Fresh Produce as a Potential Vector and Reservoir for Human Bacterial Pathogens: Revealing the Ambiguity of Interaction and Transmission

The consumer demand for fresh produce (vegetables and fruits) has considerably increased since the 1980s for more nutritious foods and healthier life practices, particularly in developed countries. Currently, several foodborne outbreaks have been linked to fresh produce. The global rise in fresh produce associated with human infections may be due to the use of wastewater or any contaminated water for the cultivation of fruits and vegetables, the firm attachment of the foodborne pathogens on the plant surface, and the internalization of these agents deep inside the tissue of the plant, poor disinfection practices and human consumption of raw fresh produce. Several investigations have been established related to the human microbial pathogens (HMPs) interaction, their internalization, and survival on/within plant tissue. Previous studies have displayed that HMPs are comprised of several cellular constituents to attach and adapt to the plant’s intracellular niches. In addition, there are several plant-associated factors, such as surface morphology, nutrient content, and plant–HMP interactions, that determine the internalization and subsequent transmission to humans. Based on documented findings, the internalized HMPs are not susceptible to sanitation or decontaminants applied on the surface of the fresh produce. Therefore, the contamination of fresh produce by HMPs could pose significant food safety hazards. This review provides a comprehensive overview of the interaction between fresh produce and HMPs and reveals the ambiguity of interaction and transmission of the agents to humans.


Introduction
Fresh produce (vegetables and fruits) consumption has increased considerably since the 1980s because of the increasing consumer demand for a healthy life, particularly in developed countries. Following FAO guidelines, 400 g of fresh fruits and vegetables should be consumed daily [1]. Fresh produce diets have been shown to protect humans from some chronic ailments including cancer, diabetes, hypertension, obesity, and cardiovascular diseases [1].
Fresh produce is one of the crucial constituents of healthy food; nevertheless, they have been linked with several seasonal or global foodborne outbreaks, causing illnesses and serious economic losses. It has been estimated that nearly 76 million cases of foodborne diseases occur yearly in the United States [2]. Salmonella enterica (e.g., S. enterica serovar Typhimurium) and Escherichia coli (e.g., E. coli O157:H7) appear to be the most prevalent Safe production methods and proper decontamination or disinfection procedures are critical steps in ensuring the food safety of ready-to-eat foods and fresh produces. Most of fresh produce is eaten raw or minimally processed and does not undergo a 'lethal' process treatment, such as cooking. In addition, disinfection and cleaning are very important processes during food processing and packaging to ensure hygienic products and food safety [33]. The efficacy of various disinfectants and sanitizing methods for reducing the burden of microbial populations on raw fruits and vegetables varies greatly. Differences in the characteristics of the surface of the fresh produce, type and physiological state of microbial cells, the method and procedure used for disinfection (e.g., temperature, contact time, pH, dosage, residual concentration, etc.), and environmental stress conditions interact to influence the activity of disinfectants and sanitizers [34]. Vigorously washing vegetables and fruits with clean water minimizes the number of microorganisms by 10-100-fold and is often as effective as treatment with 200 ppm chlorine. To date, several types of physical and chemical methods are used for the decontamination of fresh produce to prevent the infection of humans with pathogenic microorganisms [35]. Most of the commercial methods are based on chemical principles, including chlorine dioxide (ClO 2 ), ozone (O 3 ), peracetic acid, hydrogen peroxide (H 2 O 2 ), edible coatings, cold plasma, and so on [33]. Physical non-thermal decontamination methods are effective at sub-lethal temperatures, thus it minimize negative consequences on the nutritional value of food [36]. These include the application of power ultrasound, gamma irradiation, UV treatment, high hydrostatic pressure, beta irradiation, and pulsed light. They are efficient but applicable to certain types of food matrices and use more time and energy. Purely physical procedures, such as high hydrostatic pressure, are chemically secure, but they necessitate complicated and costly equipment [37], and this can affect the quality of food products [38].
Some research findings indicated that the application of physical or chemical methods fails in removing bacterial contamination from the surface of fresh produce. These phenomena are mainly associated with the internalization of the microbial agent deep inside the tissue of the plant as discussed above. Hence, a clear and comprehensive understanding of the biological and molecular interaction between HMPs and fresh produce and other associated factors are crucial to select the appropriate disinfectant method and very helpful for designing of new disinfection approach.
There is no comprehensive literature review summarizing the past and present research outputs related to the interaction between the HMPs and fresh produce. In this regard, this review discusses the interaction between fresh produce and human bacterial pathogens and related foodborne outbreaks. We reviewed the available knowledge on bacterial internalization techniques into the tissue of plants and factors that influence the overall process.

