**3. Leafy greens pre-harvest pathogen contamination: risk factors and management strategies**

#### **3.1 General considerations**

For the leafy greens grown in open fields, during pre-harvest stage, there is a constant and concomitant exposure to factors which favor the produce contamination with pathogens. While manure (i.e., improperly stored raw manure, improperly treated or composted manure) deposited nearby plating fields or without using any protective barriers, agricultural soil (manure amended or non-amended), and irrigation water, are considered main risks for the microbial safety of the leafy greens. Other factors such as the presence of domestic or wild animals, which are usually shedding the enteric pathogens via feces, and field workers are involved in leafy greens pathogen contamination [64, 65]. Proper identification and management of these factors are paramount for reducing the contamination of leafy greens in the pre-harvest stage [16, 66, 67].

*c After the removal of root hairs transferred to soil.*

*Pathogenic* Escherichia coli*: An Overview on Pre-Harvest Factors That Impact the Microbial… DOI: http://dx.doi.org/10.5772/intechopen.101552*

## **3.2 Non-amended agricultural soil: the soil-substrate management**

The non-amended agricultural soil ("soil" herein) represents a habitat for pathogenic E*. coli*, as well as for other microorganisms (pathogenic or not), and it is recognized as a potential environmental factor which could contribute to pre-harvest leafy greens contamination [68, 69]. In soil, the fate of *E. coli* (i.e., survival or die-off rates) depends on a myriad of soil properties, such as: abiotic (physico-chemical composition) and biotic properties (inherent existing microbiota), the growing soil localization (i.e., nearby unprotected animal farming operations, sewage etc.), and soil type (i.e., sandy, clay etc.) [68, 70, 71]. From an experimental standpoint, due to the difficulty to study and predict the effect of a combination of factors of influence, as well as their importance against the pathogenic *E. coli*, focus has usually been placed upon a single factor or soil component [71, 72]. Based on the experimental results, the soil-related factors which can influence the pathogenic *E. coli* survival have been divided into soil's biotic and abiotic characteristics [68, 70, 73]. Soil's biotic profile is very complex and experimental targeted studies indicated a high die-off of *E. coli* O157:H7 rates in soils containing rich microbial communities (i.e., bacteria and fungi), especially those characterized by a high metabolic diversity, and an increased *E. coli* O157:H7 concentration in sterile soils due the absence of competitive and/or predatory interactions [74]. Competition for existing nutrients, the release of secondary metabolites, such bacteriocins [75], by the microbial community, or direct antagonism could determine the fate of *E. coli* O157:H7 in soil [72, 75]. Zhang *et al.* and Majeed *et al.* confirm that the Gram-negative bacteria exhibit a greater antagonism against *E. coli* O157:H7 than the Gram-positive bacteria and are known to out-compete Gram-positive bacteria for nutrients in soil [70, 76]. Soil temperature can affect the activity of microbial communities against *E. coli* O157:H7. At 18°C the decrease of the pathogen was likely caused by enhanced antagonistic activity among soil microorganisms [74]. Also, Vidovic *et al*. confirms that *E. coli* O157:H7 declined more rapidly at 22°C compared to 4°C in autoclaved soil [77].

Since the survivability of *E. coli* O157:H7 is considered a huge risk for contaminating the leafy greens or other fresh produce, the determination of essential nutrients availability including carbon, nitrogen, trace elements, salinity, soil's pH and temperature are paramount prior to planting [78]. Zhang *et al.* found that the soil's pH influences the survival of *E. coli* O157:H7. While low pH soil values could shorten the *E. coli* O157:H7 survival to 6–7 days, in a more neutral pH *E. coli* O157:H7 could survive between 32 and 33 days. In addition, the association of an acidic soil with the richness in organic carbon could result in a prolonged survival of *E. coli* O157:H7. This experimental study indicates the fact that the soil pH influences the adsorption and desorption of soil minerals by the pathogen, nutritional availability of soil components, and heavy metal toxicity [70]. Similarly, Li and Stevens showed that the soil with low pH reduces the risk of contamination regardless the virulence of *E. coli* O157:H7 strains [79]; however, it was noted that the virulent *E. coli* O157:H7 strains survived less than the nonvirulent ones [68]. Cools *et al*. indicated that the soil's content in organic matter can be more influential on the pathogen survival than soil type [80, 81]. In this context, Brennan *et al*. found that clay loam soil has a greater nutrient availability and a fine texture which is favoring the long-term survivability of pathogenic *E. coli* [82]. In addition, the clay soil has more available micropores that favors the nutrient adsorption by the pathogen [83, 84]. For example, Fenlon *et al.* were able to isolate inoculated pathogenic *E. coli* over 4 months from clay and loam soils, and for 8 weeks from sandy soils [85]. For minimizing the long-term persistence of pathogenic *E. coli* in soil before planting, regulators and researchers are proposing several mitigation strategies (**Table 4**).


