Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials

Abstract

Effective microbial forensic analysis of materials used in a potential biological attack requires robust methods of morphological and genetic characterization of the attack materials in order to enable the attribution of the materials to potential sources and to exclude other potential sources. The genetic homogeneity and potential intersample variability of many of the category A to C bioterrorism agents offer a particular challenge to the generation of attributive signatures, potentially requiring whole-genome or proteomic approaches to be utilized. Currently, irradiation of mail is standard practice at several government facilities judged to be at particularly high risk. Thus, initial forensic signatures would need to be recovered from inactivated (nonviable) material. In the study described in this report, we determined the effects of high-dose gamma irradiation on forensic markers of bacterial biothreat agent surrogate organisms with a particular emphasis on the suitability of genomic DNA (gDNA) recovered from such sources as a template for whole-genome analysis. While irradiation of spores and vegetative cells affected the retention of Gram and spore stains and sheared gDNA into small fragments, we found that irradiated material could be utilized to generate accurate whole-genome sequence data on the Illumina and Roche 454 sequencing platforms.

A Standard Method To Inactivate Bacillus anthracis Spores to Sterility via Gamma Irradiation

“…No difference in the sensitivities of the assays employed was noted (data not shown). These results were consistent with previous reports (17,18,29). Likewise, lateral flow immunoassays specific for B. anthracis were obtained from the Defense Biological Product Assurance Office (DBPAO).…”

“…While the exact causes of the phenotypic changes are unknown, their observation is important as an indication that the dose of gamma radiation delivered is clearly suboptimal but likely approaching the lethal dose which would result in high-confidence SALs. On the basis of our results with a standard hand-held lateral-flow immunoassay, sensitivity is not substantially compromised when irradiated material is applied to the assay tickets nor is the utility of the material diminished for detection using qPCR assays, in accordance with previous work (17,18,29). We cannot exclude the possibility that spores may be damaged in ways not accounted for in this study, particularly with respect to membrane and coat components.…”

“…These larger molecular complexes would likely be more sensitive to radiation than individual proteins, since radiation sensitivity is a function of molecular target size (52). Our previous work indicated that irradiated B. atrophaeus spores lose the ability to bind malachite green spore stain (29), suggesting that irradiation can impact the integrity of the spore coat, and the loss of refractility observed in this study suggests that irradiated spores may have suffered similar damage to one or more components of the spore envelope.…”

“…Subsequent work showed that spores of various Bacillus species typically exhibited decimal reduction values ([D 10 ] the dose required to reduce the population by one log) of between 1.8 and 5.5 kGy, with marginal effects on spore ultrastructure, physical properties, and the response to antibody-or PCR-based detection assays (13,18,(26)(27)(28)(29). Because of the perceived efficacy of irradiation in yielding a safe yet responsive product that could mimic the essential properties of the live agent, irradiated spores of B. anthracis became a critical reagent for the development of systems and devices to detect agents of biological warfare and bioterrorism.…”

Review of Decontamination Techniques for the Inactivation of Bacillus anthracis and Other Spore-Forming Bacteria Associated with Building or Outdoor Materials

“…While there are well-established techniques for sterilization of materials such as foodstuffs, medical instruments, and pharmaceuticals, these techniques are typically confined for use on a small scale or may not be easily transportable (e.g., because of the use of hazardous or radioactive materials). Examples include ethylene oxide (which is flammable and carcinogenic), , as well as ionizing radiation in the forms of gamma irradiation, , X-rays, and electron beam . Although excluded from the scope of this review paper, some of the above techniques were used to decontaminate certain items following the Amerithrax attacks, and thus would be considered as decontamination tools for small, valuable, or personal items that would be sensitive to chemical exposure (e.g., cash, mail, jewelry, artwork, mechanical devices, electronics).…”