Preharvest Stress

Fruits and vegetables can be contaminated with bacterial pathogens and spoilage microorganisms by contact with feces, soil, irrigation water, improperly composted manure, air-carried dust, wild and domestic animals, and human handling [23-28]. Survival and potential proliferation of contaminants on produce depends on the type of microorganism, the type and condition of produce, and the environment (e.g., temperature, humidity). Examples of environmental stresses that may affect microorganisms on fresh produce include nutrient restrictions, temperature and pH fluctuations, water availability limitations, exposure to ultraviolet radiation, presence of organic compounds (e.g., pesticides) and metal contaminants, inhibitory plant tissue reactions, and microbiota competition, among many others.

Many foodborne pathogens have enteric origin, which could limit their ability to survive in other environments, colonize plant tissues, and compete with plant-associated microorganisms [23]. However, it is known that Salmonella can survive, adapt, and proliferate in soil, a nonhost environment characterized by its thermal variability, high osmolarity, pH fluctuations, and variable nutrient availability [28]. Brandl and Mandrell [29] reported that Salmonella Thompson was able to colonize the surface of cilantro leaves and proliferate when plants were incubated at warm temperature (30°C). In addition, it was observed that the microorganism tolerated plant dry conditions (60% relative humidity) at least as well as usual bacterial plant colonizers (e.g., Pantoea agglomerans and Pseudomonas chlororaphis). There is evidence that secretions of plant seeds can induce microbial stress. Miche et al. [30] studied the response of E. coli, containing luxCDABE reporter genes, to germinating rice seed exudates, and reported that these secretions enhanced the expression of microbial genes involved in general stress, heat shock, and oxidative stress responses. Temperature Fluctuation

Temperature variations at different stages of preharvest can affect the behavior of microbial populations present on produce. Besides influencing microbial growth, sudden fluctuations of temperature can cause heat or cold shock, and consequently may enhance the tolerance of foodborne pathogens to subsequent stresses. In this section, the effect of heat-induced stress will be discussed; cold stress will be addressed in another section of the chapter. Sublethal heat stress refers to the stress resulting from exposing a microbial population to temperatures higher than the maximum for growth and lower than that causing considerable cell death. Response to heat stress is most obvious when this stress causes minimal (less than one log) reduction in cell population.

Sublethal heat stress causes damage in the macromolecular structure of bacterial cells (e.g., protein denaturation), causing disruption of metabolic activities, which consequently affects microbial growth [31]. Microbial cells react against heat by inducing a universal protective response, generally known as the heat-shock response. This response involves the transient overexpression of heat-shock proteins that protect the cells against heat damage and other stresses. Heat-shock proteins include molecular chaperones (e.g., DnaK

and GroEL) which repair cell injury by refolding the denatured proteins [2]. Other heat-shock proteins have protease activity (e.g., ClpP), dependant on ATP, and are involved in the degradation of heat-damaged proteins [32]. Additionally, microorganisms may adapt to mild heat by modifying the fluidity of their cell membranes; this is accomplished by increasing the length or level of saturation of the membrane's fatty acids [11].

The transcription of the majority of the heat-shock proteins in E. coli is controlled by the alternative sigma factor, a32 [33]. Additionally, aE is involved in the regulation of heat-induced genes of this bacterium [34,35]. Induction of the heat-shock response in B. subtilis requires several regulatory groups including the HrcA-CIRCE system, which controls the major chaperone genes. The general stress response, controlled by aB, and the genes encoding Clp protease system are also involved in regulating the heat-shock response of this bacterium [3,33].

In addition to heat, several other stresses may trigger the synthesis of heat-shock proteins, and, as a result, induce multiple stress-protective responses. These stresses include changes in pH or osmolarity, ultraviolet irradiation, and the presence of substances such as ethanol, antibiotics, aromatic compounds, and heavy metals [36]. Synthesis of heat-shock proteins after exposure to other stresses may be attributed to the presence of a common stress sensing mechanism in the cells, which detects accumulated abnormal proteins in the cytoplasm [36-37].

