FOODBORNE DISEASE OUTBREAKS ASSOCIATED WITH FRUITS

Fruits and fruit juices are considered relatively safe because of their low pH at which most microorganisms do not grow.

However, recent foodborne disease outbreaks have shown that fruits can be carriers of pathogens (CDC 1999). About

Handbook of Fruits and Fruit Processing, Second Edition. Edited by Nirmal K. Sinha, Jiwan S. Sidhu, J´ozsef Barta, James S. B. Wu and M. Pilar Cano.

C 2012 John Wiley & Sons, Ltd. Published 2012 by John Wiley & Sons, Ltd.

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41% of foodborne illnesses in the United States were associ- ated with the consumption of produce (fruits, vegetables, and juices). The outbreaks related to contaminated produce in the United States doubled between 1973–1987 and 1988–1992 (Bean and Griffin 1990; Griffiths 2000). This can be due to increased consumption of “fresh” fruits, increase in import of fruits, and improved surveillance.

Food-poisoning outbreaks are defined as “the occurrence of two or more cases of a disease transmitted by a sin- gle food.” There are two exceptions, botulism and chemical poisoning in which one case constitutes an outbreak (CDC 1990).

There is little published information about human pathogens in raw and fresh-cut fruits and fruit juices. This is because of a large number of variables (sampling pro- cedure, location of source, number/size of samples, area or portion to be tested, etc.) and lack of optimized meth- ods for pathogen detection and isolation from fruits (FDA 2001a). Fresh whole, cut, and minimally processed fruits and juices are frequently implicated in outbreaks of bacterial food poisoning.Clostridium botulinum,Staphylococcus au- reus,Campylobacter jejuni,Listeria monocytogenes,Bacil- lus cereus,Shigella,Salmonella, andEscherichia colihave been identified as a food safety concern for fruits. Some of these microorganisms have also been responsible for food- borne outbreaks (Beuchat 1998). Use of fruit peels (Al- Zoreky 2009); chitosan biopolymer films (Kim et al. 2011);

bacteriocins (Rocha-Fleming et al. 2010) alone, or in com- bination with high-intensity pulsed electric fields (PEFs) (Mosqueda-Melgar et al. 2008; McNamee et al. 2010); UV irradiations (Gabriel and Nakano 2009); low concentrations of transcinnamaldehyde (Baskaran et al. 2010); grapefruit limonoids (Vikram et al. 2010); ozone treatment (Patil et al.

2009a); and dynamic high-pressure processing (Tahiri et al.

2006; Brinez et al. 2007) have been investigated as an alterna- tive to thermal processing to inactivate these pathogens while still retaining the maximum amounts of heat-labile nutrients in fruit juices.

C. botulinumis a Gram-positive spore-forming anaerobe that grows well in the absence of oxygen (Farber 1989). The strains ofC. botulinumcan be classified according to the toxin they produce into seven groups designated A through G.C.

botulinumtypes A, B, and E are responsible for botulism in humans. Botulism results from the consumption of a neuro- toxin that is produced by this microorganism while growing on the food. However, the rate of botulism associated with fruits is very low compared with the foods of animal origin.

This is due to the fact that most fruits are sold fresh under aerobic condition, which is not conducive for outgrowth of clostridial spores and toxins. However, safety concerns have been raised about the potential forC. botulinumtoxin in fresh- cut packaged produce (Austin et al. 1998). The high rate of respiration of both the produce and the microorganisms could create anaerobic conditions for spore outgrowth and toxin production. Thus, it is important to package produce

in a high oxygen/carbon dioxide permeable film and store at about 3◦C. Larson and Johnson (1999) tested the presence ofC. botulinumtoxin in fresh-cut cantaloupe and honey dew melons artificially inoculated withC. botulinum, and subse- quently packaged under modified atmosphere packaging and held for 9 days at 12◦C.

St. aureusis another major food-poisoning microorganism frequently involved in fruit-associated foodborne illnesses. It is a Gram-positive, facultative anaerobic coccus with a tem- perature range from 7◦C to 45◦C (Farber 1989). Food prod- ucts become contaminated withSt. aureusfrom noses, skin, or infected lesions of field workers, handlers, and processing personnel (Bryan 1980). The microorganism is also capable of producing different heat-stable enterotoxins. These entero- toxins are high-molecular-weight proteins and are produced in the lag phase of the bacterial growth (Genigeorgis 1989).

