NATIONAL ONION ASSOCIATION INTERTOX DECISION SCIENCES, LLC 600 Stewart St. Suite 1101 Seattle, WA 98101 206.443.2115 phone May 3, 2013 TABLE OF CONTENTS 1.0 Introduction ............................................................................................................................... 3 2.0 Foodborne Illness Outbreaks Associated with Onions .............................................................. 3 3.0 Onion Properties and Significance for Pathogen Contamination ............................................. 9 3.1 Onion plant structure ................................................................................................................ 9 3.2 Water activity and pH ................................................................................................................ 9 3.3 Chemical components ............................................................................................................. 10 3.3.1 3.3.2 Flavonoids................................................................................................................................ 10 Alkyl cysteine sulphoxides ....................................................................................................... 11 3.4 Anti-microbial activity of onions ............................................................................................. 11 4.0 Human Pathogens and Onions ................................................................................................ 12 4.1 E. coli O157:H7 ........................................................................................................................ 13 4.2 Salmonella spp......................................................................................................................... 14 4.3 Presence, growth and survival on onions ............................................................................... 17 4.4 Pathogen internalization ......................................................................................................... 19 4.5 Potential sources of E. coli O157:H7 and Salmonella in the primary production of onions ... 20 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5 4.5.6 Soil ........................................................................................................................................... 21 Animal manure and feces........................................................................................................ 22 Water ....................................................................................................................................... 22 Birds and insects ...................................................................................................................... 23 Workers ................................................................................................................................... 23 Equipment ............................................................................................................................... 23 5.0 Fresh-Market Dry Bulb Production Practices - Effects on Pathogen Survival and Growth ..... 24 6.0 Conclusions .............................................................................................................................. 24 7.0 Contributors ............................................................................................................................ 26 8.0 References ............................................................................................................................... 26 2 1.0 INTRODUCTION There has been several fresh produce-related foodborne illness outbreaks in the past few years including several associated with onions. Even so, none of the reported outbreaks or cases where onions were the reported vehicle has been linked to on-farm contamination. The reason there is so few incidents associated with dry bulb onions could indicate current industry practices have been effective in mitigating risk of microbial contamination and/or that onions’ intrinsic properties serve as a natural defense against the growth and survival of human pathogens. The purpose of this project was to collect, review and summarize existing research relevant to the potential for Salmonella spp. and E. coli O157:H7, two foodborne illness-related human pathogens, to contaminate dry bulb onions. The methodology included use of two primary research resources, PubMed and Google Scholar to search for published research, both peer-reviewed and non-peerreviewed. Funded by the U.S. government, PubMed is the premier repository for scientific literature comprising over 22 million citations in the fields of biomedicine, health, bioengineering, and life, behavioral and chemical sciences. We also searched the internet with Google Scholar to capture any research that was not included in PubMed. To capture unpublished research, we interviewed several scientists who work in the cooperative extension service with onion growers and/or are involved in onion-related research. The interviews provided insight into laboratory and field level research and experience. To research foodborne illness outbreaks related to onions, we used the Center for Disease Control and Prevention’s (CDC) Foodborne Outbreak Online Database (FOOD), the Foodborne Illness Outbreak Database (FIOD) sponsored by Marler Clark, and the Center for Science in the Public Interest’s Outbreak Alert! Database (OAD). FOOD is the primary source of data for both FIOD and OAD, but both databases also conduct independent searches for additional outbreak information that has not been reported to the CDC. A search of OAD revealed no additional outbreak other than those listed in FOOD, but FIOD included one additional outbreak from Canada. FIOD is additionally resourceful in that it provides links to their information sources including news releases and state health department reports. 2.0 FOODBORNE ILLNESS OUTBREAKS ASSOCIATED WITH ONIONS In conducting this research, the three foodborne illness outbreak databases described above – FOOD, FIOD, and OAD – were searched.1 Of these three databases, FOOD is the most comprehensive database. Data is available for 1998 to 2011 and data fields include the month and year of outbreak; the state where the outbreak occurred; the pathogen/agent either suspected or confirmed as responsible for the outbreak (the data includes chemical contaminants); the location of consumption; the total deaths and number of people who became ill and hospitalized; the food items associated with the outbreak (“food vehicle”); and the contaminated ingredient(s). It is important to note that many outbreaks list multiple food items as a food vehicle since the contaminated food item responsible for any particular outbreak is not always confirmed by epidemiological or laboratory methods. Even when a specific food item is confirmed as the source, the specific ingredient of food items containing multiple ingredients (e.g., salsa, potato salad) often is not determined. 1 The CDC defines a foodborne illness outbreak as an incident in which two or more persons experience a similar illness form the ingestion of a common food. 3 A search of FOOD for microbial pathogen-related outbreaks that named onions as a potential food vehicle and/or contaminated ingredient resulted in 28 outbreaks: eight with a confirmed pathogen, nine with a suspected pathogen, and 11 with neither a suspected or confirmed pathogen named (Table 1). In addition, FIOD reported an outbreak that was associated with onions that was not listed in FOOD since it occurred in Canada. In 16 of the 28 total outbreaks, onion was the only item listed as the food vehicle, but it is unclear from the data and other available information whether in any of these outbreaks, onions were confirmed as the contamination source. Norovirus was the confirmed agent in four of the outbreaks and a suspected agent in five. Salmonella enterica was confirmed as the agent in one outbreak and suspected in another. Other pathogens that were confirmed agents included E. coli O157:H7, Clostridium perfringens, and Campylobacter jejuni. Bacillus cereus was suspected in three outbreaks. In most cases the location of consumption was in a restaurant (54%). Other locations where the contaminated food was reportedly consumed included picnics, the workplace, private home, wedding reception, grocery store and camp. Two outbreaks listed multiple locations of consumption. Further investigation of individual outbreaks in published reports revealed more details for some outbreaks particularly when they involved more people (i.e., greater than 20 ill persons). The outbreak listed in FIOD and not in FOOD was attributed to E. coli O157:H7 and, based on a casecontrol analysis, associated with dining at a restaurant in Ontario, Canada, and eating onions served by the restaurant. All samples acquired from remaining onions and packaging as well as samples taken at the place of business of a grower that supplied some of