**2.2 Biofilm formation**

Leafy greens, as pathogenic biofilm carriers, pose a great threat to produce microbial safety since the biofilms poses a great resiliency towards decontamination methods applied during post-harvest processing (i.e., chemical washing solutions) [24, 25]. The general mechanisms of leafy greens contamination by pathogens' colonization takes place in phases: (a) attachment to phylloshere and/or to rhizoplane, and (b) pathogens' adaptation to environmental factors followed by survival and multiplication on the plant parts. The whole general bacterial attachmentcolonization mechanism takes pace in a similar manner for human enteric pathogens that are either environmentally shed by domestic animals and/or wildlife: cattle, sheep, pigs, goats, wild birds, deer, mice, insects, or can originate from other sources: soil, manure, irrigation waters, etc. [26]. The leafy greens' structure (leaves' roughness, leaves' surface degree of porousness, crests etc.), influences the pathogen's attachment phenomenon that results in biofilm formation. When leaves are damaged (i.e., cuts, wounds) the pathogen may further become internalized due to pathogen's multiplication in these areas where damaged plant tissue exudes inner nutrients [27]. In addition, the amount of contaminating bacteria is a factor which can affect the degree of pathogen's attachment to the leafy greens. The colonization of leafy greens, as the first stage of biofilm formation, could take place through multiple routes, such as: contaminated soil (i.e., via dust or splashes), roots, seeds, or by wetting of produce leaves (i.e., via irrigation waters) and depends on the pathogens' ability to adapt to the new environment following the attachment phase. Once colonization takes place, biofilm formation is initiated. According to Ximenes and Tarver biofilm formation on leafy greens (i.e., lettuce, spinach, basil, cilantro, green onions, and parsley) by enteric pathogens involves in several stages: (a) initial contact of *E. coli* with the leafy greens and pathogen's subsequent attachment to the produce; (b) *E.coli* cells' proliferation and cells' aggregation by the excretion of the extracellular polymeric substances – which helps the formation of the initial "matrix" where the pathogen will grow and multiply; (c) *E. coli* biofilm maturation, and (d) sporadic *E. coli* cells' dispersion or detachment into the environment and contaminate other produce from the vicinity of the "infected" produce [26, 28]. According to Beattie and Lindow, bacteria found on leaves possess two major strategies which they can apply for their attachment, growth and survival, and biofilm formation on the plant surface: (a) "*tolerance strategy*" that requires the bacteria's ability to resist exposure to environmental stresses on leaf surfaces; or (b) "*avoidance strategy*", in this case bacteria seek plant sites that are protected from those stresses. Using these bacterial strategies, a general step-by step-model of leaf colonization and biofilm formation was developed: 1. the landed bacteria on the leaf surface are randomly distributed; 2. some of bacteria will enter into the leaf via openings such as stomata while some will stay on the surface of the plant leaves and modify the local environment to fit their needs; 3. surface adhered bacteria start to multiply and to form aggregates or micro-colonies, which subsequently will develop into biofilms [29]. Subsequent to the tight adhesion on favorable sites found on plants (niches), the biofilm formation process is facing environmental factors, plant properties, and the innate plant microbiota [20]. Nevertheless, once

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

the biofilm is formed it has the capability to protects the rest of attached bacteria against environmental stressors (i.e., desiccation, UV radiation etc.), from the plant immune response, and from endogenous (plant-origin) or exogenous (indigenous microorganisms-origin) antimicrobial compounds. Studies on the attachment of human enteric *E. coli* indicate that it can rapidly adhere to a variety of growing plant tissues such leaves and roots. Surface attachment is possible due to the presence of the plant's cuticles and the plant's surface characteristics. The cuticle present on the plant surfaces favors attachment of hydrophobic molecules and any breaks in the cuticle may expose the hydrophilic structures for further attachment [30]. The characteristics of the plant's surface is also important in the microbial adhesion process. For example, the surface roughness of the plant parts depends on the nature and age of the plant, and it is important not only for adherence but also for the pathogen's survival on the produce as demonstrated for *E. coli* O157:H7 adhesion on leaves of different spinach cultivars [31]. The microbiota found on the plants is not homogenously distributed on the leaf surface, bacterial cells predominantly attaching and colonizing on specific sites of leaf surfaces such as epidermal cell wall junctions, in grooves along veins and depressions, or beneath in the cuticle [29]. Under certain factors (on-field circumstances, bacterial unspecific binding based on hydrophobic and electrostatic interactions), attachment phenomenon could be reversible. However, when the pathogen cells form the exopolymeric material, are able to fix themselves more strongly on the leafy greens, the attachment is irreversible and the pathogen cannot be removed by washing treatments [20, 32, 33].

Studies showed that both, produce and bacterial properties, are factors involved in attachment of pathogenic *E. coli*. Leafy greens surface properties (i.e., cuticles, roughness) is favoring the pathogen attachment and colonization at specific sites of leaves: base of trichomes, stomata, epidermal cell wall junctions, or in grooves existing along the produce veins and depressions [29]. The study by Takeuchi *et al.* indicated specific attachment and colonization sites the cut surfaces of lettuce are rich in water and nutrients and offer *E. coli* O157:H7 stress-protection [34]. *E. coli* strains possess an attachment-adhesion system due to its ability to produce a diversity of pili and fimbriae and non-fimbrial adhesins, that function as 'professional' adhesion systems, and flagella; these compounds could play alternative functions in attachment and adhesion stages [35, 36]. An earlier study led by Torres *et al.* showed that *E. coli* O157:H7 possesses several redundant protein adhesins and the overexpression of each adhesin alone is sufficient to promote binding to alfalfa sprouts [37]. Ximenes *et al.* indicated the importance of some bacterial hydrolytic enzymes, such as: pectinases, cellulases, proteinases, and amylases which can further enhance the ability of pathogens to invade and spread on plant tissues [26]. Several experimental studies showed that *E. coli* ability to adhere and attach varies in time and some influence factors could be the initial number of viable pathogenic cells contaminating the plant and the type of leafy green. For arugula leaves, 2 log10 CFU/g of pathogen attached after 60 min, for lettuce leaves attachment time varied between 25 and 120 min (final level of pathogenic viable cells being 1–2.5 log10 CFU/cm2 ) and for spinach approximately 3 log10/spinach leaf attached in less than 60 min [31, 38, 39].
