National Validation Study of a Cellulose Sponge Wipe-Processing Method for Use after Sampling Bacillus anthracis Spores from Surfaces

Abstract

This work was initiated to address the gaps identified by Congress regarding validated biothreat environmental sampling and processing methods. Nine Laboratory Response Network-affiliated laboratories participated in a validation study of a cellulose sponge wipe-processing protocol for the recovery, detection, and quantification of viable Bacillus anthracis Sterne spores from steel surfaces. Steel coupons (645.16 cm 2 ) were inoculated with 1 to 4 log 10 spores and then sampled with cellulose sponges (Sponge-Stick; 3M, St. Paul, MN). Surrogate dust and background organisms were added to the sponges to mimic environmental conditions. Labs processed the sponges according to the provided protocol. Sensitivity, specificity, and mean percent recovery (%R), between-lab variability, within-lab variability, and total percent coefficient of variation were calculated. The mean %R (standard error) of spores from the surface was 32.4 (4.4), 24.4 (2.8), and 30.1 (2.3) for the 1-, 2-, and 4-log 10 inoculum levels, respectively. Sensitivities for colony counts were 84.1%, 100%, and 100% for the 1-, 2-, and 4-log 10 inocula, respectively. These data help to characterize the variability of the processing method and thereby enhance confidence in the interpretation of the results of environmental sampling conducted during a B. anthracis contamination investigation.

Evaluating the long‐term persistence of Bacillus spores on common surfaces

“…It is established that germination rates of Bacillus spores are affected by temperatures with greater activation at higher temperatures (30–33°C) Granum et al ., 2013, compared to lower temps (3–8°C) (Rose et al . 2011) The temperature parameters in our study did not exceed 37°C, which is suitable for germination; therefore, our ‘activation’ results suggest the existence of independent convergent mechanisms – reproduction and breaking up of clumps – that confer behaviours similar to an ‘activation shoulder’.…”

“…Nonetheless, the data suggest that spore inactivation or adhesion may accelerate over time or under reduced relative humidity due to pronounced concavity observed in all curves fitted across 0–1038 days. An alternate explanation is that the recovery percentage decreases as the number of spores decreases, which is suggested by the lower recoveries reported by CDC and discussed further in the next section of this study (Hodges et al ., 2010; Rose et al ., 2011). Despite these uncertainties, long‐term quantitative data on Bacillus spore persistence are scarce, and the results presented here may prove useful until additional multiyear persistence experiments can be conducted (Greenberg et al ., 2010).…”

“…In contrast to our finding, the US Centers for Disease Control and Prevention (CDC) documented lower recoveries in the range of 16–55% for the recovery of B. anthracis Sterne spores from stainless steel fomites, 2 h after application, using a swabbing technique similar to the one described here (Hodges et al ., 2010). Another recovery study by the CDC reported about 30% recovery for B. anthracis Sterne spores from steel when recovered using sponges, 2 h after application (Rose et al ., 2011). It is however not certain why the recovery in our study was so high compared to others, but it should be noted that, in this study, we applied ≥ 100 times as many spores (~ 2.5 × 10 6 ) in order to allow measurement of high log 10 reductions over much longer periods of time (Hodges et al ., 2010; Rose et al ., 2011).…”

“…Another recovery study by the CDC reported about 30% recovery for B. anthracis Sterne spores from steel when recovered using sponges, 2 h after application (Rose et al ., 2011). It is however not certain why the recovery in our study was so high compared to others, but it should be noted that, in this study, we applied ≥ 100 times as many spores (~ 2.5 × 10 6 ) in order to allow measurement of high log 10 reductions over much longer periods of time (Hodges et al ., 2010; Rose et al ., 2011). Additionally, if limited fissures or crevices on the surface of the fomites could effectively sequester spores, applying more spores would lead to higher recovery percentages, as a smaller fraction of the total number of spores would be shielded from the swabbing technique.…”

“…Based on previous theoretical studies, the following spores of B. anthracis surrogates were used for this work – B. anthracis Sterne (attenuated non‐pathogenic strain of B. anthracis ) , B. cereus (common foodborne pathogen sharing morphological similarities with B. anthracis ) , B. thuringiensis (commonly used to produce a toxin used as an insecticide that is morphologically and genetically similar to B. anthracis ) and B. atrophaeus (most commonly used surrogate) (Carrera et al ., 2007; Greenberg et al ., 2010; Tufts et al ., 2013). While the recovery of spores from surfaces has been studied (Hodges et al ., 2010; Rose et al ., 2011; Calfee et al ., 2013), to date, no published studies have experimentally compared different surrogates of pathogenic B. anthracis spores under indoor conditions. Therefore, an objective of this work was to compare long‐term persistence models among the spores of B. anthracis and its surrogates, to aid in selection of a suitable surrogate for B. anthracis spores for the future.…”