EPA/600/R-22/058 | October 2022 www.epa.gov/emergency-response-research United States Environmental Protection Agency oEPA Bacillus anthracis Surrogates Enhanced for Use in Environmental Studies: A Review Literature Review Office of Research and Development Homeland Security Research Program ------- Bacillus anthracis surrogates enhanced for use in environmental studies: a review Literature Review EPA Homeland Security and Materials Management Division Technical Lead Person: M. Worth Calfee, Ph.D. U.S. Environmental Protection Agency Office of Research and Development Center for Environmental Solutions and Emergency Response Homeland Security and Materials Management Division Research Triangle Park, NC 27711 Prepared by: Denise Aslett, Ph.D. and Ahmed Abdel-Hady Ud COPS Contract 68HERC20D0018 ii ------- Disclaimer The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development's National Homeland Security Research Center, funded and managed this investigation through Contract No. 68HERC20D0018 with Jacobs Technology, Inc. (Jacobs). This report has been peer and administratively reviewed and approved for publication as an Environmental Protection Agency document. It does not necessarily reflect the views of the Environmental Protection Agency. No official endorsement should be inferred. This report includes photographs of commercially available products. The photographs are included for purposes of illustration only and are not intended to imply that EPA approves or endorses the product or its manufacturer. EPA does not endorse the purchase or sale of any commercial products or services. Questions concerning this document or its application should be addressed to the following individual: M. Worth Calfee, Ph.D. Homeland Security Materials Management Division Center for Environmental Solutions and Emergency Response U.S. Environmental Protection Agency 109 T.W. Alexander Drive Research Triangle Park, NC 27711 Telephone No.: (919) 541-7600 E-mail Address: Calfee.Worth@epa.gov ------- Forward The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the Nation's land, air, and water resources. Under a mandate of national environmental laws, the Agency strives to formulate and implement actions leading to a compatible balance between human activities and the ability of natural systems to support and nurture life. To meet this mandate, EPA's research program is providing data and technical support for solving environmental problems today and building a science knowledge base necessary to manage our ecological resources wisely, understand how pollutants affect our health, and prevent or reduce environmental risks in the future. The Center for Environmental Solutions and Emergency Response (CESER) within the Office of Research and Development (ORD) conducts applied, stakeholder-driven research and provides responsive technical support to help solve the Nation's environmental challenges. The Center's research focuses on innovative approaches to address environmental challenges associated with the built environment. We develop technologies and decision-support tools to help safeguard public water systems and groundwater, guide sustainable materials management, remediate sites from traditional contamination sources and emerging environmental stressors, and address potential threats from terrorism and natural disasters. CESER collaborates with both public and private sector partners to foster technologies that improve the effectiveness and reduce the cost of compliance, while anticipating emerging problems. We provide technical support to EPA regions and programs, states, tribal nations, and federal partners, and serve as the interagency liaison for EPA in homeland security research and technology. The Center is a leader in providing scientific solutions to protect human health and the environment. This report summarizes the findings from a literature survey intended to understand the state of the science behind Bacillus anthracis surrogates, specifically surrogates intended for use in outdoor field studies. A unique set of requirements exists for surrogate bacterial strains, when released into the environment. In addition to being nonpathogenic, surrogates should approximate the behavior of Bacillus anthracis with regards to survival in the environment, resistance to chemical and physical decontamination measures, transport by environmental forces (e.g., rain, wind, groundwater, surface water) and recovery by sampling methods. The surrogate should also be distinguishable from naturally present organisms during analysis. This report outlines those considerations, documents existing bacterial strains that may have utility as a Bacillus anthracis surrogate, analytical advancements that may be used to enhance detection in environmental samples, and genetic alterations that could further improve detection. This information is intended to support field-scale testing of remediation capabilities and enhance our Nation's ability to respond and recover to biological contamination incidents. Gregory Sales, Director Center for Environmental Solutions and Emergency Response iv ------- Contents Acknowledgments vi Executive Summary vii Introduction 1 Spore Property Considerations 5 Spore Detection Considerations 11 Common Bacillus anthracis Surrogates 19 Bacillus anthracis Sterne 19 Bacillus atrophaeus 21 Bacillus thuringiensis 22 Summary of Properties for Bacillus anthracis and Selected Surrogates 26 Bacillus anthracis Surrogates Enhanced for Environmental Sampling 30 Bacillus thuringiensis subsp. kurstaki HD-1 enhanced with an acrystalliferous phenotype 30 Enhanced strains of acrystalliferous Bacillus thuringiensis BMB171 34 Stable genetic insertions in Bacillus thuringiensis subsp. kurstaki HD-1 37 Non-viable DNA-barcoded aerosol test particles 40 B. anthracis Sterne enhanced with fluorescence genes 41 Bioluminescent reporter phage 42 Summary of Bacillus anthracis Surrogates Enhanced for Environmental Sampling 47 Bacillus anthracis Surrogates for Environmental Sampling - Desired Characteristics, Recommendations, and Conclusion 51 References 56 Appendix A - Methods 83 List of Tables Table 1.0 - Summary List of Enhanced Bacillus anthracis Surrogates viii Table 2.0 - Summary List of Desired Characteristics for Bacillus anthracis Surrogates ix Table 3.0 - Characteristics of Bacillus anthracis and Common Surrogates 28 Table 4.0 - Detailed List of Enhancements for Bacillus anthracis Surrogates 48 Table 5.0 - Desired Characteristics for Bacillus anthracis Surrogates 51 v ------- Acknowledgments This effort was directed by the principal investigator from the Homeland Security Materials Management Division, with support of a project team consisting of staff from the U.S. Environmental Protection Agency and Jacobs Technology. The contributions of the following individuals have been a valued asset throughout this effort: U.S. Environmental Protection Agency (EPA) M. Worth Calfee, Ph.D., Principal Investigator; U.S. EPA Office of Research and Development, Center for Environmental Solutions and Emergency Response, Homeland Security and Materials Management Division, RTP, NC. Anne Mikelonis, Ph.D., P.E., Environmental Engineer, U.S. EPA Office of Research and Development, Center for Environmental Solutions and Emergency Response, Homeland Security and Materials Management Division, RTP, NC. Erin Silvestri, U.S. EPA Office of Research and Development, Center for Environmental Solutions and Emergency Response, Homeland Security and Materials Management Division, Cincinnati, OH. Sanjiv R. Shah, Ph.D., U.S. EPA Office of Research and Development, Center for Environmental Solutions and Emergency Response, Homeland Security and Materials Management Division, Washington, DC. Jacobs Technology Inc. Denise Aslett, Ph.D. Ahmed Abdel-Hady Mariela Monge Brian Ford Kathleen May Abderrahmane Touati, Ph.D. vi ------- Executive Summary The 2001 dissemination of virulent Bacillus anthracis spores through the U.S. Postal Service (USPS) and subsequent clean-up efforts identified significant shortcomings in consequence management of biological contamination incidents. Validated sampling and decontamination measures for Bacillus spores were largely unavailable at that time. Research to improve sampling, analysis, and decontamination strategies for common building materials such as carpet, wood, concrete, and glass was accelerated. However, research gaps remain for samples originating from complex environmental matrices, such as soil, ground water, or vegetation. To address these gaps, significant research studies still need to be executed. Such studies are usually conducted using non-pathogenic surrogates of virulent Bacillus anthracis. Prior studies have commonly used surrogates including Bacillus atrophaeus var. globigii and Bacillus thuringensis; however, surrogates that may be easily distinguished from native organisms and safely used in outdoor settings are needed. Ideal surrogates not only have a genetic similarity to B. anthracis, but also similar physical properties such as spore size, shape, surface architecture, hydrophobicity, and charge. All of these characteristics contribute to spore interactions with environmental substrates. Spore detection and sample analysis methods must also be considered. Environmental samples may contain background organisms that often complicate analysis of samples containing B. anthracis surrogates. The traditional microbiological culture-based analytical method for B. anthracis is especially impacted by this problem. Target organisms form colonies that are often indistinguishable from those of non-target organisms making enumeration of viable spores problematic. Further, non-target organisms may outcompete germinating spores for nutrients effectively inhibiting spore growth, especially for samples with low spore concentrations. Other analysis methods, such as quantitative Polymerase Chain Reaction (qPCR) or Rapid Viability-PCR (RV-PCR), depend on detection of a molecular target (e.g., DNA) and are being developed for use with environmental samples. However, qPCR does not provide viability vii ------- data and spore coat durability often prevents efficient spore disruption and DNA release. RV-PCR includes a culturing protocol to provide viability data and eliminate spore lysis issues, but the presence of background contamination may inhibit spore germination or outgrowth. In recognition of these considerations and challenges, several surrogates have been enhanced with new properties potentially making them more compatible with outdoor release, environmental sampling, and sample analysis and detection. The purpose of this report was threefold. The first was to identify the existing enhanced surrogates and review the available literature describing their development and use. The second was to propose characteristics desired for surrogates intended for outdoor studies. And finally, the third was to propose options for obtaining a surrogate with the desired features. Table 1.0 provides a summary list of the existing B. anthracis surrogates with enhancements described in the literature. Table 1.0 - Summary List of Enhanced Bacillus anthracis Surrogates B. anthracis surrogate Enhancement(s) Significance References B. thuringiensis subsp. kurstaki HD-1 Eliminated crystal production During sporulation, crystals that are toxic to certain insects and difficult to remove from spore preparations are produced. Crystals may attach to spores and impact aerodynamics as well as physical transport through environmental matrices. Bishop & Robinson, 2014 B. thuringiensis BMB171 Introduced yellow pigment-producing genes and disrupted genes coding for sporulation regulation, small acid soluble protein production, and global regulation of virulence factors The yellow pigment provides a distinguishing phenotype on rich media. Other changes were designed for added environmental safety. When spoOA is disrupted, germinated spores are prevented from re-sporulating. And when spores lack small acid-soluble proteins and the PIcR regulator, they are potentially less likely to persist in the environment. Park etal., 2017 B. thuringiensis subsp. kurstaki HD-1 Added unique DNA identifiers known as barcodes Chromosomal insertion of barcodes provides replication stability and unique identifiers that distinguish the organism from background Bacillus, but parasporal crystal formation is retained. Genomic regions suitable for barcode insertion may be identified with a bioinformatics tool, known as barCoder. Buckley et al., 2012; Bernhards et al., 2021 viii ------- Table 1.0 - Summary List of Enhanced Bacillus anthracis Surrogates B. anthracis surrogate Enhancement(s) Significance References DNATrax Created non-viable particles Particles referred to as DNATrax (DNA Tagged Reagents for Aerosol experiments) are composed of short, customizable oligonucleotide sequences attached to maltodextrin. DNATrax particles are non-viable, providing added safety, and are similar in size and shape to B. anthracis spores. Harding et al., 2016 B. anthracis Sterne Introduced fluorescence genes B. anthracis Sterne is an attenuated B. anthracis strain that has been used in veterinary vaccine production. A constitutive Bacillus promoter plus genes responsible for production of green fluorescent protein (GFP) or red fluorescent protein (RFP) were inserted into the chromosome, providing a distinguishing phenotype for colonies growing on rich media. Su et al., 2014 phage WP (used to detect B. anthracis or B. anthracis Sterne) Introduce the luxAB bioluminescent reporter cassette Spore detection requires germination and outgrowth, as the WP phage infects only metabolically active cells then emits a detectable bioluminescence. Schofield & Westwater, 2009 While each of the existing enhanced strains have some of the characteristics desired for outdoor sampling, none of them have all. Table 2.0 provides a list of characteristics desired for surrogates intended for outdoor studies. Table 2.0 - Summary List of Desired Characteristics for Bacillus anthracis Surrogates Desired Characteristics Significance Spore size, shape, and surface properties (charge, protein content, surface adhesion, architecture, exosporium, appendages) similar to B. anthracis These properties affect spore transport and fate in the environment Absence of parasporal crystal inclusions Crystals may adhere to spores and affect transport dynamics Spore inactivation properties (UV, heat, and chemical resistance) similar to or greater than B. anthracis Inactivation strategies that are effective for the surrogate should be effective for B. anthracis Spore aerosol transport and resuspension characteristics similar to B. anthracis These properties affect spore transport and fate in the environment History of safe outdoor use Public safety Absence of virulence factors Public safety Manifests a distinguishing phenotype, such as pigmentation or fluorescence, when cultured Aids sample enumeration with conventional microbiological methods Has a molecular fingerprint that is distinct from background, non-target, heat-resistant organisms Allows for PCR and RV-PCR detection options Safe and cost-effective methods are available to extract, detect, and enumerate viable spores in samples from complex matrices, with sensitivity and specificity Surrogate selection must include consideration for detection, especially if sample enumeration of viable spores is desired Commercial availability Commercial availability is desired especially if enhanced strains cannot be obtained from an academic or government lab ------- Several options could be considered for the development of a B. anthracis surrogate possessing all or nearly all of the desired characteristics listed in Table 2.0. One option is to try recovering spores of existing B. anthracis surrogates on traditional rich media supplemented with a selective agent. Selective plating methods that exclude growth of non-target organisms could provide a safe, inexpensive, and simple way to incorporate viability data into test results. Another option is to further enhance an existing surrogate strain that has a strong safety record and history of outdoor use. For example, B. thuringiensis subsp. kurstaki HD-1 has been widely used in pesticides and has already been modified with chromosomally located bar codes. Plasmid curing methods described by Bishop and Robinson (2014) could be used to eliminate parasporal crystal formation. Second, and more difficult to achieve, would be the chromosomal addition of color- producing genes. The addition of pigmentation or fluorescence would enhance detection by traditional agar plating, especially for environmental samples that may have low concentrations of background organisms. These changes would also open the possibility of designing additional TaqMan probes that target not only the existing bar codes but also the pigmentation genes. Third, explore the use of additional genome editing technologies for surrogate enhancement. Once referred to as a bacterial immune system, the clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR- associated proteins (Cas) system has been used to modify the genomes of several Bacillus organisms. The CRISPR/Cas-9 system possibly could be used to introduce point mutations in the existing B. thuringiensis bar codes to create new, unique bar codes. Fourth, the concept of synthetic auxotrophy could be explored. In this scenario, growth of the surrogate strain would be dependent on use of a synthetic amino acid, which would be exogenously supplied in the laboratory. This approach has been demonstrated in the context of biocontainment but whether or not it could work as a selective mechanism for detecting target spores in environmentally derived samples is unknown. Finally, an x ------- immuno-capture concept could be explored. Antibodies designed to target a spore surface protein could be constructed and tagged with the small molecule biotin. A stationary