#### **Table 4.**

*Examples of mitigation strategies recommended to be applied to growing soils and adjacent lands.*

#### **3.3 Manure and manure amended soils**

In the fresh produce pre-planting and pre-harvest stages, amending the soil with organic fertilizers, such as manure, or biosolid fertilizers is a cost-effective alternative to chemical fertilizers, the later posing a great threat to humans and environment due to their potential toxicity. In farming, the use of manure is of paramount importance to enhancing the soil's fertility by primarily increasing its content of nutrients and other organic compounds required for improved production yields and agricultural sustainability. From a practical standpoint, manure is the solid part resulted after the segregation of the solid and liquid portions of the organic residual compounds from different origins (i.e., cattle, poultry, pigs etc.). Since manure has been used as an old, traditional farming practice, the advantages of using manure for soil replenishing with nutrients is well known. The studies performed over the last decades are scientifically validating the additional, multiple benefits of amending soils with manure: improving the soil's microbial diversity along with soils' agricultural properties such as soil density and structure (i.e., loosening up/breaking down the heavy soils), increment of water holding capacity [98], soil erosion, and to maintain the quality of "exhausted" soils due to the repeated use of agricultural lands—by application at the beginning of each growing season [99].

The addition of manure is performed before planting the soil and at different time periods during the fresh produce growth stages but not immediately before the harvesting stage. Manure can be applied as: solid manure (i.e., aged manure, compost, manure slurries, or manure tea). Among the identified pitfalls of soil manuring, the most important aspect is that the manure contains high levels of

*Pathogenic* Escherichia coli*: An Overview on Pre-Harvest Factors That Impact the Microbial… DOI: http://dx.doi.org/10.5772/intechopen.101552*

pathogens which can contaminate the leafy greens due to its ability to harbor and spread both animal- and human-origin pathogens including the *E. coli* O157:H7 in the farming environment. For minimizing to reduce this microbial risk, as a thumb rule, the manure must be generally added into the soil after being processed (aging or composting) and not at a stage near the produce harvesting time.

As a route of produce contamination, internalization of *E. coli* O157:H7 has been found highly prevalent on leafy greens, including lettuce leaves, when the soil has been fertilized with contaminated manure possibly due to the intake of the pathogen up through the leaves via the produce's root system [50]. Ekman *et al.* found that the *E. coli* can survive in manure amended soils and the viable *E. coli* O157:H7 numbers were declining by at least 3 logs after 50 days of manure application to the soil, regardless of the season of application [100]. Maximizing the time between manure application and harvest stage of the leafy greens is one avenue to allow the natural reductions of the target pathogen into the soil. Additionally, in manure amended soils, existing pathogens can colonize the seedlings during germination, or transfer from the manure amended soil to the leafy greens through water splashing (during irrigation or rain) or through soil dust [101, 102]. Islam *et al.* found more than 10 CFU/g *E. coli* O157:H7 on parsley and lettuce even when these produces were harvested after 160 and 70 days, respectively, when soil was amended with manure containing log10 7 CFU/g *E. coli* [49]*.* In an experimental transfer "soil-to-crops" of *E.coli* O157:H7-inoculated manure, Suslow predicted that, once the contaminated manure was incorporated into the soil, a 99% reduction of *E. coli* O157:H7 viable population could take place after 60–120 days depending on soil type but also on other factors yet to be determined [103]. Later, other several other factors responsible for leafy greens contamination with manure pathogens were indicated by Baker and were based on the high variation of farming practices, from site to site: the use of untreated manure; the differences in manure storage methods, type of manure applied treatment including the time of manure piles resting undisturbed; the manure-handling equipment cleaning, sanitation, and segregation practices; lack of protection against wild animals of the manure sitting piles' [104]. The type of manure, aged (dried and compact) or manure slurry, and temperature could also influence the survivability of *E. coli* O157:H7. Under experimental conditions, Himathongkham found that the *E. coli* O157:H7 survival in aged cattle manure was higher at 20°C, while in fresh cattle manure slurry (1-part aged manure and 2-parts water) survivability was higher at 4°C and slightly reduced at 20°C [105]. Jiang *et al.* observed a more rapid decline of *E. coli* O157 in manure-amended unautoclaved soil at 21°C than at 5°C. This was attributed to an increase in microbial activity with temperature and consequently, greater competition for nutrients. These findings are important for elucidating the influence of temperature on *E. coli* O157:H7 survival in different types of manures used for soil fertilization [106].

