Foodborne illnesses continue to negatively affect public health. Current strategies to detect and prevent illnesses rely on prolonged enrichment protocols of 24-48 hours. While rapid methods are constantly being developed, these methods do not consider preanalytical sample processing, which is a critical first step for reliable and reproducible results. Additionally, the time to detection for an assay does not include the preparation and enrichment steps that must be completed to arrive at optimal pathogen numbers to enable detection. To address this gap, the author evaluated and refined the use of a proprietary magnetic nanoparticle functionalized with chitosan (F#1 MNPs) as a preanalytical sample processing tool to capture and concentrate foodborne pathogens from complex food matrices. Two foodborne pathogens, Listeria monocytogenes (gram-positive) and Salmonella ser. Newport (gram-negative) were used to evaluate the F#1 MNPs in strawberries, romaine lettuce, and cotto salami, representing diverse food matrices. These pathogens were chosen for this proof-of-concept study based on their significant public health impact. Chitosan electrostatically binds to the cell-surface structure of bacteria. Therefore, it is hypothesized that the F#1 MNPs also bind to the exterior of pathogens. However, the exact binding mechanism remains unknown. Due to this, all testing used cold-stressed pathogens to simulate their physiological state after food processing. First, statistical design of experiments (DOE) was used to optimize protocols for extracting ≤ 3 CFU/g of bacterial contamination in diverse matrices with only minor protocol adjustments. This study highlights the potential to standardize protocols and the ability to rapidly adjust them based on regulatory requirements for different pathogens and food matrices. Next, using the same strains and food matrices, the effect of the F#1 MNPs on pathogen enrichment was evaluated. Modifications reduced broth enrichment times to 4-12 hours without inhibiting target pathogen growth on selective agars, expediting the overall time to single-colony isolation. This is especially important for regulatory enforcement that still relies on the isolation of pathogens for downstream testing and outbreak surveillance and investigation. Finally, the use of shotgun metagenomics revealed potential applications beyond bacterial pathogens. The F#1 MNPs can also capture non-pathogenic bacteria, viruses, and fungi, which may have applications such as environmental bioindicators. This further shows the versatility of the F#1 MNPs as a preanalytical sample processing tool in a wide range of detection pipelines, such as multi-organism detection with multiplex assays, pathogen-agnostic testing, and identifying pathogens in emerging food vehicles. By streamlining pathogen extraction and concentration, F#1 MNPs offer significant potential to improve surveillance, outbreak detection and prevention, and overall food safety.
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