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Foodborne pathogenic bacteria in fresh-cut vegetables and fruits
CHAPTER
Foodborne pathogenic bacteria in fresh-cut vegetables and fruits
9 Hu¨lya O¨lmez
¨ ITAK ˙ TUB MRC Material Institute, Gebze, Kocaeli, Turkey
INTRODUCTION Driven mainly by the increasing consumer demands for fresh and healthier convenience foods, the fresh produce market has grown dramatically over the last two decades. Following the increase in consumption, on the other hand, the foodborne outbreaks associated with fresh produce have also shown a raising trend globally (CSPI, 2014; EFSA, 2011a). The proportion of fresh-produceassociated foodborne illnesses increased from 12% in the 1990s to 24% in the 2010s (Painter et al., 2013; Sivapalasingam et al., 2004). Three main factors may add to the increase in the number of reported illnesses. First, the globalization of food industry has definitely resulted in the spreading of foodborne outbreaks between countries and even continents, resulting in a higher number of people being affected by the same contaminated produce. Second, the changes in the agricultural and processing practices throughout the food chain which aimed at increasing the supply may have enhanced the risk for transmission of pathogens and cross-contamination. Third, the improvements in the methods of microbial detection and identification, as well as in surveillance techniques, have also contributed to the increase in the number of reported illnesses. Fresh produce was reported to be one of the main vehicles of foodborne outbreaks in Europe, accounting for 8% of all the foodborne outbreaks in 2011 (EFSA, 2011a). In the United States, the produce category was linked to the largest number of foodborne illnesses associated with foodborne outbreaks between the periods of 1998 2007 and 2002 2011, constituting 23% and 24% of all illnesses, respectively (CSPI, 2011). The produce category surpassed the poultry, beef, and seafood categories, accounting for 667 outbreaks and 23,748 illnesses, most of which were associated with vegetables (10,806) (CSPI, 2014). The most commonly identified causative agents were Salmonella spp., norovirus, and pathogenic Escherichia coli (Painter et al., 2013). The main reason for having fresh-cut fruits and vegetables as one of the major vehicles of foodborne P. Kotzekidou (Ed): Food Hygiene and Toxicology in Ready-to-Eat Foods. DOI: http://dx.doi.org/10.1016/B978-0-12-801916-0.00009-1 © 2016 Elsevier Inc. All rights reserved.
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outbreaks associated with foodborne pathogens is that they are often consumed raw after minimal processing. There is no kill step in the processing which can assure the elimination of the pathogenic bacteria associated with fresh produce. Once contaminated in the field, it is not possible to completely eliminate the pathogenic bacteria from the fresh-cut produce. Therefore, identification of the main sources of contamination and transmission routes of pathogenic bacteria to fresh produce is a key for managing the food safety risks associated with fresh produce.
SOURCES AND ROUTES OF PATHOGEN CONTAMINATION IN FRESH-CUT PRODUCE Fresh produce may be contaminated with pathogenic bacteria via different routes at different points throughout the preharvest and harvest/postharvest stages (Fig. 9.1). Current contamination sources in the field include manure, irrigation water, soil, fecal contamination, contaminated seeds, geographical location, and climate change. The postharvest steps including the harvesting itself, precooling, processing operations, washing and sanitizing, storage packaging, and transfer
Sources of fresh produce contamination
Preharvest sources
• Soil and manure • Irrigation water and method of irrigation
• Water used as a carrier of • • • • •
pesticides and hormones Air (dust) Fecal contamination Contaminated seeds Climate change Geographical location
FIGURE 9.1 Main sources of fresh produce contamination.
Harvest/postharvest sources • Harvesting equipment • Human handling • Air (dust) • Cooling water • Storage • Sorting • Unit operations (coring, cutting, peeling, etc.) • Washing/sanitizing • Packaging • Transfer
Sources and Routes of Pathogen Contamination in Fresh-Cut Produce
may lead to pathogen contamination if not controlled properly according to the good manufacturing practices. Irrigation water and the method of irrigation have been identified as one of the main sources of contamination (Park et al., 2012). The use of contaminated surface water or untreated sewage water results in high numbers of fecal coliforms and leads to the transmission of pathogenic bacteria to the fresh produce. Due to the direct deposition of the contaminated water onto the edible parts of the produce, spray irrigation systems pose a higher risk of pathogenic bacteria contamination compared to other irrigation methods such as surface irrigation and drip irrigation (Aruscavage et al., 2006; Fonseca et al., 2011; Van der Linden et al., 2013). Studies showed that E. coli O157:H7 can persist for 2 20 days on spray-irrigated lettuce leaves (Erickson et al., 2014; Solomon et al., 2003). Moreover, the repeated spray irrigation may lead to the internalization of E. coli O157:H7 in leafy vegetables such as spinach and parsley (Erickson et al., 2014). Animal manure used as a soil amendment also represents a source of pathogenic bacteria contamination to fresh produce. The pathogenic bacteria including E. coli O157:H7, Salmonella, Listeria monocytogenes, and Campylobacter jejuni can survive for 45 175 days in soil depending on the soil characteristics (salinity, total nitrogen, etc.), moisture level, and temperature (Holley et al., 2006; Lang and Smith, 2007; Nicholson et al., 2005). Moreover, even with very low levels of contamination (100 101 CFU/g), the pathogenic bacteria are also able to survive for long periods in animal manure, depending on the temperature (Beuchat, 1999). The rate of survival is higher at lower temperatures. E. coli O157:H7 can survive in bovine feces for 70 days at 5 C, but only 49 days at 22 C (Semenov et al., 2007). Therefore, manure that has not been properly treated when used as a soil amendment may serve as a source of pathogen transmission to the fresh produce. As the soil type, moisture level, and temperature are critical factors for the survival of enteric pathogens in soil and manure, the geographical location and the climate change also appear to be important preharvest factors for fresh fruit and vegetable contamination. Moreover, higher intensity of precipitation and rain may increase the potential of pathogenic bacteria contamination to fresh produce by enhancing the survival of the pathogens in soil and manure (Cevallos-Cevallos et al., 2012; Warriner et al., 2009). Harvesting and postharvest practices can serve as a source of contamination and may enhance both the survival and growth of the pathogenic bacteria on fresh fruits and vegetables. Contaminated harvesting equipment, containers and poor hygiene of farm workers are important factors that influence the cross-contamination of fresh produce (Buchholz et al., 2012; Matthews, 2013; Todd et al., 2008). The postharvest unit operations (coring, cutting, slicing, peeling, shredding) and even the washing/sanitizing steps cause tissue damage and render the produce more prone to bacterial contamination. The destruction of tissue and cells results
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in the release of nutrient reach exudates which facilitates both the attachment and the rapid proliferation of pathogenic bacteria. The promotion of the rapid growth of E. coli O157:H7 as a result of tissue damage in lettuce has been shown by Brandl (2008). Cut leaf surfaces represent a specific target for the attachment of Salmonella cells (Kroupitski et al., 2009b). The use of inadequately sanitized water for cooling or washing/sanitizing operations can represent a source of crosscontamination and can serve as the primary vehicle for the spread of pathogenic bacteria during fresh produce processing (Pe´rez-Rodrı´guez et al., 2014).