streptavidin matrix could then be used to capture the biotin-tagged spores, thereby exploiting one of the strongest interactions known in nature. Captured spores could then be germinated on rich media and distinguished from other non-target Bacillus organisms by the enhanced pigmentation. xi ------- Introduction Prior to the 2001 dissemination of virulent Bacillus anthracis through the U.S. Postal Service (USPS), a large accidental release of anthrax-causing spores occurred in 1979 at a military research facility in Sverdlosvsk (now known as Ekaterinburg) in the former U.S.S.R. (Meselson et al., 1994; Sahl et al., 2016). Subsequent investigations estimated that the widespread contamination caused at least 66 human deaths from inhalational anthrax (Meselson et al., 1994; Sahl et al., 2016). During the 2001 "Amerithrax" case (U.S. Department of Justice, 2010), gram quantities of B. anthracis spores released through the USPS resulted in 22 anthrax cases and five fatalities (Jernigan et al., 2002; Webb, 2003). There was also widespread public alarm, long-term facility closures, analysis of over 125,000 samples (Hughes & Gerberding, 2002; U.S. Department of Health and Human Services [U.S. DHHS], 2019), ciprofloxacin prophylaxis for at least 10,000 people (Bush & Perez, 2012), large-scale decontamination and clean-up efforts, and investigations by multiple law enforcement agencies. The combined economic burden was estimated in the billions of dollars (Bush & Perez, 2012). The attack also identified significant knowledge and research gaps in consequence management of biological contamination incidents (Franco & Bouri, 2010). Validated sampling and decontamination measures for Bacillus spores, known to persist for long periods in unfavorable conditions (Manchee et al., 1981; Sinclair et al., 2008; U.S. Army Medical Research, 2011; Wood et al., 2015), were largely unavailable in 2001 (Canter, 2008). Site characterization, remediation, and clearance strategies were further complicated by the need to address multiple surface types, such as intricate mail processing equipment, ductwork, non-porous surfaces like tile floors, and porous materials like ceiling tiles and carpet. As the sampling approaches and clean-up measures were evaluated, the need for additional methods research became apparent (Canter, 2008). In 2005, the U.S. Government Accountability Office issued a report highlighting the need 1 ------- for methods validation (Rhodes, 2005). Applied research efforts were accelerated, but many challenges associated with contaminated site assessment, sample collection, analysis, and field data interpretation remain (Fitch et al., 2003; Silvestri et al., 2017). Especially challenging is the need to understand these issues within the context of complex environmental matrices such as air, soil, and water. For example, B. anthracis spores released during a World War II era biowarfare experiment on Scotland's Gruinard Island made the island uninhabitable for decades (Manchee et al., 1981; Wynn, 1982). Bombs containing virulent B. anthracis spores were detonated near ground level, and sheep tethered downwind of the detonation site were monitored for inhalational anthrax and mortality. As recently as the 1980s, soil samples showed that viable spores were detected over a ~2.6-hectare area surrounding the detonation point, with the highest concentration of spores (~3.1 x 103 CFU g"1 soil) detected at the impact site of one anthrax bomb released from an aircraft (Manchee et al., 1994). A large-scale soil decontamination effort was attempted, which primarily consisted of soaking the topsoil with over 2 million liters of 5% formaldehyde in seawater. Areas surrounding the suspected aerial bomb site were decontaminated with 38% formaldehyde to the depth of bedrock. Two months after formaldehyde application, soil sampling showed that some areas with known pre-decontamination spore viability still contained recoverable spores (3 to 132 spores g 1 soil). At least one hot spot in the detonation area still had a very high contamination level (10s spores g 1 soil) at a depth of 50 cm. These areas were further treated, and re-sampling resulted in no detectable spores. Grasses were re-planted, and 40 Cheviot sheep were placed on the island to graze for five months, with no detected health impacts (Manchee et al., 1981). With Gruinard Island presumably restored, it was returned to heirs of the original owner in 1990 (Aldhous, 1990). However, monitoring and clean-up efforts took decades and had an undetermined, but significant, cost. As recently as 2011, an unrelated, non-fatal case of inhalational anthrax was detected in a 61- year old United States resident (Griffith et al., 2014). The patient was hospitalized during a 3-week trip 2 ------- though areas where animal anthrax is enzootic. During the trip through parts of North Dakota, Montana, Wyoming, and South Dakota, the patient, with no prior anthrax exposure, frequently encountered dusty conditions while driving and stopping among herds of bison and burros. A multi-agency investigation team analyzed at least 65 environmental samples from the patient's vehicle, home workshop, and garage, but B. anthracis was not detected in any of them. Environmental samples were not collected from the travel route because coverage of the entire distance (several thousand miles) was not feasible, specific suspected exposure locations were unknown, and soil sampling can be hindered by poor sensitivity and high variability. Investigators reported that these challenges limited the investigation (Griffith et al., 2014) and a B. anthracis source was not identified. Together, these two incidents highlight the difficulties associated with analysis of environmental matrices and demonstrate a need for improved sampling and detection capabilities. The individual case of inhalational anthrax demonstrates a need for techniques that offer enhanced sensitivity and reduced variability, especially for soil and other debris-laden matrices. The larger-scale Gruinard Island events illustrate the challenges associated with hazard characterization, sampling involving complex environmental matrices (e.g., soil), outdoor decontamination methods, and post-treatment clearance sampling. Gruinard Island is relatively remote and was uninhabited at the time of the military experiments, but if a large-scale, outdoor spore release, such as one associated with bioterrorism, occurred in an inhabited area, the decontamination strategy would be far more complicated. Along with research that has been dedicated to remediation of materials found in indoor settings, there is a clear need to develop new, effective, and cost-effective clean-up strategies for large outdoor areas which encompass an even wider and more complex variety of matrices. Soil, water, and vegetation all contain high loads of background organisms that may interfere with detection and enumeration of the target organism. Soil core samples for the Gruinard Island assessments were primarily evaluated with the use of rich media supplemented with polymyxin, lysozyme, disodium ethylene-diamine tetraacetate, and 3 ------- thailous acetate, known as PLET (Knisely, 1966). The PLET media, which is selective for B. anthracis, was combined with heat treatment (65 °Cfor 1 hr) to reduce background interference. However, this media may not be compatible with non-pathogenic B. anthracis surrogates. Fully virulent B. anthracis is a Tier I Select Agent (Rotz, et al., 2002; U.S. DHHS, 2018). Possession, use, storage, or transfer of this organism requires registration with the CDC (U.S. DHHS, 2009). Work with high culture concentrations or activities which may promote aerosolization must be performed in a Biosafety Level 3 (BSL-3) facility (U.S. DHHS, 2009), and environmental release experimentation with this agent is prohibited. To mitigate these logistical constraints, minimize public health risk, and reduce the hazards of working directly with a pathogen, surrogate organisms which share important non-pathogenic properties with B. anthracis have been used and described in the literature. Greenburg et al. (2010) reviewed the historical use of eight potential B. anthracis surrogates: B. atrophaeus, B. cereus, B. subtilis, B. thuringiensis, B. anthracis Sterne, B. megaterium, B. mycoides, and Geobacillus stearothermophilus (Greenberg et al., 2010). B. atrophaeus has been used most widely, followed by B. cereus, B. subtilis, B. thuringiensis, and B. anthracis Sterne, in that order. Surrogate preference and selection depend on not only practical considerations such as safety, availability, detection options, and cost, but also on how well the surrogate's genetic and physical properties align with those of B. anthracis and the importance of those similarities to the research objectives (Greenberg et al., 2010). Physical parameters such as size, shape, spore surface architecture, and density are all important experimental considerations. Other properties such as cell surface hydrophobicity and surface charge are principal contributors to spore interactions with environmental substrates (Chen et al., 2010; Husmark & Ronner, 1992; Ronner et al., 1990; Tufts et al., 2014; White et al., 2014), specifically affecting both attachment and detachment mechanisms and therefore substantially impacting spore movement (White et al., 2012). Consideration of all these features is especially important for tests designed to assess spore mobility, fate, and persistence in outdoor settings. 4 ------- The purpose of this report was threefold. The first was to identify the existing enhanced surrogates and review the available literature describing their development and use. The second was to propose characteristics desired for surrogates intended for outdoor studies. And finally, the third was to propose options for obtaining a surrogate with the desired features. Spore Property Considerations Spore surface hydrophobicity reflects the tendency for the spore to leave an aqueous environment for preferred interactions with hydrophobic entities (White et al., 2014). It can be affected by surface structures such as extracellular proteins, oligosaccharides, and appendages (Chen et al., 2010; Doyle et al., 1984; Faille et al., 2010; Husmark & Ronner, 1992; Lequette et al., 2011; Ronner et al., 1990; Tauveron et al., 2006; Tufts et al., 2014; White et al., 2014). Hydrophobicity may be assessed using water contact angle measurements and hydrophobic interaction chromatography (HIC) (Ahimou et al., 2001), but for spores, it has most commonly been measured with hexadecane partitioning assays, known as Bacterial Adhesion to Hydrocarbon (BATH) or Microbial Adhesion to Hydrocarbon (MATH) assays (Rosenberg, 1984, 2006; Rosenberg et al., 1980; Zoueki et al., 2010). The method is based on mixing cells or spores suspended in aqueous buffer with a liquid hydrocarbon such as n-hexadecane, n- octane, or p-xylene (Rosenberg et al., 1980). After mixing, the phases are allowed to separate and spores having an affinity for the liquid hydrocarbon will partition to that phase. Spectroscopy can be used to quantify the change in aqueous phase absorbance, with decreases observed for organisms having a hydrophobic character. Use of these principles, however, to extract B. anthracis spores from liquid food matrices had limited success (Leishman et al., 2010), as the degree of partitioning can be affected by assay conditions such as buffer strength, pH, heat treatment, hydrocarbon type, and phase separation time. Even with these known limitations, general trends among similar microbial strains have been noted (Doyle et al., 1984; White et al., 2014; Wiencek et al., 1990). Spores belonging to the Bacillus 5 ------- cereus family of bacteria, such as B. cereus, B. anthracis, and B. thuringiensis, generally have been observed to be more hydrophobic than other B. anthracis surrogates such as B. subtilis or B. atrophaeus (Ankolekar & Labbe, 2010; Buhr et al., 2008; Doyle et al., 1984; Faille et al., 2010; Husmark & Ronner, 1992; Koshikawa et al., 1989; White et al., 2014; Wiencek et al., 1990). White et al. (2014) designed a study to specifically address the limitations on data comparisons imposed by MATH method variability (White et al., 2014). Using consistent conditions regarding spore preparation, buffer type and strength, pH, and assay protocol, spore surface hydrophobicity for B. anthracis Sterne was compared to several common B. anthracis surrogates including B. thuringiensis subsp. israelensis and B. atrophaeus var. globigii (White et al., 2014). Spore preparations were added to either dechlorinated tap water (DTW) or a potassium phosphate monobasic (KH2P04) buffer with a concentration of 150, 91.5, 9.15, or 0.915 mM (each adjusted to pH 8), then measured for % retention to octane. B. anthracis Sterne had the highest measured hydrophobicity (~ 104 to 107%), which was relatively constant across all buffer strengths tested. B. thuringiensis subsp. israelensis exhibited less hydrophobicity (~ 68 to 78%), and also remained relatively constant across buffer strengths. However, the hydrophobicity measurements for B. atrophaeus var. globigii decreased as buffer strength decreased (~ 83% in 150 mM KH2P04 to ~44% in DTW), perhaps reflecting differences in innate surface architecture (White et al., 2014). How well these measurements inform design of outdoor experiments or predict spore behavior in the environment — where a variety of surface types, aqueous solutions, and chemicals may be present — is unknown. Electrostatic charge on the spore surface develops when surface molecules become ionized by protonation or deprotonation (Desai & Armstrong, 2003). Spore surface charge can be affected by the presence of extracellular structures as well as external conditions such as pH, ionic strength, and organic load (White et al., 2012; White et al., 2014). And like hydrophobicity, surface charge affects spore interactions with external entities and may impact spore movement and surface adhesion in the environment (Faille et al., 2010; Husmark & Ronner, 1992; Mikelonis et al., 2020; White et al., 2012; 6 ------- White et al., 2014). Spore surface charge is typically represented by measurements of electrophoretic mobility (EPM), and instruments which combine dynamic light scattering and electrophoretic mobility are often used for analysis (White et al., 2012). Spore suspensions are placed in a cell containing two electrodes, and when an electrical field is applied across the electrodes, spores having a surface charge migrate to the oppositely charged electrode (Desai & Armstrong, 2003) with a velocity that can be measured. From these measurements, the zeta potential is calculated using the Helmholtz- Smoluchowski equation (Husmark & Ronner, 1992). Zeta potential is commonly used to characterize spore surface charge (Ankolekar & Labbe, 2010; Faille et al., 2010; Husmark & Ronner, 1992; White et al., 2012; White et al., 2014) and may help anticipate the spore's tendency to either aggregate or remain dispersed in solution. For example, at pH 7, wide variations in zeta potential have been observed for spores of different Bacillus strains, with B. anthracis 9131 (-20.26 mV) more similar to B. thuringiensis 407 (-28.47 mV) and B. thuringiensis 7138 (-26.00 mV) than to type strain B. subtilis 7145 (-46.81 mV) (Faille et al., 2010). In general, zeta potential values with magnitudes greater than 40 mV are considered indicative of electrostatic stability and resistance to aggregation (Pochapski et al., 2021); therefore, B. subtilis may be expected to behave differently in solution than B. thuringiensis. Surface charge differences, however, may be mitigated by the composition of the solution. For example, intrinsic spore surface properties can be overpowered by components such as salts and metals found in stormwater and dominate spore adhesion processes (Mikelonis et al., 2020). White et al. (2012) also evaluated B. anthracis Sterne 34F2 and some B. anthracis surrogates such as B. thuringiensis subsp. israelensis and B. atrophaeus var. globigii under comparable conditions and reported the EPM instead of the calculated zeta potentials (White et al., 2012). Spores were generated under the same culture conditions, purified, and stored at 4 °C in sterile deionized water until use. EPMs were measured by adding purified spores to either DTW or KH2P04 buffer with a concentration of 150, 91.5, or 9.15 mM. The pH was adjusted from 2 to 10 in single-pH-unit increments 7 ------- and the EPM trends, while different for each strain, demonstrated little variation above pH 6. Below pH 6, the EPMs became more positive as pH decreased. Across the tested pH range and buffer strengths, B. anthracis Sterne generally had the most positive EPM, averaging -0.42 pim cm V"1 s"1; followed by B. thuringiensis subsp. israelensis and B. cereus, with average EPMs of -1.20 and -1.07 pim cm V 1 s"1, respectively. B. atrophaeus var. globigii, having an average EPM of -2.84 pim cm V"1 s"1, was most distant from B. anthracis Sterne. But again, how well these bench measurements in clean buffers inform design of outdoor experiments or predict spore behavior in the environment is not clear. Spore surface architecture also may affect spore behavior in the environment, and one surface feature of considerable interest is the exosporium. The exosporium is an irregularly shaped proteinaceous structure (Bozue et al., 2015; Henriques & Moran, 2007; Stewart, 2015) that is not present in all Bacillus strains. It is, however, commonly associated with the B. cereus family of bacteria, which includes B. anthracis and B. thuringiensis (Stewart, 2015). The exosporium consists of a basal layer which anchors hair-like projections known collectively as a nap (Hachisuka et al., 1966; Kailas et al., 2011). The nap is present over the entire surface of the exosporium and is principally composed of a collagen-like glycoprotein known as BcIA (bacillus