Erin Silvestri

Recent literature review of soil processing methods for recovery of Bacillus anthracis spores

Identifying virulent Bacillus anthracis within soil is a difficult task due to the number and diversity of other organisms and impeding chemical constituents within soil. Regardless of the detection assay, the initial sample must be processed efficiently to ensure that debris, chemical components, and biological impurities do not obstruct downstream analysis. Soil sample processing protocols can be divided into two general types: indirect and direct. There are two requirements for successful indirect isolation of B. anthracis from soil samples: dissociate the spores from the soil particles and physically separate the free spores from the soil particles. Adding an aqueous carrier medium to a soil sample creates a sample slurry for easier manipulation. Centrifugation, high specific gravity separation, immunomagnetic separation, filtration, and settling have been used to physically separate spores from soil. Direct processing utilizes a soil sample without first separating the spores from the bulk sample and falls under two principal types: culturing on B. anthracis selective agar and bulk DNA extraction. Direct and indirect processing steps each have associated advantages and disadvantages. The objective of this review was to consolidate information acquired from previous research, focusing primarily on data gleaned in the last decade, on the processing of soils contaminated with B. anthracis. As shown in this review, an optimized soil-processing protocol with a known recovery rate and associated confidence intervals is needed. A reliable processing protocol would allow for multiple investigators and laboratories to produce high-quality, uniform results in the event of a B. anthracis release.

Performance of a Novel High Throughput Method for the Determination of VX in Drinking Water Samples

VX (O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothioate) is a highly toxic organophosphorus nerve agent, and even low levels of contamination in water can be harmful. Measurement of low concentrations of VX in aqueous matrixes is possible using an immunomagnetic scavenging technique and detection using liquid chromatography/tandem-mass spectrometry. Performance of the method was characterized in high-performance liquid chromatography (HPLC)-grade water preserved with sodium omadine, an antimicrobial agent, and sodium thiosulfate, a dechlorinating agent, over eight analytical batches with quality control samples analyzed over 10 days. The minimum reportable level was 25 ng/L with a linear dynamic range up to 4.0 μg/L. The mean accuracies for two quality control samples containing VX at concentrations of 0.250 and 2.00 μg/L were 102 ± 3% and 103 ± 6%, respectively. The stability of VX was determined in five tap water samples representing a range of water quality parameters and disinfection practices over a 91 day period. In preserved tap water samples, VX recovery was between 81 and 92% of the fortified amount, 2.0 μg/L, when analyzed immediately after preparation. Recovery of VX decreased to between 31 and 45% of the fortified amount after 91 days, indicating hydrolysis of VX. However, the preservatives minimized the hydrolysis rate to close to the theoretical limit. The ability to detect low concentrations of VX in preserved tap water 91 days after spiking suggests applicability of this method for determining water contamination with VX and utility during environmental remediation.

Observations on the Migration of Bacillus Spores Outside a Contaminated Facility During a Decontamination Efficacy Study

The fate and transport of Bacillus anthracis spores in indoor and outdoor environments is not well understood. The Bio-Response Operational Testing and Evaluation exercise evaluated decontamination technologies in a twostory building experimentally contaminated with Bacillus atrophaeus subspecies globigii spores. The Bio-Response Operational Testing and Evaluation project provided a means to evaluate the potential for the spores dispersed inside the building to migrate to the outside as well as to investigate a new method for processing soils contaminated with Bacillus spores. Duplicate sterile sand samples were placed within the tent covering the building, but outside the building itself, near entrances, exits, and high-traffic areas to assess migration and deposition of newly disseminated spores. The sand samples were utilized during three stages of the decontamination study: before spore dissemination, after spore dissemination, and after decontamination of the building. In addition, two sets of sand samples placed within the building provided positive controls. Results from two different building decontamination approaches were studied. Results were tabulated as presence or absence rather than as a quantitative figure. There was no significant association among positive samples and the location of the samples around the building. There was a significant association between the different stages of each decontamination study and the number of detectable samples. The results of this study demonstrate the potential for spores to migrate out of a contaminated building and the importance of considering migration when assessing the scope of a contamination incident

Evaluation of swabs and transport media for the recovery of Yersinia pestis.