the onions to the restaurant tested negative for E. coli O157:H7 (NBPSDHU, 2009). In 2005, a Salmonella Newport outbreak with multiple locations of consumption occurred in New York and listed both onions and tomatoes as food vehicles. In further investigation, it appears that this outbreak was attributed to sliced tomatoes from grocery stores and restaurants (NYSDH, 2007). In 2009 another Salmonella Newport outbreak occurred with diners at a restaurant in Kansas. A case-control analysis found a positive association with three food items – sandwiches, German potato salad, and tossed salad. Common ingredients among the three items were raw onions and lettuce. Food samples were not analyzed and workers did not report having symptoms of salmonellosis, but inspection of the restaurant revealed numerous violations including lack of backflow prevention devices on a sink, an icemaker, and dishwasher and improper cold holding temperatures for perishable foods. Although the lettuce and onions came from one nationwide foodservice supply company, no other Salmonella Newport cases with matching strain were identified during the same timeframe leading to the conclusion that the contamination most likely originated at the restaurant (KSDHE, 2009). In the California Department of Health Services’ (DHS) report on foodborne disease outbreaks in 1999-2000, a norovirus outbreak in 2000 that left 69 people ill did not name onions as the food vehicle as did the CDC in FOOD. According to the California DHS report, confirmed vehicles included taco bar items (which may include onions), iced tea, fresh fruit, raw tuna fish, and chicken / tuna sandwiches. It also stated that 47% of all the confirmed norovirus outbreaks in 1999-2000 were due to an infected or ill food handler (CADHS, 2004). An Campylobacter outbreak at two separate locations in Oklahoma listed onions as one food vehicle item in a fajita lunch prepared by a restaurant. An investigation by the Oklahoma State Department of Health found that a greater risk of illness was associated with meat consumption. The report also cited other potential contributing factors as improper handling of meat, questionable cooking practices for meat, lack of proper sanitation, and lack of proper hand washing procedures (OKSDH, 2006). 4 In 1983, a botulism outbreak was associated with sautéed onions on a patty-melt sandwich served at a restaurant in Peoria, IL (MMWR, 1984; MacDonald, 1985).2 Although the original batch of sautéed onions was not available for culture or toxin testing, botulinal toxin was detected from a sample of a discarded foil wrapper used by one of the patients to take a patty-melt home. Botulinal spores were cultured from five of 75 skins of whole onions taken from the restaurant. No other ingredients of the sautéed onions contained botulinal toxin or spores. The onions were said to have been prepared daily with fresh whole onions, margarine, paprika, garlic salt, and a chicken-base powder; they were held in an uncovered pan covered in melted margarine on a warm stove below 140°F and were not reheated before serving (MMWR, 1984). According to the MMWR report, sautéed onions had never before been associated with botulism, and in a search for additional cases showed no other reports of botulism outbreaks associated with onions since this 1983 incident. However, this particular outbreak is frequently cited in the literature and recounted in the media, and sautéed onions are often listed as a potential food safety hazard. Review of the outbreaks with potential or confirmed association with onions provided no evidence linking the contamination to the farm or storage or packing facility. 2 Botulism is caused by Clostridium botulinum, a soil-associated, spore-forming anaerobic bacteria that produces neurotoxins four of which (Type A, B, E and F) cause disease in humans. Botulinal spores are believed to be one of the most durable cells in nature, able to survive in a dormant state without nutrients and demonstrating resistance to desiccation, high temperatures, irradiation, strong acids, and chemical disinfectants for an undetermined amount of time. They easily become airborne and are widely distributed throughout the environment (Todar, 2012). When environmental stresses are relieved, spores germinate and once again become vegetative cells capable of actively growing and dividing. Control of this organism in food products requires destruction of the spores through formulation, temperature control, or a combination of these factors. 5 Table 1. Foodborne illness outbreaks related to onion consumption Genus Species Year Month State 2009 July MO 2009 May ID Norovirus 2008 Oct Ontario E. coli O157:H7 2008 March MN Norovirus 2008 Oct Status Location of Consumption Camp; Other; Private Home Restaurant "Fast-food"(drive Confirmed up service or pay at counter) Restaurant other or unknown type Restaurant Suspected other or unknown type Total Ill Total Hospitalizations Total Deaths 25 0 0 onion, unspecified 10 0 0 chicken salad 235 26 5 0 Food Vehicle Onion 0 onion, unspecified 0 chocolate, unspecified; multiple cheeses, pasteurized; olive, unspecified; onion, unspecified; other vegetable *****; parmesan cheese, unspecified; pesto AZ Wedding reception 18 Restaurant other or unknown type 5 Workplace, not cafeteria 13 0 0 onion 9 0 0 fried onions/onion 2007 Jan FL 2007 May WI Norovirus Confirmed 2006 April MN Norovirus Confirmed Restaurant 6 0 onion, unspecified Contaminated Ingredient lettuce; onion; tomato 2006 Dec OH Norovirus 2005 June OH Norovirus 2005 July NY 2005 Nov FL Salmonella enterica Newport Campylobacter jejuni other or unknown type Restaurant Confirmed other or unknown type rings, unspecified Confirmed Private home Grocery store; Picnic; Private home; Confirmed Restaurant other or unknown type Restaurant other or unknown type May OK 2004 August MI 2004 Sept PA Norovirus Suspected 2004 Nov CA Norovirus Suspected 2002 May CA Salmonella enterica Suspected 2001 July FL 2 0 4 0 0 27 2 0 28 1 0 Restaurant other or unknown type 2 0 0 Restaurant other or unknown type 8 1 0 4 0 0 2 1 0 2 1 Private home Restaurant other or unknown type Restaurant other or 7 dips, unspecified; fried onions/onion rings, unspecified fried onions/onion rings, unspecified onion, unspecified; tomato, unspecified Root; Vinestalk e.g. tomato fried onions/onion rings, unspecified 3 Workplace, not Confirmed cafeteria 2005 11 fajita, beef; fajita, chicken; onions; peppers, unspecified fried onions/onion rings, unspecified; salad, unspecified romaine lettuce, unspecified; tomato (see fruit); Vidalia onion multiple foods; onions fried onions/onion rings, unspecified beef, other; fried onions/onion rings, Root unknown type unspecified 2000 August KS 2000 Oct FL Bacillus cereus Suspected 2000 Oct CA Norovirus Suspected 1999 April MN Norovirus Suspected 1999 May IL Clostridium perfringens 1999 Feb Florida Bacillus cereus 1998 Sept MI 3 1998 May MI 2 1998 December FL 1998 FL April Bacillus cereus Picnic Restaurant other or unknown type Restaurant other or unknown type 21 Other 9 0 0 190 0 0 3 0 0 Confirmed Picnic Suspected Suspected Restaurant other or unknown type Restaurant other or unknown type 8 2 0 onion, unspecified 4 onion, unspecified 69 onion, unspecified 2 0 0 2 0 0 green beans, unspecified; onion, unspecified mushrooms, unspecified; onion, unspecified onion, unspecified onion, unspecified ground beef, hamburger; onion, unspecified cake, cheese; fried onions/onion rings, unspecified onion, unspecified Root 3.0 ONION PROPERTIES AND SIGNIFICANCE FOR PATHOGEN CONTAMINATION At any given point in the farm to fork supply chain, intrinsic and extrinsic factors can affect the type and level of microorganisms associated with onions. These microorganisms lead to the inevitable decay of onions and influence both the rate and type of decay. Beuchat (2002b) describes how a food’s intrinsic properties and the environment interactive with and affect populations of microorganism on fresh produce: Surfaces of fruits, stems, roots, florets, and leaves, for example, are each characterized by unique microenvironments that influence colonization of bacteria, yeasts, and molds, as well as attachment of these microorganisms, parasites, and viruses. The environment in which plants are grown imposes extrinsic factors that influence survival and growth of associated surface microflora, whereas intrinsic parameters such as the nature of the epithelium and protective cuticle, tissue pH, and the presence of antimicrobials dictate which groups of produce may be more likely than others to harbor certain types of microorganisms … The range of microorganisms recovered from raw fruits and vegetables at harvest most often reflects the microflora present in the field, orchard, grove, or vineyard at the time of harvest. Climatic and agricultural determinants affecting the microbial ecosystem at harvest include geographical location, history of precipitation, wind, irrigation practices, pre-harvest, harvest, and post-harvest practices, and the presence of insects, animals, and birds. As Beuchat describes, intrinsic properties of food play an important role in the ability of a microorganism to survive and grow, but need to be considered in the context of the external environment, which may be highly variable. In this section, the intrinsic properties of onions that are thought to affect microbial growth are discussed. 