2008 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).
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.
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. This project is aimed at developing faster detection methods for the food industry and regulatory agencies.
To reduce the dependence on culture enrichment, methods for concentration of foodborne pathogens were developed. A novel filter able to remove particulates (< 10 micrometers) was used in conjunction with conventional filters for rapid concentration of E. coli O157:H7 and L. monocytogenes from large volumes of homogenized food and the filter-captured pathogens were detected by PCR. Other studies involved the use of immunomagnetic beads (IMBs), microscopic magnetic beads with attached antibodies, to capture pathogens in food. IMB performance varied between bead type and the method used to attach the antibody, mixtures of IMB showed the most consistent behavior, and a cocktail of IMBs was developed for improved capture of E. coli O157:H7. A method was developed using L. monocytogenes-specific IMBs for the capture and microbiological detection of L. monocytogenes from foods containing a large excess of other Listeria, which normally interferes with detection. Common spoilage bacteria (Brochothrix, Carnobacterium, and Serratia spp.), which interfere with IMB pathogen capture, were characterized for growth in ground chicken at 5°C. Remarkably short doubling times (~ 4-5 h) and long recovery times (12-24 hr) were observed. Brochothrix grew in multicellular structures which attach to immunomagnetic beads. We have begun characterizing these multicellular structures.
Progress was made toward the development of antibody and DNA microarrays for the detection of multiple pathogens. DNA-based microarray development involved the design and synthesis of a comprehensive set of DNA probes targeting unique genes in E. coli O157:H7, Salmonella, Campylobacter, and L. monocytogenes as well as evaluation of methods for printing slides, isolating and labeling DNA, hybridization, and scanning. The development of antibody microarrays has progressed to allow simultaneous detection of E coli O157:H7, Salmonella, and a protein toxin surrogate. Studies are progressing on development of a mixed culture enrichment medium suitable for microarray applications.
Environmental factors such as signaling chemicals and the presence of non-pathogenic E. coli were found to affect the production of harmful toxins by E. coli O157:H7. The origin of this effect and its possible application to minimize the pathogenic effects of E. coli O157:H7 are being investigated.
Multiplex PCR assays were designed for the detection and differentiation the three pathogenic species of Yersinia. Y. pestis-specific IMBs were used in conjunction with a Y. pestis plating media to improved the specificity of detection of this pathogen.
Simultaneous detection and differentiation of foodborne Campylobacter spp. Campylobacter is a leading cause of food-related gastrointestinal illness in the developed world. While there are 17 closely related species of Campylobacter, C. jejuni, C. coli, and C. lari are most often associated with foodborne human illness. We have developed and tested a rapid PCR assay which is able to simultaneously detect and differentiate these 3 Campylobacter species in a single reaction. In testing against 29 strains of Campylobacter and 20 strains of non-Campylobacter species, the assay was found to be both very specific and very sensitive. The assay was also evaluated using both artificially and naturally contaminated chicken samples. These results demonstrated the feasibility of the developed PCR assay for rapid detection and differentiation of C. jejuni, C. coli, and C. lari in food samples. This developed PCR assay will be useful to regulatory agencies in doing baseline studies necessary for public health-based risk assessment and establishing regulatory guidelines. (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.)
Identification of most probable foodborne non-pathogenic bacteria that bind non-specifically to immunomagnetic beads. Most foods contain many harmless bacteria that interfere with the detection of very low numbers of disease causing bacteria (pathogens) that may be present. One example is the non-specific binding of harmless bacteria to immunomagnetic beads (IMBs, microscopic magnetic particles coated with antibodies that bind to specific pathogens) used to capture pathogens from foods. We developed and applied a sampling method to identify bacteria that non-specifically bind to IMBs. The bacteria were identified by determining the DNA sequence of a common gene. The most frequently isolated bacteria were species of Brochothrix, Carnobacterium, and Serratia. We discovered that the non-specific binding of these harmless bacteria was dependent on the type of food and the type of IMB used, and that these bacteria bind to the IMBs by different mechanisms. Apparently compounds in the food are involved in the organism-to-IMB attachment. This work underscores the need for researchers and regulatory agencies to develop and evaluate unique procedures for the IMB capture of pathogens for each type of food. Furthermore, this work demonstrates that the best approach to understanding the attachment of background bacteria to capture surfaces is to better understand the biology of these organisms. (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.)
Sensitive and rapid detection of Salmonella in Egg components;
The US Department of Agriculture has estimated that the annual per capita consumption of eggs in America is now about 260. According to the Center for Disease Control and Prevention, however, consumption of Salmonella Enteritidis (SE) contaminated raw or undercooked eggs has led to about 118,000 cases of illness yearly. This health concern has prompted the need to develop fast, specific and sensitive methods to detect the presence of SE in eggs. Conventional microbiological and immunological tests for Salmonella, including SE, from eggs take 5-7 days and are labor intensive. We have developed a rapid and sensitive approach that involved the specific capture of SE by immunomagnetic beads (IMBs, microscopic magnetic particles coated with antibodies that bind to SE) followed by detection of the IMB captured SE by a very sensitive time-resolved fluorescence method. We also demonstrated that the capture of SE in whole egg, egg white and egg yolk varied depending upon the type of IMBs used. The information is valuable for regulatory agencies and food safety laboratories to develop practical, automated and bead-based methodologies to detect SE in eggs (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
Gehring, A.G., Albin, D.M., Reed, S.A., Tu, S., Brewster, J.D. 2008. An antibody microarray, in multiwell plate format, for multiplex screening of foodborne pathogenic bacteria and biomolecules. Analytical and Bioanalytical Chemistry. 391:497-506.
Irwin, P.L., Nguyen, L.T., Chen, C. 2008. Binding of non-target microorganisms from food washes to anti-Salmonella and anti-E. coli O157 immuno-magnetic beads: minimizing the errors of random sampling in extreme dilute systems. Analytical and Bioanalytical Chemistry. 391:515-524.
Tu, S., Reed, S.A., Gehring, A.G., He, Y. 2008. Detection of Salmonella Enteriditis from Egg Components Using Different Immunomagnetic Beads and Time-resolved Fluorescence. Journal of Food Analytical Methods. Available:http://www.springerlink.com/content/68381p3740355j10/
Irwin, P.L., Nguyen, L., Chen, C., Paoli, G. 2008. Binding of Nontarget Organisms to Anti-Salmonella and Anti-E. coli O157 Immunomagnetic Beads: Most Probable Composition of Background Eubacteria. Analytical and Bioanalytical Chemistry. 391:525-536.