FOODBORNE OUTBREAKS ASSOCIATED WITH PATHOGENIC BACTERIA IN FRESH-CUT FRUITS AND VEGETABLES Summaries of the foodborne outbreaks associated with pathogenic bacteria in fresh fruits and vegetables in Europe (2007 2011) and in the United States (2006 2015) are given in Tables 9.1 and 9.2, respectively. In both regions, most of the fresh-produce-associated outbreaks were linked with Salmonella spp., followed by pathogenic E. coli. In fact, in all the fresh-produce-associated foodborne outbreaks viruses rank first followed by Salmonella and E. coli O157:H7 as the causative agent for both regions (CDC, 2015; EFSA, 2013). The other pathogenic bacteria identified as the causative agent in fresh-produce-associated outbreaks in the given periods include Shigella spp., Yersinia spp., Bacillus spp., Clostridium perfringens, and Staphylococcus aureus. Sprouted seeds and leafy greens were the main food vehicles in most of the fresh-produce-associated outbreaks. The use of contaminated seeds was determined to be the major cause of sprout-associated outbreaks (EFSA, 2011a; Emberland et al., 2007; Werner et al., 2007). In the reported periods, Salmonella was the most common bacterial agent, accounting for 71% of the outbreaks associated with bacteria in Europe, and 60% in the United States (Tables 9.1 and 9.2). Moreover, 86% of the human cases in the selected multistate outbreaks in the United States and 31% of the human cases in Europe were linked to Salmonella contamination. The multistate outbreak associated with Salmonella Saintpaul contaminated cantaloupes (Munnoch et al., 2009) and Salmonella spp. contaminated alfalfa sprouts (Compton et al., 2008) in Australia are other examples of Salmonella spp. linked fresh produce outbreaks with more than 200 human cases. A wide variety of fresh produce has been associated with Salmonella-linked outbreaks, sprouts, leafy greens, cucumbers, and melons being the major ones. The higher ratio of Salmonella-associated fresh produce outbreaks is partly linked to the ability of Salmonella to internalize within the plant and proliferate to high numbers under favorable conditions (Golberg et al., 2011; Schikora et al., 2008). Pathogenic E. coli was the second major causative agent of the bacteriaassociated outbreaks mainly linked to the consumption of contaminated sprouts and leafy greens for the last decade (Tables 9.1 and 9.2). E. coli O157:H7 was
Foodborne Outbreaks Associated with Pathogenic Bacteria
Table 9.1 Summary of Strong Evidence Foodborne Outbreaks Associated with Pathogenic Bacteria in Fresh Fruits and Vegetables in Europe (2007 2011) Food Vehicle
Causative Agent
Number of Outbreaks
Leafy greens Sprouted seeds Watermelon Tomatoes Baby spinach
Salmonella spp. Salmonella spp.
7 11
438 521
29/0 76/1
Salmonella Newport Salmonella Strathcona Salmonella Java and others Salmonella spp. (S. Enteritidis, S. Newport, S. Napoli, S. Java) Salmonella spp. (S. Weltevreden, S. Bareilly, S. Kottbus) Salmonella spp. (S. Weltevreden, S. Stanley, S. Bovismorbificans) S. Newport
1 1 2
17 43 189
8/0 0/0 0/0
5
249
31/0
4
275
35/1
4
114
3/0
2
126
35/0
E. coli O104:H4
1
3830
Bacillus spp.
7
343
0/0
Bacillus cereus S. sonnei Shigella sonnei Yersinia pseudotuberculosis Clostridium perfringens
1 1 1 1
2 46 145 50
0/0 4/0 5/0 10/0
1
2
NR/NR
S. aureus
1
42
NR/NR
Lettuce
Bean sprouts
Alfalfa sprouts
Mung bean sprouts Fenugreek seed sprouts Spices and herbs Lettuce Fresh basil Carrots Carrots Mixed fresh herbs Bean sprouts
Human Cases
Hospitalizations/ Deaths
2381/54
Data obtained from EFSA (2013).
the most prevalent strain mostly associated with leafy greens, especially lettuce, and sprouts. Lettuce was the food vehicle most commonly linked to E. coliassociated outbreaks in the United States, causing eight of the 46 total E. coli outbreaks between 2004 and 2012 (Callejo´n et al., 2015). The high percentage of enterohemorrhagic E. coli (EHEC) and Salmonella-associated outbreaks in the United States during the last decade can partly be explained by the increased
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Table 9.2 Summary of Strong Evidence Foodborne Outbreaks Associated with Pathogenic Bacteria in Fresh Fruits and Vegetables in Selected Multistate Outbreaks in the United States (2006 2015) Number of Outbreaks
Human Cases
Hospitalizations/ Deaths
Salmonella spp. (S. Newport, S. Saintpaul S. Poona) Salmonella spp. (S. Saintpaul S. Newport, S. serotype I 4, S. Enteritidis) Salmonella Enteritidis Salmonella Typhimurium Salmonella Saintpaul Salmonella Braenderup Salmonella spp. (S. Typhimurium S. Litchfield) Salmonella Agona Salmonella Typhi
3
1196
230/1
4
444
50/0
1
115
28/0
1
183
22/0
1
1442
286/2
1
127
33/0
2
212
110/3
1 1
106 9
10/0 7/0
E. coli O157:H7
1
33
7/0
E. coli O157:H7
1
33
13/0
E. coli O121, E. coli O26 E. coli O157:H7, E. coli O145 E. coli O157:H7 L. monocytogenes
2
48
16/0
2
84
45/0
1 1
199 5
102/3 5/2
L. monocytogenes L. monocytogenes
1 1
35 147
34/7 NR/33
Food Vehicle
Causative Agent
Cucumber
Alfalfa sprouts
Bean sprouts Tomatoes Jalapeno and serrano peppers Mangoes Cantaloupes
Papayas Frozen mamey fruit pulp Ready-to-eat salads Spinach and spring mix blend Raw clover sprouts Romaine lettuce Fresh spinach Mung bean sprout Caramel apples Cantaloupes
Data obtained from CDC (2015).
Attachment, Internalization, and Biofilm Development
prevalence of these pathogens in leafy green vegetables between 2007 and 2013 in California, where more than 70% of the US total leafy green vegetable production is done (Karp et al., 2015). The EHEC prevalence in leafy greens in California was found to increase from ,0.1% of samples in 2007 to 2.5% in 2013. The worst fresh-produce-associated outbreak case during the last decade was the large outbreak of hemolytic-uremic syndrome (HUS) caused by Shiga-toxin-producing E. coli O104:H4 centered in Germany in 2011, due to the consumption of contaminated fenugreek seed sprouts (Grad et al., 2012). As listed in Table 9.1, infecting nearly 4000 persons, mainly in Germany, it caused more than 2300 cases of hospitalization, more than 900 cases of HUS, and resulted in 54 deaths (EFSA, 2011a,b; Grad et al., 2012). In fact, this outbreak is a clear example of how global food trade can result in the spreading of a contaminated product between countries resulting in high numbers of people being affected. Listeria outbreaks linked to fresh produce are not as frequent as Salmonellaand E. coli-associated outbreaks and mainly affects vulnerable groups such as pregnant women, the elderly, infants, and immunocompromised individuals. Although rare, it is an important cause of human illness, especially in the United States. In fact, although L. monocytogenes was identified as the causative agent in only three multistate outbreaks in the United States during the last decade, it was responsible for the highest death rate in fresh-produce-associated outbreaks (Table 9.2). The multistate outbreak of listeriosis linked to whole cantaloupes affected 147 persons in 28 states and resulted in 30 deaths and one miscarriage (Laksanalamai et al., 2012). In general, Bacillus spp., Shigella spp., Yersinia spp., Clostridium spp., and S. aureus were associated with higher numbers of fresh-produce-linked outbreaks in Europe than in the United States (Tables 9.1 and 9.2) (Callejo´n et al., 2015). Bacillus spp. were linked to eight outbreaks, in seven of which spices and herbs were the food vehicle and only one was linked to lettuce. The rest of these pathogenic bacteria were associated only with one or two specific outbreak cases with much lower reported human cases.