Review Methodology
All published articles were searched in international databases, including Scopus, Medline (PubMed), Web of sciences, and Embase. The last search was done on 20 December 2022, and the English language used was while searching. The keyword search terms were a combination of the following: foodborne pathogens, green vegetables, fresh produce, pathogen internalization, reservoir, and vector. Additional searches were done for common foodborne bacteria, including E. coli O157:H7, Salmonella, and Listeria monocytogenes. The authors further narrowed the search for studies in bacteria internalization systems by searching for the names of specific types of systems, such as stomata, rhizosphere, plant tissue damage, and factors that determine the internalization process, such as biofilm, bacterial curli, flagella, cellulose, pili/fimbriae, plant surfaces, nutrient content, plant microbial flora, and foodborne outbreaks. For the present review, studies were excluded if they focused on plant bacterial pathogens or human pathogens other than bacterial pathogens. Based on these criteria, 15 papers were identified for discussion. In addition, the references of each article were reviewed to complement other studies ( Figure 1). pathogens. Based on these criteria, 15 papers were identified for discussion. In addition, the references of each article were reviewed to complement other studies ( Figure 1).

Bacteria-Fresh Produce Interaction (Bacterial Internalization Methods)
HPMs internalization and attachment are multi-step procedures, which depend upon environmental, bacterial, and plant factors ( Figure 2). HMPs' entrance into plant tissues through natural openings (stomata, lenticels), roots, or wounds has been reported in several studies ( Figure 2) [39]. Internalization facilitates HMPs to avoid adverse environmental conditions in plant tissue to get rid of UV and other environmental attacks and to get nutrients and water-rich niches inside the plants. Internalization could also shield HMPs from surface decontamination chemicals applied by consumers or companies [40].

Bacteria-Fresh Produce Interaction (Bacterial Internalization Methods)
HPMs internalization and attachment are multi-step procedures, which depend upon environmental, bacterial, and plant factors ( Figure 2). HMPs' entrance into plant tissues through natural openings (stomata, lenticels), roots, or wounds has been reported in several studies ( Figure 2) [39]. Internalization facilitates HMPs to avoid adverse environmental conditions in plant tissue to get rid of UV and other environmental attacks and to get nutrients and water-rich niches inside the plants. Internalization could also shield HMPs from surface decontamination chemicals applied by consumers or companies [40].

Stomata
The stomatal pore is an abundant natural opening in the leaf epidermis [41] that helps in gas exchange essential for photosynthesis and is also a major route for bacteria internalized into the leaf interior (phloem, xylem, and intercellular space). Several electron microscopy studies revealed the interaction between HMPs on or near guard cells ( Figure 3). For example, in a study conducted by Golberg and colleagues, S. enterica serovar Typhimurium SL1344 entered the iceberg and arugula lettuce through the stomata and localized in the sub-stomatal space [25]. In this study, despite the partial opening of stomata, no internalization of SL1344 was observed in parsley where most cells were found on the surface of the leaves [25]. Cells of S. enterica serovar Typhimurium MAE110, E. coli O157:H7 [17], and enteroaggregative E. coli [42] were linked to stomata in tomato, spinach, and Arugula leaves, respectively. Studies have shown that Shiga toxigenic E. coli (STEC) O157:H7 requires the type III secretion system (TTSS) to colonize stomata [17]. The STEC TTSS sec N mutants showed deficiency in the colonization of the plant stomata while K12 strains (non-pathogenic) containing a plasmid encoding the enterocyte effacement (LEE) pathogenicity island (effector genes and TTSS) were internalized more efficiently.

