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Development of an One-Tube Colorimetric Assay for Detention of Escherichia Coli O157:H7 in Food Animals

Investigators
Goodridge, Lawrence
Institutions
University of Wyoming
Start date
2005
End date
2008
Objective
The overall goal of this proposal is the development of an assay for the rapid detection of Escherichia coli O157:H7. This research addresses the current limitations of the available cultural, immunological and molecular methods for detection of E. coli O157:H7.

The detection system will consist of two components, including a reporter bacteriophage genetically modified to carry a thermophillic beta-galactosidase gene, and a colorimetric substrate for the beta-galactosidase.

The specific objectives of this study are to 1) develop the test in an easy to use format that incorporates the reporter phage and substrate in a single device. 2)To increase the sensitivity of the assay by optimizing the substrate to enzyme ratio present in the device, concentrating the E. coli O157:H7 cells with immunomagnetic separation, including a chemical modulator of beta-galactosidase activity into the assay, and optimizing the reporter phage. 3) Evaluate the effectiveness of the assay by testing artificially contaminated bovine feces. 4) Evaluate the effectiveness of the assay by testing animals infected with E. coli O157:H7.

More information
NON-TECHNICAL SUMMARY: Diarrhea remains one of the main causes of morbidity and mortality worldwide, and E. coli O157:H7 is a major cause of diarrheal disease in the U.S.A. Fecally contaminated food and water are the sources of E. coli O157:H7 infections in humans. Cattle and other ruminants have been established as natural reservoirs for E. coli O157:H7, and these food production animals play a significant role in the epidemiology of human infections. The overall goal of this work is the development of an assay for the rapid detection of Escherichia coli O157:H7 in food production animals, as a preliminary step to decrease contamination of meat with E. coli O157:H7.

APPROACH: Objective 1. To produce a T4 lacZ+ reporter phage, a T4 reporter phage template will be constructed by producing a T4 phage with amber mutations in genes 37 and 38 (tail fiber genes), followed by integration of a p22-lacZ fusion into a non-essential region of the T4 chromosome. To produce an E. coli O157:H7 reporter phage, the distal tail fiber locus of phage PP01 (specifically infects E. coli O157:H7) will be amplified by PCR The amplified fragment will be cloned into a suitable plasmid. The plasmid will be transformed into E. coli JM109 by electroporation. Following infection with phage T4, chimeric phage which replace their tail fiber genes with the plasmid borne tail fiber genes will be isolated and propagated.

Objective 2. We will investigate the utilization of several other colorimetric beta-galactosidase substrates and analyze their activity, in an attempt to investigate the possibility of increasing the sensitivity of the assay, by employing the most sensitive colorimetric substrate. The colorimetric substrates that will be investigated include: 6-chloro-3-indoyl-B-D-galactopyranoside, 5-bromo-6-chloro-3-indoyl-B-D-galactopyranoside, 5-iodo-3indoyl-B-D-galactopyranoside, and N-methylindoyl-B-D-galactopyranoside. In addition, we will evaluate the use of automated IMS to rapidly concentrate and purify the target bacteria from complex matrices. The compound, 1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene has been demonstrated as a positive irreversible modulator causing a rise of up to 186% in E. coli Beta-galactosidase activity. We will evaluate the ability of this compound to increase the activity of the Beta-galactosidase within the assay format.

Objective 3. An evaluation of the one-tube reporter phage assay will be accomplished, in an attempt to validate the test. In this part of the research, the one-tube assay (including IMS) will be compared to IMS followed by plating onto Sorbitol MaConkey (SMAC) agar.

We are proposing two experiments to evaluate the one-tube assay. Experiment 1: To compare the one-tube assay to IMS followed by plating on bacteriologic media, a tetracycline resistant isolate of E. coli O157:H7 will be inoculated into fecal samples collected from 10 mature steers. Inoculated feces will be mixed with up to 250 mls of tryptic soy broth and stomached for 30 s before samples are taken. The homogenized mixture will be subjected to automated IMS, and the concentrated sample will be subjected to the one-tube assay.

Experiment 2: We will investigate two different sampling methodologies to detect E. coli O157:H7 in the live animal. Fecal samples obtained from an infected animal will be compared to the production of a composite sample of feces from each pen. The production of a composite fecal sample for testing will significantly reduce the number of tests to be accomplished, and will also solve the problem of intermittent shedding of the pathogen by animals. In this testing scenario, cattle located in a pen that test positive would be separated from those cattle from pens that test negative, prior to slaughter.