ATTACHMENT, INTERNALIZATION, AND BIOFILM DEVELOPMENT BY FOODBORNE PATHOGENIC BACTERIA ON FRESH-CUT FRUIT AND VEGETABLES The increase in the numbers of Salmonella- and E. coli O157:H7-associated outbreaks involving fresh produce during the last decade increased the interest in highlighting the mechanisms of interactions of these pathogens with fresh produce. Many studies revealed that both Salmonella and E. coli O157:H7 can attach to the surface and internalize within the tissue of a variety of fresh produce. The extent of attachment and internalization depend on many factors including
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the route of contamination and internalization, the nature of the epidermis, tissue pH, the growth phase of the plant, as well as extrinsic factors imposed by the environment of the plant (Beuchat, 2002; Erickson et al., 2010a; Golberg et al., 2011; Zheng et al., 2013). Both endophytic and epiphytic colonization have been reported for foodborne pathogenic bacteria in fresh produce. The mechanisms of attachment for epiphytic colonization mainly include curli, fimbriae, flagella, and biofilm formation (Kroupitski et al., 2013; Lee et al., 2015; Neal et al., 2012; Rossez et al., 2014; Salazar et al., 2013). Fig. 9.2 summarizes the molecular mechanisms facilitating the attachment and colonization of leafy green vegetables by enteric bacterial pathogens (Martı´nez-Vaz et al., 2014). The first step in the contamination of fresh produce, that is, the initial attachment of pathogens to the pyhllosphere or rhizosphere tissue, includes cell surface structures (curli, fimbriae, and flagella), followed by biofilm formation. Functional genomic studies revealed that the genes involved in cell surface structures, virulence, motility, and biofilm formation mediate the plant bacteria interactions. The attachment of E. coli O157:H7 to leafy greens involves multiple mechanisms influenced by the leaf properties, damage to the leaf, and water hardness (Lee et al., 2015). Compared with cellulose-deficient cells, cellulose producing E. coli O157:H7 attach more to lettuce surface, but not to spinach. The level of colanic-acid-producing cells changes depending on the water hardness, being greater in higher water hardness (Lee et al., 2015). The genes involved in curli production (csgA and csgB) and those involved in biofilm
FIGURE 9.2 Molecular mechanisms mediating the colonization of leafy greens by human pathogens (Martı´nez-Vaz et al., 2014).
Attachment, Internalization, and Biofilm Development
formation (bbsA and ybiM) are upregulated in E. coli O157:H7 upon interaction with intact lettuce leaf surfaces (Fink et al., 2012). The expression of the virulance factor O-antigen in E. coli O157:H7 enhances attachment to iceberg lettuce, possibly by altering the hydrophobicity and cell surface charge (Boyer et al., 2011). Curli producing E. coli O157:H7 attach more strongly to spinach and lettuce leaf surfaces (Macarisin et al., 2012; Patel et al., 2011). On the other hand, cellulose, a constituent of extracellular matrix, was found to be dispensable for the attachment of E. coli O157:H7 to spinach (Macarisin et al., 2012). Similarly, the deletion of the curli expressing agfBA gene was more effective on the transfer and survival of S. Typhimurium on parsley, compared to the deletion of cellulose expressing bcsA gene (Lapidot and Yaron, 2009). Moreover, the putative stress regulatory gene, ycfR, and the sirA gene involved in biofilm formation in S. Enterica are essential for attachment to intact spinach leaf and grape tomatoes, and survival during postharvest minimal processing (Salazar et al., 2013). Several studies showed that enteric pathogens interact with salad leaves by mechanisms that frequently involve flagella. Flagellum is the major adhesin linking enterotoxigenic E. coli (Shaw et al., 2011) and EHEC (Xicohtencatl-Cortes et al., 2009) to lettuce, basil, and spinach leaves. Moreover, in enteroaggregative E. coli, the binding to the guard cell of stomata is flagella-mediated, whereas AFF (aggregative adherence fimbriae) pilus mediates the binding to the epidermis (Berger et al., 2009b). In fact, Salmonella serovars use strain-specific mechanisms to attach to salad leaves. Unlike S. Typhimurium, the attachment of S. Seftenberg to basil, lettuce, rocket, and spinach leaves is mainly mediated via flagella (Berger et al., 2009a). The occurrence of naturally formed microbial biofilms on fresh produce (alfalfa, clover, broccoli, sunflower sprouts, lettuce) have been demonstrated in previous studies (Fig. 9.3) (Fett, 2000; Olmez and Temur, 2010; Rayner et al., 2004).
FIGURE 9.3 Scanning electron micrograph images of natural biofilms of mixed microbial populations on the surface of raw lettuce leaves stored overnight below 10 C after harvest (Olmez and Temur, 2010).
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FIGURE 9.4 Scanning electron micrographs of stomata on baby spinach leaves inoculated with E. coli O157:H7 and stored at 4 C for 0 h (A), 24 h (B), 48 h (C), or 72 h (D). Intrusion and development of cell populations peripheral to and within the stomatal wells are indicated by arrows (Niemira and Cooke, 2010).
Artificially inoculated foodborne pathogenic bacteria have also been shown to form biofilms on fresh produce surfaces (Fig. 9.4) (Annous et al., 2005; Lapidot et al., 2006; Niemira and Cooke, 2010; Olmez and Temur, 2010). The biofilm-forming ability of bacterial pathogens is highly correlated with the attachment strength and persistence on the fresh produce surface (Kroupitski et al., 2009b; Patel et al., 2013). Solomon et al. (2005) found that curli- and cellulose-deficient S. Enterica isolates are low biofilm formers, supporting the correlation between attachment strength and biofilm formation. The endophytic colonization occurs through natural openings (stomata), damaged tissue of rhizosphere or phyllosphere, and chemotaxis to metabolites found in plant exudates or within the plant (Fig. 9.4) (Bartz et al., 2015; Critzer
Challenges in Fresh-Cut Fruit and Vegetable Safety
and Doyle, 2010; Erickson et al., 2010a,b; Golberg et al., 2011; Klerks et al., 2007; Kroupitski et al., 2009a; Neal et al., 2012). Different mechanisms are responsible for the internalization of pathogens within the plant tissue during the pre- and postharvest stages. At the preharvest level, the serovar cultivar interaction has a significant effect on the type of colonization, whether it will be epiphytic or endophytic, as well as on the level of colonization. Chemotaxis studies revealed that S. enterica serovars actively move toward root exudates of a specific lettuce cultivar (Tamburo), as the genes involved in chemotaxis and virulence are activated by the root exudates of this cultivar (Klerks et al., 2007). They also reported that among the five Salmonella enterica serovars only S. enterica serovar Dublin was able to colonize endophytically. The method, duration, and direction of irrigation have an important effect on the degree of internalization and persistence. Internalization of E. coli O157:H7 accompanied with higher persistence occurred in lettuce leaves spray-irrigated on the abaxial side compared to the leaves irrigated on the adaxial sides (Erickson et al., 2010b). At the postharvest level, washing, vacuum cooling, and hydrocooling processes can present an opportunity for the internalization of pathogenic bacteria at various infiltration sites (stomata, stem scar, cut edges, etc.) (Li et al., 2008). Infiltration is enhanced when using water cooler than the produce, applying long immersion periods, using surfactants, and with the presence of cut edges and damaged tissue (Beuchat, 2002; Bordini et al., 2007; Eblen et al., 2004; Erickson, 2012; Fatemi et al., 2006; Janes et al., 2005). The incidence of Salmonella internalization varies highly depending on the type of leafy greens, being highest in iceberg lettuce and arugula leaves, moderate in basil and lettuce, and lowest in parsley and tomato leaves (Golberg et al., 2011).
