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

Abstract

In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 ϫ 10 10 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D 10 ) values and irradiation doses required to ensure sterility (D SAL ) to the point at which the probability of detecting a viable spore is 10 Ϫ6 . Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques. IMPORTANCEThe inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.

Combining spore germination and heat inactivation to decontaminate materials contaminated with Bacillus anthracis spores

“…For example high concentrations of irradiated dead spores (≥9 log 10 spores per ml) subsequently interfered with the recovery of any remaining live spores for spore populations that were not sufficiently irradiated because Alr is not inactivated during irradiation (Cote et al . ). The data herein indicate that the highest practical confidence for recovery of any live spores within an irradiated spore sample would include a preliminary incubation at 15–25°C, ≥24 h (or longer) in recovery medium.…”

Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR/Cas9 System

“…No equivalent NHEJ system has been identified experimentally the B. cereus group strains investigated in the current study, although NHEJ-associated proteins (such as Ku-like proteins) have been identified by homology analysis. According to published research, NHEJ-mediated repair mechanisms in B. anthracis are inefficient compared with those found in B. subtilis (Cote et al, 2018). Therefore, strains with non-specific cleavage might not survive in the absence of homologous arms to repair the genomic DNA breaks.…”

Rapid Microscopic Detection of Bacillus anthracis by Fluorescent Receptor Binding Proteins of Bacteriophages

“…Sporulation and subsequent spore purification of B. anthracis and other Bacilli was done as previously described [ 33 ] with slight modifications. A colony of a fresh overnight culture of B. anthracis (or other Bacilli) was used to inoculate 50 mL sporulation medium containing 0.8% nutrient broth (Merck KGaA) amended with 0.05 mM MnCl 2 , 0.7 mM CaCl 2 and 1.0 mM MgCl 2 [ 34 ] in 500 mL baffled flasks.…”