Assessment of E. coli as an indicator of microbial quality of irrigation waters use for produce.

The goals of this project are to evaluate currently available detection methods for the accurate assessment of Escherichia coli contamination in irrigation waters and provide guidance for interpretation of results through a revised risk based E.coli standard. Currently, there is concern that the false positive rate of E.coli detection may be high in these waters giving false indications of the level of risk from enteric pathogens. This may result in unnecessary costly interventions as well as inaccurate perception of risk among consumers. We propose evaluating three different commercial systems for E.coli detection in irrigation waters and assessing false positive rates by use of molecular technologies. As a secondary objective to evaluating E.coli as a reliable indicator, we propose using a Quantitative Microbial Risk Assessment (QMRA) to assess the use of E.coli as an accurate indicator of food safety risk using data collected in the first stage of this project coupled with existing information found in the scientific literature. Ultimately this work will offer recommendations towards the most reliable methods to be used by the produce industry to assess irrigation water contamination as well as a scientific risk-based E.coli guideline that growers can use to protect public health.

The goals of this project are to assess currently used methods for the detection of Escherichia coli in irrigation waters used in Arizona and Southern California, and to provide guidelines for a revised E. coli standard for irrigation waters used for produce. Currently, there is concern that the false positive rate of E.coli detection may be high in these waters giving false indications of the level of risk from enteric pathogens. This may result in unnecessary costly interventions (e.g. disinfection of the water, attempts to limit wildlife access, etc) as well as inaccurate perception of risk among consumers. For this reason it is essential to determine the rate of false positive detection of E.coli in waters used for produce irrigation. E. coli detection methods were originally developed for assessment of treated drinking water quality and not surface/irrigation waters. Recent research by our group and others has indicated that high temperatures and elevated salinity may result in false positives rates as high as 40% in Arizona and similar climates. The first objective of this project will be accomplished by evaluating three commercially available methods for E. coli detection to test irrigation waters from three agricultural areas (Yuma and Maricopa, AZ and Imperial Valley, CA) and assessing false positive rates utilizing Polymerase Chain Reaction (PCR) and sequencing of the bacterial isolates. This unique study will thus determine the usefulness of current detection methods for the accurate assessment of E.coli contamination in irrigation waters and provide guidance for interpretation of results. The assessment and confirmation work will, however, have little value without applying these data to the currently proposed E. coli guidelines used by the produce industry. Currently, no microbial indicator standards exist for irrigation waters used for produce production in the United States. It has been suggested by the produce industry that the bathing water standard guideline (126 E.coli/100 ml) established by the United States Environmental Protection Agency (USEPA) be used. This guideline was developed from epidemiological studies of bather exposure in recreational waters and has no direct relationship to risk associated with infection or illness rates that might result from produce irrigation waters. Therefore, as a secondary objective to evaluating E. coli as a reliable indicator, we propose using a Quantitative Microbial Risk Assessment (QMRA) to assess the risk from consumption of leafy greens following irrigation with waters containing various levels of E.coli. The QMRA will consider method of irrigation, irrigation timing from harvest time, and other environmental factors that may influence indicator organism or pathogen. This will be done using water quality data collected in the first stage of this project coupled with existing information found in the scientific literature. This effort will result in a suggested E.coli guideline for irrigation waters which reflects irrigation and harvesting practices and is based on human health risk.

Phase 1: 
1. Determine the best method (most reliable, ease of use, low false positive rate) for E. coli detection in irrigation waters based on the comparison of three methods currently available for the detection of E. coli in irrigation waters. 
2. Determine influence of temperature and salinity (and other environmental factors) on false positive rates of these three methods for accurate E. coli detection in irrigation waters. 

Phase 2: 
1. Develop an exposure scenario (model) for E. coli in irrigation waters taking into consideration the type of irrigation method, the irrigated crop, the transfer rate of E. coli to the crop, and the E. coli survival post-irrigation. 
2. Estimate the risk of illness from ingestion of various levels of E. coli from the proposed irrigation scenarios. 
3. Develop a simple, user friendly guideline (program or graph) for estimating risk of infection from the different irrigation scenarios (e.g., different levels of E. coli deposited, different crops irrigated). These guidelines will be compared to risks associated with the current guideline of 126 CFU/100 mL.

• Results reveal E. coli in irrigation waters in all agricultural areas sampled, including exceedances of the LGMA guideline of 126 E.coli per 100 mL. All three methods have identified E.coli in irrigation waters, but methods including MI agar, and IDEXX Colilert Quanti-Tray®, have shown the most straightforward results for interpretation, while blue colonies on m-ColiBlue24® broth plates are typically not well defined, making it difficult to differentiate between a single colony or multiple colonies, which could over- or underestimate the E. coli in the sample. 

• Our study indicates that there are significant differences between E. coli counts measured using m-ColiBlue24® and those measured using IDEXX Colilert Quanti-Tray®; and between those measured using IDEXX Colilert Quanti-Tray® and MI methods. However, there are no significant differences between E. coli counts measured using MI Agar and IDEXX Colilert Quanti-Tray®. 

• The IDEXX Colilert Quanti-Tray® performed with the highest rate of accuracy with 49% of the time calling a true positive followed by MI Agar and mColiBlue24® broth at 33% and 29% respectively. 

• Each of the three methods seemed to have elevated False Positive rates indicating the difficultly in accurately assessing E. coli concentrations. This could be due heavily to analyst interpretation and points towards the need in methods to be straight forward and user friendly. False positive rates ranged from 53% to 71% with m-ColiBlue24® broth performing the worst. 

• According to the QMRA, if irrigation water has E. coli density of 126 per 100 ml (or 12.6 E. coli per 10 ml), and based on Stine et al. (2005), 0.00011 of the 126 E. coli per 100 ml (or 12.6 E. coli per 10 ml) will be transferred to lettuce for furrow irrigation system and 8.8 x 10-7 of the 126 will be transferred to lettuce for subsurface drip irrigation system. That corresponds to a risk of GI illness of 1.1 in 100,000 for furrows and 9 in 100,000,000 for subsurface irrigation system. 

• For sprinkler irrigation system and based on Stine et al. (2011), 0.011 of the 126 E. coli per 100 ml (or 12.6 E. coli per 10 ml) will be transferred to lettuce resulting in a risk of GI illness of 1.1 in 1,000. 

• Irrigation water containing 126 E. coli per 100 ml for lettuce would appear to present a minimal risk for furrow and subsurface drip. However, further research on contamination of lettuce by spray irrigation appears warranted to reduce uncertainty in the risk estimate.