CHALLENGES IN FRESH-CUT FRUIT AND VEGETABLE SAFETY Pathogenic bacteria associated with fresh-cut fruits and vegetables are able to proliferate to high numbers once deposited onto the injured parts of fruits and vegetables. E. coli O157:H7 was shown to grow 11-fold over 4 h of incubation after inoculation onto cut lettuce stems (Brandl, 2008). Harvesting and postharvest operation may cause significant damage to the tissue of fruits and vegetables, which can promote the growth of pathogenic bacteria. Moreover, microarraybased whole-genome transcriptional profiling studies revealed that genes involved in E. coli O157:H7 attachment, virulence, DNA repair, resistance to oxidative stress, and resistance to antimicrobials are upregulated when the cells are exposed to the exudates of the damaged plant tissue (Kyle et al., 2010). The enhancement of DNA repair ability, resistance to oxidative stress and antimicrobials also increase the ability of the enteric pathogens to evade the current sanitization treatments, as the fresh-cut industry mainly relies on the use of oxidative agents for the sanitization of produce and wash waters (Martı´nez-Vaz et al., 2014).
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This may be one of the main reasons for increasing numbers of enteric pathogenic bacteria-associated outbreaks involving fresh-cut produce and represents a critical challenge for the fresh produce industry. Attachment of bacterial cells to inaccessible sites, like stomata and cavities of the leaf, are also likely responsible for the inefficacy of sanitizing treatments and represents another challenge for the fresh-cut industry since none of the current methods is able to reach subsurface cells. Therefore, there is a need for the development of produce and wash water sanitization methods able to inactivate internalized bacteria and which are based on inactivation mechanisms that can overcome the issue of increased resistance to oxidative stress and improved DNA repair ability.
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Fink, R.C., Black, E.P., Hou, Z., Sugawara, M., Sadowsky, M.J., Diez-Gonzalez, F., 2012. Transcriptional responses of Escherichia coli K-12 and O157:H7 associated with lettuce leaves. Appl. Environ. Microbiol. 78, 1752 1764. Fonseca, J., Fallon, S., Sanchez, C., Nolte, K., 2011. Escherichia coli survival in lettuce fields following its introduction through different irrigation systems. J. Appl. Microbiol. 110, 893 902. Golberg, D., Kroupitski, Y., Belausov, E., Pinto, R., Sela, S., 2011. Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs. Int. J. Food. Microbiol. 145, 250 257. Grad, Y.H., Lipsitch, M., Feldgarden, M., Arachchi, H.M., Cerqueira, G.C., FitzGerald, M., et al., 2012. Genomic epidemiology of the Escherichia coli O104: H4 outbreaks in Europe, 2011. Proc. Natl. Academy Sci. 109, 3065 3070. Holley, R.A., Arrus, K.M., Ominski, K.H., Tenuta, M., Blank, G., 2006. Survival in manure-treated soils during simulated seasonal temperature exposure. J. Environ. Qual. 35, 1170 1180. Janes, M.E., Kim, K., Johnson, M., 2005. Transmission electron microscopy study of enterohemorrhagic Escherichia coli O157: H7 in apple tissue. J. Food. Prot. 68, 216 224. Karp, D.S., Gennet, S., Kilonzo, C., Partyka, M., Chaumont, N., Atwill, E.R., et al., 2015. Comanaging fresh produce for nature conservation and food safety. Proc. Natl. Academy Sci. 112, 11126 11131. Klerks, M.M., Franz, E., van Gent-Pelzer, M., Zijlstra, C., van Bruggen, A.H., 2007. Differential interaction of Salmonella enterica serovars with lettuce cultivars and plant-microbe factors influencing the colonization efficiency. ISME J. 1, 620 631. Kroupitski, Y., Brandi, M.T., Pinto, R., Belausov, E., Tamir-Ariel, D., Burdman, S., et al., 2013. Identification of Salmonella enterica genes with a role in persistence on lettuce leaves during cold storage by recombinase-based in vivo expression technology. Phytopathology 103, 362 372. Kroupitski, Y., Golberg, D., Belausov, E., Pinto, R., Swartzberg, D., Granot, D., et al., 2009a. Internalization of Salmonella enterica in leaves is induced by light and involves chemotaxis and penetration through open stomata. Appl. Environ. Microbiol. 75, 6076 6086. Kroupitski, Y., Pinto, R., Brandl, M., Belausov, E., Sela, S., 2009b. Interactions of Salmonella enterica with lettuce leaves. J. Appl. Microbiol. 106, 1876 1885. Kyle, J.L., Parker, C.T., Goudeau, D., Brandl, M.T., 2010. Transcriptome analysis of Escherichia coli O157: H7 exposed to lysates of lettuce leaves. Appl. Environ. Microbiol. 76, 1375 1387. Laksanalamai, P., Joseph, L.A., Silk, B.J., Burall, L.S., Tarr, C.L., Gerner-Smidt, P., et al., 2012. Genomic characterization of Listeria monocytogenes strains involved in a multistate listeriosis outbreak associated with cantaloupe in US. PLoS One 7, e42448. Lang, N., Smith, S., 2007. Influence of soil type, moisture content and biosolids application on the fate of Escherichia coli in agricultural soil under controlled laboratory conditions. J. Appl. Microbiol. 103, 2122 2131. Lapidot, A., Romling, U., Yaron, S., 2006. Biofilm formation and the survival of Salmonella Typhimurium on parsley. Int. J. Food. Microbiol. 109, 229 233. Lapidot, A., Yaron, S., 2009. Transfer of Salmonella enterica Serovar Typhimurium from contaminated irrigation water to parsley is dependent on curli and cellulose, the biofilm matrix components. J. Food. Prot. 72, 618 623.