The Government Accountability Office report investigating the surface sampling methods used during the 2001 mail contamination with Bacillus anthracis brought to light certain knowledge gaps that existed regarding environmental sampling with biothreat agents. Should a contamination event occur that involves non-spore forming biological select agents, such as Yersinia pestis, surface sample collection and processing protocols specific for these organisms will be needed. Two Y. pestis strains (virulent and avirulent), four swab types (polyester, macrofoam, rayon, and cotton), two pre-moistening solutions, six transport media, three temperatures, two levels of organic load, and four processing methods (vortexing, sonicating, combined sonicating and vortexing, no agitation) were evaluated to determine the conditions that would yield the highest percent of cultivable Y. pestis cells after storage. The optimum pre-moistening agent/transport media combination varied with the Y. pestis strain and swab type. Directly inoculated macrofoam swabs released the highest percent of cells into solution (93.9% recovered by culture) and rayon swabs were considered the second best swab option (77.0% recovered by culture). Storage at 4°C was found to be optimum for all storage times and transport media. In a worst case scenario, where the Y. pestis strain is not known and sample processing and analyses could not occur until 72h after sampling, macrofoam swabs pre-moistened with PBS supplemented with 0.05% Triton X-100 (PBSTX), stored at 4°C in neutralizing buffer (NB) as a transport medium (PBSTX/NB) or pre-moistened with NB and stored in PBSTX as a transport medium (NB/PBSTX), then vortexed 3min in the transport medium, performed significantly better than all other conditions for macrofoam swabs, regardless of strain tested (mean 12 - 72h recovery of 85.9-105.1%, p<0.001). In the same scenario, two combinations of pre-moistening medium/transport medium were found to be optimal for rayon swabs stored at 4°C (p<0.001), then sonicated 3min in the transport medium; PBSTX/PBSTX and NB/PBSTX (mean 12-72h recovery of 83.7-110.1%).

Considerations for estimating microbial environmental data concentrations collected from a field setting.

In the event of an indoor release of an environmentally persistent microbial pathogen such as Bacillus anthracis, the potential for human exposure will be considered when remedial decisions are made. Microbial site characterization and clearance sampling data collected in the field might be used to estimate exposure. However, there are many challenges associated with estimating environmental concentrations of B. anthracis or other spore-forming organisms after such an event before being able to estimate exposure. These challenges include: (1) collecting environmental field samples that are adequate for the intended purpose, (2) conducting laboratory analyses and selecting the reporting format needed for the laboratory data, and (3) analyzing and interpreting the data using appropriate statistical techniques. This paper summarizes some key challenges faced in collecting, analyzing, and interpreting microbial field data from a contaminated site. Although the paper was written with considerations for B. anthracis contamination, it may also be applicable to other bacterial agents. It explores the implications and limitations of using field data for determining environmental concentrations both before and after decontamination. Several findings were of interest. First, to date, the only validated surface/sampling device combinations are swabs and sponge-sticks on stainless steel surfaces, thus limiting availability of quantitative analytical results which could be used for statistical analysis. Second, agreement needs to be reached with the analytical laboratory on the definition of the countable range and on reporting of data below the limit of quantitation. Finally, the distribution of the microbial field data and statistical methods needed for a particular data set could vary depending on these data that were collected, and guidance is needed on appropriate statistical software for handling microbial data. Further, research is needed to develop better methods to estimate human exposure from pathogens using environmental data collected from a field setting.

Stability of ricinine, abrine, and alpha-amanitin in finished tap water

Ricinine and abrine are potential indicators of drinking water contamination by ricin and abrin, respectively. Simultaneous detection of ricinine and abrine, along with α-amanitin, another potential biotoxin water contaminant, is reportable through the use of automated sample preparation via solid phase extraction and detection using liquid chromatography/tandem-mass spectrometry. Performance of the method was characterized over eight analytical batches with quality control samples analyzed over 10 days. For solutions of analytes prepared with appropriate preservatives, the minimum reporting level (MRL) was 0.50 μg L−1 for ricinine and abrine and 2.0 μg L−1 for α-amanitin. Among the analytes, the accuracy of the analysis ranged between 93 and 100% at concentrations of 1–2.5x the MRL, with analytical precision ranging from 4 to 8%. Five drinking waters representing a range of water quality parameters and disinfection practices were fortified with the analytes and analyzed over a 28 day period to determine their storage stability in these waters. The analytical signal from ricinine was observed to be stable for 28 days after being spiked into all tap waters investigated. The analytical signal for abrine and α-amanitin decreased within 5 h after these analytes were spiked into some drinking waters, but afterwards, remained stable for 28 days. The magnitude of the decrease correlated with common water quality parameters potentially related to sorption of contaminants onto dissolved and colloidal components within the particular water. Even with the decrease, the detectability offered by the method may be 100–1000 times greater than potential toxicological benchmarks, suggesting the utility of the method for all three analytes, with additional quality control precautions for abrine and α-amanitin.