Stomata
The stomatal pore is an abundant natural opening in the leaf epidermis [41] that helps in gas exchange essential for photosynthesis and is also a major route for bacteria internalized into the leaf interior (phloem, xylem, and intercellular space). Several electron microscopy studies revealed the interaction between HMPs on or near guard cells ( Figure 3). For example, in a study conducted by Golberg and colleagues, S. enterica serovar Typhimurium SL1344 entered the iceberg and arugula lettuce through the stomata and localized in the sub-stomatal space [25]. In this study, despite the partial opening of stomata, no internalization of SL1344 was observed in parsley where most cells were found on the surface of the leaves [25]. Cells of S. enterica serovar Typhimurium MAE110, E. coli O157:H7 [17], and enteroaggregative E. coli [42] were linked to stomata in tomato, spinach, and Arugula leaves, respectively. Studies have shown that Shiga toxigenic E. coli (STEC) O157:H7 requires the type III secretion system (TTSS) to colonize stomata [17]. The STEC TTSS sec N mutants showed deficiency in the colonization of the plant stomata while K12 strains (non-pathogenic) containing a plasmid encoding the enterocyte effacement (LEE) pathogenicity island (effector genes and TTSS) were internalized more efficiently.
Stomata also serve as an active innate immunological agent against plants and HMP in Arabidopsis leaves [43]. According to previous studies, O157:H7 cannot withstand the closure of stomata, resulting in protracted stimulation of the stomatal immune response [43]. In contrast, a recent study found that S. enterica serovar Typhimurium SL1344 migrated toward stomata and internalized without stimulating an immune response [18]. This finding suggests that apart from plant pathogens [44], some HMPs have developed strategies to subvert the stomatal defense and were internalized into the plant tissue.

Rhizosphere or Root
The rhizosphere of plants is an environment that hosts a diverse group of microorganisms, including symbionts of plants and HMPs. Fresh produce plants' root exudate is nutritionally rich and attracts S. enterica to the roots of lettuce plants [45]. Though HMPs Stomata also serve as an active innate immunological agent against plants and HMP in Arabidopsis leaves [43]. According to previous studies, O157:H7 cannot withstand the closure of stomata, resulting in protracted stimulation of the stomatal immune response [43]. In contrast, a recent study found that S. enterica serovar Typhimurium SL1344 migrated toward stomata and internalized without stimulating an immune response [18]. This finding suggests that apart from plant pathogens [44], some HMPs have developed strategies to subvert the stomatal defense and were internalized into the plant tissue.

Rhizosphere or Root
The rhizosphere of plants is an environment that hosts a diverse group of microorganisms, including symbionts of plants and HMPs. Fresh produce plants' root exudate is nutritionally rich and attracts S. enterica to the roots of lettuce plants [45]. Though HMPs cannot directly infiltrate via root cells, root cracks and lateral root emergence provide sites for the internalization of S. enterica and E. coli O157:H7 into root tissues [45,46], and in some cases between the epidermal cells [45]. A study has reported higher S. enterica colonization in the shoot-root transition area [45]. Once internalized, HMPs like the cases of Salmonella enterica can be found in the endodermis, parenchyma, vascular system, and pericycle of lettuce roots and barley's inner root cortex [45]. Thorough investigations of E. coli O157:H7 localization in root tissues indicated its colonization in the cytoplasm, cell wall, and apoplast [47]. The translocation of HMPs from roots to the phyllosphere maybe depends on the flagellum [48] or presumably via the vasculature [49]. The mechanism for the migration of HMPs from root cortex to root vasculature through Casparian strips and endodermis, and from roots to phyllosphere through vascular system remains uninvestigated. Despite surface sterilization, pathogens were observed on lettuce leaves produced from hydroponic systems cultivated with water containing S Typhimurium or E. coli O157:H7 [50].

Plant Tissue Damage
Plants are usually exposed to different agents, such as environmental mechanical stresses, humans, and herbivores, that cause damage (wounding) and open the plant for HPMs to internalize the plant tissue. Wounding offers nutrients to HMPs and enables their entry into the tissue and consequent colonization [51]. For example, one study showed that wounding facilitated the quick perpetuation of E. coli O157:H7 on lettuce. Documented findings indicated that the concentration of E. coli O157:H7 enhanced by 4.5-fold, 4-fold, and 11-fold post-inoculation (4 h) on large pieces of mechanically cut lettuce leaves that were further bruised and shredded [52].

