Evaluation of sample processing methods to improve the detection of Bacillus anthracis in difficult sample matrices

Abstract

Large area sampling approaches have been developed and implemented by the US Environmental Protection Agency (EPA) to increase sample sizes, and potentially representativeness, in outdoor urban environments (e.g., concrete, asphalt, grass/landscaping). These sampling approaches could be implemented in response to an outdoor biological contamination incident or bioterrorism attack to determine the extent of contamination and for clearance following remediation. However, sample collection over large areas often contains an extensive amount of co-collected debris and native background microorganisms that interfere with the detection of biological threat agents. Sample processing methods that utilize basic laboratory equipment amenable to field deployment were selected and applied to turbid aqueous samples (TAS) to reduce particulates and native environmental organisms prior to culture and rapid viability-polymerase chain reaction (RV-PCR) analytical methods. Bacillus anthracis Sterne (BaS) spores were spiked into TAS collected by soil grab, wet vacuum collection from an outdoor concrete surface, or storm water runoff from an urban parking lot. The implementation of a sample processing method improved the sensitivity of culture and RV-PCR analytical methods for BaS spore detection in soil and wet vacuum TAS samples compared to baseline (minimal to no field processing methods applied). For soil, when the processing method was applied, samples with 15 colony forming units (CFU)/ml (60 CFU/g) and 1.5 CFU/mL (6 CFU/g) BaS spore load were detected using culture and RV-PCR, respectively. Most notably, the processing methods greatly improved the sensitivity of the RV-PCR analytical method for the wet vacuum TAS from no detection at the 1500 CFU/mL BaS spore load level to as low as 1.5 CFU/mL BaS spore load.

Keywords: Bacillus anthracis; Rapid viability-polymerase chain reaction; Soil samples; Storm water runoff samples; Surface sampling; Wet vacuum samples.

Fig. 1

Processing methods for soil and wet vacuum TAS. Soil baseline method (SBM) and soil processing method (SPM) (a); wet vacuum processing method 1 (WV1) (b); and wet vacuum processing method 2 (WV2) (c)

Fig. 2

Relative total microbial load for soil and wet vacuum TAS. Ratio of baseline to processing method total microbial load at each Ba Sterne spore load level, with the average across all spore load levels for each processing method applied. Baseline method was compared to SPM (black bars), WV1 (light gray) and WV2 (dark gray) for each initial targeted Ba Sterne spore loads tested. The error bars represent standard deviation of the microbial load across all spore loads

Fig. 3

Positive replicates for soil and wet vacuum TAS. Soil methods are shown in panels a (culture) and b (RV-PCR) and wet vacuum methods are in panels c (culture) and d (RV-PCR). Data represent the number of positive replicates (maximum possible, 3) for culture (a, c) and RV-PCR (b, d) with baseline (black bars), processing method 1 (white bars), and processing method 2 (patterned bars) for all Ba Sterne loads tested

Fig. 4

Positive replicates for storm water TAS. Data represent the number of positive replicates (maximum possible, 3) for culture (a) and RV-PCR (b) methods. SWMB (black bars), and SWRB (white bars) are shown for each Ba Sterne spore load tested

Fig. 5

Percentage of samples that were true positive or negative and percentage of samples that were false negative for TAS. True positive or negative results (black bars) or false negative results (patterned bars) for each TAS (n = 15) and a compilation of all TAS (n = 105) for culture (a) and RV-PCR (b). Values on the graph represent the percentage of true positive or negative results for each method tested. SBM, soil baseline method; SPM, soil processing method; SWRB, storm water reference baseline method; SWMB, storm water modified baseline method; WVMB, wet vacuum modified baseline method; WV1, wet vacuum processing method 1; WV2, wet vacuum processing method 2. All represents all TAS tested