Abstract
Escherichia coli O157:H7 (EcO157), an agent of life threatening hemolytic-uremic syndrome, resides in ruminants and is released in feces at numbers as high as 10 million cells/gram. EcO157 could survive in manure for as long as 21 months, but we observed a 90% decrease in cells of an outbreak strain of EcO157 within half a day in wastewater from dairy lagoons. Although chemical, environmental and biological factors may be responsible for this decrease, we observed an 11-fold increase in native protozoa when wastewater was re-inoculated with 2×107 cells of EcO157/mL. These protozoa engulfed the green fluorescent protein labeled EcO157 within 2 hours after inoculation, but expelled vacuoles filled with live EcO157 cells within 3 days into surrounding wastewater, whereas other protozoa retained the EcO157-filled vacuoles for 7 days. EcO157 was not detected by confocal microscopy either inside or outside protozoa after 7 days. Mixed cultures of protozoa enriched from wastewater consumed EcO157 preferentially as compared to native aerobic bacteria, but failed to eliminate them when EcO157 cells declined to 104/mL. We isolated three protozoa from mixed cultures and typed them by 18S sequencing as Vorticella microstoma, Platyophyra sp. and Colpoda aspera. While all three protozoa internalized EcO157, only Platyophyra and Colpoda acted as predators. Similar to mixed cultures, these protozoa failed to eliminate EcO157 from PBS containing no other supplemental nutrients or prey. However, spiking PBS with cereal grass medium as nutrients induced predation of EcO157 by Platyophyra sp. after 3 days or enhanced predation by Colpoda after 5 days. Therefore, attempts to enrich protozoa to decrease EcO157 from dairy lagoons, may correspond to an increase in protozoa similar to Vorticella and possibly facilitate transport of bacterial pathogens to food crops grown in proximity.
Highlights
Produce was linked to 23% of 117,136 foodborne illnesses associated with outbreaks occurred during 1998 and 2007 in the United States [1]
After an initial 1-day lag, Escherichia coli O157:H7 (EcO157) populations declined rapidly with a decrease of 90% populations within 0.5 day for both inoculum levels, and after the second re-inoculation to samples treated with the higher inoculum of EcO157 (Figure 1)
most-probable number (MPN) counts indicated that each protozoan was provided initially with 14 or 11,200 cells of EcO157 for grazing and we anticipated that the higher level would support protozoan growth
Summary
Produce was linked to 23% of 117,136 foodborne illnesses associated with outbreaks occurred during 1998 and 2007 in the United States [1]. Major outbreaks associated with produce occurring in the US and in other parts of the world indicate that contamination has occurred in many cases in the field (pre-harvest), so it is critical to identify sources of pathogens in the environment and interventions for minimizing them [2]. EcO157 was isolated from watersheds, cattle herds, and wild pigs on the central coast of California throughout spring and summer months of 2004–2006 [8]. These results indicate a dynamic incidence that depends on spatial and temporal factors and possibly agricultural practices
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