The potential to manage releases of Bacillus anthracis using bioretention and a high flow media filter: Results of simulated runoff testing with tracer spores Bacillus globigii.

The threat of bioterrorism has spurred research on the decontamination and containment of different agents. Anthrax [causative agent Bacillus anthracis (Ba)] is a disease that can lead to severe infections within human and animals, particularly when inhaled. This research investigated the use of spore-contaminated simulated runoff events into stormwater control measures (SCMs), which are designed to retain and improve the quality of runoff and may have the potential to filter and contain the spores. In this study, the effectiveness of a bioretention cell (BRC) and high flow media filter (HFMF) in Huron, Ohio, were evaluated for removal of Bacillus globigii (Bg) spores (a harmless cognate of Ba). Three 4-8 mm simulated runoff events were created for each SCM using a fire hydrant and Bg spores were injected into the runoff upstream of the SCM inlets. The BRC significantly (p < 0.001) outperformed the HFMF in reducing Bg concentrations and loads, with an average load reduction of 1.9 log (∼99% reduction) compared to 0.4 (∼60% reduction), respectively. A probable critical design factor leading to these differences was the infiltration rate of the media and subsequent retention time within the filters, which was supported by similar disparities in suspended solids reductions. Differences in spore removal may also have been due to particle size distribution of the HFMF, which was more gravelly than the bioretention cell. At 3 and 6 months after the-simulated runoff tests, soil samples taken from both SCMs, yielding detectable Bg spores within the top 15 cm of media, with increased spore concentrations where ponding occurred for longer durations during the tests. This suggests that forebays and areas near inlets may be hotspots for spore cleanup in a real-world bioterrorism incident.

Passenger vehicle interior decontamination by low concentration hydrogen peroxide vapor following a wide area biological contamination incident.

AIMS: To assess low concentration hydrogen peroxide (LCHP) (H2O2) vapor dispersed with a home humidifier for its ability to decontaminate vehicle interiors contaminated with Bacillus anthracis surrogate Bacillus atrophaeus spores. METHODS AND RESULTS: Efficacy of a vaporized 3% H2O2 solution was evaluated for liquid volumes, on/off vehicle heating, ventilation, and air conditioning (HVAC) system operations, and temperatures that ranged from 5 to 27°C. Survival of the spores was assessed by quantification of remaining viable spores with efficacy quantified in terms of mean log10 reduction. Decontamination efficacy after the 6-day dwell time increased when the 3% H2O2 liquid volume was doubled, increasing from 4-of-10 to 10-of-10 nondetects (zero colonies counted using standard dilution and filter plating) inside the vehicle cabin. Recirculating cabin air through the HVAC system during decontamination decreased efficacy to 6-of-10 non-detects. While no 6-log10 reduction in viable spores was observed on the cabin filter with the cabin filter kept in place, a 6-log10 reduction was achieved after its removal and placement in the cabin during treatment. CONCLUSIONS: Results from this study allow for informed decisions on the use of LCHP vapor as an effective decontamination approach for vehicle interiors.

Evaluation of steam heat as a decontamination approach for SARS-CoV-2 when applied to common transit-related materials.

AIMS: The purpose of this study was to evaluate the efficacy of steam heat for inactivation of SARS-CoV-2 when applied to materials common in mass transit installations. METHODS AND RESULTS: SARS CoV-2 (USA-WA1/2020) was resuspended in either cell culture media or synthetic saliva, inoculated (∼1 × 106 TCID50) onto porous and nonporous materials and subjected to steam inactivation efficacy tests as either wet or dried droplets. The inoculated test materials were exposed to steam heat ranging from 70°C to 90°C. The amount of infectious SARS-CoV-2 remaining after various exposure durations ranging from 1 to 60 s was assessed. Higher steam heat application resulted in higher inactivation rates at short contact times. Steam applied at 1-inch distance (∼90°C at the surface) resulted in complete inactivation for dry inoculum within 2 s of exposure (excluding two outliers of 19 test samples at the 5-s duration) and within 2-30 s of exposure for wet droplets. Increasing the distance to 2 inches (∼70°C) also increased the exposure time required to achieve complete inactivation to 15 or 30 s for materials inoculated with saliva or cell culture media, respectively. CONCLUSIONS: Steam heat can provide high levels of decontamination (>3 log reduction) for transit-related materials contaminated with SARS-CoV-2 using a commercially available steam generator with a manageable exposure time of 2-5 s.

