Bacillus anthracis spore suspensions: determination of stability and comparison of enumeration techniques

Abstract

Aim: To determine the stability and variability in concentration of spore suspensions of Bacillus anthracis (BA) spore suspensions by comparing different methods of enumeration and to detect changes, if any, under different storage conditions. Methods and Results: Plate and microscope counts were compared to measuring the genomic equivalents based on DNA content BA spore suspensions. We developed chemical methods to extract spore DNA and extra‐spore (ES) DNA. DNA mass was determined by gel electrophoresis and QPCR assays were developed using the markers on the chromosome (rpoB) and the pXO1 plasmid (pag). The plate counts and microscope counts were very stable (for up to 900 days). The effect of freezing and the presence of additives in samples were tested for up to 300 days, and the results indicated that the additives tested and freezing did not decrease the viability or microscope counts. Conclusions: Bacillus anthracis spore suspensions can be stored for long periods of time without significant loss of viability or clumping. The content of ES DNA was variable and changed with time. Significant and Impact of the Study: The study shows that BA spore suspensions can be developed for reference materials providing a uniform basis for comparing detection equipment and results from different laboratories.

Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

“…Although Coker et al reported relative amounts of pXO1 and pXO2 of respectively 11.5 and 1.6, for the same strain we used ( B. anthracis Vollum), variation in pXO plasmid copy numbers could also result from the growth phase at which DNA was harvested [3]. Our data correspond better to the lower plasmid copy numbers reported by other authors [29,30]. Nevertheless, all reports agree that pXO1 is present in multiple copies.…”

Cross-Institute Evaluations of Inhibitor-Resistant PCR Reagents for Direct Testing of Aerosol and Blood Samples Containing Biological Warfare Agent DNA

“…This extrapolation does not account for factors such as the efficiency of spore lysis within the PCR assay chamber and/or the presence or absence of DNA on spore surfaces (25). Although by adding B. subtilis spores into the PCR we have shown that direct detection of spore DNA is possible, it may be that in this instance, the PCR thermal hold (95°C; 2 or 3 min) and subsequent cycling steps are simply releasing DNA from the exospore and are not facilitating spore lysis and thus access to endospore DNA.…”

False-Negative Rate and Recovery Efficiency Performance of a Validated Sponge Wipe Sampling Method

“…A Government Accountability Office (GAO) investigation following the 2001 anthrax incident concluded that validated sampling methods and statistical sampling designs were needed to provide confidence that there is no contamination when all sample results are negative (17, 18). This conclusion strongly reinforces the need for validated sampling methods to effectively respond to biothreats and ensure public safety.Following the 2001 anthrax incident, several research teams developed and investigated (in laboratory studies) the performance of sampling methods using swab, wipe, and vacuum collection devices for Bacillus anthracis or surrogate contaminants on different surfaces (1,2,3,4,5,6,7,13,14,15,16,22,23,25,28,34,38,40,44,45). In addition, the CDC has conducted formal validation studies on two methods for sampling nonporous surfaces: macrofoam swabs (23) and cellulose sponge wipes (39).…”