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|James B Day|
|U.S. Food and Drug Administration, USA|
|ScientificTracks Abstracts: J Nutr Food Sci|
|Statement of the Problem: Listeria monocytogenes, the causative agent of listeriosis in humans, is a gram-positive bacterium that is contracted via the consumption of contaminated foods. Various leafy green vegetables, including lettuce and spinach, have been implicated in human listeriosis cases. Molecular methods and immuno-based techniques for detection of L. monocytogenes in these food matrices can be difficult due to the presence of assay inhibiting elements. Methodology: In this study, we utilize a novel enrichment media containing activated charcoal as the key ingredient that induces overexpression and secretion of L. monocytogenes virulence proteins. The Bio-Plex suspension array system, based on Luminex xMAP technology, can then be utilized to specifically detect accumulated L. monocytogenes virulence proteins via a magnetic bead-antibody complex. Iceburg lettuce and packaged ready-to-eat spinach were treated with L. monocytogenes and incubated in preenrichment broth (Buffered Listeria Enrichment Broth) followed by incubation in charcoal activated media. The supernatant fraction was TCA precipitated and L. monocytogenes lysteriolysin O (LLO) was collected using magnetic microspheres conjugated to LLO specific antibody. A newly developed antibody that exclusively recognizes L. monocytogenes LLO was used as the biotin conjugated secondary antibody and analysis was conducted using the Bio-Plex 200 system. Findings: As few as 1 CFU/ g of L. monocytogenes were detected in both foods tested. Whole cell fractions from 14h activated charcoal enrichments were also analyzed using antibody that recognize both pathogenic and non-pathogenic Listeria species which also resulted in a detection limit of 1 CFU/ g. Internal control beads were also utilized to ensure proper instrumentation function, integrity of assay reagents and to eliminate the possibility of non-specific interactions. Conclusions & Significance: This method is the first to specifically recognize and differentiate L. monocytogenes among other nonpathogenic Listeria species in various leafy greens using immune-detection. The total presumptive detection time can be achieved in less than 24h.|
James B Day is a Research Microbiologist at the U.S. Food and Drug Administration in College Park, Maryland, where he is involved in developing detection methodologies for bacterial pathogens in contaminated foods. He has developed techniques for rapid identification of Francisella tularensis, Salmonella enterica and Listeria monocytogenes in various food matrices and recently established a novel macrophage-based assay for enrichment of intracellular bacterial pathogens for enhanced identification. He earned his PhD from the University of Miami School of Medicine (UM), where he worked on bacterial pathogenesis of Yersinia pestis. At UM, he developed a widely used system to measure virulence protein secretion and host cell translocation. He went on to complete his Postdoctoral studies at Harvard Medical School, where he worked on type III secretion mechanisms of Salmonella enterica as well as regulatory factors that control virulence protein induction.
Email: [email protected]
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