PROGRESS: 2005/12 TO 2008/11
OUTPUTS: The overall goal of this proposal is the development of an assay for the rapid detection of Escherichia coli O157:H7. This research addresses the current limitations of the available cultural, immunological and molecular methods for detection of E. coli O157:H7. The detection system will consist of two components, including a reporter bacteriophage genetically modified to carry a thermophillic beta-galactosidase gene, and a colorimetric substrate for the beta-galactosidase. The specific objectives of this study are to 1) develop the test in an easy to use format that incorporates the reporter phage and substrate in a single device. 2)To increase the sensitivity of the assay by optimizing the substrate to enzyme ratio present in the device, concentrating the E. coli O157:H7 cells with immunomagnetic separation, including a chemical modulator of beta-galactosidase activity into the assay, and optimizing the reporter phage. 3)Evaluate the effectiveness of the assay by testing artificially contaminated bovine feces. 4)Evaluate the effectiveness of the assay by testing animals infected with E. coli O157:H7. Two activities were completed with respect to the outputs of this study. Specifically,teaching and mentoring activities were conducted in the form of mentoring one Ph.D student during this project. Additionally, Tools developed during this project were used during a senior undergraduate food microbiology laboratory to teach students about biotechnology.

Events related to the outputs include 8 presentations at scientific conferences. Selected presentations are listed below. 1. The Phast Swab: Rapid Detection of Viable Foodborne Bacterial Pathogens. L. D. Goodridge. 2008. CSU Ventures and Sage Networking Event and Poster Session, Fort Collins, Colorado. 2. Development of the Phast Swab for Rapid Detection of Enterohemmorhagic Escherichia coli. L. D. Goodridge, Willford, J. 2006. 1st Texas/Evergreen Phage/Virus Genomics and Ecology Meeting, Kingsville, Texas. 3. The Phast Swab: a Method for Rapid Detection of Enterohemorrhagic Escherichia coli J. Willford, Goodridge, L. D. 2006. 6th International Symposium on Shiga Toxin (Verocytotoxin) Producing E. coli Infections, Melbourne, Australia. 4. Evaluation of Three Commercially Available Shiga Toxin Enzyme Linked Immunoassays for their ability to Detect Shiga Like Toxins in Shiga Like Toxinogenic Escherichia coli. J. Willford, Goodridge, L. D. 2006. 6th International Symposium on Shiga Toxin (Verocytotoxin) Producing E. coli Infections, Melbourne, Australia. 5. Applied uses of bacteriophages in food microbiology and safety. L. D. Goodridge. 2005. First Joint Meeting of the Rocky Mountain Virology Club and Rocky Mountain Branch, ASM. Pingree Park, Colorado 6. A Simple Colorimetric Assay for Rapid Detection of Escherichia coli O157:H7. L. D. Goodridge, Willford J. 2005. International Association for Food Protection, 92nd Annual Meeting, Baltimore, Maryland.

PARTICIPANTS: Two individuals worked on this project. Dr. Lawrence Goodridge was the principal investigator/project director,and was responsible for overseeing all aspects of the project, including research, reports, and publications. John Willford worked on the project as a graduate research assistant in pursuance of a Ph.D. which was completed in the summer of 2008. Dr. Willford conducted all experiments related to the project. One partner organization was involved in this project. Hygiena Corporation (Camarillo, CA) provided in-kind support in the form of the disposable test devices that were used in the developed rapid test.

TARGET AUDIENCES: Target audiences for this project were members of the academic community (particularly in the fields of Food Science, Animal Science and Microbiology), and commercial companies, particularly those involved in food testing and diagnostic development.

IMPACT: 2005/12 TO 2008/11
During the project a reporter bacteriophage was developed, and the genetically modified phage contributed to an outcome/impact by causing a change in knowledge. Specifically, the creation of the reporter phage allowed for the development of a vertically integrated, rapid test for Escherichia coli O157:H7, that allowed rapid detection of this foodborne pathogen on meat samples. The reporter phage, a sampling swab, bacterial growth media, E. coli O157 immunomagnetic separation (IMS)beads and a enzyme substrate for beta-galactosidase were all combined into a disposable test device. The food to be tested was swabbed, and the swab was returned to the device, followed by incubation for 8-10 hours. Any E. coli O157:H7 present in the test sample was concentrated by the IMS beads, and the growth media was removed. The IMS beads and E. coli O157:H7 bacteria were resuspended in a buffer that contained the reporter phage, and the phage infected the bacteria, forcing it to make beta-galactosidase which, when mixed with the enzyme substrate, produced a detectable signal (luminescent or colorimetric). This example of a change in knowledge led to a publication. The results of this project also led to a change in conditions in the form of a US patent (#7,244,612), that was subsequently licensed to a company (technology transfer).

Funding Source
Agricultural Research Service
Project number
WYO-00579
Accession number
205313
Categories
Bacterial Pathogens
Escherichia coli
Parasites
Natural Toxins
Commodities
Produce