Selection of E. coli surrogates with attachment and survival patterns similar to those of human pathogens associated with produce.

Despite continuing efforts to eliminate pathogen contamination of fresh produce significant outbreaks continue to occur. Therefore, a research priority is to determine the source and duration of pathogen contamination in agricultural settings (i.e., in manures, soils and on plant surfaces). In-house research to determine critical control points and identify good agricultural practices benefits from the use of indicators as surrogates for pathogens due to reduced need for safety equipment and expensive supplies. Therefore, studies are needed to identify characteristics of indicators like Escherichia coli (E. coli) that contribute to their occurrence and survival in association with produce in a manner similar to that of pathogens. In this proposed research, we will compare behavior of E. coli strains with diverse physical, chemical and biological properties to that of pathogens when both are applied to soil in either manure or irrigation water. In this way we will (1) identify survival and binding characteristics of surrogates that make them better indicators of produce associated pathogens and (2) provide science based information on survival of pathogens and indicators in soil and on fresh produce when initially present in either applied manure or irrigation water.

Despite the best efforts of industry and regulatory agencies to identify and implement good agricultural practices (GAPs) to reduce or eliminate pathogen contamination, significant outbreaks associated with fresh produce continue to occur in the U.S. and around the world. While manures and irrigation waters are crucial to crop production, these are also potential sources of manure borne bacteria. Although the majority of these bacteria are beneficial and/or harmless, there remains the potential for contamination of agricultural sites, crops and water sources with pathogens like Salmonella sp., Escherichia coli O157:H7 (E. coli O157:H7) and Listeria monocytogenes (L. monocytogenes). Studies thus far indicate that the three manure-borne pathogens have the ability to survive in secondary habitats (i.e., manures, soils and on produce) for weeks or even months. For these reasons, understanding how these pathogens survive in association with produce has become a research priority in food safety. Monitoring for fecal contamination using indicators and in particular Escherichia coli (E. coli) has a long and well-documented history. Despite this a growing body of literature suggests that current indicator organisms may be poor representatives of pathogen occurrence in any given environment. However, years of precedence, including long term use in regulated monitoring, as well as availability of specific, sensitive, user and budget friendly methods make it difficult to eliminate E. coli as a model indicator of fecal contamination. Therefore studies are needed to identify properties (chemical, physical and/or biological) of indicators that contribute to their occurrence and survival in association with produce in a manner similar to that of pathogens. In previous studies, we isolated over 1,300 E. coli from diverse manure and water sources. We found that these isolates differed significantly in genotypic and phenotypic properties; conferring this organism with versatility that contributes to its survival in environments that range from the gastrointestinal tract to soil and water sources. In this proposed research, we will build on previous studies by using this collection of environmental isolates of E. coli to compare behavior of strains with diverse physical, chemical and biological properties to that of produce associated pathogens. Studies will be conducted with pathogen contaminated manures or irrigation waters and behavior of indicator and pathogen in soils and on fresh leafy greens will be determined. Correlation of E. coli survival with that of produce-associated human pathogens in the same environment will provide insight into (1) traits important to surrogate survival and adherence in a manner similar to that of pathogens and (2) traits important to survival of both in association with produce, soils or irrigation waters. These studies will also provide science based information on survival of pathogens and indicators in soil and on fresh produce when contamination originates in either applied manure or irrigation water.

The goals of the project are to (1) provide science based information on survival of pathogens and indicators in soil and on fresh produce when initially present in either applied manure or irrigation water and (2) identify characteristics of indicators that make them better surrogates of produce associated pathogens. 

The specific objectives include: 

(1) Select E. coli surrogates from 1,346 isolates obtained from poultry, dairy and swine manure or from surface water sources (Cook et al., 2011) that have properties similar to those shown to be important for survival/binding/internalization of human pathogens on fresh produce. 

(2) Determine physical, chemical and biological properties including cell surface (electrophoretic mobility, hydrophobicity, surface charge density, extracellular polymeric substance (EPS) composition), cell size, adherence to soils, carbon utilization profiles and genetic factors (including those associated with adhesion, transport, capsular formation, iron scavenging and cell aggregation) for pathogens and indicators. 

(3) Monitor die-off of pathogens and indicators in soil and on produce following application through contaminated manure or contaminated irrigation water. 

(4) Identify E. coli strains that behave similarly to pathogens by statistical analysis of survival and adherence patterns and by correlating to previously defined physical, chemical and biological characteristics.

• Growth media produced from lettuce lysates produce physiological differences in the manner by which pathogens and surrogates adhere to surfaces. 

• Growth environment is an important consideration for any experiment that will characterize the way a pathogen or surrogate behaves. 

• E. coli isolates from similar environmental sources have a high level of strain diversity. 

• Improved pathogen surrogates can be selected, and the agricultural environment may be the best source for these isolates. 

• Generic lab strain E. coli isolates, such as the FDA strain ATCC 25922, may provide a suitable surrogate for E. coli O157:H7 in lab-scale experiments but is not otherwise recommended. 

• Pathogens and surrogates can persist on lettuce leaf surfaces at low levels and for extended periods of time, emphasizing the need for use of appropriate model organisms in the development of critical control points and best management practices. 

• The environmental strains of E. coli selected for use as surrogates in these studies exhibited survival and attachment properties on lettuce and in soil that were similar to those of the E. coli O157:H7 and Salmonella typhimurium strains. 

• Ultimately, data from these studies show that the agro-ecosystem environment (manure, soil, water) is likely a better source of surrogates for produce-associated human pathogens than are common lab strains.

 • Future research should (1) demonstrate the consistent behavior of these surrogates under relevant environmental conditions, (2) determine the range of conditions in which the surrogates can be used, and (3) evaluate the diversity and behavior of pathogens represented by the selected surrogates.