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Chemical, biological, radiological, nuclear and explosive (CBRNE) response teams are responsible for mitigating and investigating events such as the distribution of anthrax letters throughout the U...

Aims To obtain quantitative efficacy data of two ultraviolet light (UVC) technologies for surface inactivation of Bacillus anthracis Ames and Bacillus atrophaeus spores. Methods and results Spores were deposited onto test coupons and controls of four different materials, via liquid suspension or aerosol deposition. The test coupons were then exposed to UVC light from either a low-pressure mercury vapor lamp or a system comprised of light emitting diodes, with a range of dosages. Positive controls were held at ambient conditions and not exposed to UVC light. Following exposure to UVC, spores were recovered from the coupons and efficacy was quantified in terms of log10 reduction (LR) in the number of viable spores compared to that from positive controls. Conclusions Decontamination efficacy varied by material and UVC dosage (efficacy up to 5·7 LR was demonstrated). There was no statistical difference in efficacy between the two species or between inoculation methods. Efficacy improved for the LED lamp at lower relative humidity, but this effect was not observed with the mercury vapor lamp. Significance and impact of the study This study will be useful in determining whether UVC could be used for the inactivation of B. anthracis spores on different surface types.

Bioterrorism has been considered a stark reality since the mailing of Bacillus anthracis spores via the United States Postal Service right after 9/11. Spores of B. anthracis cause anthrax disease in animals and humans, and the infectious dose varies from a few (1–10) to 1000 spores, depending on the age, sex, and immunity of the affected individuals. In the event of a large-scale spore release, early detection and defining the contaminated zones are significant challenges. Furthermore, rapid and effective clean-up of contaminated sites, including building interiors, is paramount in minimizing the consequence of biological warfare agent (BWA) release, and restoration of normalcy. For the past 15 years, Chemical Biological Center lab has been focused on both early detection of bacterial pathogens and ricin toxin, and BW decontamination research. With respect to early BWA detection, field detection using molecular approaches is compromised by false-negative and false-positive outcomes. In addition, use of hand-held assays (lateral flow tickets) must contend with high limits of detection (10,000–50,000 spores). In the event of wide-area release, rapid sampling and sampling efficiency from diverse exterior (porous and nonporous) surfaces are critical challenges. Some of our recent R&D and that of our collaborators at the U.S. Environmental Protection Agency (EPA) have evaluated the effectiveness of both liquid disinfectants and gaseous fumigants on a diverse range of surfaces. The results have been informative and have filled in research gaps. For example, based on results from these studies, we know that peroxide-based chemicals will be ineffective on concrete, and free-chlorine-based approaches will be ineffective on wood-like structures. Some recent work on environmental sampling and development of novel approaches, e.g., DeconGel for BW decontamination, will be summarized. Finally, current challenges and future research directions will be discussed in light of our recent study, i.e., decontamination of vertical contaminated surfaces.

Biological warfare incidents generate both immediate and delayed hazards, potentially resulting from reaerosolization of deposited hazardous particles from surfaces. Understanding the causes and effects of the initial deposition method and environmental conditions on reaerosolization is important in hazard prediction and selection of mitigation approaches. This study was conducted to determine the amount of reaerosolization of various bacterial spores and 1 µm polystyrene latex microspheres deposited wet or dry and incubated at 20 or 80% relative humidity (RH). The organisms used in this study were Bacillus atrophaeus var. globigii (Bg), B. thuringiensis (Bt), B. anthracis ΔSterne (Ba-ΔSterne), Ba-ΔSterne ΔbclA mutant (BclA), and Ba-ΔSterne ΔcotE mutant (CotE). These organisms represent a range of spore types with different outer surfaces: spores with exosporium hairs and a basal layer (Ba-ΔSterne and Bt), spores with a basal layer (BclA), and spores with a spore coat only (no exosporium, Bg and C...

Chemical, biological, radiological, and explosive incidents produce immediate as well as delayed hazards as a result of reaerosolization of deposited particles from surfaces. Understanding reaeroso...

Following an intentional or accidental bio-warfare agent (BWA) release, environmental sample analysis is absolutely critical to determine the extent of contamination. When dealing with nonspore forming BWA (e.g., Yersinia pestis), retention of cell viability is central to such analyses. Even though significant advances have been achieved in DNA sequencing technologies, a positive identification of BWAs in environmental samples must be made through the ability of cells to form colony-forming units upon culturing. Inability to revive the cells between collection and analysis renders such studies inconclusive. Commercial kits designed to preserve the viability of pathogens contained within clinical samples are available, but many of them have not been examined for their ability to preserve samples containing suspected BWAs. The study was initiated to examine the applicability of commercial solutions aiding in retention of Y. pestis viability in samples stored under nonpermissive temperatures, that is, 40 and 37°C. While none of the tested solutions sustained cell viability at 40°C, the results show five out of 17 tested preservatives were capable of supporting viability of Y. pestis at 37°C.

