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.

Use of semi-permeable bag materials to facilitate on-site treatment of biological agent-contaminated waste.

Clean up following the wide-area release of a persistent biological agent has the potential to generate significant waste. Waste containing residual levels of biological contaminants may require off-site shipment under the U.S. Department of Transportation's (US DOT) solid waste regulations for Category A infectious agents, which has packaging and size limitations that do not accommodate large quantities. Treating the waste on-site to inactivate the bio-contaminants could alleviate the need for Category A shipping and open the possibility for categorizing the waste as conventional solid waste with similar shipping requirements as municipal garbage. To collect and package waste for on-site treatment, a semi-permeable nonwoven-based fabric was developed. The fabric was designed to contain residual bio-contaminants while providing sufficient permeability for penetration by a gaseous decontamination agent. The nonwoven fabric was tested in two bench-scale experiments. First, decontamination efficacy and gas permeability were evaluated by placing test coupons inoculated with spores of a Bacillus anthracis surrogate inside the nonwoven material. After chlorine dioxide fumigation, the coupons were analyzed for spore viability and results showed a ≥6 Log reduction on all test materials except glass. Second, filters cut from the nonwoven material were tested in parallel with commercially available cellulose acetate filters having a known pore size (0.45 µm) and results demonstrate that the two materials have similar permeability characteristics. Overall, results suggest that the nonwoven material could be used to package waste at the point of generation and then moved to a nearby staging area where it could be fumigated to inactivate bio-contaminants.

Virucidal efficacy of antimicrobial surface coatings against the enveloped bacteriophage Φ6.

AIMS: Antimicrobial coatings, for use in combination with routine cleaning and disinfection, were evaluated for their effectiveness in reducing virus concentration on stainless steel surfaces. METHODS: Twenty antimicrobial coating products, predominantly composed of organosilane quaternary ammonium compounds, were applied to stainless steel coupons, dried overnight and evaluated for efficacy against Φ6, an enveloped bacteriophage. Additionally, two peel and stick polymer-based films, a copper-based film and three copper alloys were evaluated. Efficacy was determined by comparison of recoveries from uncoated (positive control) and coated (test) surfaces. RESULTS: The results indicated that some of the coating products initially demonstrated >3-log reduction of Φ6; no direct correlation of efficacy was observed with an active ingredient or its concentration. The peel and stick films and copper alloys each demonstrated efficacy in initial testing. However, none of the spray-based products retained efficacy after subjecting the coating to abrasion with either a hypochlorite or quaternary ammonium-based solution applied in accordance with EPA Interim Guidance for Evaluating the Efficacy of Antimicrobial Surface Coatings. Of the products tested for this durability, only one peel and stick polymeric film retained efficacy; the copper alloys were not tested for their durability in this study. CONCLUSIONS: These results suggest that while some organosilane quaternary ammonium compound-based products demonstrate antiviral efficacy, more research and development is needed to understand effective formulations with sufficient durability to perform as supplements to routine cleaning and disinfection.

Decontamination of soil contaminated at the surface with Bacillus anthracis spores using dry thermal treatment.

In the event of a large, aerosol release of Bacillus anthracis spores in a major metropolitan area, soils and other outdoor materials may become contaminated with the biological agent. A study was conducted to assess the in-situ remediation of soil using a dry thermal treatment approach to inactivate a B. anthracis spore surrogate inoculated into soil samples. The study was conducted in two phases, using loam, clay and sand-based soils, as well as biological indicators and spore-inoculated stainless-steel coupons. Initial experiments were performed in an environmental test chamber with temperatures controlled between 80 and 110 °C, with and without added humidity, and with contact times ranging from 4 h to 7 weeks. Tests were then scaled up to assess the thermal inactivation of spores in small soil columns, in which a heating plate set to 141 °C was applied to the soil surface. These column tests were conducted to assess time requirements to inactivate spores as a function of soil depth and soil type. Results from the initial phase of testing showed that increasing the temperature and relative humidity reduced the time requirements to achieve samples in which no surrogate spores were detected. For the test at 80 °C with no added humidity, 49 days were required to achieve soil samples with no spores detected in clay and loam. At 110 °C, 24 h were required to achieve samples in which no spores were detected. In the column tests, no spores were detected at the 2.5 cm depth at four days and at the 5.1 cm depth at 21 days, for two of the three soils. The experiments described in the study demonstrate the feasibility of using dry thermal techniques to decontaminate soils that have been surficially contaminated with B. anthracis spores.