It is established that the contaminated, untreated, or improperly treated manure has been implicated, worldwide, as a major source of pathogenic *E. coli* O157:H7-related foodborne outbreaks due to the consumption of leafy greens and fresh produce. Therefore, efficient manure management strategies and policies are required to be established and used on-farm. The public health is the ultimate, main objective of the manure management strategies which, for being successful, require multi-pronged approaches. Once adopted, these management strategies and policies should efficiently mitigate the negative impact of manure on the environment and on the leafy greens. Epidemiological and experimental studies conducted by CDC and FDA indicated manure as a major factor in the outbreaks due to *E. coli* O157:H7 and Shiga toxin-producing *E. coli* (STEC) [107–109].

To reduce the target pathogen and minimize the risk of leafy greens contamination via use of manure, FDA established a set of Good Agricultural Practices (GAPs),


#### *\*Adapted from [110].*

*a FDA is conducting additional research, working with other researchers, and working to conduct a formal risk assessment [111].*

*b A scientifically valid controlled physical, chemical, or biological process, or a combination of scientifically valid controlled physical, chemical, and/or biological processes to meet the requirements of microbial standard for E. coli O157:H7.*

*c Relevant national standard or E. coli O157:H7 is not detected using a method that has a detection limit of 0.3 MPN (Most Probable Numbers) per 1 gram or per 1 mL if liquid (i.e., agricultural manure tea) is being sampled as analytical portion.*

#### **Table 5.**

*Application requirements and the minimum application intervals of the manure depending on their treatment status and the potential on-field contact with leafy greens\*.*

as mitigation strategies to reduce the pathogen hazard. These strategies are related to the application and use of animal-origin manure and to the minimum application intervals for leafy greens according to manure treatment types (**Tables 5** and **6**) [110, 111]. In addition, minimizing direct or indirect contact between manure and the leafy greens especially at a stage closer to the harvesting time could be used as a method to reduce the contamination with *E. coli* O157:H7 [111].

Accordingly, there are other treatments on which fresh produce growers can rely on for minimizing the pathogens hazards, such as: allowing enough passage of time in conjunction with the action of other environmental factors (i.e., environmental temperature, moisture fluctuations, and solar ultraviolet irradiation) to ensure the manure is properly aged and decomposed before first application to fields. These type of manure treatments, are known as "*passive treatments*". Its disadvantage is that the treatments are time consuming compared to the "*active treatments*" because they depend on the type and source of manure, and on the climatic factors (regional and/or seasonal). When manure aging is used as a passive treatment U.S. FDA cautioned on not confusing this process with composting process, the latter being solely applied as an active treatment [111]. In addition, produce contamination with pathogens occurs if the manure is not treated before use, or if the untreated manure does not respect the recommended application method during produce growing (**Table 5**) [110].

The accepted manure active treatments consist in the application of a scientifically controlled processes such as: physical (i.e., thermal treatment), chemical (i.e., highly alkaline digestion), biological (i.e., composting), or a combination of those so that *E. coli* O157:H7 levels satisfy the microbial accepted standard levels (**Table 6**).

Similarly, in 2017, the European Union Commission in collaboration with the European Food Safety Agency (EFSA) established Good Agricultural Practices for the application of animal manures and the minimum pre-harvest intervals that should be followed when growers use organic fertilizers for leafy greens based on manure treatment types and manure microbial quality [112].


*\*Source: [146].*

*a If the manure is not treated on-farm then: (i) Growers purchasing manure should obtain a specification sheet from the manure supplier for each shipment of manure containing information about the method of treatment, (ii) Growers should contact state or local manure handling experts for advice specific to their individual operations and regions.*

#### **Table 6.**

*Control measures for minimizing E. coli O157:H7 and other microbial hazards\*.*