3.1 Onion plant structure During production the edible bulb portion of the onion is mostly below ground. Onion leaves that appear above ground are extension of the leaves that form the buried bulb. Although a tightening of the leaves occurs to form a neck between the buried and above-ground portion of the plant, the continuous nature of the leaves means that the bulb is not completely protected from aboveground contaminants. Similar to fungal plant pathogens, human pathogens deposited on aboveground leaves via wind, irrigation water, splashing water, insects, bird and animal feces, etc., could potentially migrate to the bulb (Schwartz, 2011a). In contrast to potential vulnerability to contamination due to the opening at the neck, another aspect of the onion plant’s structure may provide protection from potential contamination. During maturation, the onion begins to make scale leaves that are dry and not easily penetrated, which may provide a barrier from contaminants. 3.2 Water activity and pH Of the multiple factors that influence the growth of microorganisms in or on food, the physicochemical properties of a particular food item — water activity, pH, and temperature— appear to be the most significant (FDA, 2012). Water activity and pH are intrinsic properties while temperature is an external factor that may vary at different points throughout production, harvest and post9 harvest handling. The growth requirements of any given microorganism are the same for a food’s exterior and interior. However, the physico-chemical properties of food that affect microorganism growth are not the same for interior and exterior portions of most fresh produce commodities. For example many fresh produce commodities are covered with skins or peels that are less favorable for pathogen growth than the inner portions where, for instance, water activity is often higher and exposure to environmental factors is reduced. Water activity is a measurement of water content and is defined by Beuchat (2002a) as the proportion of water available for biological and chemical reactions to occur. Pure distilled water has a water activity of exactly one. All other water activity is measured relative to pure distilled water. The water activity for onions ranges from 0.990 to 0.974. As seen in Table 2, onions are slightly acidic (5.3 - 5.8) with the pH varying slightly with variety.3 In their draft guidance for industry on acidified foods, the FDA refers to onions as a low acid food (FDA, 2010). Table 2. Onion properties Onion Variety 3.3 pH Water activity Yellow 5.4 – 5.6 0.990 – 0.974 White 5.4 – 5.8 Red 5.3 – 5.8 Chemical components Onions contain two classes of chemicals that have been demonstrated to have antimicrobial activity – flavonoids and alkyl cysteine sulphoxides (ACSO). 3.3.1 Flavonoids All plants that conduct photosynthesis, including onions, contain chemical compounds called flavonoids in varying amounts (Cushnie, 2005). Many beneficial effects on human health have been credited to flavonoids including antimicrobial, anti-inflammatory and antioxidant activity (Cushnie, 2005; Griffiths, 2002). Flavonoids are categorized into 13 main classes, three of which are found in onions – flavonols, anthocyanins, and dihydroflavonols. Flavonols such as quercetin and its derivatives account for the yellow and brown skins of many onion varieties and anthocyanins are responsible for the red/purple color of some varieties (Griffiths, 2002). Isolation of more than 50 different flavonoids from onions has been reported in the literature (Slimestad, 2007). Studies of onion cultivars demonstrate high variability in flavonoid levels, which may be due to genetic differences and/or growing location, climate, maturity, and harvest season variation (Galdon, 2008; Lombard, 2002; Price, 1999; Slimestad, 2007; Yang, 2004; Yoo, 2010). 3 http://www.fda.gov/Food/FoodborneIllnessContaminants/CausesOfIllnessBadBugBook/ucm122561.htm 10 3.3.2 Alkyl cysteine sulphoxides The ACSOs are the sulfur-containing precursor chemicals, which, when cleaved by enzymes, produce volatile sulfur compounds (sometimes referred to as secondary metabolites or downstream products) that are responsible for the characteristic odor and taste of onion (Griffiths, 2002). These volatile sulfur compounds are thought to be present in onions to serve primarily as a deterrent to insects, and similar to flavonoids, have been credited with anti-inflammatory, antioxidant, and antimicrobial activities when consumed by humans (Griffiths, 2002). It is important to note that enzymatic cleavage occurs only when onion tissue is ruptured and so the secondary metabolites or downstream products that are credited with the antimicrobial activity are only available after bulb tissue has been cut, damaged or otherwise disrupted (Sofos, 1998; Goldman, conversation). 3.4 Anti-microbial activity of onions There are numerous published studies investigating the antimicrobial activity of raw onions and onion products (e.g., dehydrated onion powder, onion oil) with inconsistent results. Most of these studies have been conducted outside of the U.S. (e.g., Nigeria, Slovenia, Algiers, Korea, Philippines) and published in journals that are not peer-reviewed. Many studies test extracts of raw onions for antimicrobial activity, but extraction methods vary considerably among the studies, and inconsistencies in study findings are more than likely related to differences in experimental methods as well as differences in the onions related to local growing conditions and cultivation practices. The common endpoint for the studies is inhibition of colony formation on an agar plate measured by the diameter of area surrounding the onion extract sample where no bacterial colonies form (zone of inhibition) or in a nutrient broth measured by concentration of bacteria. Studies that showed an inhibitory effect on various strains of E. coli ranged from 2 to 40 mm (Adeshina, 2011; Benmalek, 2013; Masaudi, 2012; Penecilla, 2011; Santas, 2010; Sarmiento, publication date unknown). Studies using a broth dilution method also showed inhibition of laboratory strains of E. coli (Al Masaudi, 2012; Skerget, 2009). One study conducted in Nigeria showed a dose-response effect where increasing concentrations of onion juice resulted in an increasingly inhibitory effect on Salmonella colony formation (Azu, 2007). In experiments reported as part of a master thesis, the bacteriostatic activity of onions was reported as an aside to the study’s primary objective of testing the effectiveness of a hypochlorite treatment against Bacillus subtilis in the process of making dehydrated onions. In preparing contaminated onions for the hypochlorite treatment experiments, the researcher reported that B. subtilis growth was inhibited in the ground onions making it problematic for them to evaluate the treatment (Siregar, 2004). The collective literature provides some evidence that onions have antimicrobial properties that may have the potential to inhibit the growth of some human pathogens. However, little is known about how these chemical compounds may affect onions contaminated with human pathogens. The variability in these study findings may reflect of the amount and location of these compounds in the onion plant throughout its development. Levels of these compounds not only fluctuate based on the growing environment, but as deterrents for predatory insect, these compounds “cycle through onion plants from old leaf scales to new in protecting the developing bulb and then are shunted to 11 the developing leaves and ultimately to flowers for protection during pollination. This finding demonstrates this unique medicinal character fluctuates dramatically with plant development and is likely associated with a natural flux in defense compounds” (Goldman, accessed 2013). However, as stated above, one class of these compounds, the ACSOs, are not in their antimicrobial form (i.e., have not been cleaved to form the volatile sulfur compounds) until the onion bulb tissue is ruptured. This limits how the antimicrobial effect would be applicable to the microbial safety of intact dry bulb onions. In addition, evidence from infections with bacterial plant pathogens as well as contamination with human pathogens in the laboratory (as discussed in Section 4.3) indicate that at least some bacteria are not affected and/or have been able to adapt to an onion’s seemingly intrinsic antimicrobial defenses. In addition to the uncertainties related to the laboratory methods being employed to determine antimicrobial activity, it appears that there are differences related to onion development stage and cultivar as well as cultivation and handling methods that are challenging for making an assertion about the antimicrobial activity of onions in general, let alone how this would affect contamination by human pathogen. 