References
Lee, C.-C., Chen, J., Frank, J.F., 2015. Role of cellulose and colanic acid in attachment of shiga toxin producing Escherichia coli to lettuce and spinach in different water hardness environments. J. Food. Prot. 78, 1461 1466. Li, H., Tajkarimi, M., Osburn, B.I., 2008. Impact of vacuum cooling on Escherichia coli O157: H7 infiltration into lettuce tissue. Appl. Environ. Microbiol. 74, 3138 3142. Macarisin, D., Patel, J., Bauchan, G., Giron, J.A., Sharma, V.K., 2012. Role of curli and cellulose expression in adherence of Escherichia coli O157:H7 to spinach leaves. Foodborne Pathog. Dis. 9, 160 167. Martı´nez-Vaz, B.M., Fink, R.C., Diez-Gonzalez, F., Sadowsky, M.J., 2014. Enteric pathogen plant interactions: molecular connections leading to colonization and growth and implications for food safety. Microbes Environ. 29, 123. Matthews, K.R., 2013. Sources of enteric pathogen contamination of fruits and vegetables: future directions of research. Stewart Postharvest Review 9, 1 5. Munnoch, S., Ward, K., Sheridan, S., Fitzsimmons, G., Shadbolt, C., Piispanen, J., et al., 2009. A multi-state outbreak of Salmonella Saintpaul in Australia associated with cantaloupe consumption. Epidemiol. Infect. 137, 367 374. Neal, J., Cabrera-Diaz, E., Castillo, A., 2012. Attachment of Escherichia coli O157: H7 and Salmonella on spinach (Spinacia oleracea) using confocal microscopy. Agric. Food Anal. Bacteriol. 2, 275 279. Nicholson, F.A., Groves, S.J., Chambers, B.J., 2005. Pathogen survival during livestock manure storage and following land application. Bioresour. Technol. 96, 135 143. Niemira, B.A., Cooke, P.H., 2010. Escherichia coli O157:H7 biofilm formation on romaine lettuce and spinach leaf surfaces reduces efficacy of irradiation and sodium hypochlorite washes. J. Food. Sci. 75, M270 M277. Olmez, H., Temur, S.D., 2010. Effects of different sanitizing treatments on biofilms and attachment of Escherichia coli and Listeria monocytogenes on green leaf lettuce. LWT—Food Sci. Technol. 43, 964 970. Painter, J.A., Hoekstra, R.M., Ayers, T., Tauxe, R.V., Braden, C.R., Angulo, F.J., et al., 2013. Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data, United States, 1998 2008. Emerg. Infect. Dis. 19, 407 415. Park, S., Szonyi, B., Gautam, R., Nightingale, K., Anciso, J., Ivanek, R., 2012. Risk factors for microbial contamination in fruits and vegetables at the preharvest level: a systematic review. J. Food. Prot. 75, 2055 2081. Patel, J., Sharma, M., Ravishakar, S., 2011. Effect of curli expression and hydrophobicity of Escherichia coli O157:H7 on attachment to fresh produce surfaces. J. Appl. Microbiol. 110, 737 745. Patel, J., Singh, M., Macarisin, D., Sharma, M., Shelton, D., 2013. Differences in biofilm formation of produce and poultry Salmonella enterica isolates and their persistence on spinach plants. Food Microbiol. 36, 388 394. Pe´rez-Rodrı´guez, F., Saiz-Abajo, M.J., Garcia-Gimeno, R.M., Moreno, A., Gonza´lez, D., Vitas, A.I., 2014. Quantitative assessment of the Salmonella distribution on fresh-cut leafy vegetables due to cross-contamination occurred in an industrial process simulated at laboratory scale. Int. J. Food. Microbiol. 184, 86 91. Rayner, J., Veeh, R., Flood, J., 2004. Prevalence of microbial biofilms on selected fresh produce and household surfaces. Int. J. Food. Microbiol. 95, 29 39.
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Rossez, Y., Holmes, A., Wolfson, E.B., Gally, D.L., Mahajan, A., Pedersen, H.L., et al., 2014. Flagella interact with ionic plant lipids to mediate adherence of pathogenic Escherichia coli to fresh produce plants. Environ. Microbiol. 16, 2181 2195. Salazar, J.K., Deng, K., Tortorello, M.L., Brandl, M.T., Wang, H., Zhang, W., 2013. Genes ycfR, sirA and yigG contribute to the surface attachment of Salmonella enterica Typhimurium and Saintpaul to fresh produce. PLoS One. 8, e57272. Schikora, A., Carreri, A., Charpentier, E., Hirt, H., 2008. The dark side of the salad: Salmonella Typhimurium overcomes the innate immune response of Arabidopsis thaliana and shows an endopathogenic lifestyle. PLoS One. 3, e2279. Semenov, A.V., Van Bruggen, A.H., Van Overbeek, L., Termorshuizen, A.J., Semenov, A. M., 2007. Influence of temperature fluctuations on Escherichia coli O157: H7 and Salmonella enterica serovar Typhimurium in cow manure. FEMS. Microbiol. Ecol. 60, 419 428. Shaw, R.K., Berger, C.N., Pallen, M.J., Sjoeling, A., Frankel, G., 2011. Flagella mediate attachment of enterotoxigenic Escherichia coli to fresh salad leaves. Environ. Microbiol. Rep. 3, 112 117. Sivapalasingam, S., Friedman, C.R., Cohen, L., Tauxe, R.V., 2004. Fresh produce: a growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. J. Food. Prot. 67, 2342 2353. Solomon, E.B., Niemira, B.A., Sapers, G.M., Annous, B.A., 2005. Biofilm formation, cellulose production, and curli biosynthesis by Salmonella originating from produce, animal, and clinical sources. J. Food. Prot. 68, 906 912. Solomon, E.B., Pang, H.-J., Matthews, K.R., 2003. Persistence of Escherichia coli O157: H7 on lettuce plants following spray irrigation with contaminated water. J. Food. Prot. 66, 2198 2202. Todd, E.C., Greig, J.D., Bartleson, C.A., Michaels, B.S., 2008. Outbreaks where food workers have been implicated in the spread of foodborne disease. Part 5. Sources of contamination and pathogen excretion from infected persons. J. Food. Prot. 71, 2582 2595. Van der Linden, I., Cottyn, B., Uyttendaele, M., Vlaemynck, G., Heyndrickx, M., Maes, M., 2013. Survival of enteric pathogens during butterhead lettuce growth: crop stage, leaf age, and irrigation. Foodborne. Pathog. Dis. 10, 485 491. Warriner, K., Huber, A., Namvar, A., Fan, W., Dunfield, K., 2009. Recent advances in the microbial safety of fresh fruits and vegetables. Adv. Food. Nutr. Res. 57, 155 208. Werner, S., Boman, K., Einemo, I., Erntell, M., De Jong, B., Lindqvist, A., et al., 2007. Outbreak of Salmonella Stanley in Sweden associated with alfalfa sprouts, July August 2007. Euro. Surveill. 12 (42), pii. 3291. Xicohtencatl-Cortes, J., Sa´nchez Chaco´n, E., Saldan˜a, Z., Freer, E., Giro´n, J.A., 2009. Interaction of Escherichia coli O157: H7 with leafy green produce. J. Food. Prot. 72, 1531 1537. Zheng, J., Allard, S., Reynolds, S., Millner, P., Arce, G., Blodgett, R.J., et al., 2013. Colonization and internalization of Salmonella enterica in tomato plants. Appl. Environ. Microbiol. 79, 2494 2502.
Foodborne pathogenic bacteria in fresh-cut vegetables and fruits
9 Hu¨lya O¨lmez
¨ ITAK ˙ TUB MRC Material Institute, Gebze, Kocaeli, Turkey
INTRODUCTION Driven mainly by the increasing consumer demands for fresh and healthier convenience foods, the fresh produce market has grown dramatically over the last two decades. Following the increase in consumption, on the other hand, the foodborne outbreaks associated with fresh produce have also shown a raising trend globally (CSPI, 2014; EFSA, 2011a). The proportion of fresh-produceassociated foodborne illnesses increased from 12% in the 1990s to 24% in the 2010s (Painter et al., 2013; Sivapalasingam et al., 2004). Three main factors may add to the increase in the number of reported illnesses. First, the globalization of food industry has definitely resulted in the spreading of foodborne outbreaks between countries and even continents, resulting in a higher number of people being affected by the same contaminated produce. Second, the changes in the agricultural and processing practices throughout the food chain which aimed at increasing the supply may have enhanced the risk for transmission of pathogens and cross-contamination. Third, the improvements in the methods of microbial detection and identification, as well as in surveillance techniques, have also contributed to the increase in the number of reported illnesses. Fresh produce was reported to be one of the main vehicles of foodborne outbreaks in Europe, accounting for 8% of all the foodborne outbreaks in 2011 (EFSA, 2011a). In the United States, the produce category was linked to the largest number of foodborne illnesses associated with foodborne outbreaks between the periods of 1998 2007 and 2002 2011, constituting 23% and 24% of all illnesses, respectively (CSPI, 2011). The produce category surpassed the poultry, beef, and seafood categories, accounting for 667 outbreaks and 23,748 illnesses, most of which were associated with vegetables (10,806) (CSPI, 2014). The most commonly identified causative agents were Salmonella spp., norovirus, and pathogenic Escherichia coli (Painter et al., 2013). The main reason for having fresh-cut fruits and vegetables as one of the major vehicles of foodborne P. Kotzekidou (Ed): Food Hygiene and Toxicology in Ready-to-Eat Foods. DOI: http://dx.doi.org/10.1016/B978-0-12-801916-0.00009-1 © 2016 Elsevier Inc. All rights reserved.
