Detection of Bacillus anthracis spores from environmental water using bioluminescent reporter phage

Aims We investigated the ability of a temperate Bacillus anthracis reporter phage (Wβ::luxAB-2), which transduces bioluminescence to infected cells, to detect viable spores from deliberately contaminated environmental water samples. Methods and Results Environmental water was inoculated with spores and assayed with Wβ::luxAB-2. Bioluminescent signals directly correlated with input phage and spore concentrations. A limit of detection of 101 and 102 CFU per ml within 8 h was achieved from pond and lake water, respectively. Detection was greatly simplified by minimizing sample processing steps without spore extraction. The complex endogenous microbial flora and salt content of brackish water challenged the assay, extending the detection time to 12 h for a sensitivity of 102 CFU per ml. Phage-mediated bioluminescence was strictly dependent on bacterial physiology, being significantly reduced in mid/late log phase cells. This was shown to be due to an inability of the phage to adsorb. Conclusions The reporter phage Wβ::luxAB-2 displays potential for simplified detection of viable spores from contaminated water samples within 12 h. Significance and Impact of the Study A deliberate aerosol release of spores could lead to widespread contamination, leaving large areas uninhabitable until remediation. An essential requirement of this restoration process is the development of simplified detection assays in different environmental matrices.