4.0 HUMAN PATHOGENS AND ONIONS Human pathogen survival and growth characteristics are important considerations when evaluating the food safety risks associated with dry bulb onion production practices. Two human pathogens in particular, Salmonella and E. coli O157:H7 have been linked to on-farm contamination of other fresh produce items and for this reason are examined in this report. E. coli O157:H7 and Salmonella are commonly found in the lower intestines of warm-blooded animals – their preferred hosts, but both pathogens can survive and grow outside their preferred hosts. However, survival and growth outside their preferred hosts require certain conditions (i.e., temperature, moisture, nutrients, etc.). Varying amounts of moisture are required for growth, but in general, higher water activity tends to support more microorganisms. Bacteria usually require a water activity of at least 0.91 to grow and experience optimal growth at values between 0.995 and 0.980 (Gibbs, publication data unknown). The water activity of most foods is greater than 0.95 (FDA, 1984). A microorganism will grow within a range of temperatures and pH levels although each microorganism will obtain maximum growth at some optimal temperature and pH level. Microorganisms are often classified by their optimal growth temperatures (such as psychrophile, thermophile, or mesophile) when optimal growth occurs (low, high, or moderate temperatures, respectively). When the temperature or pH moves away from the optimum in either direction, microbial growth is reduced. Microorganisms on food create a dynamic and complex microenvironment. Water activity, pH, and temperature are interactive and the resulting effect of these dynamic interactions may be inhibitory, additive or synergistic dramatically affecting the ability of a microbe to grow in or on any given food. For example, the water activity at which any given microbe will grow varies depending on other conditions such as the pH of the food matrix or the presence of a preservative like salt or nitrite. Water activity does not change much over the range of temperatures that support microbial growth, but the water activity of a solution may have a tremendous impact on the ability of heat to kill a bacterium at a given temperature (Mattick, 2000). Also when microorganisms are present on food, they affect that microenvironment. For instance, the population of microorganisms on a food item will change the pH, which may enhance or inhibit the growth of other microorganisms. 12 Pertinent physico-chemical characteristics for Salmonella and E. coli O157:H7 related to their growth and survival is summarized in Tables 3 and 4. 4.1 Escherichia coli O157:H7 Escherichia coli (E. coli) O157:H7, also called enterohemorrhagic E. coli (EHEC), is a Gram-negative, rod-shaped bacterium that has the potential to cause disease in humans. E. coli O157:H7 has been isolated from many domestic and wild animals such as cattle, sheep, pigs, horses, turkeys, dogs, and deer; some of these animals have been identified as reservoir for E. coli O157:H7 (Sargeant, 1999; Fischer, 2001; Brittingham, 1988; Chapman, 1997; Ferens, 2011; Hancock, 1998; Jay, 2007). Healthy cattle serve as a primary reservoir for E. coli O157:H7 with bovine intestinal colonization for up to three months reported in the literature (Hancock, 1998). Although E. coli O157:H7-contaminated cattle manure is believed to be the primary source of most human infections, contaminated water has also been associated with illness outbreaks (Holme, 2003; Olsen, 2002). E. coli O157:H7 is able to withstand both cold and dry conditions and can survive for months in water and manure (discussed in more detail in Section 4.4). Studies have shown that E. coli O157:H7 survives well in dried foods, especially at refrigeration temperatures and in dry environment with wide ranges of water activity and pH levels (Deng, 1998; Park, 2000; Ryu, 1999). Other studies demonstrate that while there are differences depending on the isolate, E. coli O157:H7 generally does not survive well at temperatures above 44˚C or below 8˚C. E. coli O157:H7 growth rates show little variation at pH levels between 5.5 and 7.5, but decline rapidly below 4.0. However, survival in low acid foods can occur for extended periods of time under refrigeration (Buchanan, 1997). Table 3: Growth and Survival of Escherichia coli O157:H7 (NZFSA, 2001b - unless otherwise noted) Cell growth Cell survival Temperature Optimum: 37-39˚C Minimum: 7-8˚C Maximum: 46˚C pH Water activity Optimum: 6.0-7.0 Optimum: 0.995 Range: 4.4-9.0 Minimum: 0.950 (depends on acid type e.g., organic acids more inhibitory than mineral acids) Inactivation Rapidly inactivated by heating at 71˚C (159.8˚F); Disinfectants: Chlorinated agents (dependent on organic load) Survives well in chilled and frozen foods 3.6 (temperature dependent) (Curtis, 2007) At 60˚C (140˚F), it takes 0.5-0.75 min. to kill 90%; Withstands desiccation (temperature dependent) – longer survival at lower temps Growth is inhibited at 8.5% NaCl E. coli O157:H7, can be transmitted to humans from contaminated food or water or from person-toperson. The organism causes gastroenteritis commonly referred to as hemorrhagic colitis (HC) with the hallmark symptom of bloody diarrhea and gastrointestinal symptoms as described in Table 5 13 below. The disease is caused by a toxin that is produced, not in food, but in the gut of infected people. The toxin causes severe damage to the intestinal lining of humans and can cause kidney failure. In some instances, especially in children, HC develops into a more serious, sometimes fatal, condition called hemolytic uremic syndrome (HUS). This occurs when the toxin attacks the kidneys resulting in kidney dysfunction, seizures, coma, or death. In 2011, the incidence of laboratory-confirmed E. coli O157:H7 cases in the U.S., was 0.97 cases per 100,000 people (CDC, 2011). The infective dose is unknown, but is estimated to be in the range of 10 to 100 cells. Foods with as low as 0.3-0.4 organisms per gram have reportedly been involved in outbreaks (NZFSA, 2001b). The number of organisms estimated to produce a 50% probability of disease is 5.9 x 105 (NZFSA, 2001b). Approximately 10% of children infected with E. coli O157:H7 develop HUS with a fatality rate of <10%. In the elderly, HC may develop into thrombocytopenic purpura (TTP) that involves loss of platelets, skin discoloration, fever, seizures, or strokes in addition to HUS signs and symptoms. Mortality rate for cases of TTP in the U.S. is < 5% (NZFSA, 2001b). 4.2 Salmonella Salmonella species are characteristically robust in dry environments and under refrigeration and grow in presence or absence of oxygen as well as in the presence of nitrogen. Although widely distributed in the environment, they prefer neutral environments, but can grow in slightly acidic conditions when the temperature is favorable (10–30˚C). A study of Salmonella enteritidis and typhimurium in chicken manure incubated at 20°C in aerobic conditions, showed variability in survival in relation to the water activity of the manure (Himathongkham, 1999). At water activity levels above 0.93 there was a moderate increase in growth over eight to nine hours. At water activity levels between 0.89 and 0.75 Salmonella were reduced 1,000-fold. It took 25-30 days to reduce the bacteria by a million (106) fold at water activity levels of 0.97, 0.75, 0.50 and 0.38. However, at a water activity of 0.89, Salmonella were reduced 106 fold in only 8 days. The authors suggest that the improved ability to survive at water activity levels below 0.75 may indicate that the pathogen was able to adjust to the dry conditions by entering a “dormant” state where metabolic activity required for growth slows down allowing it to merely survive. The die-off in 30 days observed at 0.97 (normally not inhibitory to growth) was attributed to an increase in pH resulting from ammonia formation by the other microorganisms present in the manure. Another study also reported the ability of Salmonella to adapt to dry environments, especially when the organism is exposed to alternating high (0.987) and low (0.893) water activity levels (Eriksson de Rezende, 2001). These studies demonstrate the complexity of interacting factors determining pathogen survival in addition to the pathogens’ robustness in adapting to adverse conditions. Salmonella are transmitted through a fecal-oral route: it is shed in feces and infects through ingestion. Animals and birds are natural reservoirs for Salmonella, with feral animals, rodents and an average of 10% of pets serving as carriers (Teplitski, 2006). Most animals infected with Salmonella are symptomless. Salmonella can survive in untreated animal waste for up to two years (Teplitski, 2006). Salmonella have been isolated from flies on farms with as many as 10 million bacterial cells isolated from one fly dropping (Teplitski, 2006). Flies may serve as indirect vector and transmit Salmonella from feces to food. Some serotypes are found only in humans (e.g., Typhi and Paratyphi B). 