Exploring improved sample recovery and processing methods targeting Bacillus anthracis spores from vegetation...

Following an accidental or intentional outdoor release of an environmentally persistent biological agent, such as Bacillus anthracis (Ba), a sampling campaign involving the collection of bulk vegetation matrices (grass, ground litter, and leaves) to determine the scope of contamination might be part of a remediation plan. These vegetative materials contain fungi, bacteria, and/or other naturally present background microorganisms that will impact target analyte recovery. This study aimed to optimize recovery of surrogate Ba spores by exploring 1) methods to separate target spores from non-target compounds, and 2) methods to reduce the number of non-target organisms present in the sample. Approaches to dislodge spores from a sample matrix demonstrated, and resulted through statistical analyses, that the sample agitation time used with a Phosphate-Buffered Saline with 0.05 % Tween 20® (PBST) solution could be reduced to 30 s compared to a previous 2-min baseline study. Over 100 individual experiments were performed involving a combination of heat, chemical, antifungal, and enzyme treatments to investigate background contamination mitigation. Despite the comprehensive evaluation, no single treatment protocol completely removed competing background microorganisms to adequately enumerate Ba surrogate spores in grass samples, as determined through visual enumeration of culture plates and through ANOVA statistical analyses. Enumerating colonies from ground litter and leaf samples was often challenging, but spores from both sample types could be successfully counted following particular treatment protocols, such as a combination of H₂O₂ and heat treatment. An alternative to traditional culture plating for ground litter and grass samples was tested through the use of Rapid Viability-Polymerase Chain Reaction (RV-PCR); however, its known limitations should be considered when selecting analysis methods. This work provides a step towards developing standardized sample collection and processing procedures for select vegetation matrices by performing a comprehensive investigation of analysis methods proven through prior literature, albeit the results demonstrated that some matrix types still result in unique challenges.