Environmental surface sampling is crucial in determining the zones of contamination and overall threat assessment. Viability retention of sampled material is central to such assessments. A systematic study was completed to determine viability of vegetative cells under nonpermissive storage conditions. Despite major gains in nucleic acid sequencing technologies, initial positive identification of threats must be made through direct culture of the sampled material using classical microbiological methods. Solutions have been developed to preserve the viability of pathogens contained within clinical samples, but many have not been examined for their ability to preserve biological agents. The purpose of this study was to systematically examine existing preservation materials that can retain the viability of Bacillus anthracis vegetative cells stored under nonpermissive temperatures. The results show effectiveness of five of seventeen solutions, which are capable of retaining viability of a sporulation deficient strain of B. anthracis Sterne when stored under nonrefrigerated conditions.

AIMS To evaluate a standard aerosolization method for uniformly depositing threat-representative spores onto surfaces. METHODS AND RESULTS Lyophilized Bacillus anthracis ΔSterne spores, coated in silica, were aerosolized into a containment chamber and deposited onto nine surface types by two independent laboratories. Laboratory A produced a mean loading concentration of 1·78 × 10(5) CFU cm(-2) ; coefficient of variation (CV) was

Aims To evaluate the use of relatively low levels of hydrogen peroxide vapour (HPV) for the inactivation of Bacillus anthracis spores within an indoor environment. Methods and Results Laboratory-scale decontamination tests were conducted using bacterial spores of both B. anthracis Ames and Bacillus atrophaeus inoculated onto several types of materials. Pilot-scale tests were also conducted using a larger chamber furnished as an indoor office. Commercial off-the-shelf (COTS) humidifiers filled with aqueous solutions of 3 or 8% hydrogen peroxide (H2O2) were used to generate the HPV inside the mock office. The spores were exposed to HPV for periods ranging from 8 h up to 1 week. Conclusions Four- to seven-day exposures to low levels of HPV (average air concentrations of approx. 5–10 parts per million) were effective in inactivating B. anthracis spores on multiple materials. The HPV can be generated with COTS humidifiers and household H2O2 solutions. With the exception of one test/material, B. atrophaeus spores were equally or more resistant to HPV inactivation compared to those from B. anthracis Ames. Significance and Impact of the Study This simple and effective decontamination method is another option that could be widely applied in the event of a B. anthracis spore release.

Disinfection kinetics of vegetative cells of Bacillus anthracis in water with free available chlorine ([FAC] 2 mg/L) and monochloramine ([MC] 2 mg/L) were established in this study. FAC disinfection was performed in chlorine demand-free phosphate buffer at pH 7 and 8 at two temperatures (5 and 25 deg. C). MC disinfection was performed in normal phosphate buffer at pH 8 at both temperatures. FAC was more effective than MC in causing cell death, which was more rapid at 25 deg. C than at 5 deg. C at both pH 7 and 8. For MC disinfection, the cell inactivation rate was more rapid at 2 deg. C. The disinfection kinetics were rapid within the first 5 min, followed by a slow cell inactivation. The results were complicated by the varying number of spores present in the test inoculums. Although protocols were developed and implemented to minimize the spore number, varying numbers of spores were observed in the different runs. The presence of spores also resulted in high variability,especially at sublethal exposure levels. Our efforts were focused on developing a new culture recipe (RVLS) to ensure aminimal number of spores and to confirm the absence of spores in the sporulation-minus strain (spo-) of B. anthracis.

Available surface sampling approaches (e.g., swabs, biological sampling kits, and wipes), especially from porous surfaces, are generally ineffective. Hydrogel is a water-based gel, which is applied as a thick viscous material on contaminated surfaces and allowed to dry into a thin film within a few hours, depending on ambient conditions. The dried film is then peeled off the surface. During the drying process, the gel encapsulates the bioagent and other contaminants. In this study, biohydrogel was used to examine the sampling efficiency and preservation of materials taken from contaminated surfaces. The biohydrogel proved to be a superior sampling tool; recovery efficiency from 3D objects (e.g., screws) and pinewood coupons was 80 greater than that of existing sampling devices for the recovery of Bacillus anthracis (Sterne) spores. Biohydrogel proved to be as good a sampling tool for painted steel, polycarbonate, pinewood, and screws as available sampling techniques. Because the biohydrogel recovered 1 of Staphylococcus aureus cells from painted steel, polycarbonate, pinewood, and screws, some components of the biohydrogel were concluded to be bactericidal. Spores of B. anthracis Sterne strain were well preserved at 4 C for over5 weeks. Over 5 logs of such spores lost their viability within 2 weeks at 25 and 37 C, indicating non-optimal preservation at such temperatures.

This project was performed to determine if clinical pathogens evolve and acquire resistance to Lysol, an all-purpose cleaner and disinfectant (U.S. Environmental Protection Agency regulation no. 777-89 [ready-to-use (RTU) 1:16 dilution]). A clinical surrogate, Escherichia coli, was used in these studies. E. coli cells were grown in the absence or presence of Lysol. The parent strain (PS) was sensitive to the presence of 1.6% RTU strength Lysol. LR50, a resistant strain that showed resistance to 50% ofthe RTU strength Lysol, was derived through progressive subculturing. (A 30-fold increase in resistance to Lysol illustrates genome plasticity and adaptation of bacterial cells.) LR50 was subcultured in Tryptic soy broth five times, and then its resistance phenotype was confirmed in the presence of 50% Lysol. Biochemical characterization revealed the presence or absence of specific polypeptides unique to LR50. Genomic sequencing was done, and some single nucleotide polymorphisms were observed to be unique to LR50. Finally, altered antibiotic resistance was determined for LR50. In a separate set of experiments, the adaptive resistance of E.coli cells to 17% Germ-X (GR17), a hand sanitizer, was also observed. GR17 PS, a resistant strain, which was capable of growing in the presence of GR17, was derived during this study.