14 When humans are infected, they typically shed the organism in their feces for five weeks with 5% of carriers shedding for 20 weeks. Some infected persons become chronic carriers shedding the bacteria for years, but the prevalence of this is believed to be less than one percent (PHAC, 2010). Table 4: Growth and Survival of Salmonella spp. (NZFSA, 2001a - unless otherwise noted) Cell growth Cell survival Temperature Optimum: 3537˚C; Minimum: 7˚C Maximum: 49.5˚C pH 3.8-9.5 (varies with temperature & strain); Optimum: 7.07.5 Cool temperatures Dependent on favor survival; acid type & survives well for temperature long periods of refrigeration Water activity > 0.99 - 0.94; Optimum: 0.99 Inactivation Moist heat: 121˚C for at least 15 min.; Dry heat: 170˚C for at least 1 hr.; Disinfectants: 2-5% phenol 1% sodium hypochlorite; 4% formaldehyde, 2% glutaraldehyde, Will die at <0.93, but may 70% ethanol, 70% propanol, take a long 2% peracetic acid, time; survive 3-6% hydrogen peroxide, longer in low moisture with quaternary ammonium compounds, high fat iodophors content (e.g., (PHAC, 2010); chocolate) At 60˚C, it takes 0.4-0.6 (Curtis, 2007) min. to kill 90% At 55˚C, it takes 4˗6 min. to kill 90% In humans, Salmonella cause two distinct diseases called salmonellosis: 1) Enteric fever resulting from invasion of the bloodstream by Salmonella typhi, and 2) acute gastroenteritis caused by ingestion of food contaminated with Salmonella spp. other than S. typhi (Todar, 2012). The latter Salmonella species produce typical gastrointestinal flu-like symptoms as described below in Table 5 below. In 2011, the incidence of laboratory-confirmed Salmonella cases in the U.S. was 16.4 cases per 100,000 people (CDC, 2011). Infective dose varies by strain, individual host characteristics such as level of stomach acidity, and susceptibility of individuals. Susceptible populations include the elderly, young children, people taking certain medications, and immunocompromised individuals. Attack rates as low as 1 cell has been reported in foodborne illness outbreaks (FDA, 2012). However, in human volunteer studies high doses were required to cause disease (Blaser, 1982). The infective dose that infected 50% of mice was 105 organisms (NZFSA, 2001a). Long term effects may occur if nontyphoidal Salmonella escape from the gastrointestinal tract to invade the body and include blood poisoning (septicemia), and other non-intestinal infections as well as reactive arthritis three to four weeks after gastrointestinal symptoms subside (FDA, 2012). 15 Table 5. Description of Illnesses Associated with E. coli O157:H7 and Salmonella4 Condition, Symptoms and Long-term Effects Incubation Duration Salmonella Nausea, vomiting, abdominal Salmonellosis cramps, minimal diarrhea, fever, headache; arthritic symptoms may follow 3-4 weeks after acute symptoms 5–72 hrs. (Usually 12–36 hrs.); Typhoid/enteri c fever: 3–60 days (usually 7– 14 days) (PHAC, 2010; PHAC, 2001) 4–7 days with acute symptoms lasting 1–2 days or longer depending on host and strain characteristics and infective dose Typhoid/enteric fever: 10–14 days if treated (PHAC, 2010; PHAC, 2001) Most strains <1% Strain dependent. Host age and health dependent Non-typhoidal salmonellosis: 102–103 cells Typhoidal salmonellosis: 105 cells by ingestion (PHAC, 2010; PHAC, 2001) E. coli O157:H7 Hemorrhagic colitis - severe Hemorrhagic abdominal cramping, diarrhea colitis (bloody); occasional vomiting, low-grade or no fever Hemolytic Uremic Syndrome (HUS): Renal failure, seizures, coma, death; affects mostly children; thrombocytopenic purpura (TTP) a version of HUS most often affecting the elderly 3–9 days (mean 4 days) 8 days TTP: <5% HUS: ~1% 10–100 organisms; 5.9 x 105 organisms/g for 50% probability of disease (NZFSA, 2001b) 4 Mortality Information was obtained from FDA’s Bad Bug Book unless otherwise noted. http://www.fda.gov/Food/FoodSafety/FoodborneIllness/FoodborneIllnessFoodbornePathogensNaturalToxins/BadBugBook/default.htm 16 Infective dose 4.3 Presence, growth and survival on onions Onion water activity and pH both fall within the optimum ranges Salmonella and E. coli O157:H7 cell growth, and studies have shown that both human pathogens are able to survive, if not grow, on onions. All, but one, of these studies were conducted in the laboratory with large numbers of pathogens used to inoculate onions under conditions meant to simulate the production environment. Since the size of a microbial population affects organism growth and survival, use of high microbial populations in laboratory experiments may not represent microbial contaminations in a production and/or packing environment. Other fresh produce studies conducted in the laboratory simulating the natural environment and cultivation and harvesting practices have not always produced the same results as field experiments, using the same treatments, under natural environmental conditions using actual cultivation and harvesting practices. Although there are some limitations in application to the natural environment, laboratory studies are an essential component of scientific research as they provide a controlled setting where factors that may potentially influence the results can be isolated. Laboratory experiments are often used to make field studies more effective and efficient. Another advantage of laboratory studies is the ability to use unattenuated strains of pathogens in experiments. Attenuated strains of pathogens are ones where the disease-causing genes have been removed. While attenuated strains may not necessarily behave in the same manner as pathogens with these genes intact (wild type), some studies have showed little difference in survival outcomes between wild type and its attenuated derivatives (Lim, 2007; Ma, 2011). Because of the threat to public health, most field studies are conducted with attenuated pathogenic strains or generic strains of E. coli as “surrogates,” which introduce uncertainty to study findings (i.e., study results may not reflect what happens in the real world). Both types of studies have limitations, but, when conducted properly, can provide information about the risk of product contamination with foodborne illness-related pathogens. Contamination of onions with E. coli O157:H7 has been demonstrated both in the laboratory and in the field. In the only field study identified in our literature search, researchers at the University of Georgia investigated the survival of E. coli O157:H7 on onions and the surrounding soil when E. coli O157:H7-contaminated manure compost or irrigation water was applied to the soil (Islam, 2005). Three weeks after planting the onion seeds in plots at the university’s farm, the researchers sprayirrigated the plot with water contaminated with 105 CFU/ml of an attenuated strain of E. coli O157:H7. Onions and soil samples were collected at the time of contaminated water application and at selected intervals for 203 days. E. coli survived in water-irrigated soil where onions were grown for 161 days. E. coli levels measured on onion samples taken seven days after plots were irrigated with contaminated water (three weeks after planting), were 1.5 log10 CFU/g, but in subsequent days declined until levels were no longer detected 63 days after irrigation. In soil samples contaminated with dairy cattle manure compost or poultry manure compost, E. coli survived 168 and 175 days, respectively. The initial concentration measured on the onions (21 days post-planting) grown in soil amended with two types of contaminated dairy manure compost or poultry manure compost was 2.0 – 2.4 log10 CFU/g and declined to non-detectable levels by day 84 and 91. If these contaminated onions had been harvested at day 140 – the approximate time when onions are harvestable in that growing region, no E. coli would have been present. Carrots were 17 also included in this study, and it is worth noting that E. coli survived significantly longer (168 days) and at higher levels (~3.4 log10 CFU/g) on carrots subject to the same experimental conditions. In their discussions, the authors suggest that this difference may be due to “the presence of high concentrations of antimicrobial phenolic compounds in onions compared to carrots.” In another experiment by the same researchers, but conducted in a laboratory growth chamber, onions were grown in soil amended with E. coli O157:H7-contaminated bovine manure compost (Islam, 2004a). Seven days post-seeding, E. coli levels on onion bulbs were approximately 3.4 log10 CFU/g and declined on all subsequent sample days until reaching 1 log10 CFU/g (detectable only be enrichment) 42 days later. A baseline study to test the survival of E. coli O157:H7 and Salmonella spp. in onions was conducted on yellow onions with an inoculum containing 6.63 log10 CFU/ml consisting of three