Factor Associated with Bacteriological Agents
The interaction between HMPs and fresh produce depends on different factors, and one of these factors are linked with the pathogen by itself. HMPs population size [53], bacteria species or strain involved [54], and the presence of bacterial cell surface appendages like pili/fimbriae, curli, flagella, and cellulose. Bacterial biofilm is also another factor that determines the plant-pathogen interaction [14]. Some of these factors are discussed below.

Biofilm
A biofilm is one of the most effective mechanisms used by HMPs to generate evasive fitness against immunologically challenging environments on or inside plants. Microbial biofilms can form on the surfaces of leaves and roots, as well as within plant tissues' intercellular spaces. Biofilms protect bacteria from desiccation, UV radiation, environmental stress, and defense immunity of plants. They also protect against antimicrobial agents produced by normal flora or by the plant itself. A microbial biofilm also generates a protective coat against disinfectants and antiseptics used during food processing [55]. A biofilm is a mechanism by which HMPs survive in a nutrient-poor microenvironment inside or on the plant surface.

Bacterial Curli
Curli are the main proteinaceous constituent of extra-cellular matrix synthesized by many enterobacterial pathogens. Curli fibers participate in cell aggregation, attachment to the plant surfaces, and biofilm formation. Curli are also involved in host cell attachment and invasion, and they are crucial inducers of the plant immune response (Figure 4). A study by Macarisin et al. [56] indicated that curli-expressing E. coli O157:H7 strains developed stronger linkage with the fresh produce leaf surface, whereas curli-deficient mutants adhered to spinach at a significantly lower population [56].
A biofilm is one of the most effective mechanisms used by HMPs to generate evasive fitness against immunologically challenging environments on or inside plants. Microbial biofilms can form on the surfaces of leaves and roots, as well as within plant tissues' intercellular spaces. Biofilms protect bacteria from desiccation, UV radiation, environmental stress, and defense immunity of plants. They also protect against antimicrobial agents produced by normal flora or by the plant itself. A microbial biofilm also generates a protective coat against disinfectants and antiseptics used during food processing [55]. A biofilm is a mechanism by which HMPs survive in a nutrient-poor microenvironment inside or on the plant surface.

Bacterial Curli
Curli are the main proteinaceous constituent of extra-cellular matrix synthesized by many enterobacterial pathogens. Curli fibers participate in cell aggregation, attachment to the plant surfaces, and biofilm formation. Curli are also involved in host cell attachment and invasion, and they are crucial inducers of the plant immune response (Figure 4). A study by Macarisin et al. [56] indicated that curli-expressing E. coli O157:H7 strains developed stronger linkage with the fresh produce leaf surface, whereas curli-deficient mutants adhered to spinach at a significantly lower population [56].

Flagella
Flagella are motility organelles, which facilitate reaching favorable habitats and serve as adhesive material to enhance their capability to attach to plant surfaces (Figure 4). The bacteriological agents adhere and irreversibly attach to the plant surface to develop microcolonies. They secrete EPS for the interactions between cells and plant surfaces. They also develop complex biofilm structures by interacting with alternative matrix components. The association of S. enterica internalization in leaves with chemotaxis and motility has been reported by Kroupitski et al. [16]. Motility-deficient flagella mutants (fliGHI::Tn10) were unable to properly attach and penetrate the lettuce leaves. It also restricted the cheY mutant defective entrance to chemotaxis.

Cellulose and Pili/Fimbriae
The extracellular matrix, cellulose is crucial for the attachment of Salmonella. A lower level of colonization was noted in bcsA (cellulose synthase) lacking S. enterica Enteritidis mutant in alfalfa sprouts as compared to wild type. However, normal colonization capability was achieved after the plasmid-based bcsA expression [57].
Adhesins containing hair-like Pili/fimbriae (P, 1, F1C, and S in E. coli) are present on the bacterial cell surface that exhibit affinity to various carbohydrates. The interaction of adhesins with mammal components is either non-specific (electrostatic or hydrophobic) or specific (binding with specific host cell receptor moieties), which carries out tropism for the adhesion with specific tissue or host [58]. Salmonella and E. coli adhesins and fimbriae (amyloid curli fimbriae) have been studied concerning their plant adhesions. Curli is known to facilitate the Salmonella and E. coli attachments to leaves and sprouts, but their inactivation effect is low.