High levels ofSt. aureusin fruits indicate poor hygiene and improper storage conditions. This pathogen is a problem in the postharvest processing of fruits because of the widespread hand contact involved in sorting, packing, and repacking of fruits. Nevertheless, this microorganism is a poor competitor and does not grow well in the presence of other microor- ganisms normally present on fruits. Mukhopadhyay et al.

(2002) reported the presence of coagulase-positive St. au- reusin 17% of sliced papaya samples examined. The authors also reported that despite the risk associated with the pres- ence of this pathogen, the microbiological counts (80–100 CFU/g) were not high enough for toxin production.

C. jejunihas been recognized as the most common cause of bacterial diarrheal disease in humans (Griffiths and Park 1990). It is a Gram-negative, spiral-shaped, microaerophilic bacterium that belongs to the Spirillaceae family (Farber 1989). Foods of animal origin are commonly implicated with Campylobacterfoodborne illness. Although there are a few reported outbreaks associated withCampylobacterin fruits, this pathogen has been isolated from a variety of vegeta- bles and has been shown to survive on sliced watermelon and papaya (Castillo and Escartin 1994; Beuchat 1998). The prevalence ofCampylobacterin fresh fruits and vegetables at the retail outlets has been reported in the Netherlands and it has been suggested as a risk factor forCampylobacterin- fections if packaged raw fruits and vegetables are consumed (Verhoeff-Bakkenes et al. 2011). Since the infectious dose of Campylobacteris low (800 cells), intervention must be put in place to destroy any pathogenic bacteria that may be present on the raw fruit (Black et al. 1988).C. jejuniexposed to 20 consecutive cycles of sublethal inactivation by three different techniques, namely, lactic acid (LA), chlorine dioxide, and intense light pulses (ILP), became undetectable after first 2–5 cycles (Rajkovic et al. 2009).

L. monocytogenesis a Gram-positive, rod-shaped, motile bacterium isolated from various foods including cheese, milk, and fresh and processed meats. In accordance with other psychrotrophic bacteria,L. monocytogenes exhibits a wide temperature range from 1◦C to 45◦C with an optimum of

35–37◦C (Farber 1989). Listerosis is the disease contracted by the ingestion of contaminated food. Although the mini- mum infectious dose is still unknown, a high number of viable cells (⬎106 CFU/g) are required to cause illness in healthy adults (Farber 1989). High-risk groups including pregnant women and their fetuses, the elderly, and immunocompro- mised individuals will show clinical symptoms of listerosis at around 103–104CFU/g (Farber 1989). The importance of Listeriaas a causative foodborne agent in fruit stems from the following: (i) the ubiquity ofListeriain the environment, (ii) the ability of the microorganism to grow in extended shelf- life refrigerated products at temperatures as low as 1◦C, and (iii) high mortality rates as high as 30% (Farber and Losos 1988). Great concerns have been expressed about fruits as major vehicles of transmission of listerosis to humans. Sev- eral reports have shown that low temperature is not a hurdle to Listeriagrowth. Conway et al. (2000) reported the growth of L. monocytogenesin apples incubated at 5◦C. Furthermore, while the pH of most fruits and fruit juices (pH⬍4) is in the range unsuitable forListeriagrowth, some fruits such as watermelon (pH 5.2–5.7) and cantaloupe (pH 6.2–6.9) may serve as good substrates forListeriagrowth. Penteado and Leitao (2004) reported low-acid fruits (melon, watermelon, and papaya) as good substrates for the growth ofL. mono- cytogenes at incubation temperatures of 10◦C, 20◦C, and 30◦C. When stored at refrigeration temperatures, the garlic cheese salad and smoked ham salad were able to support the growth ofL. monocytogenes but shrimp-tomato salad sup- ported the growth least (Skalina and Nikolajeva 2010).L.

monocytogenescan grow on the peel as well as in the pulp of persimmon (Diospyros kaki) fruit at a temperature ranging from 10◦C to 30◦C, though at much slower rate, the growth is not completely inhibited (Uchima et al. 2008). The orange juice and the minimally processed orange slices can sup- port the growth of acid-adapted cells ofL. monocytogenes (Caggia et al. 2009).L. monocytogenesexposed to 20 con- secutive cycles of sublethal inactivation by three different techniques, namely, LA, chlorine dioxide (ClO2), and ILP, has been studied for change in resistance (Rajkovic et al.

