Heather A. Colburn

Bayesian Integrated Microbial Forensics

ABSTRACT In the aftermath of the 2001 anthrax letters, researchers have been exploring ways to predict the production environment of unknown-source microorganisms. Culture medium, presence of agar, culturing temperature, and drying method are just some of the broad spectrum of characteristics an investigator might like to infer. The effects of many of these factors on microorganisms are not well understood, but the complex way in which microbes interact with their environments suggests that numerous analytical techniques measuring different properties will eventually be needed for complete characterization. In this work, we present a Bayesian statistical framework for integrating disparate analytical measurements. We illustrate its application to the problem of characterizing the culture medium of Bacillus spores using three different mass spectral techniques. The results of our study suggest that integrating data in this way significantly improves the accuracy and robustness of the analyses.

Impurity profiling to match a nerve agent to its precursor source for chemical forensics applications.

Chemical forensics is a developing field that aims to attribute a chemical (or mixture) of interest to its source by the analysis of the chemical itself or associated material constituents. Herein, for the first time, trace impurities detected by gas chromatography/mass spectrometry and originating from a chemical precursor were used to match a synthesized nerve agent to its precursor source. Specifically, six batches of sarin (GB, isopropyl methylphosphonofluoridate) and its intermediate methylphosphonic difluoride (DF) were synthesized from two commercial stocks of 97% pure methylphosphonic dichloride (DC); the GB and DF were then matched by impurity profiling to their DC stocks from a collection of five possible stocks. Source matching was objectively demonstrated through the grouping by hierarchal cluster analysis of the GB and DF synthetic batches with their respective DC precursor stocks based solely upon the impurities previously detected in five DC stocks. This was possible because each tested DC stock had a unique impurity profile that had 57% to 88% of its impurities persisting through product synthesis, decontamination, and sample preparation. This work forms a basis for the use of impurity profiling to help find and prosecute perpetrators of chemical attacks.

Estimated copy number of Bacillus anthracis plasmids pXO1 and pXO2 using digital PCR

We evaluated digital PCR (dPCR) to directly enumerate plasmid and chromosome copies in three strains of Bacillus anthracis. Copy number estimates based on conventional quantitative PCR (qPCR) highlighted the variability of using qPCR to measure copy number whereas estimates based on direct sequencing are comparable to dPCR.

Detection of agar, by analysis of sugar markers, associated with Bacillus anthracis spores, after culture.

Detection of small quantities of agar associated with spores of Bacillus anthracis could provide key information regarding its source or growth characteristics. Agar, widely used in growth of bacteria on solid surfaces, consists primarily of repeating polysaccharide units of 3,6-anhydro-l-galactose (AGal) and galactose (Gal) with sulfated and O-methylated galactoses present as minor constituents. Two variants of the alditol acetate procedure were evaluated for detection of potential agar markers associated with spores. The first method employed a reductive hydrolysis step, to stabilize labile anhydrogalactose, by converting to anhydrogalactitol. The second eliminated the reductive hydrolysis step simplifying the procedure. Anhydrogalactitol, derived from agar, was detected using both derivatization methods followed by gas chromatography-mass spectrometry (GC-MS) analysis. However, challenges with artifactual background (reductive hydrolysis) or marker destruction (hydrolysis) respectively lead to the use of an alternative agar marker. A minor agar component, 6-O-methyl galactose (6-O-M gal), was readily detected in agar-grown but not broth-grown bacteria. Detection was optimized by the use of gas chromatography-tandem mass spectrometry (GC-MS-MS). With appropriate choice of sugar marker and analytical procedure, detection of sugar markers for agar has considerable potential in microbial forensics.

Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

To evaluate the sensitivity and specificity of the BioFire Diagnostics FilmArray® system in combination with their Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft) and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores.

Residual Agar Determination in Bacterial Spores by Electrospray Ionization Mass Spectrometry

Presented here is an analytical method to detect residual agar from a bacterial spore sample as an indication of culturing on an agar plate. This method is based on the resolubilization of agar polysaccharide from a bacterial spore sample, enzymatic digestion, followed by electrospray ionization tandem mass spectrometry (ESI-MS(n)) analysis for detection of a specific agar fragment ion. A range of Bacillus species and strains were selected to demonstrate the effectiveness of this approach. The characteristic agar fragment ion was detected in the spores grown on agar that were washed from 1 to 5 times, irradiated or nonirradiated, and not in the spores grown in broth. A sample containing approximately 10(8) spores is currently needed for confident detection of residual agar from culture on agar plates in the presence of bacterial spores with a limit of detection of approximately 1 ppm agar spiked into a broth-grown spore sample. The results of a proficiency test with 42 blinded samples are presented demonstrating the utility of this method with no false positives and only three false negatives for samples that were below the detection level of the method as documented.

Analysis of carbohydrate and fatty acid marker abundance in ricin toxin preparations for forensic information.