collagen-like protein A) (Kailas et al., 2011; Stewart, 2015). The presence or absence of an exosporium is an important consideration when selecting a surrogate for environmental testing because of related effects on spore hydrophobicity (Brahmbhatt et al., 2007; Husmark & Ronner, 1992; Koshikawa et al., 1989; Ronner et al., 1990; White et al., 2014), aerodynamics (Kesavan et al., 2017; Tufts et al., 2014), and surface adhesion properties (Bozue et al., 2007; Faille et al., 2010; Husmark & Ronner, 1992; Kailas et al., 2011; Lequette et al., 2011; Williams et al., 2013). Various strains of Bacillus spores characterized by either the natural presence or absence of an exosporium were examined with hexadecane partitioning assays (Koshikawa et al., 1989). Spores with an exosporium partitioned strongly from the aqueous phase to hexadecane, indicating a higher degree of hydrophobicity than spores naturally lacking an exosporium (Koshikawa et al., 1989). In 8 ------- another study using B. anthracis Sterne spores, ultrasonication was used to remove the exosporium and Transmission Electron Microscopy (TEM) was used to verify removal. Like the naturally exosporium- deficient spores, the ultrasonicated spores displayed a significantly reduced hydrophobicity when compared to wild type (WT) spores (Williams et al., 2013). The altered B. anthracis Sterne spores were further tested in soil column flow-through experiments (Williams et al., 2013). Two soil types, each having a base composition of loam, peat, and sand in a 7:3:2 ratio, differed only in added organic and calcium content. The ultrasonicated spores without exosporia were observed to have longer column retention times, suggesting that the altered spores adhered more strongly to the tested soils than WT spores (Williams et al., 2013). The adherence difference between altered and WT spores was less pronounced in the calcium-rich soil, but both differences were statistically significant (p < 0.05) (Williams et al., 2013). Because the BcIA protein is a principal exosporium component, spore hydrophobicity and surface adhesion characteristics have also been examined when it is absent. Mutant B. anthracis Sterne spores harboring a bcIA deletion and lacking BcIA displayed significantly reduced hydrophobicity in comparison to WT, and interestingly, after heat treatment (85 °C for 10 min) there was a significant increase in hydrophobicity for the bcIA' mutant but not for WT spores (Brahmbhatt et al., 2007). The bcIA' mutant spores were also observed to have greater adherence to some extracellular proteins such as fibronectin and laminin when compared to WT (Brahmbhatt et al., 2007). Similarly, bcIA' mutants of B. anthracis Ames displayed increased adherence to bronchial epithelial cells (Bozue et al., 2007). In other work, B. anthracis Sterne spores lacking BcIA had a slightly higher retention in a porous medium of silica sand than WT spores (Chen et al., 2010), suggesting that as exterior proteins are removed and inner spore layers are exposed, adhesion to some surfaces may increase (Chen et al., 2010). On the other hand, work with B. cereus mutants harboring bcIA deletions demonstrated that the BcIA" spores had increased rather than decreased hydrophobicity (Lequette et al., 2011). Additionally, mutant spores 9 ------- attached to stainless-steel surfaces were more easily detached than WT spores, suggesting that the presence of BcIA resulted in an increased interaction between spores and the stainless-steel surface and that the underlying layers for some strains of B. cereus and B. anthracis are quite different (Lequette et al., 2011). These contrasting observations highlight the need to carefully consider experimental matrices and the selected surrogate's surface characteristics, especially when spore transport and adhesion properties are important test considerations. Along with an exosporium, the surface architecture of spores belonging to the Bacillus cereus group of bacteria may feature appendages. The notable exception is B. anthracis, as examined strains appear to be without them (Faille et al., 2010; Hachisuka et al., 1984). Appendages differ from the fine hair-like projections that make up the nap. Whereas the nap is distributed over the entire surface of the exosporium, appendages have a less uniform surface distribution and can appear in both lophotrichous and peritrichous conformations (Ankolekar & Labbe, 2010; Driks, 2007). Appendages are proteinaceous structures (Kozuka & Tochikubo, 1985), and reported numbers per spore as well as dimensions vary widely. Various reports typically list three to twenty, and even up to thirty, appendages per spore with a wide range of dimensions (Plomp et al., 2005a; Tauveron et al., 2006). For example, one TEM-based study reported an average appendage diameter of ~13.6 nm and lengths varying from 0.45 to 3.8 pim (Ankolekar & Labbe, 2010). The presence of appendages may be an important consideration when selecting a B. anthracis surrogate because, like the exosporium, it may have an effect on spore adhesion under certain conditions (Husmark & Ronner, 1992; Ottlow, 1975). Appendages may allow spores to easily aggregate at low ionic strength and provide a surface attachment advantage over appendage deficient spores (Plomp et al., 2005a). For example, when appendages were removed (by ultrasonication) from some strains of B. cereus spores, adhesion to hydrophobic glass was decreased. On the other hand, appendage removal had a lesser effect on B. cereus spore adhesion to stainless steel (Stalheim & Granum, 2001). 10 ------- Finally, while it is clear that dormant spore features are important considerations for surrogate selection, it should also be noted that the metabolizing cell characteristics may also be important considerations for B. anthracis surrogate selection. Some evidence suggests that that Bacillus organisms may transition between the vegetative and spore states in outdoor environments such as soil and upon contact with plant material (Bishop, 2014; Bishop et al., 2014; Bizzarri & Bishop, 2007; Charron- Lamoureux & Beauregard, 2019), but the potential impact of such transitions during long-term spore viability studies in outdoor settings is unknown. Spore Detection Considerations Physical spore characteristics are important considerations in selection of an appropriate B. anthracis surrogate, but effective methods for spore sample extraction, detection, and quantification must also be considered. These methods, some of which are discussed in a review by Irenge & Gala (2012), vary widely and often present extensive challenges especially for samples from complex matrices (Irenge & Gala, 2012). Culture-based methods continue to be accepted as the gold standard when the experimental goals are quantification and verification of spore viability. A culture-based method has been validated by two CDC studies (Hodges et al., 2010; Rose et al., 2011). In the Rose, et al. study (Rose et al., 2011), B. anthracis Sterne spores were inoculated onto a steel surface, then sampled with pre- moistened cellulose sponges. Spores were extracted from cellulose sponges into a buffer solution and aliquots were plated on nutrient agar to determine the number of colony-forming units (CFU) per sample. Non-target organisms (Bacillus atrophaeus and Staphylococcus epidermidis) coupled with Arizona test dust particles were incorporated to challenge spore recovery, but even with this addition, these samples contained much less background interference than samples collected from complex natural environments. An alternative cellulose sponge wipe analysis method has been described, but it also relies on culture-based enumeration (Abdel-Hady et al., 2019). Culture based enumeration methods 11 ------- may be employed to analyze samples from outdoor studies encompassing multiple surfaces, like those expected to be impacted during a wide-area bioterrorist incident (including concrete, asphalt, leaves, grass, and soil). However, it should be noted that manual enumeration of recovered spores likely will be complicated by the presence of heat-resistant, non-target, native organisms (e.g., other spore formers such as B. cereus, B. subtilis, B. brevis, and some species of Sporosarcina [Kocur et al., 1963; Siala, et al., 1973]). Colony counts may be aided by use of a surrogate with a pigmented phenotype, making it distinguishable from native organisms. Even with this advantage, crowding of target colonies by those of non-target organisms may necessitate sample dilution, which in turn negatively impacts the sampling method detection limit. Finally, and perhaps most importantly, uncertainty regarding target spore germination and outgrowth (especially for low number samples) in the presence of competition from numerous non-target organisms potentially affects colony count accuracy, reliability, and repeatability. To alleviate some of the problems associated with culture based methods, molecular methods such as polymerase chain reaction (PCR) (Bassy et al., 2018; Beyer et al., 1999; Carl et al., 1992; Cheun et al., 2003; Guidi et al., 2010; Janzen et al., 2015; Kuske et al., 1998; Makino & Cheun, 2003; Qi et al., 2001; Ryu et al., 2003; Sedlackova et al., 2017; Vahedi et. al., 2009) have been used. And since a real-time PCR assay was validated for rapid identification of Bacillus anthracis (Hoffmaster et al., 2002), development of similar molecular methods intended for B. anthracis surrogate detection and quantification has been of great interest. There are several advantages associated with this approach. In general, PCR-based methods offer better specificity, faster turnaround times, and increased sensitivity in comparison to culture-based methods (Mandal et al., 2011). Probes targeting specific nucleic acid sequences can be carefully designed so that distinguishing among organisms with similar genetics is possible (Radnedge et al., 2003). Use of a unique barcode, such as T1B1 or T1B2 (Buckley et al., 2012; Emanuel et al., 2012), provides an additional advantage. And while traditional culturing requires 24 to 48 hr for analysis results, PCR samples can be analyzed within 24 hr. However, it is also recognized that while many 12 ------- environmental samples contain PCR inhibitors (Schrader et al., 2012) such as background DNA, humic acids, metal ions, or polyphenols, PCR-based methods may still offer an advantage over traditional culture plating in terms of detection sensitivity. For example, early work with traditional PCR and ethidium bromide gels demonstrated DNA detection from B. atrophaeus var. globigii (Bg) spores seeded into four soil types containing various amounts of background DNA (0.18 to 21.3 ng g"1 soil) and humic acids (49 to 2,200 ng g"1 soil) (Kuske et al., 1998). Bg spores, ranging in concentration from 2.5 x 103 to 2.5 x 107 spores g"1 soil, were seeded into each soil type. DNA was extracted with bead mill homogenization then further purified with Sephadex spin columns to remove humic substances. For samples containing the lowest Bg spore concentration (103 spores g 1), PCR amplicons were only detected in the soil having the lowest concentrations of inhibiting factors (0.18 ng DNA g"1 soil and 49 Hg humic acid g"1 soil). In samples from all other soil types, the detection limit was either 104or 10s spores g 1 soil. Even though the assay sensitivity was impacted by the inhibitors present, the qualitative results were achieved in a short time and the detection ranges are as good or better than those achieved with traditional agar plating methods (Buttner et al., 2001). Despite the advantages of specificity and processing speed offered by molecular methods, widespread use of PCR to detect and quantify spores in complex environmental samples continues to be constrained by significant challenges. The adverse impact of PCR inhibitors on sensitivity has been demonstrated (Kuske et al., 1998), so separation of spores from the sample matrix is often the first challenge (Ryu et al., 2003; Stevens & Jaykus, 2004). Also required is uninhibited access to targeted nucleic acids (Kuske et al., 1998), and pre-PCR sample processing methods must include a reliable way to completely disrupt spores and liberate quality DNA (i.e., free of excessive shearing). Unlike vegetative bacterial cells, which are relatively easy to lyse with heat or mechanical abrasion, dormant spores are very resistant to various thermal, mechanical, and chemical assaults (Dittmann et al., 2015; Fox & Eder, 1969; Moeller et al., 2009; Mohr et al., 1991; Montville et al., 2005; Nicholson et al., 2000; Reyes et al., 13 ------- 1981; Setlow, 2006). Such remarkable durability has been attributed to multiple spore features, one of which is a complex exterior. Multiple, concentric proteinaceous layers (Brauge et al., 2018; Dittmann et al., 2015; Driks, 1999; Driks, 2002a; Driks, 2002b, 2009; Henriques & Moran, 2007; McKenney et al., 2013; Setlow, 1992, 2007) surround the DNA-containing core to create a shield of protection. Nevertheless, many spore lysis methods including heat shock (Luna et al., 2003), boiling (Buttner et al., 2004; Drago et al., 2002; Ryu et al., 2003) autoclaving (Luna et al., 2003), liquid nitrogen freeze-boil cycles (Kuske et al., 1998), French press passaging (Brauge et al., 2018), sonication (Belgrader et al., 1999; Chandler et al., 2001; Luna et al., 2003; Taylor et al., 2001) and sonication plus boiling (Rueckert et al., 2005a; Rueckert et al., 2005b) have been reported with varying degrees of success. Plating assays conducted after these procedures often show that spore lysis is incomplete and significant numbers of viable spores remain (Rueckert et al., 2005a). Lysis protocols have also been supplemented with various other methods, such as immunomagnetic capture (Blake & Weimer, 1997; Bruno & Yu, 1996; Shields et al., 2012; Thomas et al., 2013), density gradient centrifugation (Ryu et al., 2003), or germination (Guidi et al., 2010; Luna et al., 2003; Ryu et al., 2003) in an effort to improve lysis efficiencies. Immunomagnetic capture protocols typically utilize magnetic beads coated with spore-specific antibodies that are designed to capture, separate, and concentrate spores from complex matrices. Capture efficiencies vary widely. Density gradient centrifugation (e.g., sucrose with a nonionic detergent) may also be used to separate spores from hydrophobic soil particles but low (22 to 28%) spore recoveries have been reported (Dragon & Rennie, 2001). Germination protocols usually involve treating spore samples with various germination activators such as amino acids, calcium dipicolinic acid, peptidoglycan fragments, or nutrients. They are designed to stimulate germination and exploit the spore's natural physiology and promote endogenous degradation (Blankenship et al., 2015; Heffron et al., 2009; Setlow, 2014; Shah et al., 2008) of the spore's exterior, thereby making them more vulnerable to lysis. However, spore populations are heterogenous and germination rates may vary based on sample 14 ------- treatment and the germinants used (Chesnokova et al., 2009; Ghosh & Setlow, 2009; Pandey et al., 2013; Warda et al., 2017; Wei et al., 2010). Germination rates may also differ according to the number of germinant receptors, which can vary based on the media used during spore preparation (Setlow, 2013). Commercial DNA extraction kits have also been evaluated with spores (Brauge et al., 2018; Dauphin et al., 2009; Dineen et al., 2010; Mertens et al., 2014; Sedlackova et al., 2017; Thomas et al., 2013), but some plating and microscopic evaluations after the lysis steps have shown that viable spores remain (Brauge et al., 2018). DNA recoveries with the tested commercial kits, when reported, were far below the theoretical yields (Dineen et al., 2010). Bead beating, either alone or coupled with heat treatment or germination, has emerged as a frequently used, if not preferred, spore lysis option (Brauge et al., 2018; Emanuel et al., 2012; Geissler et al., 2011; Guidi et al., 2010; Jones et al., 2005; J0rgensen & Leser, 2007; Kuske et al., 1998; Priha et al., 2004; Saikaly et al., 2007; Vandeventer et al., 2011). Even so, traditional plating assays have shown that viable spores can be detected after liquid or dry bead beating (Jones et al., 2005). And while 100% lysis may not be a realistic goal, significant quantities of spores surviving the treatment add to the inefficiency of the method and therefore to the variability and uncertainty of analysis results. Electron microscopy also has demonstrated that spores can endure bead beating procedures and retain the appearance of intact coat layers (Brauge et al., 2018). While germinated spores are somewhat less resistant to mechanical lysis (Jones et al., 2005), dormant spores are very resistant to mechanical forces. This is perhaps not surprising since spore coats appear to have a high degree of redundancy. They are composed of at least 70 different types of proteins (McKenney et al., 2013), some with extensive covalent cross linkages (Driks, 1999; Driks, 2002a; Driks, 2002b; Gould & Hitchins, 1963; Henriques & Moran, 2007; Lai et al., 2003; Leggett et al., 2012; McKenney et al., 2013; Plomp et al., 2014). Even Bacillus mutants that do not produce one or more selected coat proteins have post-abrasion spore viability profiles that resemble those of wild type spores (Jones et al., 2005). And even when spore viability is lost with mechanical treatment, it may not be accompanied by a breach in 15 ------- the spore exterior, and without that, DNA release is open to question. Additionally, spore preparations may have high levels of dead cell debris, including non-negligible quantities of DNA which may be free or attached to spore surfaces. Unless spore preparations are rigorously purified (e.g., heat treatment, lysozyme treatment, density gradient centrifugation, DNase treatment) before use in spiking experiments, superficial DNA may be detected (Belgrader et al., 1999; Brauge et al., 2018; Chandler et al., 2001; Kuske et al., 1998; Vandeventer et al., 2011) and result in potential over-estimations of endogenous DNA recovery or spore presence. For example, Rueckert et al. (2005) used a qPCR assay to evaluate an untreated spore suspension and detected the equivalent of ~0.4 ng/ml of DNA, which they concluded was extraneous (Rueckert et al., 2005a). Assuming one spore has the genomic equivalent of ~6 femtograms of DNA, this would be the equivalent of at least 6 x 104 spores per ml. Spore DNA is also associated with small acid soluble proteins (SASP) (Mohr et al., 1991; Setlow et al., 1992; Setlow, 1988, 1992, 2007), which cause a conformational DNA change (Mohr et al., 1991; Setlow, 1992) and help protect spore DNA during various assaults such as wet and dry heat (Setlow, 2007). During germination, these proteins typically dissociate from DNA and are subsequently degraded into amino acids by endoprotease Gpr (Setlow, 1992, 2007). However, SASP variants having a high affinity for DNA may not dissociate and therefore could be less resistant to digestion (Setlow, 2007). Whether or not the presence of these proteins may affect PCR results is unknown. When lysis protocols are followed by separate DNA purification procedures, efficiencies can be further impacted. This becomes especially problematic for samples with low spore concentrations. PCR detection limits may be affected by not only the amount and condition of the DNA used in each reaction, but also by non-target background DNA (Kuske et al., 1998; LaMontagne et al., 2002) and by inhibition caused by abiotic and/or biological contaminants (Buttner et al., 2001; LaMontagne et al., 2002). And finally, assuming good quality, endogenous DNA is isolated, and a PCR method is used for detection and/or quantification, information regarding spore viability is either limited or not provided. 