Impact of filter material and holding time on spore sampling efficiency in water

Abstract

Bacillus anthracis and other environmentally persistent pathogens pose a significant threat to human and environmental health. If contamination is spread over a wide area (e.g. resulting from a bioterrorism or biowarfare incident), readily deployable and scalable sample collection methods will be necessary for rapidly developing and implementing effective remediation strategies. A recent surge in environmental (eDNA) sampling technologies could prove useful for quantifying the extent and levels of contamination from biological agents in environmental and drinking water. In this study, three commonly used membrane filtration materials (cellulose acetate, cellulose nitrate, and nylon) were evaluated for spore filtration efficiency, yielding recoveries from 17%-68% to 25%-117% for high and low titer samples, respectively, where cellulose nitrate filters generated the highest recoveries. A holding time test revealed no statistically significant differences between spore recoveries when analyzed at the specified timepoints, suggesting that eDNA filter sampling techniques can yield and maintain a relatively high recovery of spores for an extended period of time between filtration and analysis without a detrimental impact on spore recoveries. The results shown here indicate that emerging eDNA technologies could be leveraged for sampling following a wide-area contamination incident and for other microbiological water sampling applications.

Keywords: bacillus anthracis; bacillus atrophaeus; eDNA; filter sampling; remediation.

Figure 1.

Extraction efficiency of the three tested filter membrane materials. Mean % recovery of Bg spores (± standard deviation) at a high (target 1 × 10⁷ CFU/sample, represented by dark gray bars) and low titer (target 1 × 10⁴ CFU/sample, represented by the light gray bars), calculated by comparing the average CFU/sample recovered from each tested filter type to the average recoveries from positive control samples (liquid samples that did not undergo a filtration step).

Figure 2.

Extraction efficiency for the cellulose nitrate filters after a specified holding time at 4°C. Mean % recovery of Bg spores (± SD) at a high (target 1 × 10⁷ CFU/sample, represented by dark gray bars) and low titer (target 1 × 10⁴ CFU/sample, represented by the light gray bars), calculated by comparing the average CFU/sample recovered from the filters after the specified holding time to the average recoveries from positive control samples (liquid samples that did not undergo a filtration step but were also held for the specified holding time prior to analysis).