Analysis of environmental contamination resulting from catastrophic incidents: part 2. Building laboratory capability by selecting and developing analytical methodologies.

Catastrophic incidents can generate a large number of samples of analytically diverse types, including forensic, clinical, environmental, food, and others. Environmental samples include water, wastewater, soil, air, urban building and infrastructure materials, and surface residue. Such samples may arise not only from contamination from the incident but also from the multitude of activities surrounding the response to the incident, including decontamination. This document summarizes a range of activities to help build laboratory capability in preparation for sample analysis following a catastrophic incident, including selection and development of fit-for-purpose analytical methods for chemical, biological, and radiological contaminants. Fit-for-purpose methods are those which have been selected to meet project specific data quality objectives. For example, methods could be fit for screening contamination in the early phases of investigation of contamination incidents because they are rapid and easily implemented, but those same methods may not be fit for the purpose of remediating the environment to acceptable levels when a more sensitive method is required. While the exact data quality objectives defining fitness-for-purpose can vary with each incident, a governing principle of the method selection and development process for environmental remediation and recovery is based on achieving high throughput while maintaining high quality analytical results. This paper illustrates the result of applying this principle, in the form of a compendium of analytical methods for contaminants of interest. The compendium is based on experience with actual incidents, where appropriate and available. This paper also discusses efforts aimed at adaptation of existing methods to increase fitness-for-purpose and development of innovative methods when necessary. The contaminants of interest are primarily those potentially released through catastrophes resulting from malicious activity. However, the same techniques discussed could also have application to catastrophes resulting from other incidents, such as natural disasters or industrial accidents. Further, the high sample throughput enabled by the techniques discussed could be employed for conventional environmental studies and compliance monitoring, potentially decreasing costs and/or increasing the quantity of data available to decision-makers.

Quantitative analysis and stability of the rodenticide TETS (tetramine) in finished tap water

The determination of the rodenticide tetramethylenedisulfotetramine (TETS) in drinking water is reportable through the use of automated sample preparation via solid phase extraction and detection using isotope dilution gas chromatography-mass spectrometry. The method was characterized over twenty-two analytical batches with quality control samples. Accuracies for low and high concentration quality control pools were 100 and 101%, respectively. The minimum reporting level (MRL) for TETS in this method is 0.50 μg L−1. Five drinking waters representing a range of water quality parameters and disinfection practices were fortified with TETS at ten times the MRL and analyzed over a 28 day period to determine the stability of TETS in these waters. The amount of TETS measured in these samples averaged 100 ± 6% of the amount fortified suggesting that tap water samples may be held for up to 28 days prior to analysis.

Optimization of a sample processing protocol for recovery of Bacillus anthracis spores from soil.

Following a release of Bacillus anthracis spores into the environment, there is a potential for lasting environmental contamination in soils. There is a need for detection protocols for B. anthracis in environmental matrices. However, identification of B. anthracis within a soil is a difficult task. Processing soil samples helps to remove debris, chemical components, and biological impurities that can interfere with microbiological detection. This study aimed to optimize a previously used indirect processing protocol, which included a series of washing and centrifugation steps. Optimization of the protocol included: identifying an ideal extraction diluent, variation in the number of wash steps, variation in the initial centrifugation speed, sonication and shaking mechanisms. The optimized protocol was demonstrated at two laboratories in order to evaluate the recovery of spores from loamy and sandy soils. The new protocol demonstrated an improved limit of detection for loamy and sandy soils over the non-optimized protocol with an approximate matrix limit of detection at 14spores/g of soil. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol will be robust enough to use at multiple laboratories while achieving comparable recoveries.

Review of processing and analytical methods for Francisella tularensis in soil and water

The etiological agent of tularemia, Francisella tularensis, is a resilient organism within the environment and can be acquired in many ways (infectious aerosols and dust, contaminated food and water, infected carcasses, and arthropod bites). However, isolating F. tularensis from environmental samples can be challenging due to its nutritionally fastidious and slow-growing nature. In order to determine the current state of the science regarding available processing and analytical methods for detection and recovery of F. tularensis from water and soil matrices, a review of the literature was conducted. During the review, analysis via culture, immunoassays, and genomic identification were the methods most commonly found for F. tularensis detection within environmental samples. Other methods included combined culture and genomic analysis for rapid quantification of viable microorganisms and use of one assay to identify multiple pathogens from a single sample. Gaps in the literature that were identified during this review suggest that further work to integrate culture and genomic identification would advance our ability to detect and to assess the viability of Francisella spp. The optimization of DNA extraction, whole genome amplification with inhibition-resistant polymerases, and multiagent microarray detection would also advance biothreat detection.