16 ------- One alternative to traditional PCR methods is Rapid Viability PCR (RV-PCR) (Kane et al., 2009; Letant et al., 2010; Letant et al., 2011; U.S. Environmental Protection Agency, 2011). Because spore viability is closely linked to evaluating public health risk and effective remediation responses after a biological contamination incident, sampling methods providing viability information are desired (Raber et al., 2011). Traditional agar plating assays have been used to quantitate viable spores, but a PCR-based approach potentially offers faster turnaround and greater compatibility with semi-automation and high- throughput processing. After completion of decontamination procedures, however, the presence of residual nucleic acids may complicate the results of traditional PCR assays (Buttner et al., 2004; Letant et al., 2011). Therefore, RV-PCR methods were developed to combine the detection specificity of probe- based PCR with culturing designed to support detection of targeted viable organisms. Incorporation of culturing also helps alleviate problems associated with spore lysis inefficiency. For RV-PCR, PCR is performed both before and after culturing such that differences (ACt) in the before- and after-culturing cycle threshold values (CT T0 and CT Tf respectively) are used to indicate the presence of viable spores. For samples to be considered positive for viable spores, PCR results must meet some defined criteria. For example, in one RV-PCR protocol designed for samples containing B. anthracis Ames (Letant et al., 2011), a positive viable spore result occurs when all sample replicates provide a Ct Tf < 36 in a 45-cycle PCR amplification after 9 hr of culture incubation and a ACt > 9, indicating at least a 3-log difference. A most probable number (MPN) statistical approach (Blodgett, 2005), which has been used to enumerate microbes in food (Oblinger & Koburger, 1975), environmental water samples (Carey et al., 2006; Vester & Ingvorsen, 1998), and soil (Fredslund et al., 2001), is used as the spore quantification method. The reliability of the MPN method relies on analysis of multiple replicates (usually three or five, but up to 10) of each dilution within a dilution series (usually three 10-fold dilutions) for each sample and does not provide an absolute value (Oblinger & Koburger, 1975). Using inputs for number of dilutions, number of tubes per dilution, and sample volume, published tables (Blodgett, 2010) and 17 ------- various calculators are available to determine the sample MPN estimate (Briones & Reichardt, 1999; U.S. Environmental Protection Agency, 2013). Early work to test the feasibility of the RV-PCR method utilized aliquots from heat-treated crude cell lysates in the PCR reactions (Kane et al., 2009), but later DNA purification protocols were added (Letant et al., 2011; U.S. Environmental Protection Agency, 2011). Studies comparing RV-PCR and traditional plating sample analysis results have also employed challenges such as high concentrations of dead background spores (inactivated with chlorine dioxide) and the use of Arizona test dust spiked with non-target organisms to simulate dirty surface sampling with background contamination (Kane et al., 2009; Letant et al., 2010; Letant et al., 2011). Even with these challenges, the two methods provided similar results (within 1 log). Additional work to further clarify false negative rates (FNRs) and limits of detection (LODs) was conducted with a modified RV-PCR (mRV-PCR) approach (Hutchison et al., 2018). The mRV-PCR method employs a longer enrichment (16 instead of 9 hours) and heated cell lysis for DNA extraction without further purification. Low concentrations of B. anthracis Sterne and Bg spores were applied in liquid droplet depositions on 2- by 2-in. coupons made of glass, stainless steel, vinyl tile, or plastic. Spore concentrations ranged between 2 and 500 CFU per coupon. Coupons were sampled with Puritan macrofoam swabs and samples were evaluated by both mRV-PCR and culture. In general, the observed FNR and LOD values were lower for the mRV-PCR method than for the culture method. The effects of dust, grime, or the presence of background organisms was not tested. Further testing is needed to validate the RV-PCR methods for complex environmental samples containing both viable and inactivated spores and high loads of non-target background organisms. Since the RV-PCR method incorporates a culturing step, target spore germination and growth of nascent cells in the presence of other heat-resistant background organisms may be problematic. Germinating spores and nascent cells may be outcompeted for access to nutrients, especially for samples with low spore concentrations. Germination of target organisms also may be impacted by very high densities of non- 18 ------- viable spores, where alanine racemase enzymes, which inhibit germination by converting germination activator L-alanine to germination inhibitor D-alanine, may still be active (Cote et al., 2018). Common Bacillus anthracisSurrogates In recognition of the need to employ surrogates with characteristics similar to those of the targeted infectious agent, as well as the persistent complications arising from use of conventional microbiological methods for analysis of environmental samples, key features of commonly used B. anthracis surrogates will be summarized within this context. Work to enhance some B. anthracis surrogates with features designed to overcome certain environmental sampling and detection challenges also will be reviewed. Some of this work has been summarized elsewhere (Park et al., 2018). However, this review expands the available summary information to include additional surrogate possibilities, field trial outcomes from enhanced surrogate deployment, and advantages and disadvantages of the associated detection methodologies. Bacillus anthracis Sterne: B. anthracis Sterne has been widely used in laboratory research as a surrogate for virulent B. anthracis (Greenberg et al., 2010). The popularity of B. anthracis Sterne stems in part from its status as an attenuated B. anthracis strain that is not considered a Tier I Select Agent (Staab et al., 2017; U.S. DHHS, 2018). Virulent forms of B. anthracis contain two virulence plasmids (Read et al., 2003). Plasmid pXOl features a pathogenicity island containing three anthrax toxin genes: cya (edema factor), /e/(lethal factor), and pagA (protective antigen) (Okinaka et al., 1999a; Okinaka et al., 1999b). Plasmid pX02 carries genes (capA, capB, capC) responsible for formation of a poly-y-D- glutamic acid capsule, which helps B. anthracis cells avoid phagocytosis by human immune cells (Ezzell & Welkos, 1999; Jeon et al., 2015). B. anthracis Sterne, however, carries only the pXOl plasmid and therefore does not produce a capsule (Okinaka et al., 1999b). In this attenuated state, it has been used in vaccine preparations for livestock immunizations against anthrax worldwide (Staab et al., 2017; 19 ------- Sterne, 1946; Turnbull, 1991). Other B. anthracis strains, such as NNR-1 and NNR-2, lost the pX02 plasmid when novobiocin was included in the growth media (Green, et al., 1985). Another strain, known as B. anthracis ASterne, has been cured of the pXOl plasmid. B. anthracis ASterne has been used as a reference strain during assessments of other B. anthracis surrogates such as B. thuringiensis (Buhr et al., 2015). Aside from the difference in plasmid composition, B. anthracis Sterne can be considered representative of virulent B. anthracis in terms of spore physical characteristics and colony morphology on agar-based media. B. anthracis Sterne also has been used in bench-scale studies designed to assess susceptibility to various inactivation agents. For example, when tested in aqueous buffer under conditions commonly associated with drinking water treatment facilities (pH 7 and 5°C or 25°C), B. anthracis Sterne spores were more susceptible to inactivation by free chlorine (0.8 or 2.0 mg/liter) than spores of 6. anthracis Ames and other tested surrogates (Rice et al., 2005; Rose et al., 2005). But, in another inactivation study where spores were deposited on either silicone rubber or aluminum alloy carriers and then exposed to solutions containing sodium hypochlorite or hydrogen peroxide, B. anthracis Sterne and B. anthracis Ames were similarly inactivated (within ~1 log) (Sagripanti et al., 2007). And when spores deposited on different building materials (e.g., glass, carpet, ceiling tile) were tested under various temperature, contact time, and relative humidity (RH) conditions for methyl bromide decontamination efficacy, overall inactivation efficacies for spores of both B. anthracis Ames and B. anthracis Sterne increased under higher RH (75%) conditions, but B. anthracis Sterne spores were more resistant under this condition than B. anthracis Ames (Wood et al, 2016). B. anthracis Sterne also was used to assess inactivation kinetics in food matrices. Lag phase durations, growth rates, and maximum population densities for B. anthracis Sterne and B. anthracis Ames were compared over a range (10, 15, 25, 30, 35, 40 and 70 °C) of temperatures in samples of sterile raw ground beef (Tamplin et al., 2008). For growth rate and lag time, little difference was observed but mean population densities at 30, 35, and 40 °C were higher for B. anthracis Ames (Tamplin et al., 2008). This study provides at least 20 ------- one example demonstrating that B. anthracis Sterne could be a reasonable surrogate for virulent B. anthracis when samples involve complex matrices. However, it may not be a good candidate for outdoor release studies. Even though B. anthracis Sterne is considered non-pathogenic and can be handled in BSL-2 facilities, public relations challenges would be anticipated for its use in large-scale environmental studies. And the risks, however low, associated with genetic exchange in the environment potentially resulting in the acquisition of virulent factor genes by B. anthracis Sterne, may be prohibitive from public safety and relations perspectives (Davison, 1999; Droge et al., 1998; Greenberg et al., 2010). Bacillus atrophaeus: Bacillus atrophaeus var. globigii has been a common B. anthracis surrogate for studies with biowarfare defense or decontamination objectives (Gibbons et al., 2011; Grand et al., 2010; Mickelsen et al., 2019; Raber & Burklund, 2010). In comparison to B. anthracis, B. atrophaeus spores in aqueous buffer were reported to be slightly more resistant to free available chlorine, especially at pH ranges from 6.2 to 8.6 (Brazis et al., 1958). At a higher pH of 10.5, a similar resistance was reported for both spore types (Brazis et al., 1958). In other work where spores deposited on either metal or rubber carriers were inactivated with an unadjusted hypochlorite solution (pH ~10) or other peroxide-containing products, B. anthracis and B. atrophaeus also had similar reported sensitivities (Sagripanti et al., 2007). And although spore formation conditions can affect spore heat tolerance (Nicholson et al., 2000), spores of B. atrophaeus (ATCC 9372) were also observed in bench- scale experiments to have increased resistance to dry heat in comparison to B. anthracis Sterne, with reported Diso-values (minutes) of 2.52 ± 0.16 and 1.11± 0.06, respectively (Wood et al., 2010). Together, these resistance qualities make B. atrophaeus a conservative substitute when evaluating the projected efficacy of decontamination agents and methods targeting virulent B. anthracis. B. atrophaeus also has a pigmented phenotype when cultured on solid rich media, making its colonies morphologically distinct and easily enumerated (Burke et al., 2004; Gibbons et al., 2011; Nakamura, 1989). And like B. anthracis, B. atrophaeus is not reported to have appendages (Greenberg et al., 2010; Plomp et al., 2005b; Plomp et 21 ------- al., 2005c). However, B. atrophaeus spores differ from B. anthracis spores in several important ways. For example, the B. atrophaeus spore architecture lacks an exosporium (Buhr et al., 2008; Greenberg et al., 2010; Plomp et al., 2005b; Plomp et al., 2005c). B. atrophaeus spores also tend to be physically smaller than B. anthracis spores (Buhr et al., 2008; Carrera et al., 2007; Fricker et al, 2011), although environmental conditions and spore preparation methods can impact spore size (Plomp et al., 2005b). And finally, one nucleotide level analysis involving archival strains of B. atrophaeus demonstrated that the nearest neighbor is B. subtilis (Gibbons et al., 2011), making B. atrophaeus strains more phylogenetically distant from B. anthracis than some other surrogates such as B. thuringiensis (Greenberg et al., 2010). Bacillus thuringiensis: Because of both its genetic and physical similarities to B. anthracis, Bacillus thuringiensis (B. thuringiensis) has been extensively evaluated as a surrogate (Tufts et al., 2014). Like B. anthracis, the B. thuringiensis spore architecture features an exosporium (Ball et al., 2008; Boone et al., 2018; Terry et al., 2017; Todd et al., 2003), and the spores have similar sizes and volumes (Carrera et al., 2007; Faille et al., 2010). Other spore properties such as hydrophobicity, electrophoretic mobility, and zeta potential vary widely among Bacillus strains, but B. thuringiensis can be considered generally representative of B. anthracis in these areas (Faille et al., 2010; Sinclair et al., 2012; White et al., 2012; White et al., 2014). In one long-term (up to 1,038 days) laboratory study in which viable spore recoveries from various fomites (e.g., laminate, stainless steel) were evaluated, results for B. anthracis were more similar to those for B. thuringiensis than to other tested surrogates (Enger et al., 2018). In a spore inactivation study conducted under conditions representative of those in drinking water treatment facilities, B. thuringiensis subsp. israelensis was more resistant to treatment with free available chlorine than B. anthracis Ames, making it a conservative surrogate choice for similar decontamination tests (Rice et al., 2005). But for spores deposited on either silicone rubber or aluminum alloy carriers, B. 22 ------- thuringiensis 4055 and B. anthracis Ames had similar inactivation profiles when exposed to 5% (v/v) sodium hypochlorite with no pH adjustment (Sagripanti et al., 2007). B. thuringiensis and B. anthracis also belong to the Bacillus cereus group of closely related organisms (Ehling-Schulz et al., 2019; Keim et al., 2009; Priest, et al., 2004) and likely evolved from a common ancestor (Hill et al., 2004; Ivanova et al., 2003; Turnbull, 1999). Aside from vast differences in plasmid composition (Kolst0 et al., 2009), genetic profiles among this group are closely aligned (Helgason et al., 2000). For example, the 16S rRNA gene sequences for members of this group are 99% identical (Ash et al., 1991; Klee et al., 2006) and therefore cannot be used for discrimination (Klee et al., 2006). However, a few interesting genetic differences result in distinctive phenotypes which can be used to differentiate B. anthracis, B. cereus, and B. thuringiensis (Gillis & Mahillon, 2014). For example, characteristics like the absence of phospholipase C activity, absence of (B-hemolysis on sheep blood agar, lack of motility, susceptibility to penicillin, and lysis by bacteriophage Gamma (y) are classically examined to confirm B. anthracis identity (Koehler, 2009; Turnbull, 1999). Phospholipases and extracellular hemolysins act as important virulence factors in many pathogenic Bacillus organisms (Agaisse et al., 1999; Lereclus et al., 1996). These extracellular proteins are responsible for the characteristic phenotypes which may be observed with simple plating assays (sheep blood or chromogenic agar) (Klee et al., 2006; Peng et al., 2001). Gene expression for these proteins is typically activated during stationary phase (Lereclus et al., 1996) and is regulated by the transcriptional activator PIcR (Agaisse et al., 1999; Gohar et al., 2008; Lereclus et al., 1996; Salamitou et al., 2000; Slamti & Lereclus, 2002). PIcR is encoded by the pIcR gene. In