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outbreaks associated with foodborne pathogens is that they are often consumed raw after minimal processing. There is no kill step in the processing which can assure the elimination of the pathogenic bacteria associated with fresh produce. Once contaminated in the field, it is not possible to completely eliminate the pathogenic bacteria from the fresh-cut produce. Therefore, identification of the main sources of contamination and transmission routes of pathogenic bacteria to fresh produce is a key for managing the food safety risks associated with fresh produce.
SOURCES AND ROUTES OF PATHOGEN CONTAMINATION IN FRESH-CUT PRODUCE Fresh produce may be contaminated with pathogenic bacteria via different routes at different points throughout the preharvest and harvest/postharvest stages (Fig. 9.1). Current contamination sources in the field include manure, irrigation water, soil, fecal contamination, contaminated seeds, geographical location, and climate change. The postharvest steps including the harvesting itself, precooling, processing operations, washing and sanitizing, storage packaging, and transfer
Sources of fresh produce contamination
Preharvest sources
• Soil and manure • Irrigation water and method of irrigation
• Water used as a carrier of • • • • •
pesticides and hormones Air (dust) Fecal contamination Contaminated seeds Climate change Geographical location
FIGURE 9.1 Main sources of fresh produce contamination.
Harvest/postharvest sources • Harvesting equipment • Human handling • Air (dust) • Cooling water • Storage • Sorting • Unit operations (coring, cutting, peeling, etc.) • Washing/sanitizing • Packaging • Transfer
Sources and Routes of Pathogen Contamination in Fresh-Cut Produce
may lead to pathogen contamination if not controlled properly according to the good manufacturing practices. Irrigation water and the method of irrigation have been identified as one of the main sources of contamination (Park et al., 2012). The use of contaminated surface water or untreated sewage water results in high numbers of fecal coliforms and leads to the transmission of pathogenic bacteria to the fresh produce. Due to the direct deposition of the contaminated water onto the edible parts of the produce, spray irrigation systems pose a higher risk of pathogenic bacteria contamination compared to other irrigation methods such as surface irrigation and drip irrigation (Aruscavage et al., 2006; Fonseca et al., 2011; Van der Linden et al., 2013). Studies showed that E. coli O157:H7 can persist for 2 20 days on spray-irrigated lettuce leaves (Erickson et al., 2014; Solomon et al., 2003). Moreover, the repeated spray irrigation may lead to the internalization of E. coli O157:H7 in leafy vegetables such as spinach and parsley (Erickson et al., 2014). Animal manure used as a soil amendment also represents a source of pathogenic bacteria contamination to fresh produce. The pathogenic bacteria including E. coli O157:H7, Salmonella, Listeria monocytogenes, and Campylobacter jejuni can survive for 45 175 days in soil depending on the soil characteristics (salinity, total nitrogen, etc.), moisture level, and temperature (Holley et al., 2006; Lang and Smith, 2007; Nicholson et al., 2005). Moreover, even with very low levels of contamination (100 101 CFU/g), the pathogenic bacteria are also able to survive for long periods in animal manure, depending on the temperature (Beuchat, 1999). The rate of survival is higher at lower temperatures. E. coli O157:H7 can survive in bovine feces for 70 days at 5 C, but only 49 days at 22 C (Semenov et al., 2007). Therefore, manure that has not been properly treated when used as a soil amendment may serve as a source of pathogen transmission to the fresh produce. As the soil type, moisture level, and temperature are critical factors for the survival of enteric pathogens in soil and manure, the geographical location and the climate change also appear to be important preharvest factors for fresh fruit and vegetable contamination. Moreover, higher intensity of precipitation and rain may increase the potential of pathogenic bacteria contamination to fresh produce by enhancing the survival of the pathogens in soil and manure (Cevallos-Cevallos et al., 2012; Warriner et al., 2009). Harvesting and postharvest practices can serve as a source of contamination and may enhance both the survival and growth of the pathogenic bacteria on fresh fruits and vegetables. Contaminated harvesting equipment, containers and poor hygiene of farm workers are important factors that influence the cross-contamination of fresh produce (Buchholz et al., 2012; Matthews, 2013; Todd et al., 2008). The postharvest unit operations (coring, cutting, slicing, peeling, shredding) and even the washing/sanitizing steps cause tissue damage and render the produce more prone to bacterial contamination. The destruction of tissue and cells results
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CHAPTER 9 Bacterial pathogens in fresh produce
in the release of nutrient reach exudates which facilitates both the attachment and the rapid proliferation of pathogenic bacteria. The promotion of the rapid growth of E. coli O157:H7 as a result of tissue damage in lettuce has been shown by Brandl (2008). Cut leaf surfaces represent a specific target for the attachment of Salmonella cells (Kroupitski et al., 2009b). The use of inadequately sanitized water for cooling or washing/sanitizing operations can represent a source of crosscontamination and can serve as the primary vehicle for the spread of pathogenic bacteria during fresh produce processing (Pe´rez-Rodrı´guez et al., 2014).
FOODBORNE OUTBREAKS ASSOCIATED WITH PATHOGENIC BACTERIA IN FRESH-CUT FRUITS AND VEGETABLES Summaries of the foodborne outbreaks associated with pathogenic bacteria in fresh fruits and vegetables in Europe (2007 2011) and in the United States (2006 2015) are given in Tables 9.1 and 9.2, respectively. In both regions, most of the fresh-produce-associated outbreaks were linked with Salmonella spp., followed by pathogenic E. coli. In fact, in all the fresh-produce-associated foodborne outbreaks viruses rank first followed by Salmonella and E. coli O157:H7 as the causative agent for both regions (CDC, 2015; EFSA, 2013). The other pathogenic bacteria identified as the causative agent in fresh-produce-associated outbreaks in the given periods include Shigella spp., Yersinia spp., Bacillus spp., Clostridium perfringens, and Staphylococcus aureus. Sprouted seeds and leafy greens were the main food vehicles in most of the fresh-produce-associated outbreaks. The use of contaminated seeds was determined to be the major cause of sprout-associated outbreaks (EFSA, 2011a; Emberland et al., 2007; Werner et al., 2007). In the reported periods, Salmonella was the most common bacterial agent, accounting for 71% of the outbreaks associated with bacteria in Europe, and 60% in the United States (Tables 9.1 and 9.2). Moreover, 86% of the human cases in the selected multistate outbreaks in the United States and 31% of the human cases in Europe were linked to Salmonella contamination. The multistate outbreak associated with Salmonella Saintpaul contaminated cantaloupes (Munnoch et al., 2009) and Salmonella spp. contaminated alfalfa sprouts (Compton et al., 2008) in Australia are other examples of Salmonella spp. linked fresh produce outbreaks with more than 200 human cases. A wide variety of fresh produce has been associated with Salmonella-linked outbreaks, sprouts, leafy greens, cucumbers, and melons being the major ones. The higher ratio of Salmonella-associated fresh produce outbreaks is partly linked to the ability of Salmonella to internalize within the plant and proliferate to high numbers under favorable conditions (Golberg et al., 2011; Schikora et al., 2008). Pathogenic E. coli was the second major causative agent of the bacteriaassociated outbreaks mainly linked to the consumption of contaminated sprouts and leafy greens for the last decade (Tables 9.1 and 9.2). E. coli O157:H7 was
Foodborne Outbreaks Associated with Pathogenic Bacteria
Table 9.1 Summary of Strong Evidence Foodborne Outbreaks Associated with Pathogenic Bacteria in Fresh Fruits and Vegetables in Europe (2007 2011) Food Vehicle
Causative Agent
Number of Outbreaks
Leafy greens Sprouted seeds Watermelon Tomatoes Baby spinach
Salmonella spp. Salmonella spp.
