An official website of the United States government.
Our Phase I research generated a recombinant Bacillus anthracis reporter phage by genetically engineering the bacterial luxAB genes into the Wbeta phage genome. The recombinant Wbeta::luxAB reporter phage was viable, stable, and was able to rapidly confer a bioluminescent signal to germinating spores and vegetative cells of the attenuated B. anthracis Sterne strain. <P>The Phase II Objectives will build upon the Phase I research by demonstrating the utility of the "light-tagged" Wbeta::luxAB reporter phage to: (i) confer a bioluminescent phenotype to a panel of virulent B. anthracis strains; (ii) specifically detect B. anthracis, and (iii) detect B. anthracis on deliberately contaminated foods. <P>Technical Objective 1 will demonstrate the broad-strain utility of the Wbeta::luxAB reporter phage to detect virulent B. anthracis strains. These experiments will be performed exclusively by our collaborators at the Southwest Foundation for Biomedical Research in their Biosafety Level 3 facility. Spores from 10 virulent B. anthracis strains will be generated. The ability of the Wbeta::luxAB reporter phage to transduce a bioluminescent signal to germinating spores and vegetative cells will be assessed. The results are expected to demonstrate that the reporter phage can infect, and rapidly confer a bioluminescent phenotype to all B. anthracis strains tested.<P> Technical Objective 2 will analyze the specificity of the phage detection system. The specificity of the Wbeta::luxAB reporter for B. anthracis will be investigated by analyzing the ability of the reporter phage to detect non-anthracis Bacillus species. Ten strains each from the closely related B. cereus, B. mycoides and B. thuringiensis species will be analyzed for a bioluminescent signal response upon incubation with the reporter phage. The results are expected to demonstrate that the Wbeta::luxAB reporter phage (i) is specific for B. anthracis, and (ii) the Wbeta::luxAB reporter phage specificity (bioluminescent signal transduction) is analogous to the host-range infectivity of the wild-type Wbeta phage.<P> Technical Objective 3 will investigate the ability of the Wbeta::luxAB reporter phage to detect B. anthracis on deliberately contaminated liquid and food samples. The attenuated B. anthracis Sterne strain (exempt select agent, BSL2 organism) will be used throughout this objective. The ability of the reporter phage to detect B. anthracis spores from deliberately spiked samples of: (i) pasteurized milk; (ii) apple juice and water, and (iii) ready-to-eat spinach, will be tested and optimized. <P>The research will focus primarily on milk since this medium is considered the highest priority and the most appropriate target model. The ability of the reporter phage to transduce a bioluminescent signal to germinating spores will be determined. The signal response time, the dose-response characteristics and the sensitivity limits of detection will be assessed.
Non-Technical Summary: The nations food supply is particularly vulnerable to a bioterrorist attack. Bacillus anthracis, the causative agent of anthrax, is a Category A pathogen that is considered a high priority biological weapon. Gastrointestinal anthrax is caused by the ingestion of B. anthracis contaminated foods or liquids. B. anthracis spores, which are the infectious form of the disease, are extremely resistant to chemical and physical treatment. Thus, minimally processed foods or liquids such as milk are the primary areas of concern because they do not receive the protective benefit of cooking. The risk is compounded because gastrointestinal anthrax is very difficult to diagnose since the disease has general symptoms such as nausea, vomiting, and diarrhea; if appropriate treatment is not administered within the first 24 h after exposure, the disease can develop into a systemic form that is rapidly fatal with mortality rates exceeding 40%. Consequently, novel surveillance methodologies that can detect B. anthracis on adulterated liquids and foods are critical for enhancing food safety. The long-term goal of our research is to develop a simple and rapid B. anthracis detection kit that can be used to identify this priority pathogen on deliberately contaminated foods or liquids. Our Phase I research obtained the proof of principle results by generating a genetically engineered reporter phage that could detect B. anthracis. The reporter phage was constructed by integrating the "light" genes into the genome of a B. anthracis phage. The resulting "light-tagged" reporter phage was able to rapidly (within minutes) confer a bioluminescent (light) signal to B. anthracis. The Phase II research will build upon the Phase I research by: 1: Demonstrating that the reporter phage can detect many different forms of B. anthracis. 2: Demonstrating that the reporter phage detects B. anthracis only, and not other non-pathogenic bacteria in order to reduce the possibility of false alarms. 3: Demonstrating that the reporter phage can detect B. anthracis on contaminated liquids and foods. The research proposed in this application is significant because it will potentially save lives by providing the surveillance methodology for the identification of B. anthracis on deliberately contaminated liquids and foods. The research will enhance food safety, but will also be directly beneficial to the Federal Government for the detection of anthrax-contaminated buildings and offices, and also to the clinical community as a diagnostic tool for the detection of B. anthracis. <P> Approach: The description of the methods are described for each of the three technical objectives. 1. The effectiveness of the reporter phage to detect 10 distinct, and fully virulent B. anthracis strains will be analyzed. Spores are the infectious form of B. anthracis and the most likely form to be found on adulterated foods. Therefore, the utility of the phage detection system using spores as the starting material will be determined. Spores will be generated by incubating the cells under nutrient starvation coniditions. The ability of the reporter phage to confer a bioluminescent signal to germinating spores of 10 virulent strains will be assessed. The signal response time and the sensitivity limits of detection will be determined. The utility of the phage to confer a bioluminescent signal response to vegetative cells will also be investigated. Each experiment will be performed in triplicate. Results will be determined for significance by statistical methodology. 2. The specificity of the reporter phage for B. anthracis will be determined. The ability of the parental Wbeta phage to lyse members of the B. cereus group (B. anthracis, B. cereus, B. thuringiensis, and B. mycoides) will be compared to the ability of the Wbeta::luxAB reporter phage to detect the same strains. Wbeta will be spotted onto bacterial lawns representing 10 strains from each non-B. anthracis species and analyzed for the presence of clearing (lysis). The same strains will be assessed for Wbeta::luxAB susceptibility as defined by the ability of the reporter phage to transduce a bioluminescent signal. 3. The ability of the reporter phage to detect B. anthracis spores on deliberately contaminated foods will be assessed. Spores are the infectious form of anthrax, and the form that will be present on contaminated foods. Therefore, experiments will be performed to analyze the ability of the reporter phage to detect B. anthracis spores from deliberately spiked samples of: (i) pasteurized milk; (ii) apple juice and water, and (iii) ready-to-eat spinach. The research will focus primarily on milk since this medium is considered the highest priority and the most appropriate target model. Experiments will be conducted directly using milk aliquots or after the spores have been collected by centrifugation or filtration. The ability of the reporter phage to transduce a bioluminescent signal to germinating spores will be determined. The signal response time, the dose-response characteristics, and the sensitivity limits of detection will be assessed.