B. thuringiensis and B. cereus, PIcR recognizes a conserved promoter region (Agaisse et al., 1999) and activates multiple genes that have roles not only in virulence (Agaisse et al., 1999) but also in modulation of environmental signals such as nutrient depletion and cell density (Gohar et al., 2008). Most of these genes are also present in the B. anthracis genome, but the corresponding proteins are not expressed (Slamti et al., 2004). In B. 23 ------- anthracis, the pIcR gene has a nonsense mutation (Slamti et al., 2004), which results in a truncated protein that is unable to perform as a transcriptional activator (Agaisse et al., 1999). In the absence of a functional PIcR activator, the extracellular B. anthracis matrix lacks phospholipase C and (B-hemolysin, along with a host of other extracellular proteins found for B. cereus and B. thuringiensis, but not B. anthracis (Gohar et al., 2005). Similarly, multiple flagellar genes, while present in B. anthracis, contain frameshift mutations which disrupt the formation of a functional flagellar structure. So unlike B. thuringiensis and B. cereus, B. anthracis has a non-motile phenotype (Koehler, 2009; Rasko et al., 2004; Read et al., 2003). Additionally, many strains of B. anthracis are susceptible to penicillin, whereas B. thuringiensis and B. cereus produce (B-lactamase enzymes and are typically resistant to penicillin (Chen et al., 2003; Gargis et al., 2018; Ross et al., 2009; Turnbull et al., 2004). Finally, bacteriophages are obligate intracellular parasites which infect bacteria by interaction with host cell receptors (Salmond & Fineran, 2015). Phages have a high degree of host specificity (Knoll & Mylonakis, 2014), a characteristic making them useful for distinguishing between genetically related strains like B. anthracis, B. cereus, and B. thuringiensis. For example, the bacteriophage y infects B. anthracis but not B. thuringiensis or B. cereus (Brown & Cherry, 1955; Gillis & Mahillon, 2014). To effect replication upon infection, y employs mechanisms to disrupt the susceptible host's cell membrane (Schuch et al., 2002). As cells are lysed, observable plaques form on agar plates, providing a diagnostic tool for B. anthracis. B. anthracis and B. thuringiensis differ substantially in plasmid composition and toxin production. B. anthracis virulence genes are encoded on the pXOl and pX02 plasmids (Okinaka et al., 1999a; Okinaka et al., 1999b; Read et al., 2003). B. thuringiensis strains have large plasmids carrying genes cry, vip, and cyt which code for insecticidal proteins (Chakroun et al., 2016; Ehling-Schulz et al., 2019; Espinasse et al., 2003; Hofte & Whiteley, 1989; Ibrahim et al., 2010; Mesrati et al.,2005; Schnepf et al., 1998; Schnepf & Whiteley, 1981; Whiteley & Schnepf, 1986). During B. thuringiensis sporulation, 24 ------- proteinaceous crystals which are toxic when ingested by certain insect species (Schnepf et al., 1998) are formed. The cry and cyt genes are responsible for crystals that form in the mother cell, outside of the nascent spore, but the izip genes code for proteins that are secreted during vegetative cell growth (Bechtel & Bulla, 1976; Whiteley & Schnepf, 1986). A sip gene, which encodes a secreted protein with toxicity toward Leptinotarsa decemlineata larvae, has also been reported for B. thuringiensis strain EG2158 (Donovan et al., 2006). Liquid laboratory cultures commonly used to produce spores contain both spores and crystals (Agaisse & Lereclus, 1995; Bechtel & Bulla, 1976; Dubois, 1968; Palma et al., 2014), and various methods have been developed to separate them, especially to isolate purified crystals for further study (Mounsef et al., 2014; Pendleton & Morrison, 1966). Even when separation techniques are used, spore clumping can entrap crystals (Mounsef et al., 2014) and crystal proteins may adhere to spore surfaces (Du & Nickerson, 1996), potentially affecting the spore's aerodynamic and chemical resistance properties (Tufts et al., 2014). B. thuringiensis strains have generally emerged as the preferred B. anthracis surrogates for environmental sampling, persistence, fate and transport studies. This preference arises primarily from its history of widespread outdoor use and longstanding safety record (Ibrahim et al., 2010; Raymond & Federici, 2017; Teschke et al., 2001; Valadares de Amorim et al., 2001; Van Cuyk et al., 2011). Various strains of B. thuringiensis are the active ingredients in many commercially available biopesticide formulations (Crickmore, 2006; Roh et al., 2007; Siegel, 2001). For example, Bacillus thuringiensis subsp. kurstaki is the active ingredient in Foray® 48B (Valent Biosciences,, Libertyville, IL, Flowable Concentrate, Biological Insecticide label, https://www.valentbiosciences.com/foresthealth/wP" content/uploads/sites/5/2021/04/Valent Forav-48B-44-label.pdf. and is registered with the U.S. Environmental Protection Agency as an approved biopesticide (EPA Registration Number: 73049-427, 2018; https://www3.epa.gov/pesticides/chem search/ppls/073049-0Q427~20180501.pdf). Additionally, B. thuringiensis subsp. kurstaki does not produce (B-exotoxins, which are small, thermostable secondary 25 ------- metabolites that are secreted by metabolizing cells of some B. thuringiensis strains (Hernandez et al., 2003). (B-exotoxins are low molecular weight analogs of the nucleotide adenine, and they inhibit DNA- dependent RNA polymerase (Palma et al., 2014). They exhibit non-specific toxicity against insects and mammalian cells, so B. thuringiensis strains producing them cannot be used for biopesticides in Europe, the United States, and Canada (Palma et al., 2014). Perhaps the greatest ongoing challenges associated with wide use of unenhanced B. thuringiensis surrogates in tests involving complex matrices are detection and quantification. When grown on rich solid media, unenhanced B. thuringiensis lacks a pigmented phenotype and forms white colonies with morphologies similar to those of other ubiquitous environmental organisms. Therefore, traditional agar plating is usually not a feasible analysis option, especially for environmental samples like grass, soil, or leaves, which are rich with non-target organisms that may outcompete germinating spores for nutrients and impact enumeration data. These challenges have likely prevented the use of B. thuringiensis in some studies where they may have been the preferred B. anthracis surrogate. Sample heat treatment may be used to eliminate some non-target organisms but heat resistant background spore-formers, also present in many environmental samples, remain viable to complicate analysis. Molecular approaches also may be used to remedy these problems, but more work is needed to validate the quantitative capacity of molecular-based detection methods. Summary of Properties for Bacillus anthracis and Selected Surrogates Table 3.0 provides a summary of characteristics associated with selected B. anthracis strains for both the metabolically active cell and spore forms of the organisms. Where possible, specific strains with the noted characteristics have been listed to support the surrogate level generalization. Note however, that exceptions can most likely be found as differences in culturing conditions and sporulation media components may alter the size (Carrera et al., 2007) as well as the structure and composition of spore 26 ------- coat layers (Hitchins et al., 1972). All of these potential differences could affect spore behavior (Plomp et al., 2005c) in unknown ways and complicate efforts to compare functional characteristics across strains. 27 ------- Table 3.0 - Characteristics of Bacillus anthracis and Common Surrogates1 Organism State Characteristic Bacillus onthrocis Bacillus onthrocis Sterne Bacillus otrophoeus Bacillus thuringiensis References Metabolically Active Cell Colony morphology Creamy gray to off white; tacky, colony growth may be pulled into standing peaks with a loop; circular to irregular colonies; flat or slightly convex; entire to undulate edges; granular surface textures, ground glass appearance Same as B. onthrocis Brownish or orange pigment, depending on strain; opaque, smooth, circular colonies that may become rough and crusty in appearance as colonies dry Creamy gray to off white; colonies have butyrous consistency; granular surface textures; circular to irregular colonies; entire to undulate edges, crenate and fimbriate edges (American Society for Microbiology, 2017; Hodges et al., 2010; Logan & De Vos, 2009; Nakamura, 1989; Turnbull, 1999) Motility No No Yes Yes (Bouillaut et al., 2005; Leise et al., 1959; Logan & De Vos, 2009; Nakamura, 1989; Turnbull, 1999) Sensitive to lysis by y phage Yes Yes No (B. subtilis) No (Schofield & Westwater, 2009; Schuch et al., 2002; Turnbull, 1999) poly-y-D-glutamic acid capsule formation Yes No No No (Ezzell & Welkos, 1999; Logan & De Vos, 2009; Reif et al., 1994; Turnbull, 1999) Blood agar p-hemolysis No No No (ATCC 9372) No (B. thuringiensis subsp. kurstaki HD-1) (Carlson & Kolst0, 1993; Gibbons et al., 2011; Hodges et al., 2010; Turnbull, 1999) p-lactamase production conferring penicillin resistance No No No Variable by strain (Chen et al., 2003; Coonrod et al., 1971; Logan & De Vos, 2009; Turnbull, 1999; Bishop & Robinson, 2014) Functional global transcription regulator (PIcR) No No No [B. subtilis 168) Yes (Ehling-Schulz et al., 2019; Koehler, 2009; Lereclus et al., 1996; Slamti & Lereclus, 2002; Slamti et al., 2004) 28 ------- Table 3.0 - Characteristics of Bacillus anthracis and Common Surrogates1 Virulence plasmids pXOl, pX02 pXOl No (B. subtiiis) Multiple, may contain cry, vip, cyt genes (Carlson & Kolst0, 1993; Ehling-Schulz et al., 2019; Errington, 1988; Hofte & Whiteley, 1989; Koehler, 2009; Mesrati et al., 2005; Okinaka et al., 1999a; Okinaka et al., 1999b; Palma et al., 2014; Read et al., 2003) Dormant Spore Presence of exosporium Yes Yes No Also lacks bcIA gene Yes (Buhr et al., 2008; Greenberg et al., 2010; Sella et al., 2014; Stewart, 2015) Strains examined Appendages B. anthracis 9131 B. anthracis IID 501 No Sterne 34F2 ND B. subtiiis 1AM 1206 B. subtiiis 98/7 B. globigii No B. thuringiensis 407 B. thuringiensis 7138 B. thuringiensis subsp. israelensis ATCC 35646 B. thuringiensis 1AM 11056, 1AM 11064 Yes (DesRosier & Lara, 1981; Faille et al., 2010; Hachisuka et al., 1984; Plomp et al., 2005a; Steichen et al., 2005) Parasporal crystal formation No No No Variable by strain B. thuringiensis HD-1: Yes B. thuringiensis Al Hakam: No B. thuringiensis BMB171: No (Carlson & Kolst0, 1993; Challacombe et al., 2007; Ehling- Schulz et al., 2019; He et al., 2010; Sella et al., 2014) Strain Sizes (urn) ± SD: Mean Length2 Mean Diameter2 B. anthracis Ames 1.52 ±0.19 0.81 ±0.06 Not listed 1.49 ±0.17 0.85 ±0.08 B. atrophaeus ATCC 9372 1.21 ±0.18 0.68 ±0.11 B. thuringiensis subsp. kurstaki 1.78 ±0.19 0.86 ±0.05 (Buhr et al., 2008; Carrera et al., 2007; Fricker et al., 2011) Strain Surface Hydrophobicity (% retention to octane)3 B. anthracis Ames ND B. anthracis Sterne 34F2 104 ±3 B. atrophaeus var. globigii 44 ±2 B. thuringiensis subsp. israelensis 78 ±3 (White et al., 2014) Strain Surface Charge (represented by EPM)4 B. anthracis Ames ND B. anthracis Sterne 34F2 -0.64 ±0.05 B. atrophaeus var. globigii -1.87± 0.06 B. thuringiensis subsp. israelensis (ATCC 35646) -1.23 ±0.08 (White et al., 2012) Notes: (ND) Not determined (1) Culturing conditions (e.g., temperature, pH) and sporulation media composition may affect functional and morphological spore properties (2) Length and diameter measurements using Transmission Electron Microscopy (TEM) and Advance Microscopy Technology (AMT) software (3) Data based on n-octane partitioning assays from dechlorinated tap water (4) Data reported as |im cm V"1 s"1 based on electrophoretic mobility (EPM) measurements at pH 7 in dechlorinated tap water 29 ------- Bacillus a nth racis Surrogates Enhanced for Environmental Sampling Bacillus thuringiensis subsp. kurstaki HD-1 enhanced with an acrystalliferous phenotype: Naturally occurring acrystalliferous Bacillus strains such as B. thuringiensis Al Hakam (Challacombe et al., 2007) have been used as B. anthracis surrogates in various method development studies aimed at spore disinfection of surface materials (Buhr et al., 2013; Buhr et al., 2012; McCartt et al., 2011; Omotade et al., 2014). Bishop and Robinson (2014), however, were particularly interested in a surrogate that could be used in outdoor settings. They selected B. thuringiensis HD-1 for their work largely because of its strong safety record and widespread use as a biopesticide. To address the concern regarding potential spore-crystal interactions, they enhanced B. thuringiensis subsp. kurstaki HD-1 with an acrystalliferous phenotype (Bishop & Robinson, 2014). The new surrogate strain, ultimately termed Btcry-, lacks the plasmids that possess known insecticidal genes cry, vip, and cyt and does not produce parasporal crystals. To promote loss of the plasmids, parental organisms were cultured at an elevated temperature (42°C). Phase contrast microscopy was used to verify the absence of crystals, and insecticidal gene loss was confirmed by PCR. Further, genome sequencing data for the new strain showed that none of the generated fragments were consistent with any known cry, vip, or cyt genes. Spore preparations of the new Btcry- isolate had no detected toxicity in any of the nine invertebrate species tested, even when challenge doses were ~ 1 x 10s CFU. And as expected, no evidence of (B- exotoxin was found. To test recovery from simulated environmental samples, spores of the new strain were used to inoculate (104 CFU g 1 dry weight of soil) non-sterile grass-soil microcosms (Bishop, 2014). After two weeks, soil slurries were serially diluted and plated on a Brilliance B. cereus (BBC) agar (Oxoid, UK) supplemented with Penicillin G, Polymyxin B, and Trimethoprim to inhibit growth of Gram-negative organisms. Chromogenic substrates in the agar confer B. thuringiensis colonies with a blue-green 30 ------- appearance. As expected, the blue-green colony morphology for Btcry-, a B. thuringiensis subsp. kurstaki HD-1 derivative, was observed. Colony quantification was not reported. Because of its selective and differential qualities, BBC growth media has been suggested as a potential remedy for the ongoing difficulties associated with identification and quantification of B. thuringiensis colonies using traditional agar plating methods, especially when a large contingent of non- target organisms having similar colony morphologies is present (Bishop & Robinson, 2014). However, this media is considerably more expensive (up to 25 times) than traditional tryptic soy agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The added expense can be a major disadvantage, and perhaps prohibitive, for high-throughput sample processing. More importantly, the antibiotic supplements (Polymyxin B and Trimethoprim) used to make this product selective against non-target organisms may not support germination and outgrowth of all surrogate spores. TSA plates supplemented with Polymyxin B and Trimethoprim and inoculated with ~lx 103 purified spores of B. thuringiensis subsp. kurstaki HD-1 or B. atrophaeus var. globigii resulted in either no (B. atrophaeus var. globigii) or significantly reduced (B. thuringiensis subsp. kurstaki HD-1) colony formation (personal observation, unpublished data). In another study, use of BBC media was discontinued after sample recoveries of B. anthracis Sterne were 5 to 7 times lower than those observed with sheep blood agar (SBA) (Calfee et al., 2019). Bishop and Robinson used the antibiotic-enhanced BBC media to successfully recover Btcry- from non-sterile grass-soil microcosms after a two-week incubation (Bishop & Robinson, 2014), but whether the observed colonies originated from vegetative cells (having germinated in the plant-soil microcosms) or dormant spores is unclear. It is possible that the recoveries on BBC media came from mature cells that germinated in the root system—a phenomenon observed for B. subtilis in which vegetative cells persisted on roots for days before re-sporulating (Charron-Lamoureux & Beauregard, 2019). And although, some B. thuringiensis strains are known to secrete (B-lactamase and have documented penicillin resistance (Luna et al., 2007; Turnbull et al., 2004), this phenotype is 31 ------- typically a feature of metabolically active, mature cells. However, some spore formers such as Clostridium botulinum—which is normally susceptible to penicillin (Swenson et al., 1980)—can germinate in the presence of this antibiotic (Smoot & Pierson, 1982), but further outgrowth of the germinated cell is inhibited (Treadwell et al., 1958). Whether or not B. thuringiensis spore germination and outgrowth are affected in the presence of penicillin is unknown. Overall, the BBC media has limitations regarding quantitative analysis of complex, high background samples inoculated with B. anthracis surrogate spores. In 2016, Bishop and Stapleton reported on a field trial designed to compare the aerosol dispersal and deposition behaviors of a traditional B. anthracis surrogate with those of the newly developed, acrystalliferous Btcry- strain (Bishop & Stapleton, 2016). A misting backpack sprayer was used to disperse spores of either Btcry- or B. atrophaeus var. globigii (Bg) from one end of an open-ended barn, which provided protection from crosswinds, potential rainfall, and UV radiation. Various spore collection devices were situated along the length of the barn. These included filter collectors with an air intake rate of 900 L min 1 to monitor airborne spore densities; open trays containing a buffer solution to monitor spore settling; and arrays of horizontally oriented coupons with three different hard surfaces (metal, wood, and concrete) to monitor spore surface depositions. Spores were recovered from each collection medium and enumerated with TSA plate assays. Data were collected