Efficacy of chlorine dioxide (CD) gas generated by two distinct generation systems, Sabre (wet system with gas generated in water) and ClorDiSys (dry system with gas generated in air), was evaluated for inactivation of Bacillus anthracis spores on six building interior surfaces. The six building materials included carpet, acoustic ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. There was no statistically significant difference in the data due to the CD generation technology at a 95% confidence level. Note that a common method of CD gas measurement was used for both wet and dry CD generation types. Doses generated by combinations of different concentrations of CD gas (500, 1,000, 1,500, or 3,000 parts per million of volume [ppmv]) and exposure times (ranging between 0.5 and 12 h) were used to evaluate the relative role of fumigant exposure period and total dose in the decontamination of building surfaces. The results showed that the time required to achieve at least a 6-log reduction in viable spores is clearly a function of the material type on which the spores are inoculated. The wood and cinder block coupons required a longer exposure time to achieve a 6-log reduction. The only material showing a clear statistical difference in rate of decay of viable spores as a function of concentration was cinder block. For all other materials, the profile of spore kill (i.e., change in number of viable spores with exposure time) was not dependent upon fumigant concentration (500 to 3,000 ppmv). The CD dose required for complete spore kill on biological indicators (typically, 1E6 spores of B acillus atrophaeus on stainless steel) was significantly less than that required for decontamination of most of the building materials tested.

The quantitative Three-Step Method (TSM) for testing the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard, nonporous surface (glass) was adopted as AOAC Official MethodSM 2008.05 in May 2008. The TSM uses 5 5 1 mm coupons (carriers) upon which spores have been inoculated and which are introduced into liquid sporicidal agent contained in a microcentrifuge tube. Following exposure of inoculated carriers and neutralization, spores are removed from carriers in three fractions (gentle washing, fraction A; sonication, fraction B; and gentle agitation, fraction C). Liquid from each fraction is serially diluted and plated on a recovery medium for spore enumeration. The counts are summed over the three fractions to provide the density (viable spores per carrier), which is log10-transformed to arrive at the log density. The log reduction is calculated by subtracting the mean log density for treated carriers from the mean log density for control carriers. This paper presents a single-laboratory investigation conducted to evaluate the applicability of using two porous carrier materials (ceramic tile and untreated pine wood) and one alternative nonporous material (stainless steel). Glass carriers were included in the study as the reference material. Inoculated carriers were evaluated against three commercially available liquid sporicides (sodium hypochlorite, a combination of peracetic acid and hydrogen peroxide, and glutaraldehyde), each at two levels of presumed efficacy (medium and high) to provide data for assessing the responsiveness of the TSM. Three coupons of each material were evaluated across three replications at each level; three replications of a control were required. Even though all carriers were inoculated with approximately the same number of spores, the observed counts of recovered spores were consistently higher for the nonporous carriers. For control carriers, the mean log densities for the four materials ranged from 6.63 for wood to 7.14 for steel. The pairwise differences between mean log densities, except for glass minus steel, were statistically significant (P < 0.001). The repeatability standard deviations (Sr) for the mean control log density per test were similar for the four materials, ranging from 0.08 for wood to 0.13 for tile. Spore recovery from the carrier materials ranged from approximately 20 to 70: 20 (pine wood), 40 (ceramic tile), 55 (glass), and 70 (steel). Although the percent spore recovery from pine wood was significantly lower than that from other materials, the performance data indicate that the TSM provides a repeatable and responsive test for determining the efficacy of liquid sporicides on both porous and nonporous materials.

Chlorine dioxide gas and vaporous hydrogen peroxide sterilant have been used in the cleanup of building interiors contaminated with spores ofBacillus anthracis. A systematic study, in collaboration with the U.S. Environmental Protection Agency, was jointly undertaken by the U.S. Army-Edgewood Chemical Biological Center to determine the sporicidal efficacies of these two fumigants on six building structural materials: carpet, ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. Critical issues related to high-throughput sample processing and spore recovery from porous and nonporous surfaces included (i) the extraction of spores from complex building materials, (ii) the effects of titer challenge levels on fumigant efficacy, and (iii) the impact of bioburden inclusion on spore recovery from surfaces and spore inactivation. Small pieces (1.3 by 1.3 cm of carpet, ceiling tile, wallboard, I-beam steel, and pinewood and 2.5 by 1.3 cm for cinder block) of the materials were inoculated with an aliquot of 50 μl containing the target number (1 × 106, 1 × 107, or 1 × 108) of avirulent spores ofB. anthracisNNR1Δ1. The aliquot was dried overnight in a biosafety cabinet, and the spores were extracted by a combination of a 10-min sonication and a 2-min vortexing using 0.5% buffered peptone water as the recovery medium. No statistically significant drop in the kill efficacies of the fumigants was observed when the spore challenge level was increased from 6 log units to 8 log units, even though a general trend toward inhibition of fumigant efficacy was evident. The organic burden (0 to 5%) in the spore inoculum resulted in a statistically significant drop in spore recovery (at the 2 or 5% level). The effect on spore killing was a function of the organic bioburden amount and the material type. In summary, a high-throughput quantitative method was developed for determining the efficacies of fumigants, and the spore recoveries from five porous materials and one nonporous material ranged between 20 and 80%.

