The goal of this project is to evaluate the observed Salmonella risk to fresh produce safety in multiple surface water types. Specifically, I propose to assess Salmonella serovar viability in surface water over time, and investigate the degree of Salmonella transferability from surface water to fresh produce crops in growth chamber trials. Additionally, I plan to evaluate whether Salmonella adaptation to water environments increase the risk of water-crop transfer. Finally, in collaboration with the MDA I plan to enhance grower education by incorporating a seminar on irrigation water in Food Safety Trainings, sharing research findings and discussing the importance of balancing food safety practices with environmental stewardship. The goal of this project is to evaluate the observed Salmonella risk to fresh produce safety in multiple surface water types. Specifically, I propose to assess Salmonella serovar viability in surface water over time, and investigate the degree of Salmonella transferability from surface water to fresh produce crops in growth chamber trials. Additionally, I plan to evaluate whether Salmonella adaptation to water environments increase the risk of water-crop transfer. Finally, in collaboration with the MDA I plan to enhance grower education by incorporating a seminar on irrigation water in Food Safety Trainings, sharing research findings and discussing the importance of balancing food safety practices with environmental stewardship.
The objectives of this study are:
1) SURVIVAL: Measure the growth dynamics of multiple serovars of Salmonella in various types of surface irrigation water sources in Maryland. Fate of multiple Salmonella serovars in water samples representative of pond, non-tidal, and tidal river waters will determine whether viability in water is significantly influenced by water type or serovar. Salmonella will be quantified by both dilution plating and quantitative-PCR (q-PCR) using a standard curve that will enumerate not only culturable, but also non-culturable persistor phenotypes. Assessment of biofilm formation in these water microcosms will determine Salmonella serovar-dependent risk of biofilm formation in water distribution systems used in irrigation.
2) TRANSFERABILITY and ADAPTABILITY: Determine transferability potential of two Salmonella serovars from water to crops, and assess pre-adaptation to the plant-niche. I will assess the ability of Salmonella to establish on tomato via irrigation water contaminated with varying concentrations of this pathogen. This will investigate the relationship between Salmonella hazard in water and observed risk on plants. Biofilm formation, surface attachment, and stress response genes may be important for successful transfer of Salmonella onto plants. Gene expression analysis of these will evaluate Salmonella pre-adaptation to plant surface colonization.
3) FOOD SAFETY EDUCATION to MARYLAND GROWERS: Enhance food safety education for growers on water quality, testing requirements, and new research findings. Specifically, I will design a factsheet for online dissemination, and give seminars as part of Food Safety Trainings conducted by the MDA in collaboration with UMD. I will teach growers functional knowledge about water quality and testing, and employ scenario based case studies targeting best practices for addressing on-farm food safety risk factors. These seminars will also provide a platform to highlight the importance of implementing food safety measures while considering environmental sustainability and conservation.
Salmonella enterica has historically been a food safety concern for agriculture on the eastern shore of Maryland, with multiple research teams identifying surface waters as a possible reservoir of this organism. To combat this hazard, the Food safety Modernization act has compiled stringent guidelines for water quality to be used for growing fresh fruits and vegetables. This includes developing a water quality profile based on generic Escherichia coli levels for all irrigation sources used on produce covered by the regulation, as well as employing mitigation steps for water sources that are deemed below quality. Mitigation steps can include a wait period, employing drip tape irrigation, installing treatments in the drip line, or switching to groundwater for irrigation. While these interventions may address food safety concerns, all aforementioned actions require added cost, labor or time, and can detract from sustainable farming. If farmers choose instead to switch to other water sources such as groundwater, they can burden a precious natural resource.
While microbial safety of fresh produce is a priority, the obligation for growers to comply with current E. coli standards for water is imposed without knowledge of the “real” agricultural risk. Salmonella may or may not co-exist or correlate with E. coli levels, and the risk Salmonella poses if present in these waters is also not well understood. Is there a threshold concentration below which Salmonella is unlikely to transfer onto crops or persist in soil? Can Salmonella take up residency in surface water or is there presence more transient? Does Salmonella present in water make it more likely to establish on crops? Are these interactions water type or serotype specific? It has been previously established that culturing media for bacteria can positively influence persistence on plants, such as pre-incubation of E. coli O157:H7 with manure extracts, but impact of environmental media on Salmonella has not been established. Furthermore, preferential plant colonization of some Salmonella serovars relative to others has been documented, but whether water environments impact this remains to be investigated. These data are needed to better understand the practical farming impacts of Salmonella presence in surface irrigation waters. With new understanding, better sustainable strategies for risk management can be developed rather than resorting to expensive and energy-consuming remedial actions or use of “higher quality” water such as groundwater that might not promote good environmental stewardship.
Food Safety trainings conducted by MDA in collaboration with the University of Maryland provide a platform for interactive education on food safety issues and up-to-date research. Water standards and testing are an important component of this training, but it is often difficult to convey and translate research findings to information growers can use, since so many data gaps on irrigation water-based microbial risks remain. In addition to filling these gaps, this project intends to enhance understanding on water-based food safety risks through grower outreach at food safety trainings.
To assess Salmonella population dynamics in various water types, filter-sterilized non-tidal river, tidal river, pond, and reclaimed water samples were inoculated with S. Heidelberg, Javiana, Typhimurium, Newport and serovar 4,5,12:i:- and assessed for viability using both standard plate count and 25µM propidium monoazide (PMA) treated qPCR over 90 days.
A crystal violet attachment assay was conducted to investigate abiotic surface attachment of all serovars incubating in all waters tested for 48 h in a polystyrene 96 well plate.
To assess transfer potential onto tomatoes, strains were inoculated into non-tidal fresh water and allowed to persist for 30 days. At day 1, 5, 10, and 30 an aliquot of inoculated water was concentrated, washed, then inoculated onto cv. ‘Heinz’ Tomatoes. Tomatoes were incubated for 14 h before destructive sampling and standard plating. 30 day incubations and transfers were repeated for another non-tidal fresh water source, reclaimed water, and pond water.
To investigate genetic responses of Salmonella negotiating transition from water to tomato, a targeted RT-qPCR assay was employed. S. Newport and S. Heidelberg were incubated for 30 days in non-tidal fresh water, and assay controls were prepared as previously described. At the time of inoculation, aliquots of inoculum were fixed with RNAlater. The remaining aliquots were inoculated onto cv. ‘Red Robin’ tomatoes. Bacteria were collected after incuation for RNA extraction. Samples are being assayed for multiple genes indicative of response to environmental stresses.
S. enterica differentially persisted in water with respect to water type and serovar. Comparing viability assessment methods for Salmonella residing in water for 60 days, non-tidal fresh water samples displayed significantly different rates of decline between qpcr and standard plating methods. Regarding the culturable fraction, reclaimed water had the least decline and non-tidal fresh water had the most decline. Attachment strength varied significantly by water type; pond water supported the strongest attachment across serovars. S. Heidelberg exhibited the weakest attachment in all water types. Both S. Javiana and Heidelberg exhibited significantly higher transferability from all water types to tomatoes than from tryptic soy broth, but S. Javiana better colonized tomato fruit than S. Heidelberg. Pond water supported the least amount of transfer compared to other water across all serovars tested. Interestingly, incubation time in water significantly influenced transfer in only one type of non-tidal fresh water. Finally, S. Heidelberg exhibited the poorest transfer from water to crops regardless of water type or incubation time in water.