Detection of B. anthracis Spores and Vegetative Cells with the Same Monoclonal Antibodies

Bacillus anthracis, the causative agent of anthrax disease, could be used as a biothreat reagent. It is vital to develop a rapid, convenient method to detect B. anthracis. In the current study, three high affinity and specificity monoclonal antibodies (mAbs, designated 8G3, 10C6 and 12F6) have been obtained using fully washed B. anthracis spores as an immunogen. These mAbs, confirmed to direct against EA1 protein, can recognize the surface of B. anthracis spores and intact vegetative cells with high affinity and species-specificity. EA1 has been well known as a major S-layer component of B. anthracis vegetative cells, and it also persistently exists in the spore preparations and bind tightly to the spore surfaces even after rigorous washing. Therefore, these mAbs can be used to build a new and rapid immunoassay for detection of both life forms of B. anthracis, either vegetative cells or spores.     

Inactivation of Bacillus anthracis Spores by Liquid Biocides in the Presence of Food Residue

Biocide inactivation of Bacillus anthracis spores in the presence of food residues after a 10-min treatment time was investigated. Spores of nonvirulent Bacillus anthracis strains 7702, ANR-1, and 9131 were mixed with water, flour paste, whole milk, or egg yolk emulsion and dried onto stainless-steel carriers. The carriers were exposed to various concentrations of peroxyacetic acid, sodium hypochlorite (NaOCl), or hydrogen peroxide (H₂O₂) for 10 min at 10, 20, or 30°C, after which time the survivors were quantified. The relationship between peroxyacetic acid concentration, H₂O₂ concentration, and spore inactivation followed a sigmoid curve that was accurately described using a four-parameter logistic model. At 20°C, the minimum concentrations of peroxyacetic acid, H₂O₂, and NaOCl (as total available chlorine) predicted to inactivate 6 log₁₀ CFU of B. anthracis spores with no food residue present were 1.05, 23.0, and 0.78%, respectively. At 10°C, sodium hypochlorite at 5% total available chlorine did not inactivate more than 4 log₁₀ CFU. The presence of the food residues had only a minimal effect on peroxyacetic acid and H₂O₂ sporicidal efficacy, but the efficacy of sodium hypochlorite was markedly inhibited by whole-milk and egg yolk residues. Sodium hypochlorite at 5% total available chlorine provided no greater than a 2-log₁₀ CFU reduction when spores were in the presence of egg yolk residue. This research provides new information regarding the usefulness of peroxygen biocides for B. anthracis spore inactivation when food residue is present. This work also provides guidance for adjusting decontamination procedures for food-soiled and cold surfaces.     

Combined Effects of High Hydrostatic Pressure and Temperature for Inactivation of Bacillus anthracis Spores

Spores of Bacillus anthracis are known to be extremely resistant to heat treatment, irradiation, desiccation, and disinfectants. To determine inactivation kinetics of spores by high pressure, B. anthracis spores of a Sterne strain-derived mutant deficient in the production of the toxin components (strain RP42) were exposed to pressures ranging from 280 to 500 MPa for 10 min to 6 h, combined with temperatures ranging from 20 to 75°C. The combination of heat and pressure resulted in complete destruction of B. anthracis spores, with a D value (exposure time for 90% inactivation of the spore population) of approximately 4 min after pressurization at 500 MPa and 75°C, compared to 160 min at 500 MPa and 20°C and 348 min at atmospheric pressure (0.1 MPa) and 75°C. The use of high pressure for spore inactivation represents a considerable improvement over other available methods of spore inactivation and could be of interest for antigenic spore preparation.     

Inactivation of Spores of Bacillus anthracis Sterne, Bacillus cereus, and Bacillus thuringiensis subsp. israelensis by Chlorination

Three species of Bacillus were evaluated as potential surrogates for Bacillus anthracis for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Spores of Bacillus thuringiensis subsp. israelensis were found to be an appropriate surrogate for spores of B. anthracis for use in chlorine inactivation studies.     

Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores

There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log₁₀ reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially.     

The Ultraviolet Photochemistry and Photobiology of Vegetative Cells and Spores of Bacillus megaterium

The ultraviolet (UV) photochemistry and photobiology of spores and vegetative cells of Bacillus megaterium have been studied. The response of vegetative cells of B. megaterium appears qualitatively similar to those of Escherichia coli, Micrococcus radiodurans, and Bacillus subtilis with respect to photoproduct formation and repair mechanisms. UV irradiation, however, does not produce cyclobutane-type thymine dimers in the DNA of spores, although other thymine photo-products are produced. The photoproducts do not disappear after photoreactivation, but they are eliminated from the DNA by a dark-repair mechanism different from that found for dimers in vegetative cells. Irradiations performed at three wavelengths produce the same amounts of spore photoproduct and give the same survival curves. Variation of the sporulation medium before irradiation results in comparable alterations in the rate of spore photoproduct production and in survival.     