The efficacy of ultraviolet C (UVC) light (100-280 nm) in the decontamination of three hospital-related surfaces, namely, unpainted/painted aluminum (bed railings), stainless steel (operating tables), and scrubs (laboratory coats), was investigated. Acinetobacter baumannii cells were inoculated (10(5) or 10(3) cells) on small coupons and dried overnight in a class II biosafety cabinet. Drying resulted inor =50% loss of viability. The UVC fluence of 90 J/m2 was observed to be very effective in the decontamination of cells from all metal coupon surfaces (complete killing). However, the same fluence was ineffective in the decontamination of scrubs. The effectiveness of two other common disinfection practices, that is, 15 minutes of boiling or spraying with 70% ethanol, was investigated for the scrubs. Although ethanol treatment was ineffective, the boiling treatment was very effective (complete killing). These results establish that metal surfaces can be decontaminated with UVC irradiation and boiling treatment is effective for scrub decontamination.

As a consequence of organophosphorus (OP) toxins posing a threat to human life globally, organophosphorus hydrolase (OPH) has become the enzyme of choice to detoxify such compounds. Organophosphorus hydrolase was covalently immobilized onto a quartz substrate for utilization in paraoxon detection. The substrate was cleaned and modified prior to chemical attachment. Each modification step was monitored by imaging ellipsometry as the thickness increased with each modification step. The chemically attached OPH was labeled with a fluorescent dye (7-isothiocyanato-4-methylcoumarin) for the detection of paraoxon in aqueous solution, ranging from 10(-9) to 10(-5) M. UV-visible spectra were also acquired for the determination of the hydrolysis product of para-oxon, namely p-nitrophenol.

Enzymatic catalysis is one means of accelerating the rate of hydrolysis of G-type organophosphorus nerve agents. Here, the stereospecificity of the catalysis of cyclosarin (GF, O -cyclohexyl methylphosphonofluoridate) hydrolysis by several enzymes was investigated. Stereospecificity was not evident at 3 mM GF but was evident at 0.5 mM GF. The differential effect was apparently due to fluoride-catalyzed racemization of the substrate. Alteromonas sp. JD6.5 organophosphorus acid anhydrolase (OPAA), Alteromonas haloplanktis OPAA and the wild-type phosphotriesterase (PTE) enzymes were all found to catalyze preferentially the hydrolysis of the (+)GF isomer, as determined by GC analysis of the remaining unreacted (−)GF isomer. Acetylcholinesterase inhibition experiments showed the purified (−)GF isomer to be approximately twice as toxic as the racemic mixture. One PTE mutant, H254G/H259W/L303T, was found to reverse the native PTE stereospecificity and preferentially catalyze the hydrolysis of the (−)GF isomer, as shown by its complementation of Alteromonas sp. JD6.5 OPAA and by GC analysis of the remaining (+)GF isomer. This procedure also permitted the individual preparation of either of the two GF isomers by enzymatic degradation followed by extraction of the remaining isomer.

Organophosphorus acid anhydrolases (OPAA; E.C.3.1.8.2) are a class of enzymes that hydrolyze a variety of toxic acetylcholinesterase-inhibiting organophosphorus (OP) compounds, including pesticides and fluorine-containing chemical nerve agents. In this paper, subphase conditions have been optimized to obtain stable OPAA Langmuir films, and the diisopropylfluorophosphate (DFP) hydrolysis reaction catalyzed by OPAA in aqueous solution and at the air-water interface was studied. OPAA-DFP interactions were investigated utilizing different spectroscopic techniques, that is, circular dichroism and fluorescence in aqueous solution and infrared reflection absorption spectroscopies at the air-water interface. The characterization of OPAA and its secondary structure in aqueous solution and as a monolayer at the air-water interface in the absence and in the presence of DFP dissolved in aqueous solution or in the aqueous subphase demonstrated significantly distinctive features. The research described herein demonstrated that OPAA can be used in an enzyme-based biosensor for DFP detection.

The paraoxon hydrolysis reaction catalyzed by organophosphorus hydrolase (OPH) monolayer at the air-water interface was studied. OPH-paraoxon interactions, occurring at the two-dimensional interface, by close-packed, highly orientated OPH monolayer, were investigated by several different surface chemistry techniques; e.g. surface pressure area isotherms, atomic force microscopy (AFM), and in situ epifluorescence microscopy. The characterization of OPH Langmuir and Langmuir-Blodgett films prepared in both the presence and absence of paraoxon, demonstrated significantly distinctive feature when compared with one another. Continuous growth of the OPH aggregates is a distinct phenomenon associated with hydrolysis, in addition to the pH changes in the local environment of the enzyme macromolecules.

