Molecular-based Diagnostics of Foodborne Pathogens

NON-TECHNICAL SUMMARY: Foodborne pathogens, which lead to approximately 80 million illnesses and 5000 deaths in the United States each year, are important targets for the control of food safety and public health. In addition, resistance to antibiotics among foodborne pathogens as well as clinical isolates has steadily increased in the last two decades. This research will focus on the development of various assays, either bead-based or microarray-based, which can be used for diverse applications: simultaneous detection of multiple foodborne pathogens, molecular characterization of foodborne pathogens and detection of antibiotic resistance genes.<P>APPROACH: <BR>OBJ 1: Bioinformatics software for microarray application will be employed to design probes for target organisms - initially, E. coli O157:H7, Salmonella, Listeria monocytogenes and Staphylococcus aureus. The assay format will consist of 96-well microplate containing micro-sized beads functionalized with both oligonucleotide probes and encoding indicators. Presence of target organisms will be confirmed by measuring fluorescence signal from hybridization between probes and target DNA sequences. <P>OBJ 2: Multiple oligonucleotide probes for target organisms will be designed not only from virulence factors, toxin genes, serotype-related genes, and species-specific genes, but also from common genes using bioinformatics software. Negative controls (core gene sequence from non-target organism and buffer only) will be spotted onto the array for the control purpose. Once probe sequences for target organism are designed, customized microarrays will be printed and scanned. <P>OBJ 3: In this study, multiple oligonucleotide probes will be designed from antibiotic resistance genes and from species-specific genes using bioinformatics software. Negative controls will be spotted onto the array for the control purpose. DNA from target organisms will be purified, labeled and hybridized and the fluorescence signal from the hybridization will be measured. Designed probe sequences will be synthesized with proper modification and printed directly onto chemically modified glass slides to create DNA microarrays. Results from DNA microarrays will be compared with those from standard methods.<P>PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: Multiple target genes for detection of initial target organisms including entero-aggressive E. coli (EAEC), entero-toxigenic E. coli (ETEC), entero-pathogenic E. coli (EPEC), entero-invasive E. coli (EIEC) and entero-hemorrhagic E. coli (EHEC) have been chosen. For EAEC, aggA. aggC, and aggR genes were chosen; for ETEC, lt-1, st1a, st1b, cfa1, cs1, cs3, and ingA genes were chosen; for EPEC, eaf, bfpA, and espC genes were chosen; for EIEC, invX, ipaC, ipgD, cadC, cadBA, and clyA genes were chosen; for EHEC, stx1, stx2, ehxA, etpD, katP, espP, rfb, and espFU genes were chosen. Additionally, multiple genes common to both EPEC and EHEC were chosen; eae, espP, espA, paa, espB, and tir genes. Bioinformatics software for microarray application, AlleleID (Premier Biosoft, Palo Alto, CA) was purchased to design probes and primers for all target genes. Using AlleleID, two probes for detection and a pair of primers for PCR amplification were designed for all selected target genes. Designed probe and primer sequences were checked against GenBank Database to ensure their specificity. It was noted that some of the selected target genes for EIEC, ETEC, and EHEC showed high sequence homology with Shigella spp., Citrobacter freundii, or Salmonella spp. To solve the problem, new probes and primers are being designed for these genes which can avoid or minimize cross-reactivity with non-target organisms, and more genes are being explored to replace the genes with high sequence homology. <BR>PARTICIPANTS: Collaborated generally with Dr. Ricke and Dr. Johnson, University of Arkansas - Fayetteville.<BR> TARGET AUDIENCES: Food processing industries will be interested in the results of this project for possible implementation in their sanitation and quality assurance workflow. Consumers will benefit from improved food safety. Fellow food scientists will be interested in these results. <P>IMPACT: 2007/01 TO 2007/12<BR>
Studies described above indicated that depending on virulence-related genes for simultaneous detection of multiple pathogens might cause cross-reactivity problems. Since many of the target pathogens share high similarity in their pathogenicity, it would be hard to identify each these pathogens at the species-specific level. Alternative approach to this problem will be identification of target using pattern-recognition rather than simple yes/no response. When detecting multiple pathogens which are close to each other phylogenetically, pattern-recognition approach may prove more useful for correct identification.