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<p>The standards used by the produce industry to detect fecal contamination (by indirectly testing for indicator organisms) in irrigation waters are based on tests developed for drinking waters (rather than surface waters) and include risk threshold levels established by the Environmental Protection Agency for recreational (bathing) waters. There is little scientific evidence that this threshold is meaningful for waters used to irrigate food crops. Thus, it may not be appropriate for determining if there is a risk of contamination of crops irrigated with waters tested in this manner. We will use “next generation” sequencing to determine the identities and relative concentrations of the entire prokaryotic (bacterial) and eukaryotic (e.g., protozoan) communities in water samples to identify microbial species which are present in the samples when the presence of foodborne pathogens has been confirmed. This work will evaluate the accuracy of this threshold and hopefully result in the identification of new species or shifts in the microbial communities which are more reliable indicators of the presence of contamination of fecal irrigation waters and the presence of foodborne pathogens. This will allow the produce industry to make risk-based assessments of water quality to determine when it is safe to irrigate fields.</p>
<p>Technical Abstract: The methods used by the produce industry to detect fecal indicator organisms in irrigation waters utilize techniques developed for drinking water and may not be appropriate for determining the risk of contamination of crops. The EPA’s recreational bathing water standard of 126 E. coli/100 ml has been used as a risk threshold for irrigation waters, for which there is no scientific or statistically relevant basis; there is evidence that standard E. coli tests are inaccurate (high false + results) when used with irrigation waters. Next generation DNA sequencing can be used as an inexpensive and powerful method to identify the composition (presence and relative abundance) of the entire prokaryotic (bacterial) and eukaryotic (e.g., protozoan) microbial communities in irrigation waters. Such information can be used to correlate the presence of foodborne pathogens with either shifts in the community makeup (from that of un-impacted waters) or with the presence of specific species (i.e., potential indicator organisms).</p>
<p>We will collect 100 samples in triplicate (total of 300 samples to be evaluated) from ten disparate irrigation water canals in Yuma, AZ over two fresh produce growing seasons (October-March) and two summers (non-growing) seasons. Each sample will be evaluated using Colilert® for the detection of total coliforms and E. coli. In addition, we will perform a series of enrichment/selective procedures to culture and isolate E. coli and Salmonella with confirmation via biochemical testing. The E. coli isolates will be examined using polymerase chain reaction (PCR) assays for the presence of the shiga toxin genes, stx1 and stx2. An additional volume of irrigation water will be concentrated and examined for the presence of pepper mild mottle virus and Aichiviruses (using quantitative PCR), which are known to be present in high numbers in waters with fecal contamination, in addition to the enteroviruses (a group of viral pathogens commonly found in feces).</p>
<p>Samples will be amplified using a PCR assay targeted to the conserved 16S and 18S rRNA genes that are found in all bacteria and protozoa, respectively. The PCR products will be purified and sequenced using the Illumina MiSeq sequencing platform and the species present will be identified by comparison to sequences in published databases. A comparison of the microbial communities between samples that are positive and negative for the presence of pathogens (both culturally and via next generation sequencing) will be conducted and analyzed (using specialized software such as Mothur) to identify groups of organisms or specific species whose presence correlate well (both presence/absence and relative abundance) with the occurrence of foodborne pathogens. The presence of pathogens or shifts in the microbial communities will also be compared to other water quality characteristics and potential impacts (e.g., rainfall, human and animal influences). This work should result in the identification of species which may be used as novel indicators of the presence of pathogens rather than just fecal contamination of irrigation waters and will allow the fresh produce industry to make risk-based assessments of water quality and help to determine when it is safe to irrigate fields.</p>