2009). With repeated cycles of LA and ILP treatments (but not ClO2), producedL. monocytogenesculture with higher resistance compared with the original culture. The ability of this pathogen to adapt to mild inactivation treatments would create new challenges in risk assessment and more so in hazard analysis. Canada had experienced one of the most se- rious outbreaks of foodborne listeriosis in 2008, which has prompted the government to change regulations related to this pathogen, under which the production facilities must im- plement contact surface testing forListeria spp. and/or L.

monocytogenes(Farber et al. 2011). A rapid method for the detection ofL. monocytogenesin foods has been reported that combines the culture enrichment and real-time polymerase chain reaction (PCR) techniques, and this method is 99.44%

specific, 96.15% sensitive, and 99.03% accurate than the ISO 11290-1 standard method (O’Grady et al. 2009). Kim and

Cho (2010) have recently reported a simple, rapid procedure for the detection ofL. monocytogenesin unpasteurized fruit juice using real-time PCR without the traditional enrichment culture technique.L. monocytogeneshas been shown as one of the most PEF-resistant microorganism and it should be considered as a possible target microorganism to define pro- cess adequacy with PEF pasteurization (Saldana et al. 2010).

Bacillus cereuses are rod-shaped, Gram-positive spore for- mers that are commonly present in soil, on the surface of plant material, and in many raw and processed foods. Two distinct types of illness have been attributed to the fruits contaminated withB. cereus, a diarrheal and an emetic toxin. Fruits incrim- inated in past outbreaks include orange juice (FDA 2000).B.

cereuscan adapt to changing environments by various mech- anisms, such as signal transduction systems (two-component systems, alternative factors), and the environmental factors (temperature, carbon dioxide, redox potential, and pH). The heat resistance of B. cereus spore is positively correlated with the sporulation temperature. As a consequence of cli- mate changes that are taking place, surveillance is needed to detect changes in the epidemiology of B. cereus food- borne poisoning (Carlin et al. 2010). A new primer-probe set has been developed for the detection and quantification of spoilage and pathogenicB. cereusin food products by using real-time PCR technique (Fernandez-No et al. 2011).

Shigellaspp. are rod-shaped, Gram-negative, facultative anaerobic bacteria that have been epidemiologically associ- ated with foods or water contaminated with human feces.

Shigellosis is the collective term used for the dysentery dis- ease resulting from the infection by a species of Shigella.

There are four species of Shigella known to cause bacil- lary dysentery. These includeS. dysenteriae,S. flexneri,S.

boydii, and S. sonnei. Shigellosis infections accounted for 3.1% of the total foodborne outbreaks reported from 1988 to 1992 in the United States with most of these outbreaks being attributed to the consumption of contaminated vegeta- bles including parsley, lettuce, and vegetable salads (Davis et al. 1988; Dunn et al. 1995; Bean et al. 1997). The Food and Drug Administration (FDA) conducted an analysis of 151 cantaloupe samples imported to the United States from nine countries; 2% of these imports were positive forShigella (FDA 2001b). This pathogen is of great concern to the fruit industry because of its low infectious dose (10 cells) (Wu et al. 2000).

Nontyphoid Salmonellaspecies continue to be the most reported foodborne disease with incidence rates of 17.4 and 187 cases per 100,000 population and an estimated number of 2 million cases per year worldwide (Siliker 1982; D’Aoust 1991).Salmonellais a genus of the family Enterobacteriaceae that includes other genera such asE. coli,Shigella, andPro- teus. Salmonella species are high-temperature mesophiles that grow at temperatures from 5.2◦C to 45◦C (Farber 1989).