Other Factors
Multiple studies have elaborated on the surface charge of bacterial cells, hydrophobicity, divalent cations, and capsule production during active or passive E. coli attachment to lettuce tissues [59]. These studies presented only a minor correlation between hydrophobicity, charge, cell surface appendages, and bacterial attachment capability with lettuce. Therefore, Span85 (hydrophobic surfactant) treatment could only detach 80% E. coli O157:H7 from lettuce leaves. The surfactant was also unable to detach pathogens from the cut edges, which indicates a heterogeneous surface nature [59]. Contrarily, a linear correlation between Salmonella cell surface hydrophobicity and its attachment capability to melon fruits has been reported [60].

Plant Factors
The colonization and interaction of foodborne pathogens (e.g., Salmonella enterica and Escherichia coli) with the plant immune system have been documented in various studies [19]. Plant factors include attachment sites [61], properties of plant surfaces [62], plant nutritive constituents and growing conditions [63,64], development stage [16,65], plant's cultivar [24,26], and contamination site [66]. In some situations, like the case of STEC, the rate of internalization is dependent on multiple factors, including the plant species and tissue [67] and how plants are propagated [68].

Properties of Plant Surfaces
Most of the aerial surfaces of the plants are covered with a hydrophobic cuticle that is mainly composed of polysaccharides, waxes, and fatty acids. It favors the attachment of hydrophobic molecules, whereas hydrophilic structures become exposed at the breaking points in the cuticle [62]. This situation helps the bacteria on the root surface to enter the plant cells generally covered with polysaccharides (pectin and cellulose) and glycoproteins. Such molecules are hydrophilic and can be negatively charged in some cases [69]. The attachment strength is correlated with the charge on the plant surface [60]. However, the exact binding sites or receptors remain unknown. The study of S. Typhimurium's attachment to potato slices has revealed bacterial attachment to cell wall junctions. Bacteria were particularly noted to attach with the pectin layer at the cell wall junctions that could be the bacterial binding site [70]. Contrarily, another study has demonstrated a reduced Salmonella attachment to the components of the cell wall mainly containing pectin. Therefore, it could be deduced that pectin is less favorable for bacterial attachment than cellulose [71].
Plant surface architecture and topography are crucial for microbial adhesion. Similarly, roughness is also important for bacterial survival and adherence to plant tissues. E. coli O157:H7 adhesion to the leaves of various spinach cultivars has been investigated [27]. Plant leaves' surface roughness depends on the leaf age and plant nature. During a study, high Salmonella affinity was noted for the old artificially contaminated leaves as compared to young lettuce leaves. A higher S. Typhimurium localization near the petiole has been noted. Similarly, a high bacterial affinity to the abaxial leaf side was observed as compared to the adaxial side [25]. Cantaloupe netting fissures are favorable Salmonella attachment sites, which help in their survival against sanitizers [72].