Sampling and recovery of infectious SARS-CoV-2 from high-touch surfaces by sponge stick and macrofoam swab.

Effective sampling for severe acute respiratory syndrome 2 (SARS-CoV-2) is a common approach for monitoring disinfection efficacy and effective environmental surveillance. This study evaluated sampling efficiency and limits of detection (LODs) of macrofoam swab and sponge stick sampling methods for recovering infectious SARS-CoV-2 and viral RNA (vRNA) from surfaces. Macrofoam swab and sponge stick methods were evaluated for collection of SARS-CoV-2 suspended in a soil load from 6-in2 coupons composed of four materials: stainless steel (SS), acrylonitrile butadiene styrene (ABS) plastic, bus seat fabric, and Formica. Recovery of infectious SARS-CoV-2 was more efficient than vRNA recovery on all materials except Formica (macrofoam swab sampling) and ABS (sponge stick sampling). Macrofoam swab sampling recovered significantly more vRNA from Formica than ABS and SS, and sponge stick sampling recovered significantly more vRNA from ABS than Formica and SS, suggesting that material and sampling method choice can affect surveillance results. Time since initial contamination significantly affected infectious virus recovery from all materials, with vRNA recovery showing limited to no difference, suggesting that SARS-CoV-2 vRNA can remain detectable after viral infectivity has dissipated. This study showed that a complex relationship exists between sampling method, material, time from contamination to sampling, and recovery of SARS-CoV-2. In conclusion, data show that careful consideration be used when selecting surface types for sampling and interpreting SARS-CoV-2 vRNA recovery with respect to presence of infectious virus.

Evaluation of two methods for detection of viable Bacillus anthracis simulant spores in maritime environmental samples.

Analytical methods exist to detect biothreat agents in environmental samples during a response to biological contamination incidents. However, the coastal zone facilities and assets of the US Coast Guard (USCG), including response boats in diverse geographical areas and maritime environmental conditions, can pose complex and unique challenges for adapting existing analytical detection methods. The traditional culture (TC) and the rapid viability polymerase chain reaction (RV-PCR) methods were evaluated for their compatibility for maritime environmental surface and grab sample analysis to detect spores of Bacillus thuringiensis subspecies kurstaki (Btk), a surrogate for Bacillus anthracis. The representative samples collected from a USCG installation included surfaces, such as aluminum on boats, nonskid tread on decks of watercraft, computer touchscreens, and concrete piers, and grab samples of boat washdown water, soil, vegetation, and gravel from surrounding areas. Replicate samples were spiked with Btk spores at two to three tenfold increasing levels and analyzed. Out of a total of 150 samples collected and analyzed, the TC method gave 10 false-positive and 19 false-negative results, while the RV-PCR method-based analysis resulted in 0 false-positive and 26 false-negative results. An abundance of microbial background and particulates in some samples interfered with true results, while both methods gave similar results for samples with low microbial background and particulates. Improved and high-throughput sample processing methods are needed for analysis of complex environmental samples.

Efficacy of detergent-based cleaning methods against coronavirus MHV-A59 on porous and non-porous surfaces.