These microorganisms are ubiquitous in nature and have been isolated from several sources including irrigation wa- ter, sewage, rodents, and dust (Mackenzie and Bains 1976;

Oosterom 1991). Recently, the U.S. Environmental Protec- tion Agency (EPA) scientific advisory board panel specif- ically identifiedSalmonella,L. monocytogenes, and E. coli O157:H7 as pathogens of concern for fresh produce. Further- more, the panel recommended testing five outbreak strains in a cocktail for each pathogen when conducting challenge studies (EPA 1997). Most outbreaks of humanSalmonellosis have been linked to fresh-cut melons (Tauxe et al. 1997).

These fruits are considered as potentially hazardous foods in the FDA Food Code because of their low acidity (pH 5.2–6.7) and high water activity (0.97–0.99). Thus, accord- ing to the FDA time/temperature requirements for potentially hazardous foods, melons should be prepared under sanitary conditions and cut melons should be kept at or below 7◦C and displayed no longer than 4 hours if they are not re- frigerated (Golden et al. 1993).Salmonella have also been shown to adapt to reduced pH, and numerous outbreaks re- lated to unpasteurized juices have also been reported (WHO 1998).Salmonella enteritidiscan grow on the peel as well as in the pulp of persimmon (Diospyros kaki) fruit at a temperature ranging from 10◦C to 30◦C, though slowly with prolonged generation time, the growth is not com- pletely inhibited (Rezende et al. 2009). The PCR assay has been employed successfully to identify various Salmonella serogroups based on specific targets obtained by compar- ative genomics (Liu et al. 2011). Using multiplex PCR, two species, that is, Salmonella typhi and Salmonella ty- phimuriumhave been detected simultaneously in sliced fruits being sold by the hawkers in Malaysia and the estimated quantity ranged from 0 to 19 MPN/g, thus questioning the biosafety of the sliced fruits (Pui et al. 2011). Plant molecules such as carvacrol, transcinnamaldehyde, eugenol, and ␤- resorcylic acid as a wash treatment could effectively be used to kill the Salmonellaspp. on tomatoes, but more work is needed to evaluate the sensory and quality characteristics of tomatoes treated with these chemicals (Mattson et al. 2011).

E. coliare rod-shaped, Gram-negative, facultative anaero- bic bacteria that are part of the microflora of the intestinal tract of humans and animals. PathogenicE. coliare classified into five major groups based on their virulence properties, mecha- nism of pathogenicity, clinical symptoms, and antigenic char- acteristics (Beuchat 1998). These five groups include the fol- lowing: enterotoxigenic E. coli (ETEC), enteroinvasive E.

coli(EIEC), enteropathogenicE. coli(EPEC), enterohemor- rhagicE. coli(EHEC), and enteroadherentE. coli(EAEC).

The serotypes of major concern to fruits are the ETEC and EHEC. Sources and the mechanism of contamination are similar to those described for Shigella andSalmonella. E.

coliO157:H7 has been commonly associated with outbreaks of unpasteurized apple juice (Besser et al. 1993). Although apple juice and cider are regarded as high acidic foods (pH 3–4), E. coliO157:H7 have shown to be acid tolerant and survive in apple juice and cider for weeks in storage (Zhao et al. 1993).E. coliO157:H7 has also been shown to sur- vive in stored apple juice for up to 24 days at refrigeration

temperatures (Miller and Kasper 1994). The major factor re- lated to unpasteurized fruit juice safety is that these products receive no heat treatment.E. coliO157:H7 has been shown to grow on fresh and frozen-cut papayas held at 12◦C and it survived for 28 days at 4◦C.E. coliO157:H7 can survive on frozen-cut mango and papaya for at least 180 days; thus, these fruits are potential vectors for the transmission of this pathogen (Strawn and Danyluk 2010). These numerous out- breaks have led the FDA to take action to improve the safety of fresh and processed juice by requiring processors to use an inactivation step that would result in a 5-log reduction of the target pathogen or else place a warning label on their prod- ucts (FDA 1998a). Using spectral features with the Fourier transform infrared spectroscopy technique, various bacterial cell components such as nucleic acids, proteins, phospho- lipids, peptidoglycans, and polysaccharides, from the pure and mixed cultures of Alicyclobacillus spp. cells and the pathogenicE. coliO157:H7 cells have been detected in apple juice (Al-Qadiri et al. 2006). This Fourier transform infrared spectroscopy technique has also been employed by Al-Holy et al. (2006) to discriminateE. coliO157:H7 ATCC 35150 from other bacteria such asE. coliATCC 25522,B. cereus ATCC 10876, andListeria innocuaATCC 51742 inoculated in the apple juice. Ultrasound has been suggested as one pos- sible nonthermal technology to inactivate the pathogenicE.

coliin fruit juices (Patil et al. 2009b).

