Use of Advanced Microbiological Tools to Assess Surface Waters Used for Produce Production in Arizona Coupled with Innovative Outreach and Education

Abstract

Background: The need for new pathogen testing methods and for individualized food and water safety curricula are becoming more evident, based on the number of outbreaks in the last decade. Water quality standards are continuously evolving, from shifts in monitoring regulations to increases in approved testing methods, it can be difficult for water professionals to stay abreast of current standards. This dissertation will highlight several primary issues, and will examine possible resolutions, for current concerns related to food and water safety in Arizona, US. Though Arizona is located in the arid Southwestern US, the state is a primary producer of leafy greens and other fresh produce and as such, it is a strong location for the work described herein. Study Aims: The following research and case studies aim to: (1) Establish the utility of a novel indicator organism, Bacteroides sp. to correlate with pathogen presence in agricultural irrigation water samples collected in Southern Arizona; (2) Explore the use of a cutting-edge molecular technology, droplet digital Polymerase Chain Reaction (ddPCR), for accurate analysis of environmental samples, using laboratory experimentation and a review of current literature; and (3) Develop and deliver an innovative food safety and water quality curriculum based on the Food Safety Modernization Act Produce Safety Rule (PSR), that will meet the unique needs of American Indian farmers in Arizona and New Mexico. Methods: Aim (1) focused on comparing concentrations of the currently regulated indicator organism, Escherichia coli (E. coli) to a novel indicator Bacteroides sp. to ascertain whether the novel indicator could overcome some of the known deficiencies related to generic E. coli. Irrigation water samples (n=98) were collected from Southern Arizona farms and analyzed for both indicators at the University of Arizona in Tucson. A comprehensive dataset was collected using several methods, including Real-Time Polymerase Chain Reaction (RT-qPCR), IDEXX Colilert® enzyme substrate technology, bacterial culturing techniques, and field analyses. Data from RT-qPCR (Bacteroides sp.) was compared to Colilert® results (generic E. coli) to evaluate any relationship between the two data sets, while both methods were also compared to bacterial molecular results for pathogen (E. coli O157:H7 and STEC and Salmonella) presence. Aim (2) examined the innovative technology ddPCR using recent published studies across multiple disciplines to evaluate its effectiveness for use in environmental samples on eDNA. Ultimately, a set of considerations were developed for analysis of environmental DNA samples. Lastly, Aim (3) developed a food safety and water quality curriculum based on American Indian cultural narratives using Universal Learning Design methodologies. The curriculum was successfully utilized in three trainings across Arizona and New Mexico for American Indian communities. Qualitative analysis of participant feedback was used to determine the effectiveness of the trainings and key learnings. Results: Aim (1) data suggests that Bacteroides sp., performed equally to generic E. coli in assessing water quality, but not to the extent of proposing Bacteroides sp., as a new indicator. The data did show that generic E. coli is an unsuitable fecal indicator organism, due to an 80.6% false negative rate; furthermore, neither indicator, generic E. coli or Bacteroides sp., correlated with pathogen presence. Thus, the search for other bacteria species for accurate water quality analysis continues. Aim (2) identified areas of environmental research in which ddPCR may be useful, such as marine research. However, the larger dynamic scale of RT-qPCR compared to ddPCR indicates that RT-qPCR would be the preferred method for accurate and reproducible results. Additionally, the cost of ddPCR is greater than RT-qPCR. Lastly, the classes conducted to realize Aim (3) were deemed successful by the surveys conducted and the positive feedback provided by participants. Discussion: The multi-project approach addresses three unique research and extension efforts under the main overarching question: How can water quality evaluation strategies and education in Arizona be improved to aid in the protection of public health? All three aims identified gaps in assessment of food safety and water quality in Arizona. The outstanding false negative rate found using approved methodology for E. coli found in Aim (1) suggests that the current water quality monitoring method is not accurate and this not protective of public health. This finding is supported by recent foodborne outbreaks in the US that may be related to agricultural irrigation water quality. Aim (2) showed that, although ddPCR does provide absolute quantification, the expense of the equipment and reagents as well as the confounding of data due to the complex nature of environmental samples are problematic. Thus, using RT-qPCR in lieu of ddPCR will provide similar, or even improved, results for a fraction of the cost. Aim (3) confirmed, through the development of culturally appropriate materials and deployment of trainings to AI participants, that inclusion of tribal producers though all steps is critical to the development of food safety guidelines that will be useful to all sectors of agricultural producers. By not including tribal produces into the conversation, critical perspectives about agriculture and culture are lost. While this work only touches the surface of the inclusion of tribal growers to the development of governmental regulations, it brings attention to the unique perspectives and traditions that indigenous cultures bring to the conversation.