Science-based monitoring for produce safety: Comparing indicators and pathogens in water, soil and crops.

Fecal pathogens have been implicated in numerous disease outbreaks, as well as chronic gastrointestinal illness, in people who consume food that has been irrigated with reclaimed wastewater. With growing water scarcity problems affecting many regions, including the Middle East and the United States, the risk of produce contamination is likely to rise to increasing use of reclaimed wastewater for irrigation practices. To date, the most common water monitoring practice for fecal contamination in irrigation waters relies on culturing fecal indicator bacteria such as Escherichia coli. However, numerous studies have recently raised concerns that fecal indicators may not always correlate with levels of disease-causing fecal pathogens in water sources, including treated wastewater effluents. The inability of indicators to accurately predict whether fecal pathogens are present or absent in treated wastewater, as well as on the irrigated crops themselves, suggests that current monitoring guidelines are not adequate for protecting consumers from contaminated produce. Thus, there is a crucial need for improving produce safety monitoring strategies to ensure that the use of reclaimed wastewater in irrigation is a sustainable and safe practice in arid climates. The aim of this proposal is to examine the correlation between fecal indicators and a suite of fecal pathogens that may be found in irrigation wastewater, the irrigated soil and the cultivated plants. Specifically, the project will evaluate if current monitoring techniques that target fecal indicator organisms accurately predict the presence of fecal pathogens (bacteria, protozoa and viruses) on produce using wastewater irrigated tomatoes as our model plant cultivated in plots constructed for the purpose of this project. The specific objectives will 1) evaluate if fecal indicators in water and/or soil can accurately predict degree and type of pathogen contamination in plant tissues; and 2) test for a correlation between fecal indicators and the degree and type of pathogen contamination in irrigation water, soil, and plants. To accomplish this work, we have assembled a synergistic collaborative team with expertise in microbiology, waterborne infectious disease, and plant science. Findings from the study will have immediate application potential to agriculture management and public health policy makers by providing science-based monitoring guidelines for optimal microbial sampling strategies needed to protect consumers from exposure to disease-causing microorganisms on fruits and vegetables.

The overall goal of this research was to evaluate the correlation between fecal indicator organism and the presence of pathogens in TWW, the irrigated soil, and crops. To this end, we identified three objectives to better understand the relationship between levels of fecal indicator bacteria and presence of pathogenic fecal bacteria, protozoa and viruses in reclaimed wastewater, and in the soil and crops that are irrigated with TWW. Specifically, the project evaluated if current monitoring techniques that target fecal indicator organisms accurately predict the presence of fecal pathogens on produce using wastewater irrigated tomatoes as our model plant, cultivated in experimental field test plots. 

Objective 1. Simultaneously sample irrigation water, soil, and crops for two growing cycles at an experimental plot hosting tomato plants irrigated with secondary TWW, tertiary-TWW, and potable water. 

Objective 2. Perform physico-chemical water and soil analyses and standard microbiological testing of water, soil, and plant tissues for fecal indicator bacteria. 

Objective 3. Process water, soil, and plant samples for pathogen detection. Analyze samples for fecal enteropathogenic and opportunistic bacteria, enteric viruses, and fecal protozoa.

1. Microbial contamination on the surface of tomatoes did not appear to be associated with the source of irrigated waters; FIB contamination was not statistically different on tomatoes irrigated with TWW as compared to tomatoes irrigated with potable water. 

2. Indicator bacteria testing did not predict the presence of pathogens in any of the matrices tested. High concentrations of FIB were detected in water and on tomato surfaces from all irrigation treatment schemes, while pathogen contamination on tomato surfaces (Cryptosporidium and Salmonella) was only detected on crops irrigated with TWW. These results suggest that regular monitoring for pathogens should take place to accurately detect presence of harmful microorganisms that could threaten consumer safety. 

3. Water quality can change dramatically between the time of its release from the wastewater treatment plant and the time it is utilized for irrigation. Therefore it is important to perform water quality testing throughout the growing season at the cultivation site. 

4. Methods-based accomplishments: a. An improved method for detection of protozoal pathogens (Giardia and Cryptosporidium spp.) in soil was developed. b. Short enrichment scheme for rapid and sensitive detection of a variety of enteropathogenic bacteria using real-time PCR was evaluated. Bacterial detection was sensitive even with high numbers of heterotrophic bacteria present, as validated for Salmonella and Shigella in TWW. c. Multiplex qPCR assays were validated for a variety of bacterial targets in water, soil and tomato surface washes. 

5. An unexpected result from the study is that bacteria from the water did not appear to transfer to the soil or the crop surface. This feature has been noted in other studies such as with antibiotic-resistant bacteria (Gatica and Cytryn, 2013). The effect of soil conditions (such as soil type and other microorganism populations) on persistence of pathogenic bacteria in the soil should be better elucidated.