This study evaluated the efficacy of detergent-based surface cleaning methods against Murine Hepatitis Virus A59 (MHV) as a surrogate coronavirus for SARS-CoV-2. MHV (5% soil load in culture medium or simulated saliva) was inoculated onto four different high-touch materials [stainless steel (SS), Acrylonitrile Butadiene Styrene plastic (ABS), Formica, seat fabric (SF)]. Immediately and 2-hr post-inoculation, coupons were cleaned (damp wipe wiping) with and without pretreatment with detergent solution or 375 ppm hard water. Results identified that physical removal (no pretreatment) removed >2.3 log10 MHV on ABS, SS, and Formica when surfaces were cleaned immediately. Pretreatment with detergent or hard water increased effectiveness over wet wiping 2-hr post-inoculation; pretreatment with detergent significantly increased (p ≤ 0.05) removal of MHV in simulated saliva, but not in culture media, over hard water pretreatment (Formica and ABS). Detergent and hard water cleaning methods were ineffective on SF under all conditions. Overall, efficacy of cleaning methods against coronaviruses are material- and matrix-dependent; pre-wetting surfaces with detergent solutions increased efficacy against coronavirus suspended in simulated saliva. This study provides data highlighting the importance of incorporating a pre-wetting step prior to detergent cleaning and can inform cleaning strategies to reducing coronavirus surface transmission.

Evaluation of sample processing methods to improve the detection of Bacillus anthracis in difficult sample matrices.

Large area sampling approaches have been developed and implemented by the US Environmental Protection Agency (EPA) to increase sample sizes, and potentially representativeness, in outdoor urban environments (e.g., concrete, asphalt, grass/landscaping). These sampling approaches could be implemented in response to an outdoor biological contamination incident or bioterrorism attack to determine the extent of contamination and for clearance following remediation. However, sample collection over large areas often contains an extensive amount of co-collected debris and native background microorganisms that interfere with the detection of biological threat agents. Sample processing methods that utilize basic laboratory equipment amenable to field deployment were selected and applied to turbid aqueous samples (TAS) to reduce particulates and native environmental organisms prior to culture and rapid viability-polymerase chain reaction (RV-PCR) analytical methods. Bacillus anthracis Sterne (BaS) spores were spiked into TAS collected by soil grab, wet vacuum collection from an outdoor concrete surface, or storm water runoff from an urban parking lot. The implementation of a sample processing method improved the sensitivity of culture and RV-PCR analytical methods for BaS spore detection in soil and wet vacuum TAS samples compared to baseline (minimal to no field processing methods applied). For soil, when the processing method was applied, samples with 15 colony forming units (CFU)/ml (60 CFU/g) and 1.5 CFU/mL (6 CFU/g) BaS spore load were detected using culture and RV-PCR, respectively. Most notably, the processing methods greatly improved the sensitivity of the RV-PCR analytical method for the wet vacuum TAS from no detection at the 1500 CFU/mL BaS spore load level to as low as 1.5 CFU/mL BaS spore load.

Evaluating the Environmental Persistence and Inactivation of MS2 Bacteriophage and the Presumed Ebola Virus Surrogate Phi6 Using Low Concentration Hydrogen Peroxide Vapor.

Ebola virus (EBOV) disease outbreaks, as well as the ability of EBOV to persist in the environment under certain conditions, highlight the need to develop effective decontamination techniques against the virus. We evaluated the efficacy of hydrogen peroxide vapor (HPV) to inactivate MS2 and Phi6 bacteriophages, the latter a recommended surrogate for EBOV. The phages were inoculated onto six material types with and without the presence of whole human blood. The inoculated materials were then exposed to either a high or low concentration of HPV for various elapsed times. The phages were also recovered from positive controls at these same elapsed times, to assess environmental persistence and decontamination efficacy. Low concentration hydrogen peroxide vapor (LCHP; 25 ppm) was effective against both phages on all materials without the presence of blood at 2 h. LCHP was ineffective against the phages in the presence of blood, on all materials, even with a 3-day contact time. Higher concentrations of HPV (>400 ppm) with contact times of 24-32 h achieved approximately 2-6 log reduction of the phages in the presence of blood.

Evaluation of standardized sample collection, packaging, and decontamination procedures to assess cross-contamination potential during Bacillus anthracis incident response operations.

