Rapid bacterial testing for on-farm sampling

Due to the sensitive nature of fresh produce, bacteriological safety tests which requires days for results are not practical tools for food safety. In order for a testing plan to present a pragmatic solution, it must be low-cost, reliable, robust and deliver rapid results. Our labs have been developing diagnostics to be used in non-laboratory settings by utilizing bacteriophages. These viruses can attack specific bacteria, replicate within them and then lyse the host while releasing hundreds to thousands of additional viruses. We are proposing the development of a dipstick for the rapid detection of Salmonella spp. in agricultural samples. Following a sample pretreatment, the bacteriophages will be used to infect Salmonella spp. in the sample. Within 45 minutes, the increase in bacteriophage can be quantified with a simple lateral flow device resembling a pregnancy test. Preliminary results for our E. coli sensor suggest a very low limit of detection (<10 CFU/mL). This project will perform the additional development, optimization and validation steps needed and if successful will empower farmers to perform tests on-farm with rapid results and at low-cost. This ability will then enable more risk-based testing of agricultural waters following heavy rains or high flow.

Recently, the CDC has determined that 46% of foodborne illness is attributable to leafy green vegetables. The recent outbreaks associated with fresh fruits and vegetables have resulted in an increased interest in the ability to rapidly and cost-effectively perform on-farm validation. Test methods designed for on-farm use must be rapid, easy to use and most importantly low-cost. Currently, there are no pragmatic methods to detect bacteria which can be used in settings with low resources such as a farm. As produce-related foodborne illness continues to be a major concern, there remains a vital need to provide farmers with tools to validate the safety of agricultural water (both rinse and irrigation) which comes in direct contact with food. Testing of this water will soon be required by the Food Safety Modernization Act (FSMA), yet there exists no suitable detection method to be used on-farm. A combination of bacteriophage amplification and a dipstick assay has been designed for the detection of Salmonella spp. in agricultural water. The assay is designed to be low-cost, easy-to-use and deliver quantitative results within three hours. Successful deployment of the test will empower farmers to perform risk-based testing in addition to periodic testing without incurring significant costs. The bacteria within the agricultural water will be initially concentrated. The sample is then infected with bacteriophages (a virus that infects only bacteria) specific to Salmonella spp.. This virus is only able to infect and replicate within viable bacteria thus ensuring that non-viable bacteria are not detected. Following the full 30-40 minute replication cycle, the increase in bacteriophage is quantified with a disposable dipstick. We have been developing dipstick assays for ultra-low detection limits. Our preliminary data suggests that our fluorescent dipstick will have a detection limit ofSalmonellaspp. cells which makes the test ideal for on-farm use and appropriate federal requirements.

1. Phage screening: In this objective, bacteriophages will be screened for several performance characteristics. These factors, such as specificity and replication time, will improve the overall performance of the detection system. The screening will allow the identification of a bacteriophage(s) which will allow peak performance of the detection kit. Simultaneously, we will continue collecting produce rinse water from farms throughout New England. This water will be used as a sample matrix in all objectives. 

2. Sample preparation and separation: Researchers will be investigating the detection of Salmonella spp. from agricultural water. Because agricultural water may contain plant materials, we plan to release internalized or surface-adhered bacteria using enzymatic digestion. The preliminary data has shown this method to liquefy foods within 10-15 minutes. Formerly surface-adhered and/or internalized pathogens are then able to be separated using magnetic particles. 

3. Dipstick assembly and validation: The dipstick development will utilize the established technology which has demonstrated ultra-low detection limits. The complete assay, starting from the sampling of the agricultural samples, will be conducted and compared to current methods using local laboratories. From these experiments we will demonstrate the reliability and performance of the entire detection system.

The research project has resulted in several findings that can convey a significant benefit to produce safety. Some key takeaway points are listed below: • Enzymes significantly improve the efficiency of removing adhered bacteria from produce. By digesting the produce that bacteria are adhered to, the bacteria are more likely to enter into the liquid matrix. Additionally, the digestion of the plant material results in a partial liquefaction (~40%) of the material, which means less material is able to trap the bacterial cells during the subsequent filtration step. • Phages offer an improved method for capturing bacteria. The ability of phages to adhere to the bacterial surface in non-biological conditions results in improved capture efficiency when compared to the current state of the art (antibody-based capture). Phages therefore offer a lower-cost and more reliable magnetic separation than currently available. • Phages allow the determination of viability during detection. Although phages can adhere to non-viable bacteria, they can only infect bacteria with functional replication machinery. Therefore, by using engineered phages for the detection of bacteria, it can be assumed that the bacteria are viable and able to reach log-phase.