Fly reservoirs of E. Coli O157:H7 and their role in contamination of leafy greens.

Filth flies (house flies, blow flies) have long been associated with the transmission of human disease pathogens, but they have never been implicated to transmit these pathogens to pre-harvest plants. After the 2006 E. coli O157:H7 outbreak in spinach, our group was funded by Fresh Express to examine movement of this pathogen to leafy greens by insects. To our surprise, we found large numbers of filth flies in some commercial lettuce fields and a small number of them were tested and found to harbor E. coli O157:H7. This begs the question: what proportion of flies found in or near leafy greens production areas are carrying E. coli O157:H7 and are they capable of contaminating pre-harvest leafy greens? To answer this, we propose to test a large sample of flies collected from E. coli O157:H7 risk areas (cattle feedlots and rangeland) and also from leafy green production areas to determine if any proportion of the insects are carrying the pathogen. We will do the feedlot sampling in two areas, one in California and the other in Oklahoma. Leafy greens in Monterey and San Benito counties will be sampled with the collaboration of three industry leaders, Fresh Express, Inc., Dole Fresh Vegetables and Pinnacle Organics. Finally, we will also determine if the E. coli bacterium can be transmitted to plants through fly feces. Together, the objectives outlined in our project will help to determine if flies are a route for E. coli O157:H7 (and possibly other human pathogens) contamination of pre-harvest greens and will generate information that will be useful to growers and distributers in making risk-assessment and food safety decisions.

Filth flies are well-documented mechanical vectors of human pathogens in hospitals, food areas, and confined animal production areas. Filth flies develop in animal waste, decomposing plant matter, and feed containers, all of which can be contaminated with multiples strains of bacteria, including Escherichia coli O157:H7 and other enteric species. Fly transmission of human pathogens to unprocessed, fresh produce is less certain. Recent evidence documented the presence of E. coli O157:H7 in feral flies captured in leafy green production areas and transmission of E. coli O157:H7 to spinach under experimental conditions, but the prevalence of this pathogen in the feral fly population and propensity of flies to transmit to leafy greens is unknown. We propose to examine flies captured in several locations on or near E. coli O157:H7 reservoirs and leafy green production areas in California and Oklahoma to determine if a significant number of flies harbor the O157:H7 pathogen. Furthermore, we will test whether fly excrement can serve as a source of E. coli O157:H7 colonization of leafy greens phyllosphere. The objectives of this proposal are: 1. Capture emerging adult filth flies from manure pats deposited by cattle maintained in feedlots in California and Oklahoma and test them for E. coli O157:H7, 2. Test flies captured in Salinas Valley leafy green production areas located within a 10 mile radius of a confined cattle operation or rangeland for presence of E. coli O157:H7, and 3. Determine if E. coli O157:H7 colonization of the leaf surface occurs after fly defecation. This project addresses Research Priority # 4: Human pathogen reservoirs and vectors (other animal risk factors). We anticipate that the data collected will further define whether or not flies are a significant risk to pre-harvest contamination of leafy greens. This project will support our ongoing research collaboration with our colleagues at the University of California, Riverside.

Objective 1: Test flies in California and Oklahoma feedlots/rangeland for E. coli O157:H7 to determine: What proportion of tested flies are E. coli O157:H7 positive; what time of year are E. coli positive flies more prevalent; what proportion of water, manure, and feed samples are E. coli positive; and do feral flies transmit E. coli O157:H7 to plants. 

Objective 2: Test flies captured in leafy green production areas for E. coli O157:H7 to determine: What proportion of flies captured in leafy green fields are E. coli O157:H7 positive; what times of year are E. coli O157:H7 positive flies more prevalent. 

Objective 3: Examine flyspecks on spinach for evidence of bacterial colonization over time to determine: Do excreted bacteria form biofilms on the leaf surface; and how long to excreted bacteria remain viable on the leaf surface.

A very low proportion of flies in the Salinas area tested for E. coli O157:H7 using a standard microbiological approach were found to be positive. Only four positive flies were found in/near leafy greens in the Salinas area. We believe that our findings underestimate the number of filth flies carrying E. coli O157:H7 and perhaps other STECs and EHECs. A much higher proportion of flies captured in southern California and tested for the presence of E. coli O157:H7 specific genes by PCR were positive. The greater sensitivity and accuracy of PCR-based detection makes this diagnostic preferable to culture-based detection for the presence of bacterial DNA, but our test did not distinguish between live and dead bacteria. The primary question still remains: do flies transmit viable E. coli cells? Are these cells capable of colonizing the leaf surface of leafy greens? The data from examination of fly regurgitation spots would suggest that this is possible under optimal conditions. Bacteria acquired from inoculated manure survive the ingestion and regurgitation process. These data are preliminary, however, and must be confirmed with additional replications. Although the funding period for this project has ended, this project is not yet completed. With funds from other sources, we intend to return to the Salinas area and test more flies as they become available, specifically to complete Objective 1 goals. We are working to build connections with animal production managers so that we can obtain the samples needed to answer critical questions about E. coli sources and fly transmission. Also, the graduate student working on Objective 3 is not yet finished with her project. We anticipate that she will complete her research and publish the findings later in 2010. From the data collected, we made a five presentations at regional and national meetings and expect to publish two papers in professional journals.