Composite sampling of a Bacillus anthracis surrogate with cellulose sponge surface samplers from a nonporous surface.

A series of experiments was conducted to explore the utility of composite-based collection of surface samples for the detection of a Bacillus anthracis surrogate using cellulose sponge samplers on a nonporous stainless steel surface. Two composite-based collection approaches were evaluated over a surface area of 3716 cm2 (four separate 929 cm2 areas), larger than the 645 cm2 prescribed by the standard Centers for Disease Control (CDC) and Prevention cellulose sponge sampling protocol for use on nonporous surfaces. The CDC method was also compared to a modified protocol where only one surface of the sponge sampler was used for each of the four areas composited. Differences in collection efficiency compared to positive controls and the potential for contaminant transfer for each protocol were assessed. The impact of the loss of wetting buffer from the sponge sampler onto additional surface areas sampled was evaluated. Statistical tests of the results using ANOVA indicate that the collection of composite samples using the modified sampling protocol is comparable to the collection of composite samples using the standard CDC protocol (p  =  0.261). Most of the surface-bound spores are collected on the first sampling pass, suggesting that multiple passes with the sponge sampler over the same surface may be unnecessary. The effect of moisture loss from the sponge sampler on collection efficiency was not significant (p  =  0.720) for both methods. Contaminant transfer occurs with both sampling protocols, but the magnitude of transfer is significantly greater when using the standard protocol than when the modified protocol is used (p<0.001). The results of this study suggest that composite surface sampling, by either method presented here, could successfully be used to increase the surface area sampled per sponge sampler, resulting in reduced sampling times in the field and decreased laboratory processing cost and turn-around times.

Bacillus thuringiensis as a surrogate for Bacillus anthracis in aerosol research.

Characterization of candidate surrogate spores prior to experimental use is critical to confirm that the surrogate characteristics are as closely similar as possible to those of the pathogenic agent of interest. This review compares the physical properties inherent to spores of Bacillus anthracis (Ba) and Bacillus thuringiensis (Bt) that impact their movement in air and interaction with surfaces, including size, shape, density, surface morphology, structure and hydrophobicity. Also evaluated is the impact of irradiation on the physical properties of both Bacillus species. Many physical features of Bt and Ba have been found to be similar and, while Bt is considered typically non-pathogenic, it is in the B. cereus group, as is Ba. When cultured and sporulated under similar conditions, both microorganisms share a similar cylindrical pellet shape, an aerodynamic diameter of approximately 1 µm (in the respirable size range), have an exosporium with a hairy nap, and have higher relative hydrophobicities than other Bacillus species. While spore size, morphology, and other physical properties can vary among strains of the same species, the variations can be due to growth/sporulation conditions and may, therefore, be controlled. Growth and sporulation conditions are likely among the most important factors that influence the representativeness of one species, or preparation, to another. All Bt spores may, therefore, not be representative of all Ba spores. Irradiated spores do not appear to be a good surrogate to predict the behavior of non-irradiated spores due to structural damage caused by the irradiation. While the use of Bt as a surrogate for Ba in aerosol testing appears to be well supported, this review does not attempt to narrow selection between Bt strains. Comparative studies should be performed to test the hypothesis that viable Ba and Bt spores will behave similarly when suspended in the air (as an aerosol) and to compare the known microscale characteristics versus the macroscale response.