Langmuir–Blodgett (LB) film deposition technique was employed for the immobilization of organophosphorus hydrolase (OPH). OPH enzyme was covalently bonded to a fluorescent probe, fluorescein isothiocyanate (FITC), and used as a biological recognition element. Under optimal experimental conditions, OPH monolayers were deposited onto the surface of silanized quartz slides as LB film and utilized as a bioassay for the detection of paraoxon. Two different methods were employed for detection of paraoxon: the fluorescence quenching of the fluorescence probe (FITC) covalently bonded to OPH and the UV–vis absorption spectrum of the paraoxon hydrolysis product. The UV–vis absorption measurement demonstrated a linear relationship between the absorbance at 400 nm and the concentration of paraoxon solutions over the range of 1.0 × 10−7–1.0 × 10−5 M (0.27–27 ppm). By observing the FITC fluorescence quenching, the concentration of paraoxon can be detected as low as 10−9 M (S/N = 3). The research described herein showed that the LB film bioassay had high sensitivity, rapid response time and good reproducibility.

The secondary structure of organophosphorus hydrolase (OPH) has been studied with circular dichroism (CD) spectroscopy in the far-UV region. The effect of pH on the secondary structure of OPH solution was examined over the pH range from 3.56 to 9.60. As shown on the CD spectra, the secondary structure of OPH is well defined when the pH value is near the isoelectric point (7.6); however, it is destroyed when the pH values are increased or decreased further. This is explained by the loss of helical structure. The pH effect on CD spectra contributes to clarify the optimum pH needed to obtain a stable OPH Langmuir film at the air−water interface and its correlation to the secondary structure of the enzyme. Comparative study of the thermal treatment on the secondary structure of OPH in solution, Langmuir−Blodgett film, and dry film shows that the molecular arrangement plays a dominant role in the thermal stability of OPH. With use of the CDPro software package a quantitative estimation of the secondary structu...

We report genetic maps for diploid (D) and tetraploid (AtDt) Gossypium genomes composed of sequence-tagged sites (STS) that foster structural, functional, and evolutionary genomic studies. The maps include, respectively, 2584 loci at 1.72-cM (∼600 kb) intervals based on 2007 probes (AtDt) and 763 loci at 1.96-cM (∼500 kb) intervals detected by 662 probes (D). Both diploid and tetraploid cottons exhibit negative crossover interference; i.e., double recombinants are unexpectedly abundant. We found no major structural changes between Dt and D chromosomes, but confirmed two reciprocal translocations between At chromosomes and several inversions. Concentrations of probes in corresponding regions of the various genomes may represent centromeres, while genome-specific concentrations may represent heterochromatin. Locus duplication patterns reveal all 13 expected homeologous chromosome sets and lend new support to the possibility that a more ancient polyploidization event may have predated the A-D divergence of 6–11 million years ago. Identification of SSRs within 312 RFLP sequences plus direct mapping of 124 SSRs and exploration for CAPS and SNPs illustrate the “portability” of these STS loci across populations and detection systems useful for marker-assisted improvement of the world's leading fiber crop. These data provide new insights into polyploid evolution and represent a foundation for assembly of a finished sequence of the cotton genome.

Layer-by-layer (LbL) assembly has been utilized to fabricate an ultrathin film of polyelectrolytes. The architecture was composed of chitosan and organophosphorus hydrolase polycations along with thioglycolic acid-capped CdSe quantum dots (QDs) as the polyanion. The topography of the films was studied using epifluorescence microscopy imaging. The photoluminescence property of the functionalized QDs improved when sandwiched between the polycation layers. The enhanced optical property of QDs allowed easy monitoring of LbL growth and detection of paraoxon with high sensitivity. The presence of organophosphorus compounds was confirmed through UV−vis and emission spectroscopies.

Organophosphorus hydrolase (OPH) is capable of hydrolyzing a wide variety of organophosphorus pesticides and chemical warfare agents. However, the hydrolytic activity of OPH against the warfare agent VX is less than 0.1% relative to its activity against parathion and paraoxon. Based on the crystal structure of OPH and the similarities it shares with acetylcholinesterase, eight OPH mutants were constructed with the goal of increasing OPH activity toward VX. The activities of crude extracts from these mutants were measured using VX, demeton-S methyl, diisopropylfluoro-phosphate, ethyl parathion, paraoxon, and EPN as substrates. One mutant (L136Y) displayed a 33% increase in the relative VX hydrolysis rate compared to wild type enzyme. The other seven mutations resulted in 55-76% decreases in the relative rates of VX hydrolysis. There was no apparent relationship between the hydrolysis rates of VX and the rates of the other organophosphorus compounds tested.

Enzymes catalyzing the hydrolysis of highly toxic organophosphorus compounds (OPs) are classified as organophosphorus acid anhydrolases (OPAA; EC 3.1.8.2). Recently, the genes encoding OPAA from two species of Alteromonas were cloned and sequenced. Sequence and biochemical analyses of the cloned genes and enzymes have established Alteromonas OPAAs to be prolidases (E.C. 3.4.13.9), a type of dipeptidase hydrolyzing dipeptides with a prolyl residue in the carboxyl-terminal position (X-Pro). Alteromonas prolidases hydrolyze a broad range of G-type chemical warfare (CW) nerve agents. Efforts to over-produce a prolidase from A. sp.JD6.5 with the goal of developing strategies for long-term storage and decontamination have been successfully achieved. Large-scale production of this G-agent degrading enzyme is now feasible with the availability of an over-producing recombinant cell line. Use of this enzyme for development of a safe and non-corrosive decontamination system is discussed.