Nutrient Content and Its Location in the Plant Tissue
Microflora distribution on the leaf surface is not homogenous and bacterial cells prefer to colonize at specific sites on the leaf surface, such as stomata, trichomes base, junctions of the epidermal cell wall, grooves or depressions near veins, and beneath the cuticle [73]. These points are rich in nutrients and water and protect bacteria from stress. Plant appendages (secretory ducts or cavities) could release metabolites. Glandular trichomes outgrow from the epidermis and act as an accumulation and secretion site for various compounds including defensive proteins, Pb ions, Ca, Mn, Na, and secondary metabolites (phenylpropanoids, monoterpenoids, and essential oils). Bacterial presence on the lower leaf surface is generally higher than on the upper surface. This might be due to low radiation, a thin layer of cuticle, and high trichomes and stomata density [61]. Therefore, the conditions are much better for the growth and survival of bacteria at the lower surface of the leaf as compared to other leaf parts.
Most human pathogenic bacterial strains, including STEC, preferentially colonize the roots and rhizosphere of fresh produce plants over leafy tissue and are internalized by plant tissue, where they can persist in the apoplastic space as an endophyte [66]. The apoplast contains metabolites, such as solutes, sugars, proteins, and cell wall components [66], and as such, it provides a rich environment for many bacterial species, including both commensal bacteria and human pathogens [66].
Similar behavior of human enteric pathogens has been documented on leaves with minor differences. Salmonella enterica serovar Thompson could attach in the cell margins and around the stomata of spinach leaves where the presence of native bacteria is detected [74]. The confocal microscopy of E. coli attachment at trichomes and stomata of cut lettuce plants revealed its attachment similarity with plant pathogens [15]. The stomata serve as protective bacterial niches and nutrient sources. Golberg and colleagues [25] confirmed the preference of this niche by Salmonella cells by demonstrating their high localization within and near lettuce leaves stomata. However, Salmonella colonization around stomata is limited to only a few serovars on specific plants. Contrarily, E. coli could better attach to cut lettuce surfaces, whereas Pseudomonas fluorescens prefers to attach on intact surfaces. However, S. Typhimurium could attach to both intact and cut surfaces [75]. The localization capability of enteric pathogens at similar leaf adhesion sites with plant pathogens and natural microflora helps in their long-term survival.
Enteric bacteria penetrate the soil through fertilizers, water, or directly through roots during hydroponic growth to attach to the host plant rhizosphere. Then, they invade and move to upper plant parts [76]. In contrast to fruits and leaves, the location of these bacterial attachments was significantly different than natural microflora. Natural plant pathogens and microflora generally attach to the trichome root hairs and epidermis. Plant pathogens could rapidly bind at the wound sites and cut ends of roots, whereas their binding at root tips is poor [77]. Contrarily, E. coli strains preferably attached at alfalfa sprouts root tip, but their attachment to the roots was quite slow. However, not all the studied E. coli strains bind to root hairs [78].

Decontamination Methods Employed
E. coli and Salmonella attachment is considered an active step; however, this assumption is not supported by all the studies. In one study, for instance, only Salmonella viable cells could attach to potato slices [70]. Contrarily, the attachment levels of killed E. coli O157:H7, live E. coli O157:H7, and fluorescent polystyrene microspheres remained similar [79]. The differential results could be associated with the varying methods of bacterial inactivation. Glutaraldehyde was used to inactivate the E. coli cells, which could potentially change the bacterial adhesive features whereas various methods (thermal, ethanol, formalin, and kanamycin) were adopted to inactivate Salmonella cells [70]. Fresh produce-related pathogens are difficult to wash with antimicrobial and chlorine solutions [80]. Several studies have reported that chemicals-based washing of production-associated pathogens could be achieved from 1 (minimum) to 3 logs (maximum) [12,81]. Recently, less susceptibility to enteric pathogens has been reported against common sanitizers (chlorine) as compared to indigenous microorganisms. It suggests that the pathogens remaining after sanitizing could survive and grow on wet products with comparatively less competition [81].

Plant Microbial Flora and Bacteria-to-Bacteria Interactions
Plant microbiota could inhibit or promote enteric pathogens' establishment in plants ( Figure 5). Plant diseases affect the phyllosphere atmosphere to promote the growth of the enteric pathogen. Pectobacterium carotovorum subsp. Carotovorum co-inoculation with E. coli O157:H7 or S. enterica increased their levels by more than 10-fold in comparison to individual inoculations [82]. Resident bacteria (Erwinia herbicola and P. syringae) and plant pathogens could enhance the S. enterica survival on leaves. An S. enterica viable population on plants pre-inoculated with one of two E. herbicola strains and P. syringae was increased by 10-fold compared to controls [83]. Salmonella protection from desiccation by plant epiphytic bacteria on leaf surfaces has been reported. Recently, Potnis et al. have revealed pathogen-associated molecular pattern (PAMP)-triggered immunity suppression by a virulent strain X. perforans. X. perforans generate an S. enterica-friendly environment by inducing effector-triggered susceptibility in tomato phyllosphere. However, the S. enterica population was reduced by an avirulent strain of X. perforans, which activated the effector-triggered immunity [84]. Several investigations have reported that the presence of other microbes helps in enteric pathogen colonization in leaf environment. However, a reduction in soft rot progression and P. carotovorum subsp. carotovorum population was noted in the presence of S. enterica (enteric pathogen), which moderated the pH of the local environment [85].

