Source: GUILD ASSOCIATES, INC. submitted to
PHAGE MEDIATED DETECTION OF BACILLUS ANTHRACIS ON DELIBERATELY CONTAMINATED FRESH FOODS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
0219016
Grant No.
2009-33610-20028
Project No.
OHOK-2009-01149
Proposal No.
2009-01149
Multistate No.
(N/A)
Program Code
8.5
Project Start Date
Sep 1, 2009
Project End Date
Aug 31, 2012
Grant Year
2009
Project Director
Schofield, D. A.
Recipient Organization
GUILD ASSOCIATES, INC.
5750 SHIER-RINGS ROAD
DUBLIN,OH 43016
Performing Department
Guild BioSciences
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.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71250101100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
5010 - Food;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
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. 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. 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. 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. 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. 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.
Project Methods
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.

Progress 09/01/09 to 08/31/12

Outputs
OUTPUTS: The results of this research has been disseminated in the form of (i) publications, (ii) poster presentations, (iii) oral presentations, and direct person-to-person communication. (i) The results have been published in the Journals "Bacteriophage", "Journal of Applied Microbiology", and Journal of Visualized Experiments". (ii) The results have been presented as posters at the American Society for Microbiology (ASM) General Meeting, the ASM Biodefense conference, and the Chemical and Biological Defense Physical Science and Technology conference. (iii) The results have been presented in the form of talks at the ASM Biodefense meeting, the ASM branch meeting, and the DTRA Chemical and biological defense colloquium. (iv) The results have been shared with members of the Food Emergency Response Network (FERN) and the Ohio Department of Agriculture. PARTICIPANTS: Consultant: Dr. Alvin Fox, University of South Carolina Sub contract work: Dr. George Stewart, University of Missouri Sub contract work: Dr. Chythanja Rajanna, University of Florida Sub contract work: Dr. Natasha Sharp, College of Charleston TARGET AUDIENCES: The target audiences are the scientific community, the Department of Defense, and the Food Emergency Response Network. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our diagnostic detection technology for Bacillus anthracis is currently undergoing a validation study which is being performed by the Ohio Department of Agriculture. The results of this study will be shared with the Food Emergency Response Network. The research was successful in that we developed a phage based sensor that can detect B. anthracis in contaminated foods. Consequently, we are using this as a platform technology to develop similar phage based sensors for other bacterial pathogens such as Yersinia pestis, Shigella flexneri, Escherichia coli 0157:H7 and Campylobacter species.

Publications

  • Schofield, D. A., Sharp, N. J., and Westwater, C. 2012. Phage-based platforms for the clinical detection of human bacterial pathogens. Bacteriophage, 2(2):105-121.
  • Schofield, D. A., Molineux, I. J., and Westwater, C. 2011. Bioluminescent reporter phage for the detection of category A bacterial pathogens. Journal of Visualized Experiments, 53: 2740.
  • Schofield, D. A., and Westwater, C. 2009. Phage-mediated bioluminescent detection of Bacillus anthracis. Journal of Applied Microbiology, 107(5):1468-78.


Progress 09/01/10 to 08/31/11

Outputs
OUTPUTS: The results have been disseminated to the public, the department of defense, and the research community in the forms of a research journal article, a poster, and invited oral presentations at research conferences. Our research has been published in the Journal of Visualized Experiments. The results were disseminated in the form of a manuscript and a video detailing the experiments and results. The manuscript and video is available in PubMed under PMID number 21775956. The intended audience was the research scientific community and general public. The results were presented in a poster format at the Department of Defense Chemical Biological Defense Science and Technology Conference. The intended audience were first responders, the department of homeland security, and the food emergency response network. Two oral presentations were given on our research. The first one was at the American Society for Microbiology Biodefense and Emerging Diseases Research Meeting. The intended audience was researchers in the biodefense community, and specifically, clinical diagnosticians. The second presentation was at the Defense Threat Reduction Agency Joint Science Technology Office workshop. The intended audience were federal agencies (EPA, DTRA, U.S. Army) involved in the detection and decontamination of biological agents following a deliberate release. PARTICIPANTS: Dr. David A. Schofield is the PI on the project and supervised, planned, and conducted the experiments. Dr. George Stewart at the University of Missouri is a collaborator on the project (sub contractor). Dr. Stewart contributed by performing experiments with wild-type B. anthracis strains in a BioSafety Level 3 facility. Dr. Natasha Sharp at the College of Charleston performed sub contract work associated with the project. Dr. Sharp performed experiments designed to demonstrate the effectiveness of the phage based detector for B. anthracis. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The project is focussed on a developing a phage-based sensor for the detection of the bacterial pathogen Bacillus anthracis. The results were significant since they were published in a peer-reviewed scientific journal.