The hydrophobic interface between the allosteric and the zinc domains of the regulatory subunit of aspartate transcarbamoylase has previously been implicated in the heterotropic ATP activation of the enzyme. The present work shows that this interface also affects CTP and CTP–UTP inhibition and proposes a structural explanation for the effects. Mutant enzymes derived from nonselective mutagenesis of residues r101–r106 (residues that contribute part of the interface) displayed a variety of homotropic and heterotropic effects. The cooperative behavior of the enzymes was affected, as indicated by reduced aspartate S 0.5 values and apparent Hill coefficient values for V106L, V106L/N105S, and I103F/R102C. In addition, both ATP activation and CTP inhibition were significantly reduced and CTP+UTP synergistic inhibition was decreased in these mutants. The D104G mutant enzyme was subject to inhibition by CTP andCTP+UTP, but was not activated by ATP. Finally, the I103T mutant enzyme had an increased S 0.5 value of 11.5 mM and displayed altered effector responses: ATP acted as an inhibitor, and the CTP+UTP synergistic inhibition was reduced. Most of these allosteric variations can be explained in terms of perturbations to the “tongue and groove” hydrophobic interface between the allosteric and the zinc domains and a consequent impact on a second interface (“reg1:cat4”) between regulatory and catalytic subunits.

2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, making necessary the remediation of contaminated areas and unused materials. Although bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the metabolism of TNT have proven to be complex. The three aerobic bacterial strains reported here (Pseudomonas aeruginosa, Bacillus sp. , and Staphylococcus sp.) differ in their ability to biotransform TNT and in their growth characteristics in the presence of TNT. In addition, enzymatic activities have been identified that differ in the reduction of nitro groups, cofactor preferences, and the ability to eliminate-NO2 from the ring. The Bacillus sp. has the most diverse bioremediation potential owing to its growth in the presence of TNT, high level of reductive ability, and capability of removing-NO2 from the nitroaromatic ring.

We report on the immobilization of an OPAA enzyme on luminescent porous silicon devices, and on the utilization of this new platform to hydrolyze p-nitrophenyl-soman.

The AOAC Quantitative Three Step Method (TSM; AOAC Official MethodSM 2008.05) is validated for testing the efficacy of liquid sporicides against spores of Bacillus subtilis and Bacillus anthracis on selected hard, nonporous, and porous surfaces. The TSM uses 5 × 5 × 1 mm inoculated coupons (carriers), which are placed in 400 μL liquid sporicidal agent contained in a microcentrifuge tube. Following exposure of inoculated carriers to the test chemical and subsequent neutralization, viable spores are recovered in three fractions: A (gentle tapping), B (sonication), and C (gentle agitation). The spores in suspension are serially diluted and plated on a recovery medium for enumeration. The plate counts are summed over the three fractions to provide the number of viable spores per carrier, which is log10-transformed to generate a mean log density (LD) value across carriers. As a measure of product efficacy, a log reduction (LR) value is calculated by subtracting the mean LD for treated carriers from the mean LD for control carriers. This paper reports on the comparative evaluation of the current and modified versions of the TSM in order to support a modification to simplify the procedure. The proposed modified TSM (mTSM) consolidates fractions B and C in the same tube. Thus, the sonication (fraction B) and gentle agitation (fraction C) steps are carried out in the same tube, thereby reducing the number of tubes and associated resources and time necessary to complete the test. Glass, steel, pine wood, and ceramic tile carriers were included in the comparative study. Inoculated carriers were evaluated against two preparations of sodium hypochlorite to generate two presumed levels of efficacy (intermediate and high); the control LD and LR values associated with testing each carrier type for the TSM and the mTSM were compared. For control carriers, the mean log densities per carrier (for each carrier material) were not significantly different based on the TSM compared to the mTSM. Furthermore, the treated carrier data showed comparable LR values for the TSM and mTSM. The data provided in this report demonstrate equivalency between the TSM and mTSM and support the proposed procedural modification to consolidate fractions B and C.

A method was developed for the isolation of megabase-size DNA fromSorghum bicolor. Sorghum protoplasts were isolated from young leaf tissue, embedded in an agarose matrix as microbeads or plugs, followed by cell lysis and protein degradation. The DNA prepared by this method was larger than 1 Mb in size and readily digestible with restriction enzymes. The DNA was shown to be suitable for physical mapping, and was successfully used for the construction of BAC and YAC libraries.

A polyelectrolyte architecture was fabricated that was composed of chitosan and organophosphorus hydrolase polycations along with thioglycolic acid-capped CdSe quantum dots (QDs) as the polyanion. This film was imaged by epifluorescence microscopy. UV−vis and emission spectroscopies were used to monitor the growth of the bilayer film due to the enhanced optical property of QDs. Photoluminescence of the functionalized QDs improved when sandwiched between the polycations layers. The presence of organophosphorus compounds was confirmed through photoluminescence spectroscopy.