Plant Immunity
Plants can avoid invading microbes through a complex innate immune system [53]. Plants generate a step-one response, which is triggered by molecules of conserved pathogens and degraded/modified plant products, which are referred to as pathogen or damage-associated molecular patterns (PAMP/DAMP). The conserved PAMPs are surface structure and cell wall components, which include lipopolysaccharides, chitin, and flagellin [86]. Plant extracellular pattern-recognition receptors (PRRs) could recognize PAMPs by passing intracellular signals to launch various defense molecules for restricting pathogenic invasion. Pathogen-triggered immunity (PTI) serves as the initial defense against infections [87].
The emerging field of human pathogens on plants (HPOP) has recently received the attention of phytopathologists and plant biologists. During the last decade, studies have focused on lipopolysaccharide (LPS), flagellin, and PAMPs to assess the human pathogen interaction with plants.

Plant Immunity
Plants can avoid invading microbes through a complex innate immune system [53]. Plants generate a step-one response, which is triggered by molecules of conserved pathogens and degraded/modified plant products, which are referred to as pathogen or damageassociated molecular patterns (PAMP/DAMP). The conserved PAMPs are surface structure and cell wall components, which include lipopolysaccharides, chitin, and flagellin [86]. Plant extracellular pattern-recognition receptors (PRRs) could recognize PAMPs by passing intracellular signals to launch various defense molecules for restricting pathogenic invasion. Pathogen-triggered immunity (PTI) serves as the initial defense against infections [87].
The emerging field of human pathogens on plants (HPOP) has recently received the attention of phytopathologists and plant biologists. During the last decade, studies have focused on lipopolysaccharide (LPS), flagellin, and PAMPs to assess the human pathogen interaction with plants.

(i). LPS Perception
Plant and animal Gram-negative bacteria contain lipopolysaccharides (LPS) in the cell wall. An LPS is a well-characterized PAMP in animals, which is recognized through host Toll-like receptor 4 [88]. However, there are no known LPS-recognizing receptors in plants. Nonetheless, plants could perceive LPS derived from human pathogens leading to PTI activation. A significant stomatal closure in Arabidopsis has been reported in response to LPS purified from E. coli O55:B5, S. Minnesota R595, and Pseudomonas aeruginosa [20]. Salmonella-purified LPS is known to trigger extracellular alkalinization and ROS production in the cell suspension of tobacco [89]. However, it was unable to initiate these responses in tomato leaves [90], which suggests that LPS recognition might vary with the plant species and experimental conditions.

(ii). Flagellin Perception
Flagellin is a structural component of the bacterial flagellum that participates in bacterial motility and attachment on plants [48]. It induces plant immunity and is recognized through FLS2 receptor [19,90]. Similar to PTI elicitor flg22 [91], the flg22 epitope of S. enterica serovar Typhimurium 14,028 also serves as an efficient PAMP and elicitor of immune responses in tomato, tobacco, and Arabidopsis plants [90]. Flagellum-deficient S. enterica serovar Typhimurium 14,028 mutants colonize better on Arabidopsis, alfalfa, and wheat roots than wild-type bacterium [92]. It suggests Salmonella flagellum-based induction of plant defenses inhibit bacterial colonization on various organs.

Environmental Factors
The leaf environment is generally hostile to bacteria. The leaf surface faces rapid fluctuations in relative humidity, temperature, UV radiation, moisture (dew or rain), hydrophobicity, and nutrients [93]. Human and animal pathogens do not experience such significant fluctuations in a single day. Therefore, the survivability of these pathogens on the leaf surface environment could be questioned. However, the high occurrence of human pathogens (E. coli O157:H7 and S. enterica) in fresh produce (vegetables and sprouts) causing foodborne disease outbreaks reveals the fitness of human pathogens in the leaf environment.

Molecular Interactions
Recent studies have demonstrated the involvement of protein-encoding genes in the interaction between HMPs and their persistence in the rhizosphere and phyllosphere of leafy greens (Table 1). For instance, considerable attachment defects are not observed in flagella proteins and curli subunits encoding isogenic gene mutants [94,95]. These proteins participate in various attachment mediating pathways for the leafy green surface. These steps are performed by complex molecular connections involving single or multiple mechanisms according to the bacterial and plant genotypes. Stress response induction is crucial for pathogenic colonization and persistence under hostile plant environments. Oxidative (oxyR), nutrient limitation (rpoS) regulons, and cell envelope (pspABC) are some important stress responses in the plant environment [95,96]. These mechanisms facilitate human pathogens in adapting to reactive oxygen species, scarce nutrients, defense responses, and antimicrobial substances, which threaten their survival in plants [95,96]. The enteric bacteria conserve energy with better nutrient scavenging by altering their metabolism after stress responses.

