Human pathogens can contaminate leafy produce in the field by various routes. We hypothesized that interactions between Escherichia coli O157:H7 and spinach are influenced by the route of introduction and the leaf microenvironment. E. coli O157:H7 labeled with green fluorescent protein was dropped onto spinach leaf surfaces, simulating bacteria-laden raindrops or sprinkler irrigation, and survived on the phylloplane for at least 14 days, with increasing titers and areas of colonization over time. The same strains placed into the rhizosphere by soil infiltration remained detectable on very few plants and in low numbers (102 to 106 CFU/g fresh tissue) that decreased over time. Stem puncture inoculations, simulating natural wounding, rarely resulted in colonization or multiplication. Bacteria forced into the leaf interior survived for at least 14 days in intercellular spaces but did not translocate or multiply. Three spinach cultivars with different leaf surface morphologies were compared for colonization by E. coli O157:H7 introduced by leaf drop or soil drench. After 2 weeks, cv. Bordeaux hosted very few bacteria. More bacteria were seen on cv. Space and were dispersed over an area of up to 0.3 mm2. The highest bacterial numbers were observed on cv. Tyee but were dispersed only up to 0.15 mm2, suggesting that cv. Tyee may provide protected niches or more nutrients or may promote stronger bacterial adherence. These findings suggest that the spinach phylloplane is a supportive niche for E. coli O157:H7, but no conclusive evidence was found for natural entry into the plant interior. The results are relevant for interventions aimed at minimizing produce contamination by human pathogens.

The recent outbreak of Escherichia coli O157:H7 infection associated with contaminated spinach led to an investigation of the role of insects, which frequent fields of leafy greens and neighboring rangeland habitats, in produce contamination. Four leafy greens fields adjacent to cattle-occupied rangeland habitats were sampled using sweep nets and sticky traps. Agromyzid flies, anthomyiid flies, and leafhoppers were caught consistently in both rangeland and leafy greens production fields at all sites. An unexpected number of flies (n = 34) in the Muscidae and Calliphoridae families (known as filth flies because of their development in animal feces) were caught in one leafy greens field. A subset of these filth flies were positive (11 of 18 flies) for E. coli O157:H7 by PCR amplification using primers for the E. coli O157:H7-specific eae gene. Under laboratory conditions, house flies were confined on manure or agar medium containing E. coli O157:H7 tagged with green fluorescent protein (GFP) and then tested for their capacity to transfer the microbes to spinach plants. GFP-tagged bacteria were detected on surfaces of 50 to 100% of leaves examined by fluorescence microscopy and in 100% of samples tested by PCR. These results indicate that flies are capable of contaminating leafy greens under experimental conditions and confirm the importance of further investigation of the role of insects in contamination of fresh produce.

Filth flies are known mechanical vectors of pathogenic bacteria in hospital and restaurant settings, but their role as vectors for disseminating microbes to plants has not been demonstrated. Escherichia coli O157:H7 deposition by flies onto spinach was studied using molecular, microbiological, and microscopy techniques. Relative quantitative polymerase chain reaction studies showed that bacteria acquired by flies from contaminated cattle manure and deposited in regurgitation spots on leaves survived and multiplied. Scanning electron microscopy of the regurgitation spots of flies exposed to manure inoculated with E. coli suggested the multiplication of bacteria-like organisms within the spots. This finding implies that the bacteria were active and is consistent with a hypothesis that regurgitation spots serve as a nutrition source allowing E. coli O157:H7 to survive on the spinach phylloplane. E. coli O157:H7 persisted on fly body surfaces up to 13 days after exposure to acquisition sources, suggesting that fly cuticular surfaces are conducive to the growth of this pathogen. These results are consistent with the hypothesis of bioenhanced transmission of human pathogens by house flies and suggest that filth flies may affect the microbial safety of fresh produce.

In this study, we examined the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), larval developmental habitat within the round hay bale feeding sites on cattle pastures, and we identified three zones with distinct characteristics around two types of hay feeders (ring and cone). The parameters monitored in each zone included stable fly emergence, substrate temperature, moisture, pH, thickness of hay-manure layer, and concentration of fecal coliform bacteria (Escherichia coli and Klebsiella oxytoca) as indicators of fecal material. All measurements were conducted during the period of high stable fly prevalence (HSF) in May-June and low stable fly prevalence (LSF) in July-August to better understand the environmental factors influencing stable fly seasonality. Substrate temperature and fecal coliform concentration were the only two significantly different factors between HSF and LSF. Temperatures ranged from 21 to 25 degrees C during HSF versus 25-30 degrees C in LSF but all were within the range for successful stable fly development. Fecal coliform concentrations ranged from 4.2 x 10(3) to 4.1 x 10(4) colony-forming units (CFU)/g of the substrate during HSF and from undetectable (<10) to 100 CFU/g during LSF. Furthermore, we evaluated the effect of different hay:manure ratios (0:1, 1:1, 2:1, and 5:1) on stable fly development (egg to adult). Temperature was significantly higher and stable fly developmental time significantly shorter in all substrates containing hay when compared with that of manure alone, but no significant differences were detected in stable fly emergence among the substrates. These results strongly indicate that the fecal microbial community plays an important role in stable fly larval development in hay feeding sites and that it is the main factor behind stable fly developmental seasonality on pastures. Our results also demonstrate that animal manure mixed with hay provides conditions for faster stable fly development than manure alone; however, hay does not significantly affect overall stable fly emergence.

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