Discerning Viable from Nonviable Yersinia Pestis pgm- and Bacillus Anthracis Sterne using Propidium Monoazide in the Presence of White Powders

Abstract

Purpose of the study To develop and optimize an assay to determine viability status of Bacillus anthracis Sterne and Yersinia pestis pgm- strains in the presence of white powders by coupling propidium monoazide (PMA) treatment with real-time PCR (qPCR) analysis. Approach and results After gaining entry to intracellular space, PMA can be exported by metabolically active cells. The PMA remaining in nonviable cells binds DNA, thereby increasing qPCR assay cycle threshold (CT) values compared to untreated samples. Dye concentration, cell number and fitness, incubation time, inactivation methods, and assay buffer were optimized for a Gram-positive pathogen, B. anthracis Sterne, and a gram negative pathogen, Y. pestis pgm-. Differences in CT values in nonviable cells compared to untreated samples were consistently > 9 for both B. anthracis Sterne vegetative cells and Y. pestis pgm- in the presence and absence of three different white powders. Our method eliminates the need for a DNA extraction step prior to detection by qPCR. Significance of the Study The method developed for simultaneous detection and viability assessment for B. anthracis and Y. pestis can be employed in forming decisions about the severity of a bio threat event or the safety of food.