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Title: Survival and persistence of non-pathogenic Escherichia coli and attenuated Escherichia coli O157:H7 in soils amended with animal manure in a greenhouse environment

item WHYTE, CELIA - University Of Maryland Eastern Shore (UMES)
item GRAHAM, LORNA - University Of Maryland Eastern Shore (UMES)
item COTTON, CORRIE - University Of Maryland Eastern Shore (UMES)
item HASHEM, FAWZY - University Of Maryland Eastern Shore (UMES)
item Camp, Mary
item Millner, Patricia
item Sharma, Manan

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/15/2016
Publication Date: 6/1/2016
Publication URL:
Citation: Whyte, C., Graham, L.P., Cotton, C.P., Hashem, F., Camp, M.J., Millner, P.D., Sharma, M. 2016. Survival and persistence of non-pathogenic Escherichia coli and attenuated Escherichia coli O157:H7 in soils amended with animal manure in a greenhouse environment. Journal of Food Protection. 79(6):913-921.

Interpretive Summary: Biological soil amendments (BSA's), including manure from horses, dairy or beef cattle, and poultry litter, provide numerous benefits in conventional and organic agriculture but may also introduce enteric pathogens to the food supply. The benefits of using BSA’s must be balanced against the potential risks associated with use of raw or inadequately treated manure because of the potential transfer of fecal pathogens from soil to growing fruits and vegetables and possible resulting microbial contamination contributing to human illness. Current FDA standards propose either a 90- or 120-day intervals between application of manure to the field and the harvest of crops, with direct contact between the crop and the soil requiring the longer interval, to minimize the risk of contamination of fruits and vegetables consumed raw. Performing bacterial persistence studies with non-pathogenic surrogates in different soils amended with different sources of BSAs in greenhouses could be a practical and economical option that could assist researchers and growers in complying with regulatory requirements when seeking exceptions. This research report comprises three separate, replicated, greenhouse experiments focused on the impact of two different sizes (small and large) of containers (pots) on the survival of non-pathogenic E. coli (gEc) and attenuated E. coli O157:H7 (attO157) in three different soils types: sandy loam, silt loam soil, and clay loam soils; along with four manure amendments, either unamended (UN) or amended with poultry litter (PL), dairy manure liquid (DML), or horse manure (HM), in small (398 cm3) and large (2552.5 cm3) pots. In the small pot study (study 1) and the large pot study (study 2), results show that soil types (SL and CL) did not influence the survival of E. coli in manure-amended soils, but the PL amendment enhanced survival of all E. coli compared to their survival in unamended, dairy manure liquid-amended, and horse manure-amended soils at a statistically significant level. In study 2, all E. coli populations in all manure-amended soils were significantly lower by week 8 than in week 1, indicating that E. coli populations declined over time in large pots. In a comparison between the small and large pot study (study 3), the effect of manure amendments was the same: PL-amended soils supported higher populations of E. coli then HM- or UN-amended soils in both small and large pots, and in most cases both attO257 and gEC populations were significantly lower by week 8 than in week 1. In general, E. coli also survived at higher populations in clay soils than in silt loam soils. At the end of the comparative study (week 8), attO157 populations in small pots were significantly greater than those in large pots during this study, perhaps due to the greater moisture availability in the small pots, which had limited drainage compared to the large pots. The results shows that small pots and large pots can consistently assess the effect of manure-amendments on E. coli populations, but results from small and large pot studies may be dependent on the bacterial strains used in the study. These findings provide a unique contribution to development of guidelines for approaches to container studies conducted in greenhouses to meet FDA regulatory requirements for exceptions to standard practices relative to biological soil amendments and fresh produce safety.

Technical Abstract: Biological soil amendments (BSA's), including dairy cattle, poultry litter, and horse manure, play an important role in agriculture but may contain pathogens that can contaminate raw or ready-to-eat fruit and vegetable crops that are consumed raw. Proposed FDA standards include a 90- or 120-day interval between the application of manure and harvest of crop to minimize the risk of contamination of fruit and vegetable commodities with pathogens. Data on E. coli persistence in soils under greenhouse conditions are lacking. These studies examine the factors affecting bacterial persistence in manure-amended soils in a greenhouse setting as affected by soil type, manure amendment, strain selection, and / or pot size. Study 1 evaluated the survival of a cocktail of non-pathogenic E. coli (gEc, 3 strains) and attenuated E. coli O157:H7 (attO157, 2 strains) inoculated at either a low (4 log CFU/ml) or high (6 log CFU/ml) inoculum in small pots (398 cm3) containing different types of sandy loam (SL) or clay loam (CL) amended with either poultry litter (PL), dairy manure liquids (DML), or unamended (UN), and analyzed over 8 weeks. Populations of gEC and attO157 (2.84 – 2.88 log CFU/g) in PL-amended soils were significantly ( p < 0.05) greater than those in DML-amended (0.29 – 0.32 log CFU/gdw) or UN-amended (0.25 – 0.28 log CFU/gdw). E. coli populations survived similarly in both soil types. In study 2, a high inocula of gEC and attO157 was applied to large (2552.5 cm3) pots containing either CL or silt loam (SIL) soils and amended with PL-, horse-manure (HM-), or UN-. Similar to study 1, the soils-amended with PL supported significantly higher attO157 and gEC populations in both CL and SIL compared to HM- or UN-amended CL and SIL. Populations of gEc and attO157 in all PL- and HM-amended CL and SIL soils at week 8 were significantly lower than populations in week 0. Study 3 compared the results from a small and large pot study receiving a high inoculum conducted simultaneously. Populations of attO157 were significantly greater in small pots (1.83 log CFU/gdw) compared to in large pots (0.65 log CFU/gdw). Overall, PL-amended soils supporting higher populations of gEC and attO157 compared to HM- and UN-amended soils in both small and large pots. However, greenhouse studies using small and large sizes can be useful tools when evaluating persistence of E. coli in manure-amended soils, but small pot sizes may describe specific interactions between E. coli strain, soil and manure-amendment compared to large pot studies.