Publications

  • Schofield, D. A., Sharp, N. J., Molineux, I. J., Stewart, G. C., and Westwater, C. 2011. Bioluminescent Reporter Phage For The Detection Of Category A Bacterial Pathogens. The Chemical and Biological Defense Science and Technology Conference, Las Vegas, Nevada, November 14th-18th, 2011.
  • Schofield, D. A., Molineux, I. and Westwater, C. 2011. Anthrax and plague diagnostic identification, and antibiotic susceptibility testing using bioluminescent reporter phage. 9th ASM Biodefense and Emerging Diseases Research Meeting, Washington, DC, February 6th-9th, 2011.
  • Schofield, D. A., Molineux, I. J., Stewart, G. C., and Westwater, C 2011. Bioluminescent reporter phage for the detection of viable category A bacterial pathogens. DTRA/JSTO Chemical and biological defense: Wide area anthracis spores decontamination workshop. Falls Church, Washington DC, June 23rd-June 24th, 2011.
  • Schofield, D. A., Molineux, I. J., and Westwater, C. 2011. Bioluminescent reporter phage for the detection of category A bacterial pathogens. Journal of Visualized Experiments 53: pii:2740.


Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: Specific Aim 2 of the research has been completed. Aim 2 was to determine the specificity of the reporter phage for Bacillus anthracis. The results of the Phase II research has been presented as a poster at the American Society for Microbiology and Emerging Diseases Research Meeting, and at the Chemical and Biological Defense Science and Technology Conference. PARTICIPANTS: Dr. David Schofield is the PD of the project and is responsible for the overall project and experiments. Dr. Alvin Fox (consultant), University of South Carolina. Dr. Fox provided expertise on the B. cereus group and the species and strains for determination B. anthracis specificity. Dr. Alexander Sulakvelidze (consultant), University of Florida. Dr. Sulakvelidze has provided advice on phage propagation and manipulation, and on how to develop the phage research into a product. Dr. George Stewart (sub contract work), University of Missouri. Dr. Stewart is about to start testing the ability of the phage to detect a panel of virulent B. anthracis strains (BSL3 work). TARGET AUDIENCES: The target audiences of the research is the Food Emergency Response Network (FERN), the Department of Homeland Security, the Laboratory Response Network (LRN), the Centers for Disease Control and Prevention, and the other Department of Defense agencies (DTRA, DARPA, NAVY, ARMY). PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The species specificity of the phage detection system for Bacillus anthracis was analyzed. Thus, the ability of the reporter phage to confer a bioluminescent signal to non-anthracis Bacillus species was assessed using a panel of Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides. These species were chosen from the Bacillus genus since along with B. anthracis, they are part of the closely related B. cereus group. On the basis of genetic evidence, this group can be considered as one species. Thus, B. anthracis, B. cereus, B. thuringiensis, and B. mycoides share multiple phenotypes and traits making distinction between the species difficult. This sub-set of species is considered the most appropriate to demonstrate species-specific phage-mediated bioluminescent detection. B. cereus, B. thuringiensis, and B. mycoides strains were obtained from the American Type Culture Collection (ATCC) or the Bacillus Group Stock Center. Of the 118 different species and strains tested, only 6 strains produced a bioluminescent signal above background controls (95% specificity within the B. cereus group). Moreover, of the 6 positive strains, 2 of the strains elicited an attenuated signal that was 10-fold lower than B. anthracis, and 2 of the strains produced an attenuated signal that was 100-fold lower than B. anthracis. The species specificity of the phage detection system for B. anthracis was further analyzed against common food-borne bacterial contaminants. The ability of the phage to detect Yersinia entercolitica (10 strains tested), Salmonella enterica (10 strains tested), Shigella flexneri (3 strains tested), Shigella dysenteriae (1 strain tested), Shigella sonnei (3 strains), Shigella boydii (1 strain), and Listeria monocytogenes (10 strains) was analyzed. None of the non-anthracis Bacillus species elicited a response with the phage detection system. In conclusion, the results indicate that the phage detection system has the potential to be a highly selective test for B. anthracis.

Publications

  • Schofield, D. A., Molineux, I. and Westwater, C. 2010. Light-tagged bacteriophages for the detection of the biowarfare pathogens Bacillus anthracis and Yersinia pestis. The Chemical and Biological Defense Science and Technology Conference, Orlando, Florida, November 15th-19th, 2010.
  • Schofield, D. A., Molineux, I. and Westwater, C. 2010. Plague and Anthrax Diagnostic Bioluminescent Phage. 8th ASM Biodefense and Emerging Diseases Research Meeting, Baltimore, Maryland, February 21st-24th, 2010.