Progress 08/01/24 to 07/31/25

Outputs
Target Audience:The primary target audience(s) for this reporting period is the general food safety community including the two graduate students working on the project. While the filtration device is still being modified, efforts during this reporting period focused on general food safety and pathogen detection, including understanding the diversity of Salmonella that will be detected with the device and validating the real-time thermocycler that will be used with the FDA BAM qPCR method for real-time detection of Salmonella. Changes/Problems:No major changes or problems have occurred. There has been a slight delay in construction of the GFP-tagged Salmonella because we first needed to validate the PCR conditions necessary for the real time thermocycler in our lab (the make and model of the thermocycler listed in the FDA BAM for qPCR detection of Salmonella is different from the thermocycler that we recently acquired). The constructs for the GFP-tagged Salmonella have been designed and ordered and we will now focus our efforts on construction of the GFP-tagged S. Typhimurium. What opportunities for training and professional development has the project provided?Training: During this project period two graduate students were mentored by the PD and Co-PDs through one-on-one meetings as well as monthly group meetings with all mentors. Professional Development: One graduate student presented preliminary results from this project at one regional conference (Virginia ASM branch meeting) and one local conference (Departmental conference). The graduate student will also present the data at an international conference in July (IAFP annual meeting). How have the results been disseminated to communities of interest?Results of this research have been primarily disseminated via poster presentations at meetings (local, regional, and international). What do you plan to do during the next reporting period to accomplish the goals?Work in the next project period will focus on establishing procedures for extraction of DNA from GFP-tagged cells collected on the BEM (second membrane in filtration unit; Objective II), and then using the FAST filtration unit to concentrate cells inoculated into liquid (chicken broth) and solid matrices (chicken parts; Objective III). More specifically we will: Optimize filter design, finalize the data analysis and write the manuscript for surface modification and engineering of the FAST filtration device. Genetically engineer a GFP-taggedSalmonellastrain for visualization ofSalmonellaon the membranes of FAST Optimize protocols for extracting low levels ofSalmonellafrom chicken broth and chicken parts using the FAST filtration device, with downstream qPCR detection. Our initial goal to complete GFP tagging ofSalmonellaTyphimurium in reporting period one was not met. We instead focused our efforts in the current product periodvalidating the FDA BAM qPCR procedure forSalmonelladetection using a QuantStudio 5 qPCR thermocycler; because many research, government, and industry labs have this thermocycler (and not the two that are referenced in the standard method), validating its use for the qPCR detection method is helpful for the broader food safety research community. To ensure progress of the overall project weinstead used anE. coliGFP reporter strain to allow us to continue making progress on the experiments for objective I. We have designed and received all the materials necessary for the GFP-tag, and experiments to genetically engineer the strain are on-going.

Impacts
What was accomplished under these goals? The food industry uses a variety of testing methods to screen for harmful bacteria that could cause a consumer to become ill. One of these tools involves rapid screening of food for the presence of pathogenic bacteria, requiring a minimum of 1-2 days to produce preliminary results. To deliver high-quality, safe food, new methods are needed that allow producers to more quickly screen their food without compromising accuracy of the results. The overall goal of this project is to develop a new method to quickly extract bacteria that cause foodborne illness from a food matrix to reduce the overall time-to-results. To achieve this goal, this project will accomplish the following objectives: Obj. I:Develop debris repelling membranes that through all-dry surface modification can enable flow-through of bacteria while excluding food debris During the project period, we modified the surface chemistry of different filters with the goal of achieving a filter membrane that repels food particles (fats, proteins) while allowing bacteria to pass through the first membrane. Our results suggest that chemical modification of the membrane indeed reduces the affinity of protein and fat in commercial chicken broth to bind to, and potentially clog, the membrane. We also developed staining procedures that will allow us to visualize bacteria (first using GFP-tagged non-pathogenic E. coli), protein, and fat, that may deposit on the membrane during the filtration process. While we have a membrane that works well in-hand, we are further optimizing the surface chemistry of the filter while we continue work on constructing a GFP-tagged Salmonella strain. Obj. II:Establish procedures for DNA extraction forSalmonellacells collected by the novel bacteria extraction membrane Work for this objective is ongoing, but we have demonstrated that the membrane dissolves in a solution of acidified-phenol-chloroform with minimal agitation, making it a good option for the proposed rapid acquisition of high-quality nucleic acid (DNA and RNA) material for input into downstream detection methods (both commercially available ones and in house methods that we are developing). We have also completed validation of the qPCR method in the FDA BAM Chapter 5: Salmonella with the real-time thermocycler that we recently acquired. In the next project period, we will continue working on construction of a GFP-tagged Salmonella for assessing recovery of Salmonella from filtering. Obj. III:Demonstrate the suitability of FAST to extract low levels ofSalmonellafrom liquid and solid food matrices Work on this objective is ongoing. In the next project period, we will inoculate Salmonella into liquid (chicken broth) and solid (chicken parts) matrices and assess recovery rate of Salmonella. Overall, in this reporting period, we have engineered a suitable tandem filter apparatus that allows us to capture E. coli (used as a model) inoculated into chicken broth, with minimal losses of bacteria in the first filtration step. This device shows promise in rapidly concentrating bacteria from large volumes (up to 750 mL tested thus far) of a liquid food matrix, which should reduce the time required for food safety testing.

Publications