conserved but the N-terminal regions are divergent, particularly in the homopolymeric glutamine repeats of TB3. Northern analysis indicated that TB3 expression was highest 1 h after inducing conidial germination and 1 h before germ tubes were first observed. Expression of TB3 transcripts returned to constitutive levels by 4 h after induction of germination. TB3 complemented the cot-I mutant of Neurospora crassa, demonstrating the functional conservation of this kinase between a pathogenic and a saprophytic fungus.

Isolation, characterization, and expression of a second beta-tubulin-encoding gene from Colletotrichum gloeosporioides f. sp. aeschynomene.

Colletotrichum gloeosporioides f. sp. aeschynomene is a fungal plant pathogen of Aeschynomene virginica. A beta-tubulin-encoding gene (TUB2) from this pathogen was cloned and sequenced. The deduced amino acid sequence of TUB2 had a high degree of homology to other fungal beta-tubulins. A portion of TUB2 from a benomyl-resistant C. gloeosporioides f. sp. aeschynomene mutant was also cloned and sequenced. A point mutation resulting in a glutamic acid-to-lysine substitution at amino acid 198 likely confers benomyl resistance. The mutation is relevant for use as a selectable marker in developing a gene transfer system in C. gloeosporioides f. sp. aeschynomene. Northern (RNA) hybridizations with C. gloeosporioides f. sp. aeschynomene TUB2 and another C. gloeosporioides f. sp. aeschynomene beta-tubulin-encoding gene (TUB1) as probes showed differential expression of these genes in different cell types.

Isolation and characterization of a beta-tubulin-encoding gene from Colletotrichum gloeosporioides f. sp. aeschynomene.

Colletotrichum gloeosporioides f. sp. aeschynomene (C.g.a.) is a fungal pathogen of legumes and is used as a commercial mycoherbicide for rice and soybeans. As an initial study to potentially improve the utility of this fungus and develop a gene transfer system, a beta-tubulin (beta Tub)-encoding gene (TUB1) was isolated, cloned and sequenced. The coding sequence and deduced amino acid sequence of the C.g.a. TUB1 gene was highly homologous to the TUB1 gene of Colletotrichum graminicola. Southern hybridizations, using the C.g.a. TUB1 and C. graminicola TUB2 genes as probes, suggest that C.g.a. contains two TUB genes. Variation in both the restriction pattern and the number of TUB genes present in different formae specialis of C. gloeosporioides was evident. These observations are relevant for assessing relationships among formae specialis of C. gloeosporioides.