7 11
438 521
29/0 76/1
Salmonella Newport Salmonella Strathcona Salmonella Java and others Salmonella spp. (S. Enteritidis, S. Newport, S. Napoli, S. Java) Salmonella spp. (S. Weltevreden, S. Bareilly, S. Kottbus) Salmonella spp. (S. Weltevreden, S. Stanley, S. Bovismorbificans) S. Newport
1 1 2
17 43 189
8/0 0/0 0/0
5
249
31/0
4
275
35/1
4
114
3/0
2
126
35/0
E. coli O104:H4
1
3830
Bacillus spp.
7
343
0/0
Bacillus cereus S. sonnei Shigella sonnei Yersinia pseudotuberculosis Clostridium perfringens
1 1 1 1
2 46 145 50
0/0 4/0 5/0 10/0
1
2
NR/NR
S. aureus
1
42
NR/NR
Lettuce
Bean sprouts
Alfalfa sprouts
Mung bean sprouts Fenugreek seed sprouts Spices and herbs Lettuce Fresh basil Carrots Carrots Mixed fresh herbs Bean sprouts
Human Cases
Hospitalizations/ Deaths
2381/54
Data obtained from EFSA (2013).
the most prevalent strain mostly associated with leafy greens, especially lettuce, and sprouts. Lettuce was the food vehicle most commonly linked to E. coliassociated outbreaks in the United States, causing eight of the 46 total E. coli outbreaks between 2004 and 2012 (Callejo´n et al., 2015). The high percentage of enterohemorrhagic E. coli (EHEC) and Salmonella-associated outbreaks in the United States during the last decade can partly be explained by the increased
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CHAPTER 9 Bacterial pathogens in fresh produce
Table 9.2 Summary of Strong Evidence Foodborne Outbreaks Associated with Pathogenic Bacteria in Fresh Fruits and Vegetables in Selected Multistate Outbreaks in the United States (2006 2015) Number of Outbreaks
Human Cases
Hospitalizations/ Deaths
Salmonella spp. (S. Newport, S. Saintpaul S. Poona) Salmonella spp. (S. Saintpaul S. Newport, S. serotype I 4, S. Enteritidis) Salmonella Enteritidis Salmonella Typhimurium Salmonella Saintpaul Salmonella Braenderup Salmonella spp. (S. Typhimurium S. Litchfield) Salmonella Agona Salmonella Typhi
3
1196
230/1
4
444
50/0
1
115
28/0
1
183
22/0
1
1442
286/2
1
127
33/0
2
212
110/3
1 1
106 9
10/0 7/0
E. coli O157:H7
1
33
7/0
E. coli O157:H7
1
33
13/0
E. coli O121, E. coli O26 E. coli O157:H7, E. coli O145 E. coli O157:H7 L. monocytogenes
2
48
16/0
2
84
45/0
1 1
199 5
102/3 5/2
L. monocytogenes L. monocytogenes
1 1
35 147
34/7 NR/33
Food Vehicle
Causative Agent
Cucumber
Alfalfa sprouts
Bean sprouts Tomatoes Jalapeno and serrano peppers Mangoes Cantaloupes
Papayas Frozen mamey fruit pulp Ready-to-eat salads Spinach and spring mix blend Raw clover sprouts Romaine lettuce Fresh spinach Mung bean sprout Caramel apples Cantaloupes
Data obtained from CDC (2015).
Attachment, Internalization, and Biofilm Development
prevalence of these pathogens in leafy green vegetables between 2007 and 2013 in California, where more than 70% of the US total leafy green vegetable production is done (Karp et al., 2015). The EHEC prevalence in leafy greens in California was found to increase from ,0.1% of samples in 2007 to 2.5% in 2013. The worst fresh-produce-associated outbreak case during the last decade was the large outbreak of hemolytic-uremic syndrome (HUS) caused by Shiga-toxin-producing E. coli O104:H4 centered in Germany in 2011, due to the consumption of contaminated fenugreek seed sprouts (Grad et al., 2012). As listed in Table 9.1, infecting nearly 4000 persons, mainly in Germany, it caused more than 2300 cases of hospitalization, more than 900 cases of HUS, and resulted in 54 deaths (EFSA, 2011a,b; Grad et al., 2012). In fact, this outbreak is a clear example of how global food trade can result in the spreading of a contaminated product between countries resulting in high numbers of people being affected. Listeria outbreaks linked to fresh produce are not as frequent as Salmonellaand E. coli-associated outbreaks and mainly affects vulnerable groups such as pregnant women, the elderly, infants, and immunocompromised individuals. Although rare, it is an important cause of human illness, especially in the United States. In fact, although L. monocytogenes was identified as the causative agent in only three multistate outbreaks in the United States during the last decade, it was responsible for the highest death rate in fresh-produce-associated outbreaks (Table 9.2). The multistate outbreak of listeriosis linked to whole cantaloupes affected 147 persons in 28 states and resulted in 30 deaths and one miscarriage (Laksanalamai et al., 2012). In general, Bacillus spp., Shigella spp., Yersinia spp., Clostridium spp., and S. aureus were associated with higher numbers of fresh-produce-linked outbreaks in Europe than in the United States (Tables 9.1 and 9.2) (Callejo´n et al., 2015). Bacillus spp. were linked to eight outbreaks, in seven of which spices and herbs were the food vehicle and only one was linked to lettuce. The rest of these pathogenic bacteria were associated only with one or two specific outbreak cases with much lower reported human cases.
ATTACHMENT, INTERNALIZATION, AND BIOFILM DEVELOPMENT BY FOODBORNE PATHOGENIC BACTERIA ON FRESH-CUT FRUIT AND VEGETABLES The increase in the numbers of Salmonella- and E. coli O157:H7-associated outbreaks involving fresh produce during the last decade increased the interest in highlighting the mechanisms of interactions of these pathogens with fresh produce. Many studies revealed that both Salmonella and E. coli O157:H7 can attach to the surface and internalize within the tissue of a variety of fresh produce. The extent of attachment and internalization depend on many factors including
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CHAPTER 9 Bacterial pathogens in fresh produce
the route of contamination and internalization, the nature of the epidermis, tissue pH, the growth phase of the plant, as well as extrinsic factors imposed by the environment of the plant (Beuchat, 2002; Erickson et al., 2010a; Golberg et al., 2011; Zheng et al., 2013). Both endophytic and epiphytic colonization have been reported for foodborne pathogenic bacteria in fresh produce. The mechanisms of attachment for epiphytic colonization mainly include curli, fimbriae, flagella, and biofilm formation (Kroupitski et al., 2013; Lee et al., 2015; Neal et al., 2012; Rossez et al., 2014; Salazar et al., 2013). Fig. 9.2 summarizes the molecular mechanisms facilitating the attachment and colonization of leafy green vegetables by enteric bacterial pathogens (Martı´nez-Vaz et al., 2014). The first step in the contamination of fresh produce, that is, the initial attachment of pathogens to the pyhllosphere or rhizosphere tissue, includes cell surface structures (curli, fimbriae, and flagella), followed by biofilm formation. Functional genomic studies revealed that the genes involved in cell surface structures, virulence, motility, and biofilm formation mediate the plant bacteria interactions. The attachment of E. coli O157:H7 to leafy greens involves multiple mechanisms influenced by the leaf properties, damage to the leaf, and water hardness (Lee et al., 2015). Compared with cellulose-deficient cells, cellulose producing E. coli O157:H7 attach more to lettuce surface, but not to spinach. The level of colanic-acid-producing cells changes depending on the water hardness, being greater in higher water hardness (Lee et al., 2015). The genes involved in curli production (csgA and csgB) and those involved in biofilm
FIGURE 9.2 Molecular mechanisms mediating the colonization of leafy greens by human pathogens (Martı´nez-Vaz et al., 2014).