over five spore releases. Filter counts across all spray events and all filters were significantly (99% confidence level) lower for Btcry- (mean, 4.03 x 107 CFU) than for Bg (mean, 1.54 x 10s CFU). The highest filter counts were observed near the middle of the barn and the lowest counts were near the spray source for both strains. Filters near the barn exit, furthest away from the release source, provided higher counts for Bg than for Btcry-, indicating that the smaller, less dense Bg spores stayed airborne longer and traveled farther than the larger Btcry- spores. A corroborating pattern was observed for the settling trays. Significantly (90% confidence level) fewer Bg spores were detected (Bg mean, 7.33 x 109 CFU vs. Btcry- mean, 1.03 x 1010 32 ------- CFU) in the trays, again demonstrating the relative tendency of the smaller Bg spores to remain airborne. Recoveries across all solid surfaces were significantly (99% confidence level) higher for Btcry- (mean, 5.36 x 10s CFU m"2) than for Bg (mean, 2.81 x 10s CFU m"2). Depositions recovered from both aluminum and concrete were higher than wood for both organisms, possibly reflecting differences in surface porosities and recovery efficiencies from these material types. Differences observed for hard surface depositions may also reflect variances in spore surface structures, such as the presence (Btcry-) or absence (Bg) of an exosporium. The Btcry- surrogate was used in another field study involving hot, humid air decontamination of a hangar-enclosed C-130 aircraft (Buhr et al., 2016). Btcry- spores (~ 9.68 x 1011) were released into the cargo hold of the sealed aircraft using Micro-Jet 7401 foggers (The Fogmaster Corp., Deerfield, FL), then further disseminated with mixing fans and allowed to dry overnight. Control biological indicators, which were inoculated with ~107 naturally acrystalliferous B. thuringiensis Al Hakam spores, were placed inside the aircraft prior to dissemination of Btcry- spores. The inside of the aircraft was held at 75°C to 80°C and 70% to 90% relative humidity for seven days, then pre- and post-treatment sample results were evaluated. Overall, the study was designed to assess the efficacy of a hot, humid air decontamination approach, but it also demonstrated practical use of a B. thuringiensis strain (Btcry-) enhanced for use in environmental sampling. Viability results for the selected test conditions were similar for the newly developed Btcry- and the B. thuringiensis Al Hakam strains. To further evaluate the genetic relationship among B. thuringiensis subsp. kurstaki HD-1 (the parent strain of Btcry-) and 232 B. anthracis, B. cereus, and B. thuringiensis isolates, Amplified Fragment Length Polymorphism (AFLP) analysis (Hill et al., 2004) was performed. Consistent with the Hill et al. (2004), AFLP analysis, in which the monomorphic nature of B. anthracis strains was demonstrated, the tested B. anthracis strains clustered together in a single phylogenetic branch. The B. cereus and B. thuringiensis isolates formed additional but related branches. B. thuringiensis Al Hakam mapped within the same 33 ------- branch as the tested B. anthracis strains, whereas B. thuringiensis subsp. kurstaki HD-1 mapped to a different but closely related branch. As expected, the analysis showed that Btcry- is genetically similar to B. anthracis. In addition, tests in an environmental chamber demonstrated that Btcry- had inactivation kinetics similar to those observed for the naturally acrystalliferous B. thuringiensis Al Hakam strain, which had previously shown inactivation kinetics similar to B. anthracis ASterne under selected test conditions (Buhr et al., 2012). Enhanced strains of acrystalliferous Bacillus thuringiensis BMB171: Public access (via GenBank) to the annotated genome sequence for naturally acrystalliferous B. thuringiensis strain BMB171 (He et al., 2010) made it a feasible candidate for modification. Park et al. (2017) developed several new strains of BMB171, each with enhancements designed to address specific environmental sampling concerns such as detection in samples containing non-target organisms and the long-term persistence of spores after environmental release (Park et al., 2017). To create a readily identifiable strain (BT-001), the crtM-crtN pigment-producing genes from Staphylococcus aureus KCTC 3881 (Wieland et al., 1994) were randomly inserted via transposon mutagenesis into the BMB171 chromosome. The crtM-crtN genes produce enzymes which convert farnesyl diphosphate to 4,4'-diaponeurosporene, thereby conferring a yellow phenotype on colonies growing on solid media. Whole-genome sequencing results for BT-001 showed that the pigment- producing genes were inserted only once, disrupting a gene encoding the hypothetical protein BMB171_C4312, which was assumed to be non-essential when apparently normal culture growth and sporulation were observed for the new strain. TSA plating assays showed that the yellow colonies of non-heat-treated BT-001 were distinguishable from those of soil organisms which survived a 70 °C incubation for 30 min. Colony pigmentation is a significant advantage for analysis with traditional agar plating, but competition from numerous non-target organisms may impact germination and outgrowth 34 ------- of targeted spores, especially for low-concentration samples. Therefore, repeatable and reliable enumeration of viable spores for complex environmental samples is still a challenge. To reduce the long-term persistence of test spores in the environment, two enhancement approaches were used. First, a spoOA gene knockout circuit was introduced to prevent re-sporulation of germinated spores. Transcription of the spoOA gene is controlled by a promoter-switching mechanism (Chibazakura et al., 1991; Strauch et al., 1992) such that spoOA gene expression during vegetative cell growth is held to low levels under a promoter recognized by the housekeeping sigma factor, oA (Chastanet & Losick, 2011; Chibazakura et al., 1991; Haldenwang, 1995). But under sporulation-inducing conditions and during the transition from exponential growth to stationary phase, gene expression switches to a promoter recognized by the sporulation-associated sigma factor (oH) and SpoOA cell levels are substantially increased (Chastanet & Losick, 2011; Chibazakura et al., 1991; Haldenwang, 1995). SpoOA is phosphorylated via a phosphorelay system in response to external stimuli signaling stress or nutrient depletion (Smith, 1989). In this activated, phosphorylated form, SpoOA serves as a master regulator for sporulation and controls expression of hundreds of sporulation associated genes (Hoch, 2017; Molle et al., 2003; Piggot & Hilbert, 2004). Park et al. (2017) used the loxP-Cre recombinase system (Meinke et al., 2016) to create a sporulation-dependent spoOA gene knockout (Park et al., 2017). The lox P-Cre recombinase was inserted in the BMB171 chromosome under the control of a promoter activated during sporulation events. So, just as high levels of SpoOA are needed to fully complete formation of an endospore, the Cre recombinase mediates spoOA gene disruption, effectively preventing sporulation. Several of the new BMB171 strains had greater than 99% spoOA knockout efficiency during induced sporulation bench experiments. While the spoOA knockout feature provides added environmental safety and potentially reduces the presence of residual spores at multiple-use test sites, further testing in a field setting is needed to fully evaluate its applicability for long-term fate and transport studies. 35 ------- The second approach for reducing environmental spore persistence was directed toward small acid soluble spore proteins (SASPs). These abundant proteins saturate the core DNA and have an important role in spore resistance to multiple stresses (Setlow, 1988, 1995), such as exposure to UV light (Mason & Setlow, 1986; Moeller et al., 2009), heat (Setlow & Setlow, 1995; Setlow, 2007), and various chemicals such as hydrogen peroxide (Setlow & Setlow, 1993). SASPs are formed during sporulation, located exclusively in the spore core, and degraded during germination and outgrowth (Setlow, 1988, 2006). To create an enhanced spore strain predicted to quickly lose viability after release into the environment, Park et al. (2017) used l-Scel mediated transformations (Janes & Stibitz, 2006) to create BMB171 strains lacking one or more genes encoding SASPs (Park et al., 2017). Amino acid sequences for SspA and SspB from Bacillus subtilis 168 were used to identify BMB171_C04286 (sspA) and BMB171_C0753 (sspB). Gene knockouts were created at each of these sites. Bench-scale experiments with spores of the ssp mutants showed that they were substantially more sensitive to UV-C (78 juW/cm2) than wild type BMB171 spores, with nearly 100% of the double mutants (sspA", sspB") losing viability after a 20-s exposure. Heat sensitivity was also increased in the mutant strains. Over a 10-week period at 37 °C, spores with the double knockout (sspA", sspB") lost viability (~1 log reduction) in comparison to BMB171 wild type spores (~0.1 log reduction). With the enhanced sensitivity to UV light, spore persistence in the environment is predicted to be considerably reduced; therefore, these strains may not be feasible for long-term environmental studies designed to assess spore viability. And when environmental samples (e.g., soil) are heat treated (70 °C) to reduce growth of non-target background organisms, the increased heat sensitivity may also prevent the use of this strain for viability assays. PCR detection may be an option if these strain characteristics are desired. Strain BT-016 incorporates all three enhancements (crtM-crtN pigment producing addition, spoOA gene knockout circuit, and sspA, sspB deletions) plus deletion of the pIcR gene (Lereclus et al., 1996; Park et al., 2017). The pIcR gene encodes the transcriptional activator PIcR, which has been implicated in 36 ------- the regulation of hundreds of genes (Gohar et al., 2008) encoding degradative enzymes, cell-surface proteins, or toxins that function in intra- and extra-cellular locations (Agaisse et al., 1999; Gohar et al., 2005). For example, PIcR regulates the secretion of hemolysins and Bacillus strains (including BT-016) with p/cR deletions have a diminished hemolysis phenotype on sheep blood agar. Because of the PIcR association with virulence in B. thuringiensis strains (Agaisse et al., 1999; Salamitou et al., 2000), the pIcR gene was deleted for added safety (Park et al., 2017). To demonstrate enhanced safety for BT-016, the researchers intratracheal^ infected BALB/c mice (Potter, 1985) with spores (1 x 107 CFU/mouse) of either wild type BMB171 or modified BT-016, then analyzed lung homogenates for the presence of viable spores or cells at 1, 2, and 4 weeks post-infection (Park et al., 2017). Recoveries for modified BT- 016 were significantly lower than for wild type BMB171, possibly indicating that the modified strain was easier to clear because the sspA, sspB deletions made the spores more sensitive to phagocytic reactive oxygen species such as hydrogen peroxide (Park et al., 2017). While all of these strains need further testing to demonstrate applicability in field settings, the methods demonstrated in this work open significant opportunities for creation of strains with other desired enhancements. Stable genetic insertions in Bacillus thuringiensis subsp. kurstaki HD-1: Buckley et al. (2012) reported a unique approach to develop B. anthracis surrogates specifically designed for studies in complex outdoor environments. Using Bacillus thuringiensis subsp. kurstaki HD-1, ATCC 33679, crystal positive serotype 3a3b (B. thuringiensis subsp. kurstaki HD-1), they developed and tested a method for introducing unique, short, and stable nucleotide sequences (referred to as barcodes) into a single intergenic spacer region of the organism's chromosome. The barcodes provide specific signatures for the test organism, making it clearly distinguishable from not only naturally occurring environmental Bacillus but also from test organisms released during prior experiments. Residual experimental organisms are particularly concerning for spore formers since they can persist on building materials 37 ------- (Enger et al2018; Wood et al., 2015) or in the environment for long periods (decades) of time (Carlson et al., 2018; Van Cuyk et al., 2011). To develop the modified B. thuringiensis subsp. kurstaki HD-1 strain, potential barcode insertion sites were identified using the published genome of B. thuringiensis subsp. kurstaki strain BMB171 and screened according to a list of selection rules (Buckley et al., 2012). For example, the target region had to be located on the chromosome to maximize replication stability and the insertion point had to be in an intergenic spacer region to minimize potential disruption of coding sequences. Of the 294 intergenic spacer regions identified, three met all the selection criteria. Potential 20 base pair (bp) barcodes were screened against an unpublished B. thuringiensis subsp. kurstaki HD-1 (ATCC 33679) draft genome (M. Krepps, S. Broomall, P. Roth, C.N. Rosenzweig, and H.S. Gibbons) and sequences with any similarity to the B. thuringiensis subsp. kurstaki HD-1 chromosome were discarded. Barcoded sequences with flanking regions having homology to the targeted chromosomal regions were synthesized and cloned into a plasmid delivery vector. The barcodes were ultimately introduced into the B. thuringiensis subsp. kurstaki HD-1 chromosome by bacterial mating protocols and a series of homologous recombination events mediated by endonuclease l-Scel (Janes & Stibitz, 2006). Two unique B. thuringiensis subsp. kurstaki HD-1 strains with stable genetic inserts denoted "T1B1" and "T1B2" were created with this approach. Each 46-bp insert is composed of two sequences separated by an fcoRI restriction site, as follows: 1) a sequence that is common to both inserts, and 2) a specific sequence, either T1B1 or T1B2. Real-time PCR detection assays using SYBR-green platforms were developed to target the common tag as well as each specific tag. Assays (4 replicates) targeting the common tag detect both strains, but assays for either T1B1 or T1B2 detect only the individually targeted strain. Assays targeting either of the three tagged regions (common, T1B1, or T1B2) did not detect DNA from other Bacillus strains such as non-barcoded B. thuringiensis subsp. kurstaki, B. anthracis Ames, B. anthracis ASterne, B. atrophaeus 38 ------- subsp. globigii, B. subtilis, or B. cereus. Sensitivities for each assay were reported as 83, 83, and 8.3 genome copies for the common tag, T1B1 specific tag, and T1B2 specific tag, respectively. The T1B2 barcoded strain was subsequently used in spore release trials occurring both in a controlled indoor setting and outdoors (Emanuel et al., 2012). The indoor spore release was conducted in an ambient breeze tunnel designed to simulate outdoor wind conditions. Vinyl tiles situated in rows (5 rows of 11 tiles each) were used for spore capture and sampling. Dry T1B2 barcoded B. thuringiensis subsp. kurstaki HD-1 spores (~100 mg) were dispersed over the tiles using a fan-generated breeze. Aerosolized spores were allowed to settle overnight. The following day, wetted cotton wipes were used to collect spores from each tile surface. Samples were analyzed by PCR and culture. Reported results showed that as the distance from the seeded tile area increased, there was a consistently decreasing trend of average spore detection on the collection tiles. This inverse relationship was reported for both PCR (Ct values) and plate assay (CFU/ml) detection methods. While these results demonstrate a dose- dependent relationship between PCR detection and culture enumeration, spore quantification via PCR detection of the unique barcode is not provided. The outdoor test with T1B2 barcoded B. thuringiensis subsp. kurstaki HD-1 was conducted at Aberdeen Proving Ground, MD (Emanuel et al., 2012). A bank of 20 dry filter unit (DFU) air sample collectors were arrayed downwind of a single spore release area. The test was carried out over a 14-day period and consisted of background sampling, two spore releases each followed by DFU sampling, then a simulated air turbulence (with leaf blowers) event followed by additional DFU sampling. Barcoded T1B2 B. thuringiensis subsp. kurstaki HD-1 spores (~3.3 x 1013 spores) were released on Day 1, then non- barcoded B. thuringiensis subsp. kurstaki HD-1 WT spores (~9.5 x 1012 spores) were released eight days later. A light detection and ranging (LIDAR) system was used to monitor movement of the aerosolized spore cloud over the targeted DFU collection areas. Filters from the DFUs were collected and analyzed by PCR and culture plating. For the PCR analysis, filters were suspended in 10 ml phosphate-buffered 39 ------- saline (PBS) with 0.1% Triton X-100. Aliquots of 1 ml were subjected to a 15-min bead beating protocol to lyse spores. Following bead beating, DNA was extracted from the supernatant with a BioMek FX laboratory automation workstation and eluted in 150 |al of water. PCR reactions based on SYBR green chemistry were performed with 1 |al DNA aliquots. PCR results (Ct values) for both the common and T1B2 specific tags were compared to plate assay data (CFU ml 1). And although the PCR data was not quantitated with a standard curve, trends for both the PCR and culture plating detection signals were consistent. After the initial spore release (T1B2 barcoded B. thuringiensis subsp. kurstaki HD-1) on Day 1, there was an increase in PCR detection signals for the barcoded strain as expected. And after the second spore release (non-barcoded B. thuringiensis subsp. kurstaki HD-1) eight days later, PCR signals for the non-barcoded B. thuringiensis subsp. kurstaki HD-1 strain were much larger than for either T1B2 or the common tag. Overall, the test demonstrated that barcoded B. thuringiensis subsp. kurstaki