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Recycled water from municipal and agricultural sources is often used as an alternative to supplement increasingly scarce fresh water sources for irrigation of food crops. Due to limitations in treatment methods, recycled water often contains trace level contaminants, such as antibiotics, metals, and pathogens. During irrigation these chemical and microbial constituents are introduced to cropland, where they can either be degraded, or persist and accumulate in soil. Among the persistent constituents are antibiotic resistant pathogens (ABR pathogens) and antibiotics. <P>Pathogens can acquire antibiotic resistance genes (ABR genes) both in recycled water prior to irrigation and in soil after exposure to antibiotics introduced to soil during irrigation. ABR pathogens can gain rapid and widespread entry to food crops, colonize internally, and become endophytes. Endophytic pathogens, such as ABR Salmonella, cannot be eliminated during washing and pose a food safety concern of consuming vegetables that are eaten raw. Currently, little is known about the fate of ABR Salmonella in soil or their accumulation in fresh vegetables. This knowledge is critical in predicting the public health impacts of fresh vegetables irrigated using recycled water.<P> The overall objective of the project is to understand and minimize the accumulation of ABR pathogens in vegetables irrigated using recycled water. The central hypothesis is that accumulation of ABR pathogens occurs in vegetables that are irrigated with recycled water, and the accumulation is positively correlated with the abundance of ABR pathogens in the rhizosphere. <P>Three supporting Research Objectives have been established: 1) determine the fate of ABR Salmonella in soil after irrigation using recycled water, 2) quantify the accumulation of ABR Salmonella in lettuce, and 3) develop best management practices to minimize ABR Salmonella accumulation in lettuce. <P>Completion of Research Objective #1 will reveal the mechanism governing the proliferation of ABR Salmonella in soil after irrigation using recycled water. Given that the majority of the endophytic bacteria originate from the rhizosphere, understanding the fate of the ABR Salmonella in soil is critical in predicting the abundance of the pathogen in vegetables. <P>Research Objective #2 will generate quantitative information on the accumulation of ABR Salmonella in lettuce with respect to abundance as well as resistance level. The quantitative information is critical to evaluate the health risk of consuming fresh vegetables irrigated using recycled water. <P>Research Objective #3 will yield practical solutions that can minimize the accumulation of ABR Salmonella in vegetables. Development of these solutions is critical in promoting the use of recycled water for irrigation, because addressing food safety concerns over the accumulation of pathogens is critical in gaining public acceptance of vegetables irrigated using recycled water.
Non-Technical Summary: Water scarcity occurs to many parts of the US and the rest of the world. As a sector, agriculture consumes ~40% of the total water supplies. Given the increasing scarce water sources, alternative water sources are needed for agriculture irrigation. One of such sources is treated municipal and agriculture wastewaters, as they are abundant and inexpensive. Over the past few decades, successful examples in the US and other countries demonstrate that using recycled water for irrigation is a win-win situation. The use of recycled water saves fresh water that can be used for other purposes. In the meantime, using recycled water for irrigation reduces discharge of wastewater effluents into receiving waters. However, the use of recycled water for irrigation also raises food safety concerns. Even after proper treatment, recycled waters still contain trace level chemical and microbial contaminants. These contaminants could enter the cropland through irrigation using recycled water, and subsequently enter food vegetables. Among the contaminants are pathogens. Once entering vegetables, pathogens are hard to remove, as common washing steps will be ineffective to them. If vegetables contaminated with internalized pathogens, particularly antibiotic resistant (ABR) pathogens, are consumed by human beings, medical attention will be needed. So far, little is known about the internal accumulation of ABR pathogens in vegetables irrigated using recycled water. Therefore, the purposes of this study are to quantify the internal accumulation of ABR pathogens in vegetables irrigated using recycled water, investigate the mechanisms controlling the internalization process, and find solutions to minimize the accumulation. <P> Approach: To achieve Research Objective #1, we establish the following working hypothesis. Irrigation with recycled water introduces both Salmonella and human and/or veterinary antibiotics into soil. In the soil environment, a subpopulation of Salmonella acquires ABR genes due to the exposure to antibiotics that are biologically active and becomes antibiotic resistant. Later, the ABR Salmonella subpopulation accumulates in the rhizosphere around vegetable roots. To test the working hypothesis, we will first quantify ABR Salmonella and antibiotics in recycled water from typical municipal and agricultural sources. Surface water from the Platte River and ground water from the Ogallala aquifer will also be included in the analysis as controls. Antibiotics will be measured using LC/MS/MS, while Salmonella will be quantified using both culture-based and culture-independent methods. Then, recycled water will be applied to cropland using drip irrigation at typical irrigation rates. Subsequently, we will monitor the levels of ABR Salmonella and antibiotics in soil after repeated irrigation. For Research Objective #2, we establish the following working hypothesis. ABR Salmonella can gain entry to vegetable plants through roots, colonize the plants internally, and become endophytes. During endophytic growth, the abundance and resistance level of ABR Salmonella will increase. In the meantime, the growth of vegetables will be negatively affected by these endophytic pathogens. To test the working hypothesis, we will first monitor the abundance of Salmonella and ABR Salmonella in lettuce using a culture-based method designed for endophyte studies. Then, we will take a closer look at the resistance level of the endophytic ABR Salmonella in lettuce. Isolates grown on antibiotic-amended agar plates will be subject to MIC tests. Finally, we will correlate the abundance of endophytic Salmonella with lettuce growth and yield. To achieve Research Objective #3, the following working hypothesis is established. The accumulation of ABR Salmonella in lettuce will be positively correlated with the abundance of ABR Salmonella in the rhizosphere. Therefore, best management practices designed to minimize the abundance of the pathogen in soil can minimize the accumulation of the pathogen in vegetables. To test the working hypothesis, we will irrigate lettuce using waters with various blending ratios of recycled water to clean water. Efforts will be made to correlate the blending ratios with quantitative information on endophytic Salmonella and lettuce growth/yield. In addition, we will test irrigation schedules involving recycled water and clean water alternately. In this experiment, recycled water will be avoided at certain lettuce growth stages that are more susceptible to pathogen internalization than the others.