Sensitivities of Germinating Spores and Carvacrol-Adapted Vegetative Cells and Spores of Bacillus cereus to Nisin and Pulsed-Electric-Field Treatment

Treatment of Bacillus cereus spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage. In contrast, germinating spores were found to be sensitive to PEF treatment. Nisin treatment was more efficient than PEF treatment for inactivating germinating spores. PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated. Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment. Resistance to nisin was lost immediately when the germination process started. A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin. On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol. Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.     

Inactivation of Vegetative Cells, but Not Spores, of Bacillus anthracis, B. cereus, and B. subtilis on Stainless Steel Surfaces Coated with an Antimicrobial Silver- and Zinc-Containing Zeolite Formulation

Stainless steel surfaces coated with paints containing a silver- and zinc-containing zeolite (AgION antimicrobial) were assayed in comparison to uncoated stainless steel for antimicrobial activity against vegetative cells and spores of three Bacillus species, namely, B. anthracis Sterne, B. cereus T, and B. subtilis 168. Under the test conditions (25°C and 80% relative humidity), the zeolite coating produced approximately 3 log₁₀ inactivation of vegetative cells within a 5- to 24-h period, but viability of spores of the three species was not significantly affected.     

Fast and Sensitive Detection of Bacillus anthracis Spores by Immunoassay

Bacillus anthracis is one of the most dangerous potential biological weapons, and it is essential to develop a rapid and simple method to detect B. anthracis spores in environmental samples. The immunoassay is a rapid and easy-to-use method for the detection of B. anthracis by means of antibodies directed against surface spore antigens. With this objective in view, we have produced a panel of monoclonal antibodies against B. anthracis and developed colorimetric and electrochemiluminescence (ECL) immunoassays. Using Meso Scale Discovery ECL technology, which is based on electrochemiluminescence (ECL) detection utilizing a sulfo-Tag label that emits light upon electrochemical stimulation (using a dedicated ECL plate reader, an electrical current is placed across the microplate with electrodes integrated into the bottom of the plate, resulting in a series of electrically induced reactions leading to a luminescent signal), a detection limit ranging between 0.3 × 10(3) and 10(3) CFU/ml (i.e., 30 to 100 spores per test), depending on the B. anthracis strain assayed, was achieved. In complex matrices (5 mg/ml of soil or simulated powder), the detection level (without any sample purification or concentration) was never altered more than 3-fold compared with the results obtained in phosphate-buffered saline.     

Biochemical Studies of Bacterial Sporulation IV. Inorganic Pyrophosphatase of Vegetative Cells and Spores of Bacillus megaterium

Inorganic pyrophosphatase activities extracted from vegetative cells and spores of Bacillus megaterium were compared and found to be similar in behavior on polyacrylamide gel electrophoresis, in metal and pH requirements for activity, and in response to inhibitors. In the sporulating cell, an additional electrophoretic species of the enzyme was observed which could be partially converted to the principal form by treatment with Mn(++); both forms of the enzyme required Mn(++) for stabilization in solution as well as for activity.     

Evaluation of the Efficacy of Methyl Bromide in the Decontamination of Building and Interior Materials Contaminated with Bacillus anthracis Spores

The primary goal of this study was to determine the conditions required for the effective inactivation of Bacillus anthracis spores on materials by using methyl bromide (MeBr) gas. Another objective was to obtain comparative decontamination efficacy data with three avirulent microorganisms to assess their potential for use as surrogates for B. anthracis Ames. Decontamination tests were conducted with spores of B. anthracis Ames and Geobacillus stearothermophilus, B. anthracis NNR1Δ1, and B. anthracis Sterne inoculated onto six different materials. Experimental variables included temperature, relative humidity (RH), MeBr concentration, and contact time. MeBr was found to be an effective decontaminant under a number of conditions. This study highlights the important role that RH has when fumigation is performed with MeBr. There were no tests in which a ≥6-log₁₀ reduction (LR) of B. anthracis Ames was achieved on all materials when fumigation was done at 45% RH. At 75% RH, an increase in the temperature, the MeBr concentration, or contact time generally improved the efficacy of fumigation with MeBr. This study provides new information for the effective use of MeBr at temperatures and RH levels lower than those that have been recommended previously. The study also provides data to assist with the selection of an avirulent surrogate for B. anthracis Ames spores when additional tests with MeBr are conducted.     