Sample collection procedures and primary receptacle (sample container and bag) decontamination methods should prevent contaminant transfer between contaminated and non-contaminated surfaces and areas during bio-incident operations. Cross-contamination of personnel, equipment, or sample containers may result in the exfiltration of biological agent from the exclusion (hot) zone and have unintended negative consequences on response resources, activities and outcomes. The current study was designed to: (1) evaluate currently recommended sample collection and packaging procedures to identify procedural steps that may increase the likelihood of spore exfiltration or contaminant transfer; (2) evaluate the efficacy of currently recommended primary receptacle decontamination procedures; and (3) evaluate the efficacy of outer packaging decontamination methods. Wet- and dry-deposited fluorescent tracer powder was used in contaminant transfer tests to qualitatively evaluate the currently-recommended sample collection procedures. Bacillus atrophaeus spores, a surrogate for Bacillus anthracis, were used to evaluate the efficacy of spray- and wipe-based decontamination procedures. Both decontamination procedures were quantitatively evaluated on three types of sample packaging materials (corrugated fiberboard, polystyrene foam, and polyethylene plastic), and two contamination mechanisms (wet or dry inoculums). Contaminant transfer results suggested that size-appropriate gloves should be worn by personnel, templates should not be taped to or removed from surfaces, and primary receptacles should be selected carefully. The decontamination tests indicated that wipe-based decontamination procedures may be more effective than spray-based procedures; efficacy was not influenced by material type but was affected by the inoculation method. Incomplete surface decontamination was observed in all tests with dry inoculums. This study provides a foundation for optimizing current B. anthracis response procedures to minimize contaminant exfiltration.

Bacillus thuringiensis as a surrogate for Bacillus anthracis in aerosol research.

Characterization of candidate surrogate spores prior to experimental use is critical to confirm that the surrogate characteristics are as closely similar as possible to those of the pathogenic agent of interest. This review compares the physical properties inherent to spores of Bacillus anthracis (Ba) and Bacillus thuringiensis (Bt) that impact their movement in air and interaction with surfaces, including size, shape, density, surface morphology, structure and hydrophobicity. Also evaluated is the impact of irradiation on the physical properties of both Bacillus species. Many physical features of Bt and Ba have been found to be similar and, while Bt is considered typically non-pathogenic, it is in the B. cereus group, as is Ba. When cultured and sporulated under similar conditions, both microorganisms share a similar cylindrical pellet shape, an aerodynamic diameter of approximately 1 µm (in the respirable size range), have an exosporium with a hairy nap, and have higher relative hydrophobicities than other Bacillus species. While spore size, morphology, and other physical properties can vary among strains of the same species, the variations can be due to growth/sporulation conditions and may, therefore, be controlled. Growth and sporulation conditions are likely among the most important factors that influence the representativeness of one species, or preparation, to another. All Bt spores may, therefore, not be representative of all Ba spores. Irradiated spores do not appear to be a good surrogate to predict the behavior of non-irradiated spores due to structural damage caused by the irradiation. While the use of Bt as a surrogate for Ba in aerosol testing appears to be well supported, this review does not attempt to narrow selection between Bt strains. Comparative studies should be performed to test the hypothesis that viable Ba and Bt spores will behave similarly when suspended in the air (as an aerosol) and to compare the known microscale characteristics versus the macroscale response.

Effect of inoculation method on the determination of decontamination efficacy against Bacillus spores.

Decontamination studies investigating the effectiveness of products and processes for the inactivation of Bacillus species spores have traditionally utilized metering viable spores in a liquid suspension onto test materials (coupons). The current study addresses the representativeness of studies using this type of inoculation method compared to when coupons are dosed with a metered amount of aerosolized spores. The understanding of this comparability is important in order to assess the representativeness of such laboratory-based testing when deciding upon decontamination options for use against Bacillus anthracis spores. Temporal inactivation of B. anthracis surrogate (B. subtilis) spores on representative materials using fumigation with chlorine dioxide, spraying of a pH-adjusted bleach solution, or immersion in the solution was investigated as a function of inoculation method (liquid suspension or aerosol dosing). Results indicated that effectiveness, measured as log reduction, was statistically significantly lower when liquid inoculation was used for some material and decontaminant combinations. Differences were mostly noted for the materials observed to be more difficult to decontaminate (i.e., wood and carpet). Significant differences in measured effectiveness were also noted to be a function of the pH-adjusted bleach application method used in the testing (spray or immersion). Based upon this work and the cited literature, it is clear that inoculation method, decontaminant application method, and handling of non-detects (i.e., or detection limits) can have an impact on the sporicidal efficacy measurements.