Attachment, Internalization, and Biofilm Development
formation (bbsA and ybiM) are upregulated in E. coli O157:H7 upon interaction with intact lettuce leaf surfaces (Fink et al., 2012). The expression of the virulance factor O-antigen in E. coli O157:H7 enhances attachment to iceberg lettuce, possibly by altering the hydrophobicity and cell surface charge (Boyer et al., 2011). Curli producing E. coli O157:H7 attach more strongly to spinach and lettuce leaf surfaces (Macarisin et al., 2012; Patel et al., 2011). On the other hand, cellulose, a constituent of extracellular matrix, was found to be dispensable for the attachment of E. coli O157:H7 to spinach (Macarisin et al., 2012). Similarly, the deletion of the curli expressing agfBA gene was more effective on the transfer and survival of S. Typhimurium on parsley, compared to the deletion of cellulose expressing bcsA gene (Lapidot and Yaron, 2009). Moreover, the putative stress regulatory gene, ycfR, and the sirA gene involved in biofilm formation in S. Enterica are essential for attachment to intact spinach leaf and grape tomatoes, and survival during postharvest minimal processing (Salazar et al., 2013). Several studies showed that enteric pathogens interact with salad leaves by mechanisms that frequently involve flagella. Flagellum is the major adhesin linking enterotoxigenic E. coli (Shaw et al., 2011) and EHEC (Xicohtencatl-Cortes et al., 2009) to lettuce, basil, and spinach leaves. Moreover, in enteroaggregative E. coli, the binding to the guard cell of stomata is flagella-mediated, whereas AFF (aggregative adherence fimbriae) pilus mediates the binding to the epidermis (Berger et al., 2009b). In fact, Salmonella serovars use strain-specific mechanisms to attach to salad leaves. Unlike S. Typhimurium, the attachment of S. Seftenberg to basil, lettuce, rocket, and spinach leaves is mainly mediated via flagella (Berger et al., 2009a). The occurrence of naturally formed microbial biofilms on fresh produce (alfalfa, clover, broccoli, sunflower sprouts, lettuce) have been demonstrated in previous studies (Fig. 9.3) (Fett, 2000; Olmez and Temur, 2010; Rayner et al., 2004).
FIGURE 9.3 Scanning electron micrograph images of natural biofilms of mixed microbial populations on the surface of raw lettuce leaves stored overnight below 10 C after harvest (Olmez and Temur, 2010).
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CHAPTER 9 Bacterial pathogens in fresh produce
FIGURE 9.4 Scanning electron micrographs of stomata on baby spinach leaves inoculated with E. coli O157:H7 and stored at 4 C for 0 h (A), 24 h (B), 48 h (C), or 72 h (D). Intrusion and development of cell populations peripheral to and within the stomatal wells are indicated by arrows (Niemira and Cooke, 2010).
Artificially inoculated foodborne pathogenic bacteria have also been shown to form biofilms on fresh produce surfaces (Fig. 9.4) (Annous et al., 2005; Lapidot et al., 2006; Niemira and Cooke, 2010; Olmez and Temur, 2010). The biofilm-forming ability of bacterial pathogens is highly correlated with the attachment strength and persistence on the fresh produce surface (Kroupitski et al., 2009b; Patel et al., 2013). Solomon et al. (2005) found that curli- and cellulose-deficient S. Enterica isolates are low biofilm formers, supporting the correlation between attachment strength and biofilm formation. The endophytic colonization occurs through natural openings (stomata), damaged tissue of rhizosphere or phyllosphere, and chemotaxis to metabolites found in plant exudates or within the plant (Fig. 9.4) (Bartz et al., 2015; Critzer
Challenges in Fresh-Cut Fruit and Vegetable Safety
and Doyle, 2010; Erickson et al., 2010a,b; Golberg et al., 2011; Klerks et al., 2007; Kroupitski et al., 2009a; Neal et al., 2012). Different mechanisms are responsible for the internalization of pathogens within the plant tissue during the pre- and postharvest stages. At the preharvest level, the serovar cultivar interaction has a significant effect on the type of colonization, whether it will be epiphytic or endophytic, as well as on the level of colonization. Chemotaxis studies revealed that S. enterica serovars actively move toward root exudates of a specific lettuce cultivar (Tamburo), as the genes involved in chemotaxis and virulence are activated by the root exudates of this cultivar (Klerks et al., 2007). They also reported that among the five Salmonella enterica serovars only S. enterica serovar Dublin was able to colonize endophytically. The method, duration, and direction of irrigation have an important effect on the degree of internalization and persistence. Internalization of E. coli O157:H7 accompanied with higher persistence occurred in lettuce leaves spray-irrigated on the abaxial side compared to the leaves irrigated on the adaxial sides (Erickson et al., 2010b). At the postharvest level, washing, vacuum cooling, and hydrocooling processes can present an opportunity for the internalization of pathogenic bacteria at various infiltration sites (stomata, stem scar, cut edges, etc.) (Li et al., 2008). Infiltration is enhanced when using water cooler than the produce, applying long immersion periods, using surfactants, and with the presence of cut edges and damaged tissue (Beuchat, 2002; Bordini et al., 2007; Eblen et al., 2004; Erickson, 2012; Fatemi et al., 2006; Janes et al., 2005). The incidence of Salmonella internalization varies highly depending on the type of leafy greens, being highest in iceberg lettuce and arugula leaves, moderate in basil and lettuce, and lowest in parsley and tomato leaves (Golberg et al., 2011).
CHALLENGES IN FRESH-CUT FRUIT AND VEGETABLE SAFETY Pathogenic bacteria associated with fresh-cut fruits and vegetables are able to proliferate to high numbers once deposited onto the injured parts of fruits and vegetables. E. coli O157:H7 was shown to grow 11-fold over 4 h of incubation after inoculation onto cut lettuce stems (Brandl, 2008). Harvesting and postharvest operation may cause significant damage to the tissue of fruits and vegetables, which can promote the growth of pathogenic bacteria. Moreover, microarraybased whole-genome transcriptional profiling studies revealed that genes involved in E. coli O157:H7 attachment, virulence, DNA repair, resistance to oxidative stress, and resistance to antimicrobials are upregulated when the cells are exposed to the exudates of the damaged plant tissue (Kyle et al., 2010). The enhancement of DNA repair ability, resistance to oxidative stress and antimicrobials also increase the ability of the enteric pathogens to evade the current sanitization treatments, as the fresh-cut industry mainly relies on the use of oxidative agents for the sanitization of produce and wash waters (Martı´nez-Vaz et al., 2014).
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This may be one of the main reasons for increasing numbers of enteric pathogenic bacteria-associated outbreaks involving fresh-cut produce and represents a critical challenge for the fresh produce industry. Attachment of bacterial cells to inaccessible sites, like stomata and cavities of the leaf, are also likely responsible for the inefficacy of sanitizing treatments and represents another challenge for the fresh-cut industry since none of the current methods is able to reach subsurface cells. Therefore, there is a need for the development of produce and wash water sanitization methods able to inactivate internalized bacteria and which are based on inactivation mechanisms that can overcome the issue of increased resistance to oxidative stress and improved DNA repair ability.
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