There is substantial evidence confirming that exposure to sublethal heat increases the resistance of microorganisms to single or multiple lethal stresses. Seyer et al. [38] observed that E. coli, heated at 55°C for 105 minutes and permitted to recover, had enhanced tolerance to subsequent lethal, thermal treatments (60° C for 50 minutes). In addition, the investigators reported that internal cell concentration of the DnaK chaperone played a key role in microbial recovery and stress tolerance. Lou and Yousef [39] indicated that stressing Listeria monocytogenes with heat (45° C for 60 minutes) protected the cells to subsequent exposure to lethal concentrations of ethanol, hydrogen peroxide, and sodium chloride. Lin and Chou [40] observed a similar behavior in the same microorganism under comparable sublethal stress conditions. These researchers also indicated that thermal stress at a higher temperature and shorter time (48° C for 10 minutes) than those used by Lou and Yousef [39], protected L. monocytogenes against sodium chloride but decreased the resistance of the pathogen to lethal concentrations of hydrogen peroxide. Ultraviolet Radiation

Microorganisms are exposed to ultraviolet (UV) radiation from sunlight while present on the surface of fruits and vegetables. The main fraction of solar UV radiation that reaches the Earth's surface consists of long-wavelength UV (320-400 nm), which is usually designated as ultraviolet A (UVA). This type of radiation affects the microbial cell membrane and causes oxidation of unsaturated fatty acids. In addition, UVA participates in an oxygen-dependent reaction that involves the photosensitization of pigments, which results in the generation of reactive oxygen species (e.g., OJ) with antimicrobial activity [41]. In E. coli, UVA induces lethal and sublethal stress that may cause temporary growth inhibition, loss in phage sensitivity, and inhibition of tryptophanase induction [42,43]. There is evidence that when E. coli is treated with sublethal UVA radiation while in the stationary phase the microorganism recovers rapidly and acquires resistance to subsequent lethal irradiation treatments; however, this tolerance is not associated with the general stress response involving rpoS [41,42].

Short-wave UV radiation (200-280 nm), designated as ultraviolet C (UVC), causes damage to microbial DNA and RNA by inducing formation of pyrimidine-base dimers and DNA-protein crosslinks, which results in cell growth cessation, decreased viability, or cell death [44]. This microbicidal UVC radiation, particularly at 254 nm, has been implemented as a preservation treatment in a variety of foods; however, its application as an intervention strategy to be used alone is not recommended [11,45]. Sublethal doses of UVC may induce mutations and render cells tolerant to lethal irradiation and other stresses [46,47]. Hartke et al. [48] reported that irradiation of Lactococcus lactis with sublethal UVC (at 254 nm) induces the production of numerous protective proteins and enhances the tolerance of the microorganism to subsequent lethal treatments with heat, acid, and hydrogen peroxide. Bacterial cells can recognize damage caused to DNA as a consequence of UVC exposure and trigger a series of mechanisms to repair deleterious nucleic acid modifications. These processes require the participation of enzymes, which can be induced in the absence or presence of visible light, and these are named dark-repair and photoreactivation mechanisms, respectively [44]. Damage caused to microbial DNA can be counterbalanced by induction of the SOS response, which regulates the expression of genes involved in DNA repair [49]. Osmotic Stress

Although osmotic stress of microorganism on produce surfaces is not common at the preharvest stage, the following scenarios are likely to occur. Microorganisms may experience osmotic stress when they are exposed to saps released from bruises and wounds on produce surfaces. Dryness of microorganisms on produce surfaces also may result in osmotic stress. Exposure of surface microbiota to salts may occur with some commodities if brine flotation is used in conveying, sorting, or sizing operations. In order to survive osmotic stress, microorganisms must keep a balance between the water inside the cells and the concentration of solutes in the environment. Generally, bacterial cells use two protective mechanisms to survive hyperosmotic stress: (1) discharging the excess of solutes within the cells to the outside and (2) accumulating compatible solutes or osmolytes. In addition, microorganisms adapt to this stress by modifying their cell membranes, e.g., by increasing the ratio of trans to cis unsaturated fatty acids [2,4]. Microbial accumulation of compatible solutes is a mechanism that has been well characterized. Compatible solutes are small, polar, organic molecules that remain water soluble at relatively high concentrations without affecting intracellular structures or metabolic activities. These solutes include compounds such as carnitine, trehalose, glycerol, sucrose, proline, mannitol, glycine-betaine, and small peptides, among others [4,15]. The accumulation of compatible solutes, as a result of osmotic stress, requires the expression of proteins involved in the synthesis of the osmoprotectants or their transport systems [2,50]. The synthesis of several proteins that participate in osmolyte accumulation is under the control of the general stress response sigma factors aS and aB in E. coli and B. subtilis, respectively, which regulate the expression of chaperones and proteases [3,15]. Therefore, adaptation of microorganisms to osmotic stress may render them resistant to subsequent stresses of different types. Pretreatment of B. cereus with NaCl (1%) caused microbial resistance to subsequent lethal treatments with heat, ethanol, hydrogen peroxide, and acid. However, stress adaptation failed to protect the microorganism against a medium containing 12% NaCl [51,52]. Periago et al. [53] observed that pre-exposure of the same microorganism to osmotic stress, with 2.5% NaCl for 30 minutes, induced cell tolerance to lethal heating at 50°C. Osmotic stress of L. monocytogenes, by previous exposure to 3.5% NaCl for 2 hours, increased microbial tolerance to acid (pH 3.5), but the adaptation was strain-specific [54].