strains of each pathogen (IEH, 2011). The inoculum was injected into one inch onion cubes and incubated for two days at room temperature after which the samples were pulped and plated on selective growth media for enumeration. The results for the three onion samples ranged from 6.95–7.04 CFU/ml for E. coli O157:H7 and 6.66 to 6.85 CFU/ml for Salmonella spp. Very little, if any, growth occurred in the onion samples. This finding was interesting in comparison to growth in the positive control (buffered peptone water) with minimal nutrients – 8.11 and 8.76 CFU/ml for E. coli O157:H7 and Salmonella spp., respectively. Although whole onions were not tested, E. coli O157:H7 was isolated from fresh-cut onions during an investigation of a multi-state outbreak in 2006 associated with Taco Bell restaurants. In the course of the investigation, public health officials tested food samples from various restaurants and found yellow onions from an open bin to be positive for E. coli O157:H7 in a restaurant in New York. However, this particular strain had never been associated with human illness in the U.S., and it did not match the DNA fingerprint of the strain that was responsible for the Taco Bell-related outbreak (CDC, 2006). Our literature search located two reports of fresh produce surveys for incidence of Salmonella that included dry onion bulbs. A 2004–2005 survey of fresh produce from the Central Produce Supply Station in Mexico City for incidence of Salmonella, resulted in no positive isolates from 100 randomly selected samples of white bulb onions (Quiroz-Santiago, 2009). In preparing the samples for laboratory analysis, produce items did not receive a disinfection treatment before being homogenized so microorganisms on both the interior and exterior were assessed. Another such study conducted in New Jersey in the mid-1990s, investigated the incidence of Salmonella on decaying and injured fresh produce collected from local supermarkets. Included in the sampling were 11 dry bulb onions all of which tested negative (Wells, 1997 & 1999). However, in a laboratory experiment to test the efficacy of chlorine dioxide against Salmonella on several fresh whole and fresh-cut produce commodities including whole Vidalia onions, Salmonella survived on inoculated onions, but did not grow. Whole onions inoculated with Salmonella were held at room temperature and 35 to 64% relative humidity. After approximately 22 hours in these conditions, Salmonella levels had declined approximately 1 log10 CFU/onion from the initial concentration of 7.96 log10 CFU/onion piece to 6.95 log10 CFU/ onion piece (Sy, 2005). In considering these studies collectively, field and laboratory studies provide evidence that E. coli O157:H7 can survive on onions, at least under conditions used in the studies. Although none of the 18 survey studies reported finding Salmonella on onions, in laboratory experiments Salmonella survived on inoculated onions. 4.4 Pathogen internalization Potential routes for pathogen internalization within the onion bulb during production may occur through the upper opening at the neck and through the root system. During production plant pathogens infect onions via both routes – pathogenic fungi may enter the onion through wounds or damaged leaf tissue or the neck, while soil-borne pathogens may enter the bulb through the roots. Post-harvest infection with plant pathogens may occur during storage through closely topped or improperly dried necks or damaged bulbs (Schwartz, 2011a & 2011b). If human pathogens enter onion plants using the same mechanisms as plant pathogens is uncertain, but some evidence suggests that human pathogens inhabit plant tissue differently than plant pathogens (Teplitski, 2009). Human pathogen internalization of fresh produce has been observed in several commodities, but how the bacteria are internalized and where they are localized within the plants are inadequately understood. Lateral root emergence sites seem to be a particularly vulnerable site. Tyler and Triplett (2008) review numerous studies where E. coli O157:H7 and Salmonella were observed by confocal microscopy to invade plant roots through cracks in the lateral roots. Similar to plant pathogens, wounded or damaged plant tissue have also been a point of entry for human pathogens. Although there are a few laboratory studies investigating the internalization of pathogens with green onions, we found no published studies of pathogen internalization conducted with onions that are harvested when bulbs are mature and handled in the same manner as dry bulb onions during post-harvest activities (e.g., field and shed curing). While green onions are the same plant, young immature onion plants may be more susceptible to human pathogens. In addition, whether human pathogens would survive the curing process, storage, etc. once internalized in immature onion plants is unknown without experiments carried through to the mature plant stage with conditions reflecting dry bulb onion processing. In an investigation of pathogen internalization, researchers in India selected fresh produce items (including onions) that are commonly eaten raw or minimally processed and tested for the incidence of Salmonella and E. coli O157:H7 (Ansingkar, 2010). In order to make an assertion about internalization, the produce items were washed in water containing 70% ethanol to sterilize the exterior before testing for the pathogens. Onion samples tested negative for both pathogens. Unfortunately, the publication has minimal details about samples or analytical methods such as onion variety, sample number, the locations from where the produce was taken, etc. Ge et al. (2011, 2013), conducted experiments on green onions in the laboratory to investigate whether Salmonella when applied to the leaves would migrate into the plant. Their objective was to mimic surface contamination scenarios such as spray irrigating onion plants with contaminated water, wildlife fecal droppings, or splashing of contaminated soil onto the plant surface. They applied Salmonella in concentrations of 10, 103, and 105 CFU/onion to the onion leaves by pipetting it directly on to the leaves while protecting the soil so the inoculum would not contaminate the soil. Plants were harvested 2 days later, sanitized by ethanol and silver nitrate and cut to separate upper and lower parts. Salmonella internalization was confirmed visually by confocal microscopy and enumerated by plate count and quantitative polymerase chain reaction (qPCR). The authors assert 19 that their results indicate that Salmonella Typhimurium can be taken up through the plant surface and transported from the upper to the lower part of the plant. The two methods of enumeration, plating and qPCR, had conflicting results. With the plate count method, the level of viable internalized Salmonella Typhimurium was higher in the lower part than the level in the upper leafy part, especially when the leaves were contaminated with a high concentration of Salmonella (105 CFU/onion), whereas when using qPCR, the total internalized Salmonella Typhimurium was significantly higher in the upper part at the same contamination level. The authors concluded that this discrepancy in their results suggests that most internalized Salmonella lost viability in the upper part but survived in the lower part (qPCR detects DNA in both dead and viable cells). In another laboratory study on green onions, Neetoo et al. (2012) irrigated green onions with Salmonella enterica and E. coli O157:H7 contaminated water (concentration ~8 log10 CFU/ml – 5 x 108 CFU/ml) every other day for 15 days. The results are summarized in Table 6 below. Table 6. Concentrations of E. coli O157:H7 and Salmonella enterica measured on green onions E. coli O157:H7 (log10 CFU/g) Salmonella enterica (log10 CFU/g) Harvested plant with trimmed roots 5.7 + 0.3 5.8 + 0.1 Surface-sanitized edible parts 5.4 + 0.3 4.9 + 0.6 Roots only 6.8 + 0.3 6.1 + 0.4 Bulb only 5.9 + 0.3 5.9 + 0.6 Stem only 5.5 + 0.5 5.4 + 0.3 Leaves only 1.8 + 0.7 1.5 + 0.4 According to scientists who were interviewed for this project and do research on plant pathogens that infect onions, infection of the bulb via the root system does occur with soil-borne plant pathogens, however, infection through the above-ground portion of the leave is more common (e.g., fungi). All the interviewed researchers were unaware of studies on dry bulb onions investigating internalization of E. coli O157:H7 or Salmonella or any foodborne illness-related human pathogens through the root system. None of those interviewed believed that research in young (green) onion plants would necessarily be indicative of what would occur if experiments were carried through until the onions were mature and harvested and handled in the same manner as dry bulb onions. 