Fate and transport of viable Bacillus anthracis simulant spores in ambient air during a large outdoor decontamination field exercise.

The Wide Area Demonstration (WAD) was a field exercise conducted under the U.S. EPA's Analysis of Coastal Operational Resiliency program, in conjunction with the U.S. Department of Homeland Security and the U.S. Coast Guard. The purpose of the WAD was to operationalize at field scale aspects of remediation activities that would occur following an outdoor release of Bacillus anthracis spores, including sampling and analysis, decontamination, data management, and waste management. The WAD was conducted in May 2022 at Fort Walker (formerly known as Fort A.P. Hill) and utilized Bacillus atrophaeus as a benign simulant for B. anthracis. B. atrophaeus spores were inoculated onto the study area at the beginning of the study, and air samples were collected daily during each of the different phases of the WAD using Dry Filter Units (DFUs). Ten DFU air samplers were placed at the perimeter of the study area to collect bioaerosols onto two parallel 47-mm diameter polyester felt filters, which were then subsequently analyzed in a microbiological laboratory for the quantification of B. atrophaeus. The study demonstrated the use of DFUs as a rugged and robust bioaerosol collection device. The results indicated that the highest B. atrophaeus spore air concentrations (up to ~ 5 colony forming units/m3) occurred at the beginning of the demonstration (e.g. during inoculation and characterization sampling phases) and generally downwind from the test site, suggesting transport of the spores was occurring from the study area. Very few B. atrophaeus spores were detected in the air after several weeks and following decontamination of exterior surfaces, thus providing an indication of the site decontamination procedures' effectiveness. No B. atrophaeus spores were detected in any of the blank or background samples.Implications: Following an incident involving a release of Bacillus anthracis spores or other biological threat agent into the outdoor environment, understanding the factors that may affect the bioagent's fate and transport can help predict viable contaminant spread via the ambient air. This paper provides scientific data for the first time on ambient air concentrations of bacterial spores over time and location during different phases of a field test in which Bacillus atrophaeus (surrogate for B. anthracis) spores were released outdoors as part of a full-scale study on sampling and decontamination in an urban environment. This study advances the knowledge related to the fate and transport of bacterial spores (such as those causing anthrax disease) as an aerosol in the outdoor environment over the course of three weeks in a mock urban environment and has exposure and health risk implications. The highest spore air concentrations occurred at the beginning of the study (e.g. during inoculation of surfaces and characterization sampling), and in the downwind direction, but diminished over time; few B. atrophaeus spores were detected in the air after several weeks and following decontamination. Therefore, in an actual incident, potential reaerosolization of the microorganism and subsequent transport in the air during surface sampling and remediation efforts should be considered for determining exclusion zone locations and estimating potential risk to neighboring communities. The data also provide evidence suggesting that the large-scale decontamination of outdoor surfaces may reduce air concentrations of the bioagent, which is important since exposure of B. anthracis via inhalation is a primary concern.

Evaluation of surface sampling for Bacillus spores using commercially available cleaning robots.

Five commercially available domestic cleaning robots were evaluated on their effectiveness for sampling aerosol-deposited Bacillus atrophaeus spores on different indoor material surfaces. The five robots tested include three vacuum types (R1, R2, and R3), one wet wipe (R4), and one wet vacuum (R5). Tests were conducted on two different surface types (carpet and laminate) with 10(6) colony forming units of B. atrophaeus spores deposited per coupon (35.5 cm × 35.5 cm). Spores were deposited on the center surface (30.5 × 30.5 cm) of the coupon's total surface area (71.5 × 71.5 cm), and the surfaces were sampled with an individual robot in an isolation chamber. Chamber air was sampled using a biofilter sampler to determine the potential for resuspension of spores during sampling. Robot test results were compared to currently used surface sampling methods (vacuum sock for carpet and sponge wipe for laminate). The test results showed that the average sampling efficacies for R1, R2, and R3 on carpet were 26, 162, and 92% of vacuum sock sampling efficacy, respectively. On laminate, R1, R2, R3, R4, and R5 average sampling efficacies were 8, 11, 2, 62, and 32% of sponge wipe sampling efficacy, respectively. We conclude that some robotic cleaners were as efficacious as the currently used surface sampling methods for B. atrophaeus spores on these surfaces.