4.2.2 Postharvest Stress

Many sources of microbial contamination of produce at the postharvest stage have been identified. These include humans (i.e., workers and consumers), wild and domestic animals, insects, improperly sanitized harvesting equipment, transportation vehicles and containers, air-carried dust, wash, rinse, and cooling water, ice, processing and packaging equipment, and storage facilities, among many others [23,24,26,55-57]. Foodborne pathogens can survive on the intact outer surface of fresh fruits and vegetables, but they may not proliferate due to restriction of nutrients and water, or as a result of their inability to synthesize degradative enzymes against protective barriers covering produce. Survival and proliferation of pathogens on produce are enhanced by physical damage (e.g., punctures and bruises) of the protective epidermal barrier or the infection of the produce with pests and microorganisms [26]. Microbial stress adaptation may occur at various postharvest stages, and can involve transportation conditions, use of wash and rinse water at variable temperature, application of intervention strategies (e.g., use of sanitizers), pH fluctuations, and storage and packaging conditions. Cold Stress

Microorganisms respond to cold stress by undergoing an adaptive response known as the cold-shock response. Adaptation to cold stress involves the expression and accumulation of cold-shock proteins, which could protect the cells to subsequent freezing or against other lethal stresses [53,58,59]. Broadbent and Lin [58] observed that cold shocking L. lactis at 10° C for 2 hours increased its resistance to freezing (—60°C for 24 hours) and lyophilization. Bollman et al. [60] reported that stress-adapted E. coli O157:H7, previously cold-shocked at 10°C for 1.5 hours, had enhanced survival in several foods including milk, whole egg, and sausage when compared to the nonadapted bacterium. A previous study indicated that cold shock of B. cereus (7°C for 2 hours) increased the survival of the microorganism to subsequent lethal thermal treatment [53]. In a different study, cold shocking Clostridium perfringens at 15°C for 30 minutes increased the thermotolerance of the bacterium at 55°C [59].

The cold-shock response involves a number of physiological adjustments, which include modifications in the cell membrane fluidity via increasing the unsaturation of membrane lipids or decreasing the chain length of its fatty acids, synthesis of protective proteins that bind to DNA and RNA, and importation of compatible solutes [4]. The cytoplasmic membrane, nucleic acids, and ribosomes participate in sensing temperature variations in microbial cells, and temperature downshifts induce the synthesis of up to 50 different cold protection-associated proteins [61,62]. Microbial response to cold stress involves the overexpression of two types of proteins, the cold-shock proteins (Csps) and the cold-acclimation proteins (Caps). A sudden drop in temperature induces the rapid, and transient, synthesis of Csps. Conversely, Caps are synthesized for extended time periods under continuous microbial growth at low temperatures; the expression of both protein types, however, can overlap during stress adaptation [63,64].

The cold-shock response has been well characterized in E. coli, and its Csps fall into two classes, I and II. Class I Csps are expressed at very low levels at 37°C, and are induced and overexpressed after a temperature downshift to 15°C. These class I proteins include the major cold-shock protein, CspA (a RNA- and DNA-binding chaperone), ribosomal binding factors (e.g., RbfA, CsdA), and the transcriptional termination and antitermination factors (e.g., NusA) [4,61,65]. Class II Csps are present in cells at 37°C, and are induced at moderate levels (< 10-fold) after the cold shock. Among the induced Csps, there are recombination factors (e.g., RecA), a subunit of DNA gyrase (GyrA), and energy-generating enzymes (e.g., dihydrolipoamide transferase and pyruvate dehydrogenase) [61,64,66].