strains released in outdoor field studies could be detected by the SYBR green PCR assays reported by Buckley et al (Buckley et al., 2012). But because the reverse PCR primer anneals to a region of the WT genome, the developers expressed concern regarding asymmetric amplification. A new TaqMan probe-based platform is desired to enhance assay fidelity. Non-viable DNA-barcoded aerosol test particles: Developed at Lawrence Livermore National Laboratory, these particles are composed of short, customizable oligonucleotide sequences and maltodextrin, an FDA-approved food additive (Harding et al., 2016). The particles, referred to as DNATrax (DNA Tagged Reagents for Aerosol experiments), are generated with a spray drying process which produces a fine crystalline powder. Spray-dried particle-size distribution can vary significantly, but the DNATrax process was optimized to provide microparticle sizes similar to those associated with Bacillus spores (Carrera et al., 2007). Aerodynamic particle sizer (APS) and scanning electron microscopy (SEM) analyses showed that generated particles ranged in diameter from 1 to 5 microns (Harding et al., 2016). The particles offer several advantages for aerosol transport and fate experimentation. For 40 ------- example, the oligonucleotide sequences can be designed to minimize similarity to DNA found in common background microorganisms. It also can be customized for individual tests, thereby eliminating problems associated with pre-existing background contamination and multiple releases in a single test area. The unique oligonucleotide sequences can be detected in qPCR assays designed with TaqMan probes specific to the customized sequence. And because the particles are non-viable and biodegradable, public safety concerns can be minimized, especially for releases planned near or in occupied areas. To demonstrate DNATrax distribution in a test setting, a small-scale aerosol release was conducted after working hours in an operational indoor facility. An eductor located near one end of a hallway was used to aerosolize approximately 1 gram of DNATrax particles designed with DNA sequences found in the marine thermophile Thermotoga maritima (Latif et al., 2013). Dry Filter Unit (DFU) aerosol collectors positioned both upstream and downstream of the particle release point were used to sample the ambient air for 30 min post-release. DFU samples were analyzed using qPCR. Most particles were detected in DFUs located nearest the release point, and as the distance from the release point increased, the number of detected particles decreased. Such particle transport data could be used to inform emergency evacuation plans. And although DNATrax particles are not suitable for testing decontaminant efficacy, they may have broader applicability for studies incorporating fate and transport considerations. Additional work is needed to compare the aerosol behaviors of DNATrax particles with viable B. anthracis surrogate spores. When tested under the same conditions (e.g., temperature and humidity), are the observed aerosol transport, surface distribution, and associated recovery characteristics similar? B. anthracis Sterne enhanced with fluorescence genes: Intended for use in macrophage infection studies and antibacterial compound screening in biosafety level 3 (BSL-3) facilities, the chromosome of B. anthracis Ames was modified to provide constitutive expression of either green 41 ------- fluorescent protein (GFP) or red fluorescent protein (RFP) (Su et al., 2014). To identify an appropriate gene delivery system and a robust Bacillus promoter, proof-of-concept work was conducted with surrogate B. anthracls Sterne. Constructs containing one of the fluorescent genes and a Bacillus promoter (either Pntr or P0253 for gene GBAA_0253) with demonstrated potent activity (Bergman et al., 2007; Gat et al., 2003) were prepared. Chromosomal insertions targeted the (B-lactamase coding region encompassing blal (GBAA_2507), which is not expressed at levels sufficient to provide B. anthracis Sterne with resistance to (B-lactam compounds such as penicillin (Chen et al., 2003). B. anthracis Sterne cells transformed with either promoter and streaked on brain heart infusion (BHI) medium exhibited fluorescence visible to the naked eye, but cells expressing either GFP or RFP under the control of the promoter P0253 provided the stronger signal. Whether or not this type of added fluorescence would provide enhanced detection capability for samples from complex matrices (such as grass or soil) with high loads of background microorganisms would need to be tested. Additionally, widespread outdoor release of B. anthracis Sterne (pX01+, pX02") may be prohibitive because of regulatory and safety concerns. The enhancement methodology and gene delivery system modeled in Su et al. (2014), however, could be used as a blueprint for enhancement of other B. anthracis surrogate strains with either GFP or RFP. GFP also has been engineered into B. anthracis Sterne to study its replication potential in blow flies, and this system may be also used as a model (von Terzi et al., 2014). Finally, in other work, B. anthracis Sterne 7702 was transformed with an RFP-containing plasmid, then used to investigate a potential environmental persistence mechanism through interactions with environmental amoebas common to moist soils and standing water (Dey et al., 2012). However, this approach may not be ideal for surrogates intended for long term outdoor studies, as the introduced plasmid may not be maintained without the presence of a selective pressure (Marston et al., 2005). Bioluminescent reporter phage: As an alternative to B. anthracis bacterial surrogates which have been enhanced for detection in environmental samples, recombinant reporter bacteriophages, or 42 ------- phages, may be considered. Phages are obligate intracellular parasites which infect bacteria by interacting with host bacterial cell receptors and injecting the phage genome (Salmond & Fineran, 2015). Once in the host, one of two replication strategies may be employed depending on whether the phage is virulent or temperate (Salmond & Fineran, 2015). Virulent phages replicate via a lytic cycle whereby newly formed virus particles are released from the infected bacteria by bacterial cell lysis (Salmond & Fineran, 2015). Temperate phages, on the other hand, may adopt a more stable approach: using a process termed "lysogeny." During lysogeny, phages may integrate with the chromosome or exist in a plasmid-like state such that they are replicated with the bacterial DNA. Under conditions of stress, phages leave the lysogenic state, adopt a lytic cycle, and are released from the lysed cell as new virions (Salmond & Fineran, 2015). And because they typically have a specific host range (Knoll & Mylonakis, 2014), phages may naturally provide specific detection mechanisms for targeted bacteria (Schofield & Westwater, 2009). Numerous phages have been associated with the Bacillus cereus group of bacteria (Gillis & Mahillon, 2014). For example, work with an atypical Bacillus cereus strain W led to the identification of a naturally occurring, temperate phage W (McCloy, 1951) which was later renamed W(3 (Schofield & Westwater, 2009). Tests showed that Phage W(B specifically infected smooth, non-encapsulated forms of 171 strains of B. anthracis and two strains of B. cereus without impacting other Bacillus species such as B. megaterium or B. subtilis (McCloy, 1951; Schofield & Westwater, 2009), raising the possibility that phages may be used to help identify B. anthracis in clinical samples. Later work led to isolation of a lytic variant of temperate phage W(B, which was designated as gamma (y) phage (Brown & Cherry, 1955). Gamma phage could infect not only the smooth form of B. anthracis, but also the encapsulated form. And upon failing to infect additionally tested B. cereus strains, its potential as a diagnostic tool for B. anthracis was solidified (Brown & Cherry, 1955). A validation study confirmed these results (Abshire et al., 2005), and y phage has been used for decades as part of a diagnostic test panel to detect and 43 ------- confirm the presence of B. anthracis in clinical samples (Inglesby et al., 2002; Schuch & Fischetti, 2006). Sample aliquots are mixed with phage, then incubated overnight on agar-based media. As the bacterial cells are lysed by the phage, viral plaques form on the solid media, indicating the presence of viable B. anthracis cells. In an effort to develop more rapid B. anthracis detection methods, a homologous recombination (Alberts et al., 2002) strategy was used to introduce the luxA and luxB genes from Vibrio harveyi into a non-essential region of the W(B phage (Schofield et al., 2013; Schofield & Westwater, 2009), creating phage \Nfi::luxAB. The bacterial luciferase gene cassette (luxAB) confers a bioluminescent phenotype and has been extensively used as a bioreporter (Close et al., 2012). An aldehyde substrate such as n- decanal can be supplied exogenously, and bioluminescence is recorded as relative light units (RLU) (Close et al., 2012; Schofield et al., 2013). Initial tests with B. anthracis Sterne showed that when the \Nfi::luxAB phage was mixed with cells, a bioluminescent signal above background was detected within 20 min and observed to have a dose-dependent response (Schofield & Westwater, 2009). To detect spores, a germination protocol is employed because the phage receptor is only present on the cell form. When spores (1.6 x 10s CFU ml"1) were heat activated, mixed with \Nfi::luxAB phage (6.6 x 10s PFU ml"1), and then incubated under spore germinating conditions, a dose-dependent signal above background (spores alone or phage alone) was observed. Similar results were observed in the presence of either B. cereus or B. thuringiensis cells or spores, demonstrating that \Nfi::luxAB phage could be used to detect B. anthracis Sterne in a mixed Bacillus population. Testing against 119 Bacillus strains belonging to the B. cereus group showed that phage \Nfi::\uxAB had a 95% specificity (Schofield et al., 2013). In other tests, the detection capability of \Nfi::luxAB was challenged with samples from various food matrices (Sharp et al., 2015). Liquid samples of 2% milk, half-and-half, and baby formula were spiked with B. anthracis Sterne spores ranging in concentration from 8 x 10° to 8 x 10s CFU ml"1. After an equilibration period, germination-inducing media was added and samples were incubated at 35 °C with 44 ------- agitation for either 7 or 16 hr, then \Nfi::luxAB phage was added and samples were monitored for bioluminescence. Signal response was significantly above background (phage only) after the 7-hr enrichment for all spore inoculation levels, except the 8 x 10° CFU ml"1, which required a 16-hr enrichment for detection. A similar pattern was observed for ground beef samples inoculated with spore concentrations ranging from 3.2 x 102 to 3.2 x 104 CFU g"1 and then heat treated (65 °C, 30 min). To achieve signals significantly above background (phage only) for the lowest inoculum, the enrichment time had to be extended to 16 hr. \Nfi::luxAB in non-heat-treated ground beef samples delivered significantly lower (~50 fold) signal responses, demonstrating the potential for background flora in complex samples to reduce B. anthracis detection. A second-generation reporter phage, \Nfi::luxAB-2, was developed by enhancing the promoter region directly upstream of the luxAB cassette (Sharp et al., 2016). The modified promoter included all conserved regions of Gram-positive bacterial promoters, resulting in increased signal intensity, duration, and sensitivity when compared to the original \Nfi::luxAB. Phage \Nfi::luxAB-2 was used with samples composed of either sterile or non-sterile soil (1 g) and a B. anthracis Sterne spore inoculum ranging in concentration from 101 to 107 CFU g"1 soil. For sterile soil, tests showed that a 12-hr enrichment period provided the best sensitivity, with signal detection at 101 CFU g"1 soil (Sharp et al., 2016). But when a fixed concentration (104 CFU g"1) of B. thuringiensis spores was also added to the sterile soil samples, the \Nfi::luxAB-2 assay sensitivity was degraded to 102 CFU g"1. Further degradation of assay sensitivity was observed for non-sterile soil samples (Sharp et al., 2016). Signals significantly above background (phage only) could be detected only at much higher B. anthracis Sterne spore concentrations (10s CFU g"1 soil). Adjustments to the multiplicity of infection and a 1-hr, 70 °C heat treatment to remove background soil organisms did not improve signal characteristics. However, the addition of spectinomycin (100 ng ml"), which could be employed to reduce growth of background organisms 45 ------- because a spectinomycin resistance gene was used as a selective marker during reporter phage construction, improved detection to 104 CFU g"1 soil. The second-generation \Nfi::luxAB-2 phage was also evaluated for samples collected from an urban pond, a freshwater lake, and brackish water (Nguyen et al., 2017). Water samples were inoculated with B. anthracis ASterne spores and equilibrated at 4 °C for 16 hr. Inoculated water was then added to germination-inducing media supplemented with \Nfi::luxAB-2 phage such that final spore concentrations were 101 to 10s CFU ml"1. Bioluminescence was recorded after an 8-hr enrichment period at 35 °C. The detection limit for all samples was 102 CFU ml"1, but the brackish water samples produced inconsistent signals. Additional tests with 0.22 pim filter sterilized brackish water samples provided a more dose- dependent signal with a detection limit of 103 CFU ml"1. These experiments demonstrate the potential for the use of reporter phage technology to detect spores in complex matrices such as food, soil, and ground water. The technology can be used with minimal sample processing and minimal equipment investment, but it may not be compatible with situations requiring high-throughput sample analysis. In addition, its quantitative capacity has not been demonstrated and assay sensitivities need to be improved. Further testing is needed for samples containing interferences such as high or low pH levels, high salinity, and/or chemicals. And finally, the specificity of the enhanced reporter phages limits their use to the B. anthracis Sterne or B. anthracis ASterne surrogates. While this specificity is a great advantage for clinical diagnostics, whether or not these organisms could be easily deployed in large-scale outdoor experiments is unknown, and perhaps unlikely in light of public perceptions of safety. However, the significant work to develop these reporter phages could serve as a guide to make enhanced phages that detect other B. anthracis surrogates with a more robust history of outdoor experimentation and demonstrated safety. 46 ------- Summary of Bacillus anthracisSurrogates Enhanced for Environmental Sampling Table 4.0 provides a summary of characteristics associated with each B. anthracis surrogate enhanced for environmental sampling and detection. Associated detection methods, along with potential advantages and disadvantages related to enhanced surrogate deployment in large scale outdoor studies, are also included. 47 ------- Table 4.0 - Detailed List of Enhancements for Bacillus anthracis Surrogates Enhanced Strain Parent Strain Enhancement Characteristics Detection Method Advantages Disadvantages References Btcry- Bacillus thuringiensis HD-1 Curing protocols were used to remove plasmids encoding parasporal crystals and virulence factors Traditional agar plating on rich media or Bacillus Brilliance Agar plating • Absence of parasporal crystals which may adhere to spores and affect transport properties • Provides viable spore and enumeration data • Tested in a field setting, has history of outdoor release • Detection in complex matrices may be limited by presence of background organisms • Detection requires incubation, increased analysis time (Bishop & Robinson, 2014; Bishop & Stapleton, 2016) BT-001 Bacillus thuringiensis BMB171 Random insertion of crtM-crtN from Staphylococcus aureus KCTC 3881, conferring a yellow phenotype on BT-001 colonies Traditional agar plating on rich media • Absence of parasporal crystals which may adhere to spores and affect transport properties • Provides viable spore and enumeration data • Chromosomal insertion of genes provides stability • Yellow phenotype enhances detection with traditional agar plating • Detection in complex matrices may be limited by presence of background organisms • Requires incubation, increased analysis time • Field testing needed (Park et al., 2017; Park et al., 2018) SpoOA knockout Bacillus thuringiensis BMB171 Insertion of the loxP-Cre recombinase under control of a promoter activated during sporulation to mediate disruption of spoOA gene, so once formed, spores cannot re-sporulate Traditional agar plating on rich media • Absence of parasporal crystals which may adhere to spores and affect transport properties • Provides viable spore and enumeration data • Chromosomal insertion of genes provides stability • Potentially reduced spore persistence at multiple-use test sites • Detection in complex matrices may be limited by presence of background organisms • Requires incubation, increased analysis time • Field testing needed (Park et al., 2017; Park et al., 2018) SASP deletions Bacillus thuringiensis BMB171 Gene disruptions were created in regions coding for small acid-soluble proteins BMB171_C04286 (sspA) and BMB171_C0753 (sspB) Traditional agar plating on rich media • Absence of parasporal crystals which may adhere to spores and affect transport properties • Provides viable spore and enumeration data • Potentially reduced spore persistence at multiple-use test sites • Enhanced spores lose viability after short exposure to UV-C light, potentially reducing suitability for use in long term environmental field trials • Detection in complex matrices may be