4.5 Potential sources of E. coli O157:H7 and Salmonella in the primary production of onions Pathogenic bacteria such as E. coli O157:H7 and Salmonella may be present in all segments of the onion supply chain from the water used to irrigate to the hands of shoppers and workers in the 20 market place and consumers in their homes. In this section, we discuss potential sources of pathogenic bacteria and mitigation measures used in controlling or monitoring these pathogens in the onion during production and harvest. Some unique onions characteristics that affect microbial growth are: They are a subterranean crop. They are covered with one to two layers of scale leaves They may be stored for months before consumption or processing. Potential production-related sources of pathogenic bacteria include soil, composted manure and other soil amendments, irrigation water, water used to mix agricultural chemicals, feces of wild and domestic animals, insects, and humans (Beuchat, 2002b; Gorny, 2006). Factors that affect the presence and/or levels of these pathogens in the onion production environment, may be locationand/or grower-specific, and include the following: Agricultural and management practices Climate, season and weather Environmental conditions such as soil type and water sources. Use of land adjacent to current production Previous use of production fields In 2010, the dry bulb onion industry addressed these issues and others in their Commodity Specific Food Safety Guidelines for the Dry Bulb Onion Supply Chain. 4.5.1 Soil Because of their subterranean growing environment, onions are in contact with many soil-dwelling microbes, including both plant pathogens and soil pathogens. In addition soil dwelling pathogens may reach the leaves from water splashing off the soil during overhead irrigation or rain. If leaves are injured or damaged (i.e., from hail during a storm), they are more vulnerable to contamination due to splashing. However, because of the layered structure of the onion plant, bulb tissue is open to the environment via the plant neck leaving the potential that even without damage, pathogens could access the bulb with splashing. Studies provide evidence that some soil types may support growth of human pathogens better than other soil types. Natvig et al. (2002) investigated the transfer of Salmonella typhimurium from two types of soil amended with bovine manure to radishes grown in controlled-environment chambers. Salmonella enterica was detected more often on unwashed radishes grown in silty clay soil, than on radishes grown in loamy sand soil. In addition, they found that washing radishes grown in loamy sand soil was more effective at reducing levels of Salmonella enterica than for radishes grown in silty clay soil (Natvig, 2002). Ibekwe et al. (2011) tested the survival of E. coli O157:H7 (initial concentration of 104) in clay and sand soil and found that in sandy soil the bacteria levels dropped rapidly between day 10 and 30, but survived at a low level (~1 log10 CFU/g) after day 30 until it was no longer detected at the experiment’s end (60 days). In clay soil at the same initial inoculum level, E. coli levels declined less rapidly and were still measured at ~3.0 log10 CFU/g on day 30, but dropped to nondetectable levels by day 60. In another study by the same group, E. coli survived at significantly higher levels in clay soil than in sandy soil at 12 days post-inoculation (Ibekwe, 2006). In a study to test the persistence of E. coli O157:H7 in different soil types, Ma et al. (2011) reported 21 the time needed to reach the detection limit (100 CFU/g) in loamy sand, sandy loam, and silty clay soils to be 32, 80, and 110 days, respectively. In their study of E. coli O157:H7 persistence in soil, Gagliardi and Karns (2002) also concluded that E. coli persisted longer in clay soil. 4.5.2 Animal manure and feces Animals both wild and domestic may serve as potential reservoirs for human pathogens. Various human pathogens including E. coli O157:H7 and Salmonella have been isolated from cattle, deer, pigs, birds, sheep, dogs, horses, and flies and reported in the scientific literature (Fenlon, 1981; Sargeant, 1999; Fischer, 2001; Brittingham, 1988; Chapman, 1997; Hancock, 1998; Jay, 2007). Pathogens may persist in manure for weeks or even months depending on environmental conditions. Low temperatures, moist conditions and adequate nutrients optimize survival. Wang et al. (1996) investigated the survival and growth characteristics of E. coli O157:H7 in bovine feces at different temperatures and found that at 37°C the bacterium survived for 42 and 49 days when inoculated at 103 and 105 CFU/g, respectively. At the same inoculation levels, but at 22°C, survival was 49 and 56 days, respectively. Near the study’s end, feces at both temperatures had only about 10% moisture levels with a water activity <0.5. In another study, E. coli O157:H7 survived in unaerated stockpiles of sheep manure for 21 months and 4 months in aerated stockpiles. The same study reported an E. coli O157:H7 survival rate in aerated bovine manure stockpiles of 47 days (Kudva, 1998). USDA researchers in Maine tested chicken, cow, pig and liquid dairy manures (LDM) for naturallyoccurring Listeria monocytogenes, Salmonella, and E. coli O157:H7. The results show: Listeria monocytogenes was detected in two of six samples of liquid dairy manure and one of six cow manure samples; Salmonella was detected in two out of six chicken manure samples and three out of six cow manure samples: generic E. coli was detected in all six samples regardless of the type of manure; and E. coli O157:H7 was not detected in any of the manure types (Liao, 2003). As part of this same study, the researchers measured the level of L. monocytogenes, Salmonella, E. coli O157:H7and generic E. coli in soil in a potato field sprayed with LDM and in potato tubers grown in the LDM-amended soil. Approximately 10 weeks after LDM application, L. monocytogenes became undetectable in soil. Of the 120 soil samples analyzed, Salmonella and E. coli O157:H7 were not detected. L. monocytogenes, Salmonella spp. and E. coli O157:H7 were not detected in any of the 20 potatoes collected during two potato-growing seasons (Liao, 2003). 4.5.3 Water Most human pathogens can survive in water for varying lengths of time depending on several factors such as temperature, salinity, dissolved oxygen, pH, the amount of pathogen present, predation, exposure to UV light and nutrient availability (Merge, 2002). The viability of most pathogenic bacteria in water decreases over time. Salmonella spp., for example, generally survive less than 30 days at 20–30˚C (Steele, 2004). In experiments testing the survival of E. coli O157:H7 in autoclaved municipal water, reservoir water and water from two recreational lakes at 8, 15 or 25°C, the bacterium survived longer in all three water types at 8°C, and the least amount of time at 25°C. At 8°C populations declined from 1 to 2 log10 CFU/ml in 91 day. At 25°C, populations decreased below the detection limit between day 49 and 84 in all but the autoclaved municipal water. 22 With onions given water is used for irrigation and agricultural chemical mixing, the source of the water should be considered when evaluating potential risks. For example, since surface water comes from an open environment, it is more susceptible to contamination than ground water and therefore a potential risk. There are numerous studies investigating the transfer of pathogenic bacteria and viruses from irrigation water to crops grown on or near the soil surface. As a subterraneous vegetable, these studies may have minimal application to onions; however, it is feasible that onions could be contaminated by the penetration of contaminated irrigation water into the soil. As discussed above, Islam et al. (2005) reported finding E. coli O157:H7 on onions and carrots for weeks following irrigation with artificially contaminated water. They also did similar experiments with Salmonella on carrots and radishes and had similar results (Islam, 2004b). 4.5.4 Birds and insects Insects and birds can act as vectors of human pathogens transmitting pathogens from a contaminated source to onions curing in the field after harvest or in open sheds (Olsen, 1998). Insects such as various types of flies have been shown to transmit human pathogens to food in various environments. A fly can mechanically transmit human pathogens from various sources by carrying the pathogens on its body as well as through their regurgitation and excretions (De Jesus, 2004; Talley, 2009; Vriesekoop, 2010; Wasala, 2013). Janisiewicz et al. (1999) demonstrated that fruit flies can transmit E. coli O157:H7 to wounds in apples as well as from apple to apple. In addition to being vectors, birds can serve as reservoirs of Salmonella and E. coli O157:H7 (Fenlon, 1985; Gorski, 2011; Hancock, 1998; LeJeune, 2008; Shere, 1998). Birds flying over uncovered onions curing in the field pose a risk of contamination; however, numerous studies in the U.S. and abroad show a low rate of fecal contamination in birds (Brittingham, 1988; Fenlon, 1985; Foster, 2006; Gorski, 2011; Hancock, 1998; LeJeune, 2008; Shere, 1998; Wallace, 1997). One study of Salmonella in birds on dairy farms in California showed a 2.5% infection rate (Kirk, 2002). 