**2.3 Internalization**

Experimental studies indicated that there are many mechanisms used by *E. coli* O157:H7 to contaminate and internalize both the leafy greens root and leaves tissues [40–42]. From the roots, the pathogen can pass to the leaves by using the produce's vascular system or can penetrate the produce internal tissues using the existing wounds or other natural "openings" of the leaf system [42–44]. While due to the difficulty to study the pathogen internalization in the natural growing plant environment, internalization has been extensively studied in systems that mimic the natural environment, many factors which can promote produce contamination are yet to be clarified. Although the leafy greens possess physical and chemical defense mechanisms to restrict the internalization of pathogens under certain circumstances the produce defense can be disrupted either by biological or mechanical means and *E. coli* O157:H7 access to produce inner tissues is favored [45–47]. Once the pathogen penetrates the produce inner tissues it can potentially evade the produce defense systems by adapting to the plant environment and becomes internalized [48].

Generally, it has been accepted that the internalization of pathogens depends on several factors, such as: (a) plant type, age, and exposure time to pathogen; (b) produce growing system (i.e., soil, hydroponic, aquaponic), (c) the level of contamination of produce with the pathogen, (d) the type and the degree of roots or leaves injury, (e) length of time given to the pathogen to spread from injured roots to the mature leaves etc. (**Table 2**) [40].

For soil-grown plants, internalization was observed as a sporadic phenomenon and with low incidence. Usually, the contaminated soil, could have a little to no influence on the noted internalization, soil presenting a relatively low risk of internalization as compared to other produce growing systems (i.e., hydroponic or aquaponic systems). Generally, the soil-grown produces are protected by environmental stressful conditions which are not favoring the pathogen internalization [40, 49]. The pathogen internalization in soil-grown leafy greens remains controversial: while several studies on leafy greens (lettuce or spinach) grown on contaminated soil have shown that internalization of *E. coli* O157:H7 could occur [50, 51] other researchers found little to no pathogen internalization in soil-grown produce [52]. When pathogen internalization in leafy greens grown in soil was observed, the incriminated factors were either the root damage during growth or soil's microbial profile lacking the microorganisms that may compete with the pathogen [53]. Despite the extensive experimental studies, there are many possible factors which can interact together in promoting the pathogen internalization, and it remains controversial whether *E. coli* O157:H7, when introduced through soil or irrigation water, could internalize the edible parts of the mature produce. For example, the specific role of produce type in bacterial internalization is very difficult to assess in detail given the multiple existing interfering variables. In this regard, it was found that *E. coli* O157:H7 was able to internalize into inoculated seeds of cress, spinach, and lettuce [54]. In spinach plants, internalization was observed in the root tissue or seedlings but not in mature leaves [55]. Plant roots appear to be preferred by the pathogen as attachment and entrance site, and the roots contamination was reported to be dependent on roots health status (healthy, non-damaged roots versus damaged roots) and on the degree of pathogen contamination level [40]. While produce roots are getting mature, the differences in the produce developmental stages may also influence the ability of *E. coli* O157:H7 to interact with the produce, the pathogen could be enabled to enter the produce leaves by traveling through the root system [56]. Hora *et al.* [55] found that the degree of *E. coli* O157:H7 internalization of the spinach roots depends on the type of roots damage and produce age but it does not favor the internalization of leaves (**Table 3**).

When the produce contamination occurs, produce age, produce exposure to pathogen, and contact length of time with the pathogen can result in possible internalization of the pathogen [40]. Produce leaf's age has been shown to influence the growth and survival of *E. coli* O157:H7; young lettuce leaves were found to be associated with a greater risk of pathogen contamination and internalization [21].


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

## **Table 2.**

*Examples of Escherichia coli O157:H7 internalization status in leafy greens grown in different environments\*.*

As an example, in spinach grown under greenhouse conditions, the internalization of *E. coli* O157:H7 in the leaves is rare and mostly is taking place from outside of the produce to the inside if the plant surface is exposed to a heavy contamination with the pathogen [57]. Some studies show that hydroponic systems favor a greater


*a Roots without damage; spinach plant was not removed from soil.*

*b Seminal root was severed from 5-week-old spinach plants; plants were repotted.*

*d Roots inoculated with nematodes (Meloidogyne hapla); plant age-14 days.*

#### **Table 3.**

*The degree of E. coli O157:H7 internalization of spinach roots and leaves following different types of root damages\*.*

internalization of leafy greens compared to soil-growing system [58–60] and the water, as a growing environment, is indicated as the main source of produce contamination via pathogen uptake by roots [61, 62]. In aquaponic growing systems, under certain circumstances, STEC *E. coli* can internalize both roots and the plant leaves. STEC *E. coli* first internalizes the roots which are mechanically injured due to manipulation during transplanting. Subsequently internalization into the leaves occurs when the pathogen is given sufficient time to spread into the plant shoots and into mature leaves. Internalization of STEC *E. coli* into the whole plant grown in aquaponic system seems to be dependent on the plant age at the time of root injury: if the infection takes place during the early stage of plant development the STEC *E. coli* internalization in the whole young plant is favored [63].

Although the variability of the published experimental results is great, several conclusions can be reached in relationship with leafy greens pathogen internalization: (a) the produce growth environment plays an important role in pathogen internalization; (b) internalization is a plant-pathogen specific interaction; (c) health status of the roots does not enable the uptake of pathogen into produce, and (d) the presence of internalized pathogens into roots of plants is cannot be used as an indicator for pathogen internalization in leaves and does not directly correlate with internalized pathogens in the produce leaves.
