The Role of Biofilms as a Reservoir for Foodborne Pathogens in Irrigation Systems

Non-Technical Summary: <BR>Shiga-toxin producing Escherichia coli (STEC) and Salmonella species are responsible for approximately 61% of all produce-associated illnesses. California ranks first in the nation for the production of leafy greens and Arizona ranks third in the nation in the production of fresh market vegetables, all of which are grown by irrigated agriculture. Several multi-state outbreaks have been linked to contaminated irrigation waters; nevertheless, pathogens (disease causing microorganisms) are usually only found sporadically in water and thus it is often difficult to determine the source of contamination. One area that has been overlooked is the possible contribution of biofilms within irrigation infrastructure as reservoirs for these foodborne pathogens. Biofilms are thin layers of microorganisms that form at surface-water interfaces in aquatic environments in both natural and engineered systems. They form on pipe surfaces, rocks, aquatic plants, filters, shower and spray devices, and at almost any other water-surface interface. They are characterized by the presence of a visible oslimyo layer. Biofilms aid the persistence and survival of microorganisms by a number of mechanisms including an increased ability to survive under low nutrient conditions and enhanced resistance to disinfectants and other adverse environmental conditions. Under the proposed project, we will collect biofilm samples from irrigation water canals, pumps, sprinkler heads, gaskets, pipe sections, and siphon hoses from systems in four irrigation districts in Yuma, AZ and surrounding areas during the entire growing and harvesting season. These samples will be evaluated for the presence and levels of STEC and Salmonella. We will also assess several treatment/disinfection methods for their effectiveness at removing biofilms in irrigation pipes. The results from this research will then be used to create a mathematical model to estimate the magnitude of the risk and the probability of illness caused by exposure to these pathogens based on their occurrence in biofilms. In addition to these efforts, a comprehensive outreach and educational program will be developed to regularly disseminate the information from this work to field workers, growers, producers, and processors in the fresh produce industry. The outreach programs will provide information regarding specific measures to prevent or eliminate biofilms to diverse clientele within the commercial produce industry. All information from the project results will be incorporated into an Irrigation Best Management Practices (BMPs) manual for producers. In addition, we will provide education for field managers and workers related to basic microbiology concepts to engage them in understanding the irrigation and water management choices available and to motivate them into becoming emissaries for active risk management. <P> Approach: <BR> Irrigation systems in Yuma, AZ will be evaluated during growing and harvesting seasons. Equipment will be swabbed with dampened sterile sponges or cotton swabs and scraped with a sterile scalpel blade. Biofilm and water samples will be collected from canals and pumps using sterile sponges/scalpel blades. Each canal will be sampled using a sponge/spatula just below the water surface and 1 liter of water will be collected. All samples will be centrifuged and the cell pellets then dispersed in buffered peptone water which will then be diluted (10-fold) in triplicate and enriched by incubating for 12 h at 37C. After incubation, each sample will be diluted 10-fold in Tetrathionate broth for Salmonella or EC broth for STEC assessment and incubated for 24 h at 37C. Samples from the Tetrathionate and the EC will be used to inoculate XLD agar and sorbitol-MacConkey (SMAC) agar plates, respectively, and incubated for 24h at 37C or 42.5C. Sorbitol-positive and sorbitol-negative colonies on the SMAC plates will be tested for indole production. Putative Salmonella and E. coli will be quantified using a standardized 3-sample Most Probable Number (MPN) table. Salmonella will be confirmed by agglutination with polyvalent antisera to somatic and flagellar antigens and by PCR using primers to the invA gene. Presumptive E. coli O157:H7 will be confirmed using O157 and H7 latex agglutination assays. Other E. coli isolates will be tested for shiga toxin genes by multiplex polymerase chain reaction (PCR). Salmonella isolates will be sent to APHIS for serotyping. STEC isolates will be sent to the E. coli Reference Center for serotyping. Pipes with biofilms will be cut into 6 sections each measuring 5 feet in length and various interventions will be assessed: 1) control with no intervention, 2) pipe flushed with irrigation water, 3) pipe allowed to dry in the field for 14 days and then flushed with irrigation water, 4) pipe flushed with chlorine, and 5) pipe flushed with chloramines. The pipes will be sampled and total bacteria (no enrichment step) as well as STEC and Salmonella will be enumerated as before. We will develop exposure models using QMRA to estimate risks from irrigation water contamination from biofilms in irrigation infrastructure. Information on the occurrence and survival of pathogens on produce and the level of contamination caused by different types of irrigation will be utilized. This will provide quantitative guidance on acceptable exposures relative to acceptable levels of risk. Interventions will be evaluated to determine their efficacy in meeting the acceptable risk level. We will conduct outreach and extension activities that will bring researchers and stakeholders together to discuss relevant issues and to convey the results from our research via workshops, field days, a bi-monthly informational text/email, and the development of a best management practices (BMP) manual. We will also develop and implement an educational program to provide information related to general microbiology, food safety, and the potential role of biofilms in foodborne illness to producers and processors of fresh produce.