The following Technical Objectives will be pursued to demonstrate the proof of concept of a Bacillus anthracis-specific phage, genetically engineered with the Vibrio harveyi luxAB genes, to detect B. anthracis. Technical Objective 1 will integrate the luxAB genes into a non-essential region of the B. anthracis Wbeta phage to generate a B. anthracis luxAB reporter phage. Technical Objective 2 will perform feasibility studies to demonstrate that the recombinant Wbeta::luxAB phage can effectively be used as a B. anthracis detection system.
NON-TECHNICAL SUMMARY: Bacillus anthracis, the causative agent of anthrax, is a category A bioterrorist pathogen that is classed as the most likely weapon used in a bioterrorist attack. Although this pathogen is not a common contaminant on foods, the deliberate contamination of ready to eat foods is of vital concern since: <ul>
<li>(i) anthrax spores, which are the infectious form, are resistant to chemical and physical insult and are difficult to decontaminate using conventional food sanitizers; <li>(ii) the 'ready to eat' contaminated food will not be cooked, and therefore, will not experience the protective benefit of heating, and <li>(iii) gastrointestinal anthrax, caused by the ingestion of contaminated foods, is very difficult to diagnose and without antibiotics, results in high mortality. </ul>Consequently, detection methodologies that can quickly, and specifically detect the presence of B. anthracis on deliberately contaminated foods are urgently needed. This proposal will generate the proof of principal studies for a novel 'light producing' phage that specifically detects B. anthracis on contaminated foods. The aim is to generate a phage detection system that: <ul>
<li>(i) bioluminesces if B. anthracis is present; <li>(ii) detects viable cells only; <li>(iii) requires only minimal exogenous consumables; <li>(iv) will be environmentally friendly and cheap to produce, and <li>(v) can be visually assessed using a hand held illumination device.</ul> This research will enhance food safety, but will also be directly beneficial to the Federal Government for the detection and decontamination of anthrax-contaminated buildings and offices.
APPROACH: Technical Objective 1 will clone the luxAB genes into an expression cassette under the transcriptional and translational control of preferred Bacillus expression sequences. The expression cassette will be flanked by Wbeta phage DNA. LuxAB will be integrated into a non-essential region of the Wbeta genome by homologous recombination based on a double cross over event. Recombinant Wbeta::luxAB will be identified and isolated based on the ability of infected cultures to emit light. luxAB integration will be verified by diagnostic agarose gel electrophoresis and PCR. The 'fitness' of the recombinant phage will be compared to the wild-type phage. Technical Objective 2 will generate spores from the attenuated B. anthracis Sterne strain and Wbeta::luxAB, in conjunction with spore germinating agents, will be analyzed for its ability to infect B. anthracis and produce a light signal. To demonstrate that the phage will detect viable cells only, bioluminescence assays will be performed using viable and non-viable spores. The sensitivity limits of detection, the signal response time, and the dose-response characteristics in relation to the number of spores present will be determined. The thermal stability of LuxAB proteins in B. anthracis will be examined at different temperatures. Since the phage is expected to be used in a non-laboratory environment, Wbeta::luxAB will be analyzed for its ability to remain infective at different pH's, at different temperatures, and over extended storage periods.