Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log 10 on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log 10 . The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.Sprout producers in North America, Asia, and Europe have faced numerous Salmonella enterica and Escherichia coli O157:H7 outbreaks since 1995 (2, 17). Several of the outbreaks were traced to sprouts grown from seeds contaminated with low levels of human pathogens (2,15,19,20,27).As a result of the many recent outbreaks traced to sprouts and guidance by a U.S. Food and Drug Administration document (2), many producers now sanitize their sprout seeds with 20,000 ppm of calcium hypochlorite before sprouting and test each crop of sprouts for S. enterica and E. coli O157:H7. Epidemiological evidence suggests that this sanitation protocol may prevent some, but not all, outbreaks (2, 5), and experimental sanitation of naturally contaminated alfalfa seeds did not eliminate S. enterica from the seeds (23). To date, no seed sanitation method has been shown to eliminate S. enterica or E. coli O157:H7 from laboratory-contaminated seeds (4,13,14,19,24,25). In addition, the recommended calcium hypochlorite method does not kill or remove all naturally occurring nonpathogenic bacteria from alfalfa seeds, suggesting that bacteria on seeds may be in locations inaccessible to calcium hypochlorite treatment (7).Sprouts present an unusual food safety predicament compared to other fresh produce because bacteria, including S. enterica and E. coli O157:H7, may multiply by several logs on the sprouting seeds during the first few days of germination (1,22,23). Experiments with radish and alfalfa sprouts grown from laboratory-contaminated seeds have demonstrated that human pathogens may be present between plant cells inside sprouts, where they can resist decontamination treatments (9, 12). Washing sprouts in water only slightly reduces the number of bacteria found on the sprouts, and green sprouts are usuall...

Aim:  To determine the survival of Escherichia coli O157:H7 in dairy wastewater from on‐site holding lagoons equipped with or without circulating aerators. Methods and Results:  Survival was monitored in dairy lagoon microcosms equipped with or without scale‐size circulators. Both laboratory strains of E. coli O157:H7 and an isolate of E. coli H7 from wastewater had poor survival rates and none proliferated in water from waste lagoons with or without circulators. Furthermore, the decline of E. coli O157:H7 was not enhanced in those microcosms equipped with circulators. Strain variation in survival was observed in both circulated and settling waters. The decline rate of E. coli O157:H7 Odwalla strain increased proportionately with the inoculum load. Escherichia coli failed to establish itself in wastewater even after four sequential inoculations simulating continuous faecal input into the lagoon. The native aerobic bacteria survived longer with a decimal reduction time of 21·3 days vs either introduced or native E. coli, which declined rapidly with decimal reduction time of 0·5–9·4 days. Conclusions:  Escherichia coli O157:H7 failed to establish and proliferate in dairy wastewater microcosms equipped with or without circulating aerators. Significance and Impact of the study:  This study furthers our knowledge of pathogen survival in wastewater, and suggests that proper management of wastewater before its use in irrigation is essential to reduce pathogen transfer to crops.

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.

At least 14 separate outbreaks of food poisoning attributed to either Salmonella enterica or Escherichia coli O157:H7 have been traced to sprouts in the past decade. Seeds contaminated with human pathogens caused most of these outbreaks, thus many sprout growers are now treating alfalfa seeds with the sanitizing agent, calcium hypochlorite (Ca[OCl]2), prior to sprouting. The efficacy of alfalfa seed sanitation varies between seed lots and between seeds within each lot. Alfalfa seeds from different seed lots were sorted by type in an effort to determine if certain seed types carry more aerobic bacteria than other seed types. Seeds with a wrinkled type, characteristic of lygus bug damage, had significantly higher levels of culturable aerobic bacteria and were more difficult to sanitize than smooth, healthy seeds. After sanitation, wrinkled alfalfa seeds that had been inoculated with S. enterica ser. Newport carried significantly higher levels of Salmonella Newport than smooth seeds. If S. enterica is present on wrinkled seeds in naturally contaminated seed lots, it may be difficult to chemically sanitize the seed lot. Removal of the wrinkled alfalfa seeds from the seed lots, perhaps by adapting color sorting equipment similar to that used to sort rice grains and other seeds, should reduce the level of aerobic bacteria in seed lots and may result in lower levels of human pathogens on contaminated alfalfa seeds.