A variant of Bacillus thuringiensis subsp. kurstaki containing a single, stable copy of a uniquely amplifiable DNA oligomer integrated into the genome for tracking the fate of biological agents in the environment was developed. The use of genetically tagged spores overcomes the ambiguity of discerning the test material from pre-existing environmental microflora or from previously released background material. In this study, we demonstrate the utility of the genetically “barcoded” simulant in a controlled indoor setting and in an outdoor release. In an ambient breeze tunnel test, spores deposited on tiles were reaerosolized and detected by real-time PCR at distances of 30 m from the point of deposition. Real-time PCR signals were inversely correlated with distance from the seeded tiles. An outdoor release of powdered spore simulant at Aberdeen Proving Ground, Edgewood, MD, was monitored from a distance by a light detection and ranging (LIDAR) laser. Over a 2-week period, an array of air sampling units collected samples were analyzed for the presence of viable spores and using barcode-specific real-time PCR assays. Barcoded B. thuringiensis subsp. kurstaki spores were unambiguously identified on the day of the release, and viable material was recovered in a pattern consistent with the cloud track predicted by prevailing winds and by data tracks provided by the LIDAR system. Finally, the real-time PCR assays successfully differentiated barcoded B. thuringiensis subsp. kurstaki spores from wild-type spores under field conditions.

A significant gap in technology preparedness exists in the U.S. Federal response to wide-area contamination resulting from the release of biological agents such as Bacillus anthracis spores. In 2001, release of just a few letters containing anthrax spores resulted in the contamination of several building interiors, including the U.S. Postal and Distribution Centers (Brentwood, Washington, DC; Trenton and Jersey City, NJ) and American Media Inc. (Boca Raton, FL). Despite heavy contamination levels of several building interiors, remediation of building interiors was achieved successfully by fumigation with chlorine dioxide (CD) or vapor hydrogen peroxide (VHP). A wide-area release and contamination of building exteriors and the outdoors would likely exhaust the national remediation capacity. Cleanup could take years and lead to incalculable financial drain because of a delay in effective response. Additionally, agencies responsible for the mitigation of contaminated sites are exploring alternative methods for decontamination including combinations for the disposal of contaminated items, source reduction by vacuuming, mechanical scrubbing, and pH-adjusted bleach pressure wash. If proven effective, a pressure wash-based removal of anthrax spores from building surfaces with readily available equipment will significantly increase the readiness of federal agencies to meet the daunting challenge of restoration and cleanup efforts following a wide-area biological release.

The quantitative Three-Step Method (TSM) for testing the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard, nonporous surface (glass) was adopted as AOAC Official Method 2008.05 in May 2008. The TSM uses 5 x 5 x 1 mm coupons (carriers) upon which spores have been inoculated and which are introduced into liquid sporicidal agent contained in a microcentrifuge tube. Following exposure of inoculated carriers and neutralization, spores are removed from carriers in three fractions (gentle washing, fraction A; sonication, fraction B; and gentle agitation, fraction C). Liquid from each fraction is serially diluted and plated on a recovery medium for spore enumeration. The counts are summed over the three fractions to provide the density (viable spores per carrier), which is log10-transformed to arrive at the log density. The log reduction is calculated by subtracting the mean log density for treated carriers from the mean log density for control carriers. This paper presents a single-laboratory investigation conducted to evaluate the applicability of using two porous carrier materials (ceramic tile and untreated pine wood) and one alternative nonporous material (stainless steel). Glass carriers were included in the study as the reference material. Inoculated carriers were evaluated against three commercially available liquid sporicides (sodium hypochlorite, a combination of peracetic acid and hydrogen peroxide, and glutaraldehyde), each at two levels of presumed efficacy (medium and high) to provide data for assessing the responsiveness of the TSM. Three coupons of each material were evaluated across three replications at each level; three replications of a control were required. Even though all carriers were inoculated with approximately the same number of spores, the observed counts of recovered spores were consistently higher for the nonporous carriers. For control carriers, the mean log densities for the four materials ranged from 6.63 for wood to 7.14 for steel. The pairwise differences between mean log densities, except for glass minus steel, were statistically significant (P0.001). The repeatability standard deviations (Sr) for the mean control log density per test were similar for the four materials, ranging from 0.08 for wood to 0.13 for tile. Spore recovery from the carrier materials ranged from approximately 20 to 70%: 20% (pine wood), 40% (ceramic tile), 55% (glass), and 70% (steel). Although the percent spore recovery from pine wood was significantly lower than that from other materials, the performance data indicate that the TSM provides a repeatable and responsive test for determining the efficacy of liquid sporicides on both porous and nonporous materials.

The organophosphate acid anhydrolase (OPAA) is a member of a class of bimetalloenzymes that hydrolyze a variety of toxic acetylcholinesterase-inhibiting organophosphorus compounds, including fluorine-containing chemical nerve agents. It also belongs to a family of prolidases, with significant activity against various Xaa-Pro dipeptides. Here we report the X-ray structure determination of the native OPAA (58 kDa mass) from Alteromonas sp. strain JD6.5 and its cocrystal with the inhibitor mipafox [N,N'-diisopropyldiamidofluorophosphate (DDFP)], a close analogue of the nerve agent organophosphate substrate diisopropyl fluorophosphate (DFP). The OPAA structure is composed of two domains, amino and carboxy domains, with the latter exhibiting a "pita bread" architecture and harboring the active site with the binuclear Mn(2+) ions. The native OPAA structure revealed unexpectedly the presence of a well-defined nonproteinaceous density in the active site whose identity could not be definitively established but is suggestive of a bound glycolate, which is isosteric with a glycine (Xaa) product. All three glycolate oxygens coordinate the two Mn(2+) atoms. DDFP or more likely its hydrolysis product, N,N'-diisopropyldiamidophosphate (DDP), is present in the cocrystal structure and bound by coordinating the binuclear metals and forming hydrogen bonds and nonpolar interactions with active site residues. An unusual common feature of the binding of the two ligands is the involvement of only one oxygen atom of the glycolate carboxylate and the product DDP tetrahedral phosphate in bridging the two Mn(2+) ions. Both structures provide new understanding of ligand recognition and the prolidase and organophosphorus hydrolase catalytic activities of OPAA.