Role of the gerP Operon in Germination and Outgrowth of Bacillus anthracis Spores

Germination of Bacillus anthracis spores occurs when nutrients such as amino acids or purine nucleosides stimulate specific germinant receptors located in the spore inner membrane. The gerP_ABCDEF operon has been suggested to play a role in facilitating the interaction between germinants and their receptors in spores of Bacillus subtilis and Bacillus cereus. B. anthracis mutants containing deletions in each of the six genes belonging to the orthologue of the gerP_ABCDEF operon, or deletion of the entire operon, were tested for their ability to germinate. Deletion of the entire gerP operon resulted in a significant delay in germination in response to nutrient germinants. These spores eventually germinated to levels equivalent to wild-type, suggesting that an additional entry point for nutrient germinants may exist. Deletions of each individual gene resulted in a similar phenotype, with the exception of ΔgerPF, which showed no obvious defect. The removal of two additional gerPF-like orthologues was necessary to achieve the germination defect observed for the other mutants. Upon physical removal of the spore coat, the mutant lacking the full gerP operon no longer exhibited a germination defect, suggesting that the GerP proteins play a role in spore coat permeability. Additionally, each of the gerP mutants exhibited a severe defect in calcium-dipicolinic acid (Ca-DPA)–dependent germination, suggesting a role for the GerP proteins in this process. Collectively, these data implicate all GerP proteins in the early stages of spore germination.     

Formaldehyde Solution Effectively Inactivates Spores of Bacillus anthracis on the Scottish Island of Gruinard

Gruinard Island was heavily contaminated with the spores of virulent Bacillus anthracis during biological weapons trials in World War II. However, an extensive survey in 1979 showed that most of the island was not contaminated. In the early 1980s, a more intensive survey revealed that the contamination was largely confined to the top 8 cm of the soil in a 2.6-ha area of the 211-ha island. Small-scale tests showed that the spores could be inactivated by drenching the soil with fluid biocides. A solution of 5% formaldehyde in seawater applied by surface spray to each square meter of ground was shown to be the most effective treatment and was utilized for large-scale decontamination of the affected areas. Following this treatment, extensive sampling revealed that most of the spores of B. anthracis had been inactivated. Isolated pockets of surviving spores were treated further. A flock of sheep was then allowed to graze over the entire island for 5 months; none contracted anthrax.     

Biochemical Studies of Bacterial Sporulation and Germination XIII. Adenylate Kinase of Vegetative Cells and Spores of Bacillus subtilis

The spore and vegetative cell adenylate kinases of Bacillus subtilis, purified about 1,000-fold, proved indistinguishable by several physical and functional tests, including polyacrylamide gel electrophoresis, DEAE cellulose chromatography, and specificity toward substrates. Adenylate kinase activity in cell extracts, followed throughout growth and sporulation, was found to reach a maximum near the end of exponential growth, remain at that level during sporulation, until shortly before the appearance of refractile forms, and then decline, along with total protein, during the subsequent maturation of the spores. The enzyme, stable in extracts of exponential growing cells, was unstable in extracts of sporulating cells, presumably as a result of degradation by protease(s) appearing after the end of exponential growth.     

Response of Clostridium perfringens Spores and Vegetative Cells to Temperature Variation

The vegetative cells and spores of four strains of Clostridium perfringens were examined to determine the effect of lowered and elevated temperatures. Spores were produced by following the method of Ellner, and vegetative cells were obtained from thioglycolate cultures. After exposure to freezing or refrigeration temperatures (-17.7 and 7.1 C, respectively), only small numbers of the vegetative cells were recovered. After similar treatment, 16 to 58% of the spores were recovered. Essentially no vegetative cells and few spores survived holding at 80 C for 10 min. Although all strains were isolated from food, only one strain of the four studied had its origin in a food-poisoning outbreak, and it had been carried on laboratory media for approximately 10 years.     

Spermidine Biosynthesis During Germination and Subsequent Vegetative Growth of Bacillus megaterium Spores

Spermidine biosynthesis was extremely low early in germination of Bacillus megaterium spores and the spermidine level remained constant. Rapid synthesis began after 130 min and thereafter accounted for the increase in spermidine level which began at this time. Biosynthesis was greatly (>84%) diminished by exogenous spermine or spermidine. Arginine and ornithine were both converted efficiently into spermidine, but arginine was the more immediate precursor as shown by isotope competition studies and by the absence of ornithine decarboxylase and the presence of arginine decarboxylase. Exogenous putrescine was not incorporated into spermidine, although it was taken up rapidly and degraded.     

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

A single-chain Fv (scFv) antibody was developed and applied for efficient and specific detection of Bacillus anthracis spores. The antibody was isolated from a phage display library prepared from spleens of mice immunized with a water-soluble extract of the outer membrane of the B. anthracis spore (exosporium). The library (7 × 10⁶ PFU) was biopanned against live, native B. anthracis ATCC Δ14185 spores suspended in solution, resulting in the isolation of a unique soluble scFv antibody. The antibody was affinity purified and its affinity constant (3 × 10⁸ ± 1 × 10⁸ M⁻¹) determined via flow cytometry (FCM). Preliminary characterization of scFv specificity indicated that the scFv antibody does not cross-react with representatives of some phylogenetically related Bacillus spores. The potential use of scFv antibodies in detection platforms was demonstrated by the successful application of the soluble purified scFv antibody in enzyme-linked immunosorbent assays, immunofluorescence assays, and FCM.