Produce Safety and Microbiology Research Unit, United States Department of Agriculture, Agriculture Research Service, Western Regional Research Center, Albany, California, United States of America.
PLoS One. 2010 Dec 20;5(12):e15671. doi: 10.1371/journal.pone.0015671.
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 2×10(7) 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 10(4)/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.
产志贺毒素大肠杆菌 O157：H7（EcO157）是一种可引起严重溶血性尿毒综合征的病原体，存在于反刍动物中，其粪便中的数量高达 1000 万个细胞/克。EcO157 可以在粪便中存活长达 21 个月，但我们观察到，在奶牛场泻湖废水的一半时间内，一种暴发菌株 EcO157 的细胞数量减少了 90%。尽管化学、环境和生物因素可能是导致这种减少的原因，但我们观察到，当废水重新接种 2×10(7)个 EcO157/mL 细胞时，原生原生动物增加了 11 倍。这些原生动物在接种后 2 小时内吞噬了标记有绿色荧光蛋白的 EcO157，但在 3 天内将充满活 EcO157 细胞的空泡排放到周围废水中，而其他原生动物则将充满 EcO157 的空泡保留了 7 天。7 天后，通过共聚焦显微镜既没有在原生动物内也没有在原生动物外检测到 EcO157。从废水富集的原生动物混合培养物优先消耗 EcO157，而当 EcO157 细胞数量下降到 10(4)/mL 时，无法将其消除。我们从混合培养物中分离出三种原生动物，并通过 18S 测序将其分型为 Vorticella microstoma、Platyophyra sp.和 Colpoda aspera。虽然这三种原生动物都能内化 EcO157，但只有 Platyophyra 和 Colpoda 表现出捕食行为。与混合培养物一样，这些原生动物也无法从不含其他补充营养物或猎物的 PBS 中消除 EcO157。然而，向 PBS 中添加谷物草培养基作为营养物质，可在 3 天后诱导 Platyophyra sp.捕食 EcO157，或在 5 天后增强 Colpoda 的捕食作用。因此，试图从奶牛场泻湖中富集原生动物以减少 EcO157 的方法可能会导致类似于 Vorticella 的原生动物数量增加，并可能促进食源性细菌病原体向附近种植的农作物的传播。