Outbreaks Associated with Fresh Produce Consumption
The raw consumption of fresh produce without eliminating pathogens could lead to foodborne diseases [2]. E. coli STEC and S. enterica could cause fresh produce-related outbreaks of foodborne diseases (Table 2). Green leafy vegetables are the most common reservoirs for E. coli STEC infections including the "big six". Contaminated water (drag water from cattle lots or water contaminated by other sources) is the most common source of contamination [100]. The E. coli O104:H4 outbreak in 2011 in North Germany is a recent example. A new strain of E. coli O104:H4 has caused the highest hemolytic uremic syndrome frequency and mortalities in a single outbreak. Imported fenugreek seeds from Egypt were most likely the outbreak source [101]. In the USA, 1779 foodborne outbreaks occurred from 2004 to 2010 with a confirmed etiology and food vehicle, and 9.2% (163) were fresh produce-related outbreaks [102]. An amount of 27.6% (45) of 163 produce-related outbreaks occurred in different U.S states. The same food contamination in various states is known as a multistate outbreak. From 2010 to 2017, 1797 foodborne outbreaks occurred in the U.S., where 12.7% (228) were related to fresh produce [102]. In 2020, a multistate outbreak of E. coli O157:H7 infections was associated with leafy greens, including romaine lettuce. A total of 40 individuals were infected across 19 states, leading to 10 reported hospitalizations and 4 cases of hemolytic uremic syndrome. Epidemiologic investigation identified leafy greens as the probable source of the outbreak; however, no specific type or brand of leafy greens was able to be identified. However, of 23 ill people interviewed, 22 reported eating leafy greens, wherein 15 consumed romaine.

Conclusions and Future Directions
Accumulating research findings indicate that fresh produce is one of the potential reservoirs of HMPs that cause disease in humans. Although there is no simple or single solution to this problem, one crucial aspect is to understand how human pathogens can penetrate and survive in plant tissues. This will lead to the discovery of a novel control approach and, consequently, reduce the rate of human infection.
As we have discussed in this review, several factors determine the survival of HMPs and their internalization (successful penetration) into the plant tissues. These factors could be either intrinsic (factors associated with the fresh produce) or extrinsic factors that are linked to the nature of the fitness of the HPMs, as well as the surrounding environment. Most of these factors positively or negatively contribute to the transmission of the pathogens to humans and determine the outcome or severity of the disease.
Some bacterial species may induce stronger immunity depending on how efficiently plants can recognize their system and trigger defense responses. Some bacteria may also evolve some unknown mechanisms to avoid recognition by the plant's immune system and can penetrate and survive inside the plant tissue. Hence, a deep practical investigation is needed to broaden the understanding of plant-HMP interaction. In addition, like human pathogens, plant immune systems are diverse and need to be fully explored to understand the real interaction between them. This hidden mechanism may contribute to the internalization of foodborne human pathogens and the subsequent transmission of the agents to humans. Some plant pathogens or normal flora may support or suppress the activity of the human pathogenic bacteria, either inside or outside the tissue of the plant. Hence, this kind of interaction should be investigated at the molecular level.
In this review, we have demonstrated that the internalization of HMPs in plant tissues combined with their prolonged survival using nutrients and other essential substances creates a very serious public health risk. In the long run, this problem could be solved by developing novel physical and/or chemical disinfectants that can reach the target agent without damaging fresh produce's nutritional value and the safety of humans. In addition, intense research is needed to know about the efficacy of currently available disinfectants and antiseptics items of the dose, concentration, duration of action, and other related factors that determine their efficacy. Homemade decontaminating agents should also be investigated for their efficacy in reducing the microorganisms found on the surface of the fresh produce and the efficacy of those approaches can be assessed in combination with commercially available disinfectants. Moreover, there are very limited research outputs related to the intercellular interaction between pathogens and the fresh produce. Hence, intensive research works are needed to explore the ambiguity of the interaction and its consequence on human health.