In spite of the evidence of the protective effects of cold shock against multiple stresses, other researchers indicated that previous exposure to low temperatures sensitized L. monocytogenes [67,68] and Vibrio parahaemolyticus [69] to subsequent thermal treatments. As discussed earlier in this chapter, exposing microorganisms to a stress may lead to their adaptation or sensitization to more severe stresses. This variable behavior of pathogens in response to cold stress should be considered when treating produce that has been previously refrigerated to antimicrobial processes such as surface pasteurizing. Acid Stress

Foodborne bacteria usually encounter drastic pH variations in the environment, and are exposed to acidic conditions while present in foods, during processing, and when they invade the gastrointestinal tract of animals and humans [70]. Acidification is a common food preservation method, in which organic acids (e.g., acetic, propionic, and lactic) are produced during fermentation or added as preservatives to foods. These weak acids, in their nondissociated form, are capable of diffusing into microbial cells; once inside the cytoplasm, they dissociate and decrease the intracellular pH, which results in disruption of metabolic activities. Acid stress of foodborne microorganisms results from the combination of the biological effect of low pH and the direct effect of weak acids [15].

Microorganisms have developed strategies to respond to acid stress by inducing a protective response known as the acid-tolerance response (ATR) [71]. Microbial cells develop an ATR when exposed to a moderately low pH (e.g., 4.5 to 5.5), and this results in the induction of proteins that protect the cells against lethal acid conditions (e.g., pH < 4). In addition, cells respond to acid environments by modifying their membrane composition, increasing proton efflux and amino acid catabolism, and by synthesizing enzymes involved in DNA repair [3,4].

In Salmonella Typhimurium, two different acid adaptation systems are recognized: these are the log-phase and the stationary-phase ATR [71]. Logphase ATR is triggered when cells are grown under moderately acid conditions, and involves the synthesis of acid-shock proteins under the control of aS, the signaling protein, PhoP, and the iron regulator, Fur [15,70]. The stationary-phase ATR consists of aS-independent and aS-dependent mechanisms. The response independent of aS requires acid induction, and involves the participation of the response regulator, OmpR, to control the synthesis of acid-shock proteins. The induction of the ATR dependent on aS does not require previous exposure of the microorganism to acid, and it is triggered by entry of the cells into stationary phase [3,4,71]. Therefore, the latter ATR involves the induction of the general stress response, which is associated with multiple stress adaptation. Wong et al. [72] reported that V. parahaemolyticus, pretreated in acid medium (pH 5.0 to 5.8), showed increased resistance to treatments with low salinity and heat (45°C). In a different study, Rowe and Kirk [73] indicated that exposing pathogenic E. coli to acid shock (pH 4 for 1 hour) enhanced microbial tolerance against subsequent lethal treatments with osmotic stress (20% NaCl) or heat at 56°C.

Microorganisms grown under mild acid conditions are more resistant to lethal acid environments, as well as to other lethal stresses, than those grown at neutral pH [71]. Tosun and Gonul [74] indicated that Salmonella Typhimurium, grown at pH 5.8, developed tolerance to lethal doses of heat, salt, and organic acids, but not to cold shock. Ryu and Beuchat [75] observed that acid-adapted E. coli O157:H7, grown under gradual pH reduction in a medium containing 1% glucose, showed enhanced tolerance to thermal treatments (52° C) in apple cider and orange juice. In a different study, Bacon et al. [76] reported that stress-adaptation of Salmonella spp., grown under gradually increasing acid conditions (i.e., in a medium containing 1% glucose), caused cross-protection against lethal heat treatments. Listeria monocytogenes, growing under similar gradually increasing acid conditions, or previously treated at pH 5.0 to 5.5 for 90 minutes, showed enhanced survival to a lethal acid medium (pH 3.5). Nonetheless, exposure to other stress conditions including high osmolarity, heat, and cold was unable to protect the microorganism against acid [77]. These results may have implications in the washing of fruit since many of the commonly used washing agents are acidic in nature. Application of these agents in a manner that sensitizes, rather than hardens, the pathogens to other stresses would improve the safety of produce. Oxidative Stress