Raw fruits may also harbor many nonbacterial pathogens of public health concerns including viruses and parasites.

These pathogens are usually not part of the natural mi- croflora of fresh produce and are primarily introduced by symptomatic food handlers via fecal-oral route. Secondary modes of parasite and viral transmission to produce include sewage, water-containing untreated sewage, and sludge from primary or secondary municipal water treatment facilities (Beuchat 1998). Fresh produce consumed raw or minimally processed, such as fruits and certain vegetables, can be car- riers of pathogenic bacteria and viruses (Newell et al. 2010).

An increasing proportion of fruit-associated foodborne outbreaks have been recently traced to viruses. Hepatitis A virus (HAV) and the small round structured viruses (SRSVs) are the most common viral contaminants of fresh produce.

Historically, HAV has been recognized as the major cause of viral foodborne outbreaks. However, more recently, SRSVs have emerged as potential pathogen in fresh produce. O’Brien et al. (2000) reported that SRSVs represented 20% of the to- tal produce (vegetable, fruits, salads) associated outbreaks during 1992–1999 in England and Wales. Hepatitis A and SRSVs have been linked to outbreaks of strawberries and raspberries (Niu et al. 1992; CDC 1996a). Brassard et al.

(2011) have developed a method for the simultaneous re- covery of bacteria and viruses from contaminated water and spinach using a combination of conventional microbi- ology, PCR and RT-PCR. This combined method can be ap- plied to identify the responsible agent for the foodborne ill- nesses. HAV is another foodborne enteric virus that has been

associated with raw fruits such as red berries (Deeboosere et al. 2010). Various RNA-extraction kits with RT-PCR or RT-qPCR are now available for the detection of HAV in food samples (Bianchi et al. 2011). NoVs are now recognized as one of the leading causes of foodborne illnesses. Although efforts have been made to culture this virus, no reliable cul- ture method is available at this time. The detection of this virus in soft red fruits has been evaluated using molecular methods such as RT-PCR (Stals et al. 2011). Cranberry juice and cranberry proanthocyanidins have shown potential for controlling foodborne viral diseases, but their mechanism of action is not known (Su et al. 2010).

Parasites includingGiardia,Cryptosporidium parvum, and Cyclospora cayetanensis have been historically associated with waterborne outbreaks (Rose and Slifko 1999). How- ever, in recent years, these pathogens have also emerged as potential risk for foodborne illness and a concern to the fresh produce industry. The CDC reported that these para- sites contributed to 2% of the foodborne outbreaks between 1988 and 1992 in the United States (CDC 1996b). However, these numbers could be misleading due to the limitations of improved methods for recovery and detection of parasites in foods. Contamination of fruits with parasites occurs when crop irrigation water becomes contaminated with sewage or untreated wastewater or by runoff from nonpoint sources.

Thurston-Enriquez et al. (2002) studied the occurrence of GiardiaandCryptosporidiumin irrigation water in the United States and several Central American countries. They reported that 60% and 36% of irrigation water samples tested positive forGiardiaandCryptosporidium, respectively. In a survey conducted in Norway, out of 475 fruit and vegetable sam- ples examined, 6% were found to be positive forGiardiaand Cryptosporidium. NoCyclosporawere detected in any of the samples. Furthermore, water samples were also positive for those two parasites, indicating that irrigation and wash water are the sources of parasite transmission to fruits (Robertson and Gjerde 2001). Outbreaks linked to these protozoan par- asites include fruit salad, apple cider, and raspberries (Tauxe et al. 1997; CDC 1997; Anon 2000). An extensive review of various methods for rapid detection of foodborne microbial pathogens in comparison with traditional methods is given by Mandal et al. (2011).