The effects of environmental conditions on persistence and inactivation of Brucella suis on building material surfaces.

AIMS: The purpose of this study was to evaluate the effects of environmental conditions and material type on persistence and inactivation of Brucella suis. METHODS AND RESULTS: Brucella suis (approx. 1 × 10(8) CFU) was spiked onto surfaces (glass, aluminium and wood) by liquid inoculation. Persistence was evaluated over 56 days at 22 ± 2°C, 40 ± 15% r.h. and 5 ± 3°C, 30 ± 15% r.h. In addition, three readily available decontaminants (pH-adjusted bleach, 70% ethanol and 1% citric acid) were evaluated for their effectiveness at inactivating Br. suis on these materials. Decontaminations were conducted following 0 and 28 days exposure to the two conditions. Results indicated that Br. suis can persist on environmental surfaces for at least 56 days. Persistence was highest at low temperature. Decontamination was most challenging on wood with all three decontaminants. CONCLUSIONS: Following a Br. suis contamination incident, passive decontamination (through attenuation) may not be feasible, as this organism can persist for months. In addition, the results suggest that some sporicidal decontaminants may be ineffective on materials such as wood, even for vegetative biological agents such as Br. suis. SIGNIFICANCE AND IMPACT OF STUDY: This study aids incident commanders and remediation experts to make informed decisions regarding decontamination after a biological contamination incident.

Formaldehyde Vapor Characteristics in Varied Decontamination Environments.

INTRODUCTION: This effort investigated formaldehyde vapor characteristics under various environmental conditions by the analyses of air samples collected over a time-course. This knowledge will help responders achieve desired formaldehyde exposure parameters for decontamination of affected spaces after a biological contamination incident. METHODS: Prescribed masses of paraformaldehyde and formalin were sublimated or evaporated, respectively, to generate formaldehyde vapor. Adsorbent cartridges were used to collect air samples from the test chamber at predetermined times. A validated method was used to extract the cartridges and analyze for formaldehyde via liquid chromatography. In addition, material demand for the formaldehyde was evaluated by inclusion of arrays of Plexiglas panels in the test chamber to determine the impact of varied surface areas within the test chamber. Temperature was controlled with a circulating water bath connected to a radiator and fan inside the chamber. Relative humidity was controlled with humidity fixed-point salt solutions and water vapor generated from evaporated water. RESULTS: Low temperature trials (approximately 10°C) resulted in decreased formaldehyde air concentrations throughout the 48-hour time-course when compared with formaldehyde concentrations in the ambient temperature trials (approximately 22°C). The addition of clear Plexiglas panels to increase the surface area of the test chamber interior resulted in appreciable decreases of formaldehyde air concentration when compared to an empty test chamber. CONCLUSION: This work has shown that environmental variables and surface-to-volume ratios in the decontaminated space may affect the availability of formaldehyde in the air and, therefore, may affect decontamination effectiveness.

Decontamination of Bacillus Spores with Formaldehyde Vapor under Varied Environmental Conditions.