Comparison of surface sampling methods for an extended duration outdoor biological contamination study.

Bacillus anthracis, the causative agent for anthrax, is a dangerous pathogen to humans and has a history as a bioterrorism agent. While sampling methods have been developed and evaluated for characterizing and clearing contaminated indoor sites, the performance of these sampling methods is unknown for use in outdoor environments. This paper presents surface sampling data for Bacillus atrophaeus spores, a surrogate for B. anthracis, from a 210-day outdoor study that evaluated the detection and recovery of spores using five different sampling methods as follows: sponge sticks, 37-mm vacuum filter cassettes, residential wet vacuums, robotic floor cleaners, and grab samples of soil, leaves, and grass. The spores were applied by spraying a liquid suspension onto the surfaces. Both asphalt and concrete surfaces were sampled by all the surface sampling methods, excluding grab sampling. Stainless steel coupons placed outdoors were additionally sampled using sponge sticks. Sampling methods differed in their ability to collect detectable spores over the duration of the study. The 37-mm vacuums and sponge sticks consistently detected spores on asphalt through day 37 and robots through day 99. The wet vacuums detected spores on asphalt for days 1 and 4, but not again until day 210. On concrete, all samplers detected spores until day 210 except for sponge stick samplers that detected spores only up until the day 99 time point. For all sampling methods, spore recoveries were higher from concrete than from asphalt surfaces. There was no statistically significant difference in recoveries of sponge sticks and 37-mm vacuums from either asphalt or concrete surfaces. Processing of grab samples was challenging due to non-target background microorganisms resulting in high detection limits for the samples.

Alternative fast analysis method for cellulose sponge surface sampling wipes with low concentrations of Bacillus Spores.

Environmental sampling is a critical component of the post decontamination verification process following a bioterrorism event. The current work was performed to produce a less labor-intensive method for processing cellulose sponge-wipes used for sampling areas potentially contaminated with low concentrations (i.e., post-decontamination) of Bacillus anthracis spores. An alternative fast-analysis processing method was compared to the processing protocol validated by the Centers for Disease Control and Prevention (CDC) for the Laboratory Response Network (LRN). Glazed tile coupons (1102 cm2) were inoculated with 50, 500, or 5000 spores of Bacillus thuringiensis subsp. kurstaki (Btk), then sampled with cellulose sponges. Sampling was limited to a 25- by 25-cm area and performed in the same manner as the CDC sampling method. Samples were then processed using either the alternative "Fast Analysis" method or the "CDC method". Three different analysts repeated the tests at each concentration utilizing each method. Mean recoveries, labor time, and potentially hazardous waste produced were compared for the two methods. The mean percent recoveries and standard errors for the samples processed using the "CDC method" were 39.9 ±â€¯6.7, 43 ±â€¯7.6, and 36.8 ±â€¯10.1 for the 5000, 500, and 50 spore loading levels, respectively; compared to 54.2 ±â€¯12.9, 64.2 ±â€¯21.7, and 45.2 ±â€¯8.6 for the "Fast Analysis" method. At each titer tested the "Fast Analysis" method resulted in a statistically significant higher percent recovery. Furthermore, analysts processed samples utilizing the "Fast Analysis" method in less than half the time and generated half as much potentially hazardous waste compared to the "CDC method".

A novel gateway-compatible binary vector series (PC-GW) for flexible cloning of multiple genes for genetic transformation of plants.

The rapidly advancing field of plant synthetic biology requires transforming plants with multiple genes. This has sparked a growing interest in flexible plant transformation vectors, which can be used for multi-gene transformations. We have developed a novel binary vector series, named the PC-GW series (GenBank: KP826769-KP826773), for Agrobacterium-mediated plant transformation. The PC-GW vectors use the pCAMBIA vector backbone, and contain NPTII, hpt, bar, mCherry or egfp genes as selectable markers for plant transformation. In a modified multiple cloning site (MCS) of the T-DNA region, we have placed the attR1, attR2 and ccdB sequences for rapid cloning of one to four genes by Gateway™-assisted recombination. In addition, we have introduced four meganuclease sites, and other restriction sites for multi-gene vector construction. Finally, we have placed a CaMV 35S promoter and a 35S terminator on the 5' and 3' ends of the MCS. The CaMV 35S promoter is flanked by PstI restriction sites that can be used to replace it with another promoter sequence if needed. The PC-GW vectors provide choices for selectable markers, cloning methods, and can accommodate up to eight gene constructs in a single T-DNA, thereby significantly reducing the number of transformations or crosses needed to generate multi-transgene expressing plants.