One challenge in the forensic analysis of ricin samples is determining the method and extent of sample preparation. Ricin purification from the source castor seeds is essentially a protein purification through removal of the nonprotein fractions of the seed. Two major, nonprotein constituents in the seed are the castor oil and carbohydrates. We used derivatization of carbohydrate and fatty acid markers followed by identification and quantification using gas chromatography/mass spectrometry (GC/MS) to assess compositional changes in ricin samples purified by different methods. The loss of ricinoleic acid indicated steps for oil removal had occurred, and a large decrease of ricinoleic acid was observed between unextracted mash and solvent extracted and protein precipitate preparations. Changes to the carbohydrate content of the sample were also observed following protein precipitation. The differential loss of arabinose relative to mannose was observed indicating the removal of the major carbohydrate fraction of the seed and enrichment of the protein content. When the data is combined and multivariate principle component analysis is applied, these changes in fatty acid and carbohydrate abundance are discriminating enough to be indicative of the preparation method used for each sample.

The effect of growth medium on B. anthracis Sterne spore carbohydrate content.

The expressed characteristics of biothreat agents may be impacted by variations in the culture environment, including growth medium formulation. The carbohydrate composition of B. anthracis spores has been well studied, particularly for the exosporium, which is the outermost spore structure. The carbohydrate composition of the exosporium has been demonstrated to be distinct from the vegetative form containing unique monosaccharides. We have investigated the carbohydrate composition of B. anthracis Sterne spores produced using four different medium types formulated with different sources of medium components. The amount of rhamnose, 3-O-methyl rhamnose and galactosamine was found to vary significantly between spores cultured using different medium formulations. The relative abundance of these monosaccharides compared to other monosaccharides such as mannosamine was also found to vary with medium type. Specific medium components were also found to impact the carbohydrate profile. Xylose has not been previously described in B. anthracis spores but was detected at low levels in two media. This may represent residual material from the brewery yeast extract used to formulate these two media. These results illustrate the utility of this method to capture the impact of growth medium on carbohydrate variation in spores. Detecting carbohydrate profiles in B. anthracis evidentiary material may provide useful forensic information on the growth medium used for sporulation.

Determination of post-culture processing with carbohydrates by MALDI-MS and TMS derivatization GC-MS

Biological materials generally require stabilization to retain activity or viability in a dry form. A number of industrial products, such as vaccines, probiotics and biopesticides have been produced as dry preparations. The same methods and materials used for stabilizing commercial microbial products may be applicable to preserving biothreat pathogens in a dry form. This is a likely step that may be encountered when looking at samples from terrorism attempts since only spores, such as those from Bacillus anthracis, are inherently stable when dried. The stabilizers for microbial preparations generally include one or more small carbohydrates. Different formulations have been reported for different industrial products and are often determined empirically. However sugar alcohols (mannitol and sorbitol) and disaccharides (lactose, sucrose and trehalose) are the common constituents of these formulations. We have developed an analytical method for sample preparation and detection of these simple carbohydrates using two complementary analytical tools, MALDI-MS and GC-MS. The native carbohydrates and other constituents of the formulation are detected by MALDI-MS as a screening tool. A longer and more detailed analysis is then used to specifically identify the carbohydrates by derivatization and GC-MS detection. Both techniques were tested against ten different types of stabilization recipes with Yersinia pestis cell mass cultured on different media types used as the biological component. A number of additional components were included in these formulations including proteins and peptides from serum or milk, polymers (e.g. poly vinyl pyrrolidone - PVP) and detergents (e.g. Tween). The combined method was characterized to determine several figures of merit. The accuracy of the method was 98% for MALDI-MS and 100% for GC-MS. The repeatability for detection of carbohydrates by MALDI-MS was determined to be 96%. The repeatability of compound identification by GC-MS was determined by monitoring variation in retention time, which is vital for identification of isomeric carbohydrates. The figures of merit illustrate an effective and accurate method for mono and disaccharide detection independent of formulation. This meets our primary goal for method development as small carbohydrates are among the most common stabilizers employed.

Detection of Acetone Processing of Castor Bean Mash for Forensic Investigation of Ricin Preparation Methods

Abstract:  Samples containing the toxic castor bean protein ricin have been recently seized in connection with biocriminal activity. Analytical methods that enable investigators to determine how the samples were prepared and to match seized samples to potential source materials are needed. One commonly described crude ricin preparation method is acetone extraction of crushed castor beans. Here, we describe the use of solid‐phase microextraction and headspace analysis to determine whether castor beans were processed by acetone extraction. We prepared acetone‐extracted castor bean mash, along with controls of unextracted mash and mash extracted with nonacetone organic solvents. Samples of acetone‐extracted mash and unextracted mash were stored in closed containers for up to 109 days at both room temperature and −20°C, and in open containers at room temperature for up to 94 days. Acetone‐extracted bean mash could consistently be statistically distinguished from controls, even after storage in open containers for 94 days.