Foodborne microorganisms are exposed to oxidative stress, which may be induced endogenously as a result of microbial metabolism or exogenously due to treatments that increase the levels of reactive oxygen species, i.e., hydrogen peroxide (H2O2), superoxide anion (O2), hydroxyl radical (HO"), and singlet oxygen (1O2). Similarly, microbial oxidative stress can be triggered by conditions that lead to depletion of protective antioxidant molecules or enzymes. Reactive oxygen species can be generated during processing as a result of radiation, presence of heavy metals, or treatments with oxidizing sanitizers. Reactive oxygen species are deleterious to microorganisms, and can cause extensive damage to their cellular components such as lipids, proteins, and nucleic acids; this negatively affects cell functionality and reduces its viability [78-80]. Microorganisms respond to oxidative stress by synthesizing (1) protective proteins (e.g., glutathione reductase, thioredoxin 2) and other organic molecules (e.g., methylerythrol, cyclopyrophosphate) with antioxi-dant capacity or (2) proteins that participate in repairing oxidative damage (e.g., exonuclease III and endonuclease IV), specifically repairing deleterious modifications affecting nucleic acids [2,81,82].

In E. coli, response to oxidative stress caused by H2O2 and O2_ is under the control of oxyR and soxRS regulons, respectively [79,81,83]. Genes controlled by oxyR include those encoding the hydroperoxidase I (HPI), glutaredoxin, glutathione reductase, NADPH-dependent alkyl hydroperoxide reductase, and a protective DNA-binding protein (Dps) [83,84]. The regulon soxRS controls the expression of genes encoding Mn-superoxide dismutase (Mn-SOD), endonuclease IV, glucose-6-phosphate dehydrogenase, fumarase, aconitase, and ferredoxin reductase, among others [80,83]. In unstressed cells, both proteins OxyR and SoxR are present in an inactive form. During oxidative damage, e.g., by exposure of cells to H2O2, OxyR senses the stress and is activated by the formation of intramolecular disulfide bonds [81,84]. There is evidence that the colanic acid polysaccharide produced by many strains of E. coli O157:H7 protects the microorganism against oxidative stress and other environmental conditions such as acid, heat, and osmotic stresses [85]. Van der Straaten et al. [86] reported that RamA, a protein synthesized in response to oxidative stress in Salmonella Typhimurium, could be involved in antibiotic resistance and virulence.

Produce microbiota are often exposed to oxidative stress. Sanitizers that may be used in washing produce (e.g., chlorine, chlorine dioxide, and ozone) undoubtedly lead to oxidative stress, which may trigger stress adaptation among these microorganisms. Metal ions in washing water and oxygen in package headspace are additional factors that may contribute to the oxidative stress adaptation of microorganism on produce. Minimal Processing

Recently there has been an increase in consumer demand for high-quality and safe foods with fresh-like attributes. Minimally processed fruits and vegetables can be defined as products that are processed with methods (e.g., low-level irradiation and active packaging) that achieve food preservation and safety while causing minimal quality modifications or alteration of the fresh characteristics compared to produce treated by conventional food preservation treatments [87]. Applying minimal processing involves using preservation factors singly or in combination. Therefore, minimal processing may be considered an implementation of the ''hurdle concept'' which refers to the application of mild preservation factors (i.e., hurdles) in combinations, either in sequence or simultaneously, to enhance microbial inactivation by additive or synergistic effects [88,89].

Combination of sublethal stresses, although potentially acting syner-gistically to inactivate microorganisms in foods, could lead occasionally to stress adaptation and cross-protective responses [11,90]. Examples of cross protection were reported by Lou and Yousef [39] who observed that stressing L. monocytogenes with heat (45°C for 60 minutes) protected the cells to subsequent exposure to lethal concentrations of ethanol, hydrogen peroxide, and sodium chloride. During food processing, microorganisms are treated with sublethal stresses sequentially rather than simultaneously. Therefore, microbial exposure to a sublethal stress could harden the microorganisms and protect them against subsequent treatment factors or hurdles. Consequently, stress hardening could pose limitations to the possible benefits of the hurdle concept. However, careful application of minimal processing could alleviate the consequences of stress adaptation of microbiota in produce.

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