Percolation and Survival of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Soil Amended with Contaminated Dairy Manure or Slurry

The effect of cattle manure and slurry application on percolation and survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium was investigated for different soil depths after the addition of water. Four treatments were chosen for the first set of experiments: (i) addition of inoculated farmyard manure on the soil surface, (ii) mixing of inoculated farmyard manure with the top 10 cm of soil, (iii) addition of inoculated slurry on the soil surface, and (iv) injection of inoculated slurry into the top 10 cm of the soil. Homogeneity of water distribution in the soil profile was confirmed by a nondestructive nuclear magnetic resonance method. Survival data were fitted to a modified logistic model, and estimated survival times were compared. In the second set of experiments, pathogen-inoculated farmyard manure or slurry was applied to soil columns with 1-month-old lettuce plants. More pathogen cells percolated to greater depths after slurry than after manure application. Survival of E. coli O157:H7 was significantly longer in soil with slurry than in that with manure, while survival of Salmonella serovar Typhimurium was equally high with manure and slurry. The densities of the pathogens were not different in the rhizosphere compared to the bulk soil with manure, while the densities were higher by 0.88 ± 0.11 and 0.71 ± 0.23 log CFU per g (dry weight), respectively, in the rhizosphere than in bulk soil after slurry application. Our results suggest that surface application of manure may decrease the risk of contamination of groundwater and lettuce roots compared to injection of slurry.In the last 10 years food-borne disease outbreaks have increasingly been associated with the consumption of fresh vegetables and fruits contaminated with human pathogenic bacteria (3, 31). A significant number of the outbreaks were attributed to Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Bovine manure and slurry are the main environmental sources of these pathogens, with average concentrations between 10³ and 10⁴ CFU per g (dry weight) (gdw) of manure or slurry (24), but the density can be as high as 10⁷ CFU gdw⁻¹ of manure (10).Utilization of organic manures such as farmyard manure and slurry is the most economic and practical option for improving soil quality while providing as well an additional source of nutrients for growing plants. This is especially true for organic farms, where synthetic fertilizers cannot be used. Both organic and conventional soils can be fertilized with liquid slurry and/or farmyard manure. However, farmyard manure is more frequently used at organic farms.The survival of E. coli O157:H7 and Salmonella serovar Typhimurium is thought to be better in slurry than in farmyard manure (24; also A. V. Semenov, L. van Overbeek, N. Hidayah, A. J. Termorshuizen, and A. H. C. van Bruggen, submitted for publication) but is also dependent on the way manure or slurry is applied to agricultural fields (24). Survival of the pathogens may range from several days (turned composted manure) to more than a year (nonaerated manure) (9, 19). This broad difference in survival times is caused by various abiotic factors such as temperature (30, 38), presence of oxygen (A. V. Semenov, et al., submitted), and chemical composition (5) as well as by biological factors (e.g., microbial community composition) (5, 16, 30). The presence of plant roots is often neglected in controlled experiments although root exudates may support survival of human pathogens by providing a supply of easily available nutrients (18). Moreover, it has been shown that E. coli O157:H7 and Salmonella serovar Typhimurium may become associated with the surface of plants growing in soil amended with contaminated manure (15, 23) and may even be internalized by the plants (7, 18, 20, 32).When microorganisms are introduced on or in soil, their movement is mainly determined by the flow of percolating water (13). Water flow and the ultimate distribution of bacteria in soil are affected by soil texture, pH, temperature, and the structure of the root system in soil (17). Like other bacteria, E. coli O157:H7 and Salmonella serovar Typhimurium are able to move through the soil profile with water after rainfall or irrigation and can even reach the groundwater (2, 21). In field experiments, 20% of E. coli cells applied with contaminated slurry to the field were found in drain water (37). This water can contaminate plants when it is used for irrigation. Since E. coli O157:H7 can survive in well water up to 65 days (1), there is a high risk that private water supplies could be contaminated with enteric pathogens.Laboratory transport studies can mimic bacterial transport in field conditions only to a certain extent. The natural heterogeneity in field soil leads to the appearance of cracks and macropores through which water flow may occur while relatively homogeneous soil is commonly used in laboratory experiments. This may lead to underestimations of the movement of enteropathogens through the homogenized and possibly compacted soil. On the other hand, the presence of artificial boundaries (the so-called “wall effect”) and unexpected cracks may lead to overestimations of the movement of water and bacteria through the soil in mesocosms. The wall effect can be minimized by inserting sandpaper against the inner wall of soil columns while cracks can be minimized by careful packing of the soil. Nuclear magnetic resonance (NMR) can be used to check the homogeneity of water distribution in a soil column. NMR is a nondestructive and noninvasive spectroscopic method to measure static and dynamic water behavior in heterogeneous substrates (35). The data received from magnetic resonance images can give information about the spin density and spin relaxation values that reflect the interaction of water with the soil. These measurements have been proven to be highly correlated with water content in soils (35).While it was shown that water is the most important dispersal factor for percolation of bacteria in different types of soil (13, 36) as well as for percolation of enteropathogens under various management practices (11), the movement and distribution of E. coli O157:H7 and Salmonella serovar Typhimurium in soil after application of manure and slurry are still unclear. It is also not clear if and how survival of enteric pathogens is influenced by the depth of the soil where they end up after transport through the soil.The objectives of our study were the following: (i) to determine the extent of percolation of water and E. coli O157:H7 and Salmonella serovar Typhimurium from contaminated manure or slurry through a soil column, (ii) to determine the influence of application methods of manure and slurry on percolation and survival of these pathogens at different depths in a soil column, and (iii) to determine the influence of plant roots on percolation and survival of the pathogens, applied with manure or slurry, at different depths in bulk soil and the rhizosphere.