4.5.5 Workers Worker hygiene is addressed in commodity-specific food safety guidelines including the food safety guidelines for dry bulb onions. Workers who are infected with pathogenic bacteria and handle onions can potentially transmit the pathogens to the onions (Todd, 2009). Salmonella was isolated from swabbing of workers hands on a farm in Mexico where it was also found in the irrigation water and harvested cantaloupes and peppers (Gallegos-Robles, 2008). Gloves, if not handled properly, can also be a source of cross-contamination (Todd, 2010). According to Todd et al. (2007) 24 foodborne outbreaks with 8,580 cases of illness were attributed to workers in food processing plants from 1927−2006. Due to different harvesting practices, onion harvesting may be laborintensive or largely mechanical. However, even with mechanical harvesting, workers have direct contact with onions during sorting activities in the field and/or in the packinghouse. 4.5.6 Equipment Equipment used in harvesting that is not properly cleaned and/or sanitized (e.g. equipment used for animal transport or spreading raw, non-composted manure) can lead to the transference of fecesassociated pathogens to onions during post-harvest activities. Transport equipment and containers, conveyor belts, scales, and all contact surfaces in packing facilities may also be sources of contamination if not properly cleaned and sanitized. Studies have shown that surfaces constructed 23 from various materials (e.g. glass, Teflon, Formica, stainless steel, copper, polyvinyl chloride, polythene, glazed ceramic) are capable of supporting pathogens survival (Todd, 2009). 5.0 FRESH-MARKET DRY BULB PRODUCTION PRACTICES - EFFECTS ON PATHOGEN SURVIVAL AND GROWTH In the production of dry bulb onions, the practice that potentially has the most impact on the survival and growth of pathogens is the curing process. After bulbing, onions are mechanically removed from the soil either by lifting or undercutting and remain in the field for curing either on the soil or in burlap bags for anywhere from a day to weeks depending on the local production climate (Boyette, 1992; Fritz, 2010; OSU, 2004; Pelter, 2003; Smith, 2011; Swift, 2011;). After field curing, the curing process continues in sheds usually using forced air ventilation (@ 2–3 cubic feet of air per minute per each cubic foot of onions) and heat as needed to reach a certain temperature or to speed the drying process (Fritz, 2010). Optimal shed curing temperatures reportedly range from 75–90°F with relative humidity of 60–75%. Although these temperatures are below the optimal growing temperatures of E. coli O157:H7 (98.6–101.5°F) and Salmonella spp. (95–98.6°F), both pathogens have a broad range of temperature at which growth can occur, and both would no doubt survive at these temperatures. When onions are properly cured, they lose 3 to 5% of their weight and have one to two layers of scale leaves on the exterior (Fritz, 2010). Although many nonpathogenic bacteria are susceptible to desiccation, some strains of Salmonella and pathogenic E. coli have demonstrated tolerance for dry conditions in the laboratory setting. In experiments by Chang et al. (2005), Salmonella and E. coli O157 survived for 15 days at 77 and 95°F. However, even though drying may not kill some Salmonella and pathogenic E. coli, it remains to be seen whether the combination of desiccation and exposure to ultraviolet light during the field curing process may work in combination or synergistically to contribute to human pathogen inactivation. Optimal temperature for storing onions varies during the season, but is generally between 32 and 41°F, which is well below the minimum temperature (44.6°F) required for E. coli O157:H7 and Salmonella growth, but both pathogens can survive for long periods of time in refrigeration temperatures (NZFSA, 2001a & 2001b). However, without direct measurements it is difficult to assess how either pathogen would survive at the lower temperatures in combination with the low pH and low water activity of onion leaf scales. 6.0 CONCLUSIONS A search of three foodborne illness outbreak databases for reported outbreaks related to onions revealed no reports of outbreaks associated with on-farm contamination of dry bulb onions. Review of the outbreaks with potential or confirmed association with onions provided no evidence linking the contamination to the farm or storage or packing facility. Intrinsic onion properties play an important role in the ability of a microorganism to survive and grow, but need to be considered in the context of the external environment, which may be highly variable. The layered leaf structure of an onion may make it vulnerable to human pathogen contamination during production (i.e., access to bulb portion through neck opening), but the formation of leaf scales on the bulb during maturation, when intact may prevent human pathogen access to the edible bulb portion of the onion. 24 Onions contain two classes of chemicals demonstrated to have antimicrobial activity, and published reports provide some evidence that onions have antimicrobial properties that may have the potential to inhibit the growth of some human pathogens. However, study findings are highly variable, and in general, reveal little about how these chemicals may affect potential onion contamination. In addition, evidence from infections with bacterial plant pathogens as well as contamination with human pathogens in the laboratory studies indicate that at least some human pathogens are not affected by and/or have seemingly been able to adapt to an onion’s intrinsic antimicrobial defenses. Studies show differences in the levels of these compounds related to onion development stages and cultivars as well as cultivation and handling methods. Therefore it is challenging to make general conclusions about onion antimicrobial activity, let alone how this activity would affect the risk of contamination by the two human pathogens discussed. Laboratory and field studies using onions inoculated with E. coli O157:H7 and Salmonella spp. demonstrate that at least under the study conditions, these pathogens can survive on onions. Evidence of their ability to grow is limited and inconclusive. No studies of E. coli O157:H7 or Salmonella internalization in dry bulb onions were identified. There are a few studies demonstrating E. coli O157:H7 internalization in green onions, however, due to differences in the stage of plant development as well as in cultivation and post-harvest handling practices, application of the studies’ findings to dry bulb onions is questionable. Studies such as Islam et al. (2004a, 2004b, 2005) demonstrate the importance of commodityspecific research on the persistence of foodborne illness-related pathogens on fresh produce. In those studies persistence of E. coli O157:H7 in soil and on the produce itself was dependent on the commodity: E. coli O157:H7 persisted longer on carrots (>15 weeks) and in soil in which carrots were grown than on onions (<7 weeks) and in soil in which onions were grown. Levels of E. coli O157:H7 on carrots were also higher than on onions. Although, in our search of the literature, no data was found that measured human pathogen persistence during onion curing and storage processes, a similar commodity-specific response to those conditions may occur. And if pathogen persistence is commodity-specific, then persistence on commodities in the same genus as onions – e.g., garlic, may not be the same as persistence on onions. Many of the antimicrobial studies tested both onions and garlic and most found substantially different results. Because there are data gaps and unanswered questions about human pathogen internalization such as, if internalization does occur, how long do pathogens persist and under what conditions, further onion-specific research would be useful. Little is known about the persistence of human pathogens on the onion scale leaves and the effect of the curing process and/or storage conditions, if scale leaves are contaminated. Additional studies on irrigation water sources and types of irrigation would provide valuable information about the potential to contaminate onions if they are irrigated with contaminated water and whether pathogens would persist through harvesting and storage. 25 7.0 CONTRIBUTORS The following researchers and extensionists were interviewed for this report: Irwin Goldman, Ph.D., Department Chair and Professor, Dept. of Horticulture, University of Wisconsin-Madison Stuart Reitz, Ph.D., Professor and Extension Faculty, Department of Crop and Soil Science, Oregon State University Brenda K. Schroeder, Ph.D., Assistant Professor, Department of Plant Pathology, Washington State University Howard F. Schwartz, Ph.D., Professor and Extension Faculty, Department of Bioagricultural Sciences and Pest Management, Colorado State University 8.0 REFERENCES Adeshina GO, Jibo S, Agu VE, Ehinmidu JO. 2011. 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