Rickettsia rickettsii transmission by a lone star tick, North Carolina.

Only indirect or circumstantial evidence has been published to support transmission of Rickettsia rickettsii by Amblyomma americanum (lone star) ticks in North America. This study provides molecular evidence that A. americanum ticks can function, although most likely infrequently, as vectors of Rocky Mountain spotted fever for humans.

Identification of tertiary butyl alcohol (TBA)-utilizing organisms in BioGAC reactors using 13C-DNA stable isotope probing.

Biodegradation of the gasoline oxygenates methyl tertiary-butyl ether (MTBE) and ethyl tertiary-butyl ether (ETBE) can cause tertiary butyl alcohol (TBA) to accumulate in gasoline-impacted environments. One remediation option for TBA-contaminated groundwater involves oxygenated granulated activated carbon (GAC) reactors that have been self-inoculated by indigenous TBA-degrading microorganisms in ground water extracted from contaminated aquifers. Identification of these organisms is important for understanding the range of TBA-metabolizing organisms in nature and for determining whether self-inoculation of similar reactors is likely to occur at other sites. In this study (13)C-DNA-stable isotope probing (SIP) was used to identify TBA-utilizing organisms in samples of self-inoculated BioGAC reactors operated at sites in New York and California. Based on 16S rRNA nucleotide sequences, all TBA-utilizing organisms identified were members of the Burkholderiales order of the ß-proteobacteria. Organisms similar to Cupriavidus and Methylibium were observed in both reactor samples while organisms similar to Polaromonas and Rhodoferax were unique to the reactor sample from New York. Organisms similar to Hydrogenophaga and Paucibacter strains were only detected in the reactor sample from California. We also analyzed our samples for the presence of several genes previously implicated in TBA oxidation by pure cultures of bacteria. Genes Mpe_B0532, B0541, B0555, and B0561 were all detected in (13)C-metagenomic DNA from both reactors and deduced amino acid sequences suggested these genes all encode highly conserved enzymes. One gene (Mpe_B0555) encodes a putative phthalate dioxygenase-like enzyme that may be particularly appropriate for determining the potential for TBA oxidation in contaminated environmental samples.

Isotopic fractionation of methyl tert-butyl ether suggests different initial reaction mechanisms during aerobic biodegradation.

Carbon isotopic enrichment factors (epsilonC) measured during cometabolic biodegradation of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) by Pseudonocardia tetrahydrofuranoxydans strain K1 were -2.3 +/- 0.2 per thousand, -1.7 +/- 0.2 per thousand, and -1.7 +/- 0.3 per thousand, respectively. The measured carbon apparent kinetic isotope effect was 1.01 for all compounds, consistent with the expected kinetic isotope effects for both oxidation of the methoxy (or ethoxy) group and enzymatic SN1 biodegradation mechanisms. Significantly, delta13C measurements of the tert-butyl alcohol and tert-amyl alcohol products indicated that the tert-butyl and tert-amyl groups do not participate in the reaction and confirmed that ether biodegradation by strain K1 involves oxidation of the methoxy (or ethoxy) group. Measured hydrogen isotopic enrichment factors (epsilonH) were -100 +/- 10 per thousand, -73 +/- 7 per thousand, and -72 +/- 20 per thousand for MTBE, ETBE, and TAME respectively. Previous results reported for aerobic biodegradation of MTBE by Methylibium petroleiphilum PM1 and Methylibium R8 showed smaller epsilonH values (-35 per thousand and -42 per thousand, respectively). Plots of Delta2H/Delta13C show different slopes for strain K1 compared with strains PM1 and R8, suggesting that different mechanisms are utilized by K1 and PM1/R8 during aerobic MTBE biodegradation.