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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
The ultimate goal of this project is to develop rapid, specific, and sensitive biosensor-based assays for diverse pathogenic bacteria which can be widely adopted in applications ranging from simple field tests to high speed, high throughput laboratory screening assays. To meet this goal, several objectives will be pursued: 1) Develop specific, high affinity biorecognition reagents for food-borne pathogens and toxins. 2) Develop rapid and effective means to separate and concentrate targeted pathogens without carryover of background organisms. 3) Develop integrated assay systems based on multiple target biosensor platforms.

1b.Approach (from AD-416)
The primary objective of the proposed research is to develop biosensor processes that are capable of detecting multiple pathogens of food safety and food security concern. We plan to concentrate our research on a few selected pathogens: E. coli O157:H7, Listeria monocytogenes, Salmonella and Yersinia spp. Unless otherwise indicated, all experiments conducted with cells of Yersinia spp. or other biosafety level 3 (BSL3) pathogens will be conducted in house using non-virulent BSL2 surrogate strains. Collaborative arrangements have been made for evaluation of the developed methods with virulent strains. Methods will generally be developed with culture media as the sample matrix, and then extended to food samples containing the target pathogen. The efficacy of developed methods will be primarily tested in ground beef, ready-to-eat meats and liquid eggs. Modification of the plan to include other pathogens and foods will be determined by ARS needs. To facilitate the progress of planned research, we will seek useful advice and/or input from our colleagues in other Research Units at the Eastern Regional Research Center (ERRC).

3.Progress Report
Traditional methods for the detection of foodborne pathogens require 2 or more days to complete and involve the growth of microorganisms in selective culture media (enrichment) followed by microbiological and biochemical characterization of putative pathogenic isolates. Furthermore, each of these traditional methods is designed for the detection of a single pathogenic species. This project is aimed at developing more rapid methods for the detection of the very low numbers of pathogens from food and the development of methods to detect multiple pathogenic species simultaneously. The traditional enrichment methods used allow the selective growth of a single species which precludes the use these culture methods for the detection of multiple species. Methods are being investigated for the simultaneous culture enrichment of multiple microbial pathogens. Several media formulations have been investigated and progress has been made on the development of a culture medium that allows the growth of 4 different pathogenic bacteria to levels sufficient for detection. In addition, a new approach was developed using culture enrichment in a water-in-oil emulsion containing growth media in the aqueous phase. Each droplet in this emulsion contains zero or one bacterial cell, and acts as an isolated compartment in which bacteria can grow without competition or interference from other microorganisms. To date, simultaneous enrichment of two pathogens (E. coli O157:H7 and L. monocytogenes) to detectable levels has been demonstrated, but this approach can provide a universal method for enrichment of any target set. Antibody-based immuno-biosensor methods were developed for the detection of one or more pathogens. A combined immunomagnetic bead (IMB) / time resolved fluorescence (TRF) method was developed for the simultaneous detection of E. coli O157:H7 and Salmonella. The method employed IMBs covered with antibodies to both E. coli O157:H7 and Salmonella for simultaneous capture and separate antibodies coupled to unique lanthanide molecules for TRF detection. The assay was optimized and tested with pure culture and is ready to be used to detect both organisms from a food matrix. A similar IMB/TRF assay was applied to the detection of Salmonella Typhimurium from peanut butter. The assay results are promising and enrichment methods aimed at reducing interference caused by the high fat content of peanut butter are being investigated. DNA-based methods were developed for the detection and differentiation of multiple pathogens. For effective and reliable detection of foodborne pathogens, a multiplex real-time PCR combined with multi-pathogen enrichment strategy was developed to simultaneously detect E. coli O157, Salmonella, and Listeria monocytogenes. Evaluation and application of the assay in food is currently in progress. In addition a multiplex real-time PCR assay was developed for the simultaneous detection and differentiation of the 3 pathogenic species of Yersinia from food.

1. Multi-pathogen detection was achieved by using DNA Microarray: E. coli O157:H7, Salmonella, Campylobacter jejuni, and Listeria monocytogenes are the four most serious bacterial foodborne pathogens. At present most methods for detection of foodborne pathogens require 2 or more days to complete and are designed to detect a single pathogen. We have developed a more rapid DNA-based microarray for effectively screening all four of these important pathogens from food. Combined with amplification of target sequences, the microarray unambiguously distinguished these four prominent foodborne pathogens in a single array with high sensitivity. The cost of the detection microarray has been substantially reduced by designing and fabricating 12 identical arrays on each chip, which can be used for screening up to 12 independent samples. The application of the micro array was established in fresh meat samples.

2. The production of Shiga-like toxins by pathogens is affected by environmental factors: Using a sandwiched immunoassay for detection of Shiga-like toxins (SLTs) produced by Escherichia coli O157:H7, we showed that the presence of E. coli K-12 conditioned media decreased the production of SLTs by E. coli O157:H7. Apparently, E. coli K-12 cells produces signaling chemicals that inhibit the production of SLT by E. coli O157:H7. This type of cell-to-cell signaling is known as quorum sensing and some of the molecules involved have been previously identified (referred to as autoinducers 1 and.
2)and are known to regulate the expression of virulence genes. The possible molecular identity of the autoinducer secreted by E. coli K-12 and the potential application to minimize the pathogenic effects of E. coli O157:H7 are being investigated.

3. Demonstrated that the goal to detect one bacterial cell or DNA fragment per tested volume is only reasonably achievable using a binomial approach (presence or absence: In order to accurately determine the concentration of pathogenic bacteria in foods it will be necessary to detect as few as 1 entity (DNA molecules or bacterial cells) per sample because the level of pathogenic bacteria in foods is very small. To this end we developed/tested a simple model for accurately predicting purely stochastic errors associated with random sampling at extreme dilutions in order to determine the minimum sample size (n or number of technical replicates) necessary to accurately quantify such low levels of pathogenic organisms in food products. At these extreme dilutions we found that the change in sampling error with respect to n is minimized only as when n is made unreasonably large. This result argues that pathogen detection is most economically performed, even using highly sensitive techniques such as PCR, when some form of organism cultural enrichment is utilized to increase cell numbers resulting in an essentially binomial response (a plus/minus result). Thus, in theory at least, using a method capable of detecting a single target [e.g., a specific gene PCR-based assay (+ or –)] one could quantitatively detect anywhere from 1 to 100,000 bacteria per mL depending on the initial concentration of the pathogen.

4. Showed that nanoparticles (nP) inhibit bacterial growth: Some types of nanoparticles (nPs) have been observed to have antibacterial properties. The effect of antibacterial nPs on the bacterial growth was therefore examined. Bacterial growth normally has a lag time in which minimal growth of bacteria occurs. After passing the lag time, exponential growth of bacteria to a maximum density. We have found that nPs induce a maximal change in T of about 14-19 hrs depending upon the type of nP used. These results are important because they demonstrate that these nPs do not kill bacterial cells and demonstrate a method by which growth inhibition via increased lag phase can be measured.

6.Technology Transfer

Number of the New/Active MTAs (providing only)2

Review Publications
Albin, D.M., Gehring, A.G., Reed, S.A., Tu, S. 2009. Apparent Thixotropic Properties of Saline/Glycerol Drops with Biotinylated Antibodies on Streptavidin-Coated Glass Slides: Implications for Bacterial Capture on Antibody Microarrays. Sensors. 9:995-1011.

Brewster, J.D. 2009. Large-volume filtration for recovery and concentration of E. coli O157:H7 from ground beef. Journal of Rapid Methods and Automation in Microbiology. 17:242-256.

Last Modified: 8/27/2015
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