Introduction: This study investigated formaldehyde decontamination efficacy against dried Bacillus spores on porous and non-porous test surfaces, under various environmental conditions. This knowledge will help responders determine effective formaldehyde exposure parameters to decontaminate affected spaces following a biological agent release. Methods: Prescribed masses of paraformaldehyde or formalin were sublimated or evaporated, respectively, to generate formaldehyde vapor within a bench-scale test chamber. Adsorbent cartridges were used to measure formaldehyde vapor concentrations in the chamber at pre-determined times. A validated method was used to extract the cartridges and analyze for formaldehyde via liquid chromatography. Spores of Bacillus globigii, Bacillus thuringiensis, and Bacillus anthracis were inoculated and dried onto porous bare pine wood and non-porous painted concrete material coupons. A series of tests was conducted where temperature, relative humidity, and formaldehyde concentration were varied, to determine treatment efficacy outside of conditions where this decontaminant is well-characterized (laboratory temperature and humidity and 12 mg/L theoretical formaldehyde vapor concentration) to predict decontamination efficacy in applications that may arise following a biological incident. Results: Low temperature trials (approximately 10°C) resulted in decreased formaldehyde air concentrations throughout the 48-hour time-course when compared with formaldehyde concentrations collected in the ambient temperature trials (approximately 22°C). Generally, decontamination efficacy on wood was lower for all three spore types compared with painted concrete. Also, higher recoveries resulted from painted concrete compared to wood, consistent with historical data on these materials. The highest decontamination efficacies were observed on the spores subjected to the longest exposures (48 hours) on both materials, with efficacies that gradually decreased with shorter exposures. Adsorption or absorption of the formaldehyde vapor may have been a factor, especially during the low temperature trials, resulting in less available formaldehyde in the air when measured. Conclusion: Environmental conditions affect formaldehyde concentrations in the air and thereby affect decontamination efficacy. Efficacy is also impacted by the material with which the contaminants are in contact.

Pseudomonas aeruginosa PqsA is an anthranilate-coenzyme A ligase.

Pseudomonas aeruginosa is an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. The Pseudomonas quinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. The pqsABCDE operon has been identified as being necessary for PQS production, and the pqsA gene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway in P. aeruginosa, we have characterized the function of the pqsA gene product. Extracts prepared from Escherichia coli expressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of anthranilate. Inhibition of PQS formation in vivo was observed for the chloro- and fluoroanthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling in P. aeruginosa.

Evaluation of sampling methods for Bacillus spore-contaminated HVAC filters.

The objective of this study was to compare an extraction-based sampling method to two vacuum-based sampling methods (vacuum sock and 37mm cassette filter) with regards to their ability to recover Bacillus atrophaeus spores (surrogate for Bacillus anthracis) from pleated heating, ventilation, and air conditioning (HVAC) filters that are typically found in commercial and residential buildings. Electrostatic and mechanical HVAC filters were tested, both without and after loading with dust to 50% of their total holding capacity. The results were analyzed by one-way ANOVA across material types, presence or absence of dust, and sampling device. The extraction method gave higher relative recoveries than the two vacuum methods evaluated (p≤0.001). On average, recoveries obtained by the vacuum methods were about 30% of those achieved by the extraction method. Relative recoveries between the two vacuum methods were not significantly different (p>0.05). Although extraction methods yielded higher recoveries than vacuum methods, either HVAC filter sampling approach may provide a rapid and inexpensive mechanism for understanding the extent of contamination following a wide-area biological release incident.

Inactivation of vegetative bacterial threat agents on environmental surfaces.

Following a wide-area biological terror attack, numerous decontamination technologies, techniques, and strategies will be required for rapid remediation. Establishing an understanding of how disinfectants will perform under field conditions is of critical importance. The purpose of this study was to determine the efficacy of several liquid decontaminants, when used to inactivate vegetative biological agents on environmental surfaces. Aluminum, carpet, concrete, glass, and wood coupons were inoculated with 1×10(8) CFU of Burkholderia mallei, Francisella tularensis, Vibrio cholerae, or Yersinia pestis. Using spray-based application methods, decontamination was then attempted with pH-adjusted bleach, 1% citric acid, 70% ethanol, quaternary ammonia, or Pine-Sol®. Results indicated that decontamination efficacy varied significantly by decontaminant and organism. Materials such as wood are difficult to decontaminate, even when using sporicides. The data presented here will help responders develop efficacious remediation strategies following a large-scale contamination incident.