limited by presence of background organisms; increased heat sensitivity may exclude use of heat treatment to reduce background • Requires incubation, increased analysis time • Field testing needed (Park et al., 2017; Park et al., 2018) BT-016 Bacillus thuringiensis BMB171 Includes yellow phenotype, SpoOA knockout circuit, SASP deletions, and PIcR deletion Traditional agar plating on rich media • Absence of parasporal crystals which may adhere to spores and affect transport properties • Provides viable spore and enumeration data • Added safety associated with reduced production of potential virulence factors • Yellow phenotype enhances detection with traditional agar plating • Deletion of pleiotropic regulator PIcR may have unknown and undemonstrated effects on spore behavior and cell growth (Park et al., 2017; Park et al., 2018) 48 ------- Table 4.0 - Detailed List of Enhancements for Bacillus anthracis Surrogates Enhanced Strain Parent Strain Enhancement Characteristics Detection Method Advantages Disadvantages References T1B1, T1B2 barcodes Bacillus thuringiensis HD-1, serotype 3a3b Short, unique nucleotide sequences inserted in intergenic region of chromosome SYBR-green based qPCR, traditional agar plating, or potentially RV-PCRwith development of TaqMan probe targeting T1B1 or T1B2 • Chromosomal insertion of genes provides stability • Unique bar code sequences allow qPCR detection and distinction of test organism from Bacillus background • qPCR detection does not require Incubation, resulting in potentially faster analysis times • RV-PCR could be used with semi-automation to provide culturing (for viability) and potentially faster analysis times • Has a history of field use and demonstrated safety profile • Presence of parasporal crystals, which may adhere to spores and affect transport properties, but plasmid curing could alleviate this issue • qPCR detection does not currently provide spore viability or enumeration data, but RV-PCR may be an alternative • Efficient and reliable DNA extraction from spores are significant challenges for qPCR detection, but could be potentially overcome with RV-PCR culturing • If used RV-PCR, there is potential for spore growth inhibition in presence of large background contingent • Use of RV-PCR and Most Probable Number method for spore quantification is not standardized (Buckley et al., 2012; Emanuel et al., 2012) DNATrax Non-viable barcoded aerosol test particles Short, unique, customizable nucleotides attached to maltodextrin, an FDA approved food additive TaqMan qPCR • DNATrax particle size and shape are similar to those of Ba spores • Unique bar code sequences allow qPCR detection and distinction of test organism from background • Customizable bar code sequences allow multiple releases in a single area • qPCR detection does not require Incubation, resulting in potentially faster analysis times • Amenable to automated sample processing • Has been tested in an indoor field setting • Use of a non-viable particle minimizes public safety concerns • Spore viability data not available • Transport and adhesion characteristics of DNATrax particles may differ significantly from those of viable Bacillus spores under similar field conditions, such as temperature and humidity • DNA extraction methods may require further optimization for complex environmental samples such as soil and ground water • DNATrax particle stability in and recovery from environmental samples (e.g., soil) may be compromised by degradation or extra-cellular nucleases of native soil organisms • Outdoor field tests are needed (Harding et al., 2016) GFP and RFP Bacillus anthracis Sterne Fluorescent protein genes inserted in chromosome in naturally non-expressed p- lactamase coding region, GFP or RFP expressed under control of constitutive promoter P0253 Fluorescence observable by naked eye using Brain Heart Infusion agar, or fluorescence microscopy • Chromosomal insertion of genes provides stability • Provides viable spore and enumeration data • qPCR or RV-PCR detection assay targeting RFP or GFP could be designed • Fluorescence observable by naked eye • Detection in complex matrices may be limited by presence of background organisms • Requires incubation, increased analysis time • Stability and reproducibility of RFP or FGP signal in environmental samples needs to be tested • Outdoor field tests are needed (Su et al., 2014) 49 ------- Table 4.0 - Detailed List of Enhancements for Bacillus anthracis Surrogates Enhanced Strain Parent Strain Enhancement Characteristics Detection Method Advantages Disadvantages References Bioluminescent reporter phage Phage Wp, which is specific for B. onthrocis Modified with iuxAB bioluminescent reporter cassette Relative light units (RLU) recorded after addition of an aldehyde substrate such as n-decanal • Phage detection of target B. onthrocis is specific, even in presence of other Bacillus strains • Provides evidence of spore viability because phage only infects metabolically active cells with appropriate phage receptor • Current technology only applicable to B. onthrocis Sterne or ASterne surrogates which have limited potential for large scale outdoor studies • Detection in complex matrices limited by presence of background organisms • Spore quantification data not provided by current assays (Schofield et al., 2013; Schofield & Westwater, 2009; Sharp et al., 2016; Sharp et al., 2015) 50 ------- Bacillus a nth racis Surrogates for Environmental Sampling™ Desired Characteristics, Recommendations, and Conclusion While it should be recognized that a single surrogate may not be applicable for all test conditions and experimental goals, a summary of desired characteristics for a B. anthracis surrogate intended for environmental sampling/detection studies is shown in Table 5.0. Existing surrogates meeting each desired characteristic are also listed. Table 5.0 - Desired Characteristics for Bacillus anthracis Surrogates Desired B. anthracis Surrogate Characteristic Applicable Surrogate Spore size, shape, and surface properties (charge, protein content, surface adhesion, architecture, exosporium, appendages) similar to virulent B. anthracis strains • B. thuringiensis (some strains have appendages) • B. anthracis Sterne, ASterne Absence of parasporal crystal inclusions • B. thuringiensis subsp. kurstaki HD-1, enhanced strain Btcry' • B. thuringiensis subsp. Al Hakam • B. thuringiensis BMB171 • B. atrophaeus var. globigii • B. anthracis Sterne, ASterne Spore inactivation properties (UV, heat, and chemical resistance) similar to or greater than B. anthracis • B. atrophaeus var. globigii • B. thuringiensis • B. anthracis Sterne, ASterne Spore aerosol transport and resuspension characteristics similar to B. anthracis • B. anthracis Sterne, ASterne • B. thuringiensis subsp. kurstaki HD-1, enhanced strain Btcry' • B. thuringiensis subsp. Al Hakam • B. thuringiensis BMB171 History of safe outdoor use • B. thuringiensis subsp. kurstaki HD-1 Absence of virulence factors • B. thuringiensis subsp. kurstaki HD-1, enhanced strain Btcry' • B. thuringiensis BMB171, enhanced strain BT-001 • B. thuringiensis subsp. Al Hakam • B. thuringiensis BMB171 • B. atrophaeus var. globigii • B. anthracis ASterne 51 ------- Table 5.0 - Desired Characteristics for Bacillus anthracis Surrogates Desired B. anthracis Surrogate Characteristic Applicable Surrogate Manifests a distinguishing phenotype, such as pigmentation or fluorescence, when cultured. Appropriate genes should operate under a constitutive Bacillus promoter, be codon optimized for expression in the surrogate, have an appropriate Bacillus transcription terminator, and be stably incorporated into the chromosome such that normal growth is not affected. • B. atrophaeus var. globigii • B. thuringiensis BMB171, enhanced strain BT-001 • B. anthracis Sterne, enhanced with GFP or RFP Has a molecular fingerprint that is distinct from background, non- target, heat resistant organisms such as Bacillus subtilis and Bacillus cereus and is stably maintained over generations of laboratory spore production • B. thuringiensis subsp. kurstaki HD-1, enhanced strains with genetic bar codes T1B1 or T1B2 • B. thuringiensis BMB171, enhanced strain BT-001 • D NATrax Safe and cost-effective methods are available to extract, detect, and enumerate viable spores in samples from complex matrices, with sensitivity and specificity Challenges exist with all currently available surrogates and enhanced strains Several options can be considered for the development of a B. anthracis surrogate possessing all or nearly all of the desired characteristics listed in Table 5.0. One option is to try recovering spores of existing B. anthracis surrogates on traditional rich media supplemented with a selective agent. One of the key challenges associated with environmental sampling studies is the presence of non-target organisms. These organisms may outcompete targeted germinating spores for nutrients and thereby inhibit their growth. Selective plating methods that exclude growth of non-target organisms could provide a safe, inexpensive, and simple way to incorporate viability data into test results. Historical approaches to isolate Bacillus species such as acetate selection (Travers et al., 1987), antibiotic selection (Bishop & Robinson, 2014), and 50% v/v ethanol incubations (Logan & De Vos, 2009) may offer a means for recovery of targeted Bacillus surrogates from environmental samples, but these methods need to be tested with germinating spores. For example, metabolically active cells of some Bacillus strains grow in the presence of penicillin, but the use of this approach to specifically recover and enumerate known concentrations of dormant spores needs to be tested first in sterile laboratory conditions. If spores germinate and develop colonies on rich media in the presence of the selective agent, then further testing with known spore concentrations in complex environmental samples could be pursued. Such a simple selective-plating approach combined with sample heat treatment may reduce background 52 ------- contamination to levels that provide enhanced sample resolution, thereby making surrogate identification and viability quantification by traditional agar plating more reliable. Another option is to further enhance an existing surrogate strain that has a strong safety record and history of outdoor use. For example, B. thuringiensis subsp. kurstaki HD-1 has been widely used in pesticides and has already been modified with chromosomally located bar codes. The modified strains have also been field tested. Several additional enhancements to this strain would provide a surrogate with most of the desired characteristics listed in Table 5.0. First, plasmid curing methods described by Bishop and Robinson (2014) and used to develop enhanced Btcry' (Bishop & Robinson, 2014) could be used to eliminate parasporal crystal formation in the T1B1/T1B2 bar-coded B. thuringiensis strain. PCR could be used to verify removal of the toxin genes and SEM could be used to examine spore preparations for the absence of crystals. Second, and more difficult to achieve, would be the chromosomal addition of color-producing genes in bar-coded B. thuringiensis. The techniques employed by Park et al. (2017) provide random chromosomal insertions (Park et al., 2017), so the undesired interruption of key metabolic genes is likely. Use of this technique, while effective, would require extensive screening to make sure the pigmented strain does not have altered or undesirable sporulation, germination, and growth kinetics. Another option is to explore the allelic exchange mechanism described by Janes and Stibitz (2006) and used to modify B. anthracis. This approach allows more selective gene placement within the chromosome, but analysis would be required to select a non-essential insertion region. Potential color- producing gene candidates include the following: the crtM-crtN genes, which encode a yellow pigment from Staphylococcus aureus KCTC 3881, employed by Park et al. (2017); the GFP or RFP insertions used to transform B. anthracis by Su et al. (2014); or perhaps selected genes from the pig cluster responsible for production of the red prodigiosin secondary metabolite found in Serratia marcescens (Harris et al., 2004; Thomson et al., 2000). Selected genes could be synthesized and codon-optimized for expression in 53 ------- Bacillus thuringiensis. Synthesized genes would be flanked by synthesized DNA sequences including a robust, constitutive promoter, such as GBAA_0253 (Su et al., 2014), and a terminator sequence specific to Bacillus. The addition of pigmentation or fluorescence would enhance detection by traditional agar plating, especially for environmental samples that may have low levels of background organisms (e.g., aerosols or some liquid runoff samples). Once these genes are introduced, TaqMan assays targeting either these new sequences or the existing T1B1/T1B2 bar-coded regions could be designed. This would open the possibility of using RV-PCR for sample processing, detection, and semi-quantification. Third, explore the use of additional genome editing technologies for surrogate enhancement. Once referred to as a bacterial immune system, the clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) system (Adli, 2018) has been used to modify the genomes of B. subtilis (Westbrook et al., 2016), B. thuringiensis BMB181 (Tan et al., 2019), B. anthracis (Wang et al., 2019), and B. cereus (Wang et al., 2019). The CRISPR/Cas-9 system was used to introduce a point mutation in the B. cereus pIcR gene, resulting in loss of hemolytic and phospholipase activities (Wang et al., 2019). The possibility for using this system to modify the existing genetic bar codes in B. thuringiensis T1B1/T1B2 and create new unique tags could be explored. And if genes associated with appendage production are identified, CRISPR/Cas-9 may be used to disrupt these genes to eliminate the possibility of spore appendages. Fourth, the concept of synthetic auxotrophy could be explored. It has been discussed by Park et al. (2018), specifically in the context of biocontainment (Park et al., 2018). In this scenario, growth of the surrogate strain would be dependent on use of a synthetic amino acid, which would be exogenously supplied in the laboratory. This approach has been demonstrated using Escherichia coli, a synthetic phenylalanine-derived amino acid, and multiplex automated genome engineering (Rovner et al., 2015). While synthetic auxotrophy is a promising biocontainment strategy, whether or not it could work as a selective mechanism for detecting target spores in environmentally derived samples is unknown. 54 ------- Finally, an immuno-capture concept could be explored. Antibodies designed to target the BcIA spore surface protein (Nuttall et al., 2011) could be constructed and tagged with the small molecule biotin. Streptavidin-lined filters could then be used to capture the biotin-tagged spores, thereby exploiting one of the strongest interactions known in nature (i.e., biotin and streptavidin) to separate the tagged spores from background organisms (Chivers et al., 2011). Captured spores could then be germinated on rich media and distinguished from other non-target Bacillus organisms by the enhanced pigmentation. In conclusion, this review provides a description of B. anthracis surrogates that have been modified for detection in environmental samples. In most cases, details regarding the methods used to generate the selected enhancements were included. This approach was used so that the reader may gain an appreciation for the enormous amount of work involved and to explain the rationale for selection of a particular enhancement. In addition to information regarding specific strain enhancements, experimental results with the new strains were also provided where available. 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The specific search terms were as follows: Detection OR Quantification methods "environmental samples of Bacillus anthracis" Detection OR Quantification "Bacillus anthracis surrogates" Detection OR Quantification "surrogate biological warfare agents" Detection OR Quantification "Bacillus thuringiensis" Detection OR Quantification "Bacillus atrophaeus OR globigii" "Bacillus atrophaeus OR globigii OR thuringiensis" antibiotic marker "Bacillus atrophaeus OR globigii OR thuringiensis" fluorescent marker "Bacillus atrophaeus OR globigii OR thuringiensis" luminescence "Bacillus atrophaeus OR globigii OR thuringiensis" radiolabeled These terms were expanded as resulting articles led to additional information. When evaluating scientific and technical information for use in this review, the following five general assessment factors, as outlined in the EPA General Assessment Factors for Evaluating the Quality of Scientific and Technical Information (EPA/100/B-03/001).20, were considered: • Soundness: The extent to which the scientific and technical procedures, measures, methods, or models employed to generate the information is reasonable for, and consistent with, the intended application. • Applicability and Utility: The extent to which the information is relevant for the intended use. • Clarity and Completeness: The degree of clarity and completeness with which the data, assumptions, methods, QA, and analyses employed to generate the information are documented. • Uncertainty and Variability: The extent to which variability and uncertainty (quantitative and qualitative) related to results, procedures, measures, methods, or models are evaluated and characterized. • Evaluation and Review: The extent of independent verification, validation, and peer review of the information or of the procedures, measures, methods, or models. 83 ------- vvEPA United States Environmental Protection Agency PRESORTED STANDARD POSTAGE & FEES PAID EPA PERMIT NO. G-35 Office of Research and Development (8101R) Washington, DC 20460 Official Business Penalty for Private Use $300 ------- |