The secondary structure of the organophosphorus acid anhydrolase (OPAA) Langmuir monolayer in the absence and presence of diisopropylfluorophosphate (DFP) in the subphase was studied by infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated IRRAS (PM-IRRAS). The results of both the IRRAS and the PM-IRRAS indicated that the alpha-helix and the beta-sheet conformations in OPAA were parallel to the air-water interface at a surface pressure of 0 mN.m-1 in the absence of DFP in the subphase. When the surface pressure increased, the alpha-helix and the beta-sheet conformations became tilted. When DFP was added to the subphase at a concentration of 1.1 x 10(-5) M, the alpha-helix conformation of OPAA was still parallel to the air-water interface, whereas the beta-sheet conformation was perpendicular at 0 mN.m-1. The orientations of both the alpha-helix and the beta-sheet conformations did not change with the increase of surface pressure. The shape of OPAA molecules is supposed to be elliptic, and the long axis of OPAA was parallel to the air-water interface in the absence of DFP in the subphase, whereas the long axis became perpendicular in the presence of DFP. This result explains the decrease of the limiting molecular area of the OPAA Langmuir monolayer when DFP was dissolved in the subphase.

The secondary structure of organophosphorus hydrolase (OPH) at the air-water interface was studied using polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The shape and position of the amide I and amide II bands were used to estimate the surface conformation and orientation of OPH. The PM-IRRAS results indicated that the enzyme did not unfold for the range of surface pressure used (0-30 mN/m). At low surface pressures, the signal of amide I was very weak and the intensity was almost the same as amide II. Upon further compression, the PM-IRRAS signal and the ratio of the intensity of amide I and amide II both increase, implying an increased interfacial concentration of the enzyme. From the amide I/amide II ratio and the band position, it was deduced that the enzyme adopts a conformation which gives a higher occupied surface at low surface pressure and rotates to a more vertical orientation at high surface pressures. The compression and decompression of the OPH monolayer indicated that the fingerprint of the secondary structure at the air-water interface was reversible. PM-IRRAS was also used to investigate the pH effect of the subphase on the secondary structure of OPH. The secondary structure of OPH at the air-water interface was well defined when the pH of the subphase was near its isoelectric point (IP, pH 7.6). However, it adopted a different orientation when the subphase pH values were higher or lower than the IP with formation of random coil structure. The hydrolysis of organophosphorus compound paraoxon by OPH was also studied at the air-water interface by PM-IRRAS. The pH effect and the interaction with paraoxon both seem to orientate the enzyme more in the plane of the interface and to produce random coil structure.

In this paper, we first report a novel biosensor for the detection of paraoxon based on (CdSe)ZnS core-shell quantum dots (QDs) and an organophosphorus hydrolase (OPH) bioconjugate. The OPH was coupled to (CdSe)ZnS core-shell QDs through electrostatic interaction between negatively charged QDs surfaces and the positively charged protein side chain and ending groups (-NH2). Circular dichroism (CD) spectroscopy showed no significant change in the secondary structure of OPH after the bioconjugation, which indicates that the activity of OPH was preserved. Detectable secondary structure changes were observed by CD spectroscopy when the OPH/QDs bioconjugate was exposed to organophosphorus compounds such as paraoxon. Photoluminescence (PL) spectroscopic study showed that the PL intensity of the OPH/QDs bioconjugate was quenched in the presence of paraoxon. The overall quenching percentage as a function of paraoxon concentration matched very well with the Michaelis-Menten equation. This result indicated that the quenching of PL intensity was caused by the conformational change in the enzyme, which is confirmed by CD measurements. The detection limit of paraoxon concentration using OPH/QDs bioconjugate was about 10(-8) M. Although increasing the OPH molar ratio in the bioconjugates will slightly increase the sensitivity of biosensor, no further increase of sensitivity was achieved when the molar ratio of OPH to QDs was greater than 20 because the surface of QDs was saturated by OPH. These properties make the OPH/QDs bioconjugate a promising biosensor for the detection of organophosphorus compounds.

The development of DECON GREEN (Trademark) from its inception to the present is described, and efficacy data for VX, GD, TGD, HD, THD, and anthrax are presented. Examples of consumer products containing the identical or similar ingredients of DEC ON GREEN (Trademark) are given. The efficacy data reveals the tremendous decontamination efficacy afforded by a solvent-based, material-penetrating decontaminant. However, materials susceptible to agent absorption and absorption of the decontaminant are apt to suffer deleterious effects - the inevitable price of thorough decontamination.