2007 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. 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).
1935-42000-058-00D - To prevent outbreaks of foodborne illness, the food industry and regulatory agencies need simple and cost-effective methods to detect harmful bacteria (pathogens) in foods. The methods must be rapid, sensitive, and selective. Traditional methods require growth of microorganisms in selective culture media (enrichment) and take 2 days or more. This project is aimed at developing faster biosensor-based methods. A novel filtration method was developed for the concentration of E. coli O157:H7 ground beef homogenates from 250 to 0.1mL. Further applications of this method will obviate the need for enrichment. Immunomagnetic bead-based (IMB) methods for pathogen capture and concentration were coupled with a fluorescence-based detection of Salmonella in liquid eggs and the simultaneous detection of E. coli O157:H7 and its toxins from ground beef. Another study involving IMBs determined that the non-specific binding of the harmless bacteria present in food that can interfere with the capture of pathogens was dependent on the type of food and was variable with different lots of commercial IMBs. Biosensors utilize biorecognition elements, antibodies or DNA molecules, to determine the presence of a specific pathogen in a sample. A molecular technique (phage display) for the selection of specific antibody fragments (scFvs) was applied to pathogenic bacteria. Progress this year included:.
1)the selection scFvs that bind to Francisella tularensis, 2)the characterization of previously isolated scFvs that bind to Yersinia pestis, and.
3)the development of an optical biosensor using scFvs that bind to Listeria monocytogenes. Furthermore, scientific collaborations have been established for the selection of scFvs by another method (yeast surface display) and for the selection of DNA or RNA biorecognition elements (aptamers). Progress was made toward the development of both antibody and DNA microarrays for the simultaneous detection of multiple pathogens. Various methods for the attachment of antibodies to glass microarray slides were evaluated. Once antibody binding was optimized, a rapid (<4 hours) fluorescent antibody assay was developed in a microarray format yielding a level of detection of approximately 10,000 cells/mL. By attaching different antibodies onto a single glass slide in an array of spots, an antibody microarray can be constructed to allow the simultaneous detection of multiple pathogens. A similar approach can be used to construct a DNA hybridization microarray using pathogen specific DNA sequences. An extensive literature search and bioinformatics approaches were applied to identify unique DNA sequences from several foodborne pathogens. These DNA sequences will serve as the basis for a DNA-based microarray assay for the simultaneous detection of multiple pathogens from an individual food sample. 1935-42000-058-03S – Specific Cooperative Agreement with the University of Arkansas, “Nanoparticle-based Fluorescent Biosensor for Rapid Detection of Listeria monocytogenes in Foods.” Progress is monitored via e-mail and phone. For a complete report on the progress of this subordinate project, see the corresponding annual report.
Isolate and concentrate pathogens to speed detection. Detection of low levels of pathogens in food requires 6 to 24 hours of growth in selective microbiological culture media (enrichment) to allow the target bacteria to multiply and reach a detectable level. Much faster results could be obtained if a means of isolating and concentrating the target organism from a large volume of food sample is available. A rapid filtration process was developed using information on the size distribution of particles in homogenized beef, chicken, turkey, and frankfurters versus the retention of pathogens (E. coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium) on various filter media. Using a novel combination of filters, E. coli O157:H7 was isolated from 250 mL of ground beef homogenate and concentrated to a volume of 0.1 mL in 30 minutes with high efficiency. When coupled with selective microbiological plating media, this approach allowed the detection of E. coli O157:H7 from ground beef in less than 24 hours. When coupled with an appropriate biosensor, this approach will allow detection of E. coli O157:H7 and other pathogens without prior enrichment, reducing detection time to 4-8 hours. (This project is aligned to National Program - 108, Food Safety, Action Plan Component 1.2.1 Pathogens, Toxins and Chemical Contaminants Postharvest: Detection and Validation)
Optimization of the conditions for simultaneous detection of E. coli O157:H7 and shiga-like toxins. To assure the safety of foods, it is desirable to simultaneously detect the pathogenic bacteria and their toxins from the same sample. An immunomagnetic bead (IMB) method was developed to detect pathogenic E. coli O157:H7 and their shiga-like toxins (SLTs). To achieve this goal, antibodies specific for E. coli O157:H7 and SLTs were coupled to magnetic beads of various sizes, and the antibodies were labeled with europium and samarium fluorescent probes. The bacteria and toxins were captured from ground beef using IMBs, and the fluorescent antibodies were then used to reveal the presence of captured E. coli O157:H7 and SLTs. The method was able to simultaneously detect both the bacterium and the toxin, but the results indicated that the detection of SLT was significantly lowered by the presence of non-target bacteria. In addition to its basic physiological significance (i.e., total bacterial population effects on the growth of SLT-producing E. coli), this observation indicated a practical limitation in the detection of SLTs in foods. (This project is aligned to National Program - 108, Food Safety, Action Plan Component 1.2.1 Pathogens, Toxins and Chemical Contaminants Postharvest: Detection and Validation)
5.Significant Activities that Support Special Target Populations
|Number of new CRADAs and MTAs||4|
|Number of active CRADAs and MTAs||5|
|Number of non-peer reviewed presentations and proceedings||12|
|Number of newspaper articles and other presentations for non-science audiences||1|
Paoli, G., Kleina, L.G., Brewster, J.D. 2007. Development of listeria monocytogenes-specific immunomagnetic beads using a single-chain antibody fragment. Foodborne Pathogens and Disease. 4:74-83
Brewster, J.D. 2007. Lattice boltzmann simulations of three dimensional fluid flow on a desktop computer. Analytical Chemistry. 79:2965-2971.
Gehring, A.G., Albin, D.M. 2007. Protein-Based Microarray for the Detection of Pathogenic Bacteria. Journal of Rapid Methods and Automation in Microbiology. 15:49-66
Tu, S., Gehring, A.G., Paoli, G. 2007. Detection of salmonella in liquid eggs by immunomagnetic capture and time-resolved fluorescence. Journal of Rapid Methods and Automation in Microbiology. 15:107-119
Paoli, G., Brewster, J.D. 2007. Identification of the surface antigen recognized by a listeria monocytogenes-specific phage-displayed antibody fragment and its presence in different physiological conditions. Journal of Rapid Methods and Automation in Microbiology. Vol. 4(1):74-83.
Feder, I.E., Wijey, C., Paoli, G., Crawford, C.G., Tu, S. 2007. Immunomagnetic-electrochemiluminescent detection of escherichia coli 0157 in ground beef after pre-enrichment in brilliant green bile broth, modified escherichia coli broth with or without novobiocin, or gram negative broth. Journal of Rapid Methods and Automation in Microbiology. 15:92-106.
Gehring, A.G., Albin, D.M., Irwin, P.L., Reed, S.A., Tu, S. 2006. Comparison of enzyme-linked immunomagnetic chemiluminescent with usf das bam method for the detection of escherichia coli o157:h7. Journal of Microbiological Methods. 67:527-533.