- Key Facts
- Outbreaks and surveys documenting pathogen contamination
- Pathogen detection
- Harvesting and postharvest handling practices
- Pre-harvest sources and fate of contamination
- Practices for prevention of contamination in the field and during harvest
- Post-harvest interventions to minimize contamination and cross-contamination
- Romaine lettuce accounts for 30% of the lettuce consumed in the United States. Between 1990 and 2009, per capita consumption increased from 1.2 to 7.7 pounds per person.
- Although contamination of marketplace product is rare, from 2010 to 2013, three outbreaks totaling more than 700 illnesses were associated with Romaine lettuce.
- The majority of product produced and consumed in the U.S. is currently from California and Arizona. The bulk of romaine lettuce is hand harvested although mechanical harvesters are used by some operations.
- Small numbers of bacterial pathogens may survive on field lettuce for one or more weeks after contamination.
- Chlorine or other sanitizers are used in wash water to prevent cross-contamination of lettuce.
Lettuce (Lactuca sativa L.) is a popular vegetable that is widely grown and consumed throughout the world. China is the largest producer of lettuce, contributing 55% to the world’s total production by weight. By contrast, the United States contributes only about 16% to total lettuce production worldwide. The primary type of lettuce produced and consumed in China is a stem lettuce (L. sativa L. var. angustana), while in the U.S. there are three main lettuce types produced: iceberg (L. sativa L. var. capitata), romaine (L. sativa L. var. longifolia), and leaf (L. sativa L. var. crispaa). These three lettuces account for 54.1%, 30.1%, and 15.8% of U.S. production, respectively.(40)
Lettuce comes in a variety of colors, sizes and shapes. Romaine lettuce is exceptionally crisp, slightly bitter, and is characterized by long, narrow leaves with thick ribs. In addition, the leaves are typically upright to form an elongated head (top of the head may or may not close over the inner leaves) with commercial heads typically weighing approximately 0.75 kg (40). Romaine lettuce is well known as the primary ingredient in Caesar salad but in foodservice arenas, it is now also included in a variety of sandwich wraps as well as entrée and Greek salads. It is also included in the supermarket sections as prepackaged romaine hearts and in a wide variety of prepackaged salads. Although not as popular as iceberg lettuce, from 1990 to 2009, the per capita consumption of romaine lettuce increased from 1.2 to 7.7 pounds per person. (47)
Traditionally, lettuce had not been considered a very nutritious product primarily due to its high water content (ca. 95%). However, nutrient composition and bioactive compounds vary among lettuce types. Romaine lettuce, in particular, has nutritional benefits due to its low sodium content and its dietary contribution of fibers, folate, vitamin C, carotene, lutein, and phenolic compounds. (21)
Romaine lettuce is typically grown under conditions where contamination with enteric pathogens (Salmonella, Escherichia coli O157:H7, Listeria monocytogenes, Cyclospora cayetanensis, etc.) is possible and hence, depending on the level of contamination as well as the health of the individual, consumption of the raw product could lead to illness, if not death. Based on this potential for contamination, this article is therefore intended to detail recent outbreaks in which romaine lettuce has been implicated as well as to tabulate a number of surveys documenting the extent to which romaine lettuce in the marketplace is contaminated. Upon presenting this information, the article will then proceed to give a brief overview of the common practices applied to grow, harvest, and distribute romaine lettuce for retail distribution and risks as well as preventative measures associated with such practices.
Outbreaks and surveys documenting pathogen contamination
Foodborne outbreaks from contaminated fresh leafy vegetables have been increasingly recognized in many parts of the world. (4,16)
Moreover, within the U.S., there have been several high profile outbreaks that have been specifically associated with the consumption of romaine lettuce (see table below).
Although outbreaks are cause for concern, they serve only as examples of how the production system has not been appropriately managed to mitigate food safety risks. In fact, based upon pathogen prevalence surveys conducted on romaine lettuce in the marketplace (see table below), the industry, as a whole, has appropriate safeguards in place such that minimal risk exists for consumers to encounter a contaminated product.
It is noteworthy to mention that the majority of survey data only reports that enteric pathogens, such as Salmonella and E. coli O157:H7, would have been present or absent. Hence, they rarely give any indication as to the numbers present which, together with the virulence associated with the isolate(s) present, would affect the risk of becoming ill if that produce item were consumed raw. Variations in the methods used for detection of the pathogens in lettuce also exist with different surveys, and as discussed in the next section, the data may only be signifying that a viable pathogen has been present but not whether it is still viable and hence able to cause illness.
For decades, the primary means for detection of bacterial pathogens in foods and clinical samples took days to perform and relied on culture-based assays and biochemical tests for verification. Detection of viruses and parasites, on the other hand, was even more tedious in that isolation of the pathogen from the sample was required prior to its detection. Then assays employing molecular tools came to the forefront. As a result, the ability to detect pathogens in food took less time and epidemiologists, with tools such as pulsed field gel electrophoresis (PFGE), were able to match the isolates recovered from the patient, food, and contamination source. More recently with increased accessibility and decreased costs, whole genome sequencing (WGS) is now routinely used as a replacement for PFGE in many public health and research laboratories. As an early example of the utility of WGS, a L. monocytogenes isolate recovered from a patient was highly related by WGS to an isolate recovered from a romaine lettuce-containing product. (18)
As pathogen detection assays have evolved over the years, it is important to be cognizant that they still may have limitations and as such, the results may be biased. For example, a popular method of detection, the polymerase chain reaction (PCR) assay amplifies DNA that is specific to the pathogen, with pathogen quantification being based on rates of amplication of the DNA. However, this method is incapable of confirming whether the DNA derives from viable or nonviable cells as DNA does not degrade rapidly from intact nonviable cells. Hence, amplication of mRNA (RT-PCR) has been touted to detect only viable cells under the assumption that these bacterial transcripts are sensitive to degradation by intra- and extra-cellular RNases and would not be present shortly after death. Unfortunately, such an assumption has recently been proven false for detection of E. coli O157:H7 on field-grown romaine lettuce that would have been subjected to low-moisture conditions. (19) In any event, when viable cells are present but in low numbers, to circumvent inhibitors present in sample extracts that prevent PCR amplification enzyme activity, enrichment is necessary to have sufficient quantities of either DNA or mRNA present. Under these conditions, the assay then becomes qualitative and there is no way to know whether only one cell is present or thousands.
In the U.S. over a 20-year span (1992-2012), yields of romaine lettuce increased from 31.7 to 35.6 t/ha for an average increase of 12%. Although shifts in market use (e.g., reduced proportion of the head harvested for romaine lettuce hearts) negated some of the increased yields, changes in agronomic practices (e.g., wide beds and high plant density) and more uniform and disease- and pest-resistant cultivars were largely responsible for the increased yield gains. (40)
Romaine lettuce is classified as a cool-season crop and is grown in moderate climates with optimal day-time temperatures of 7 to 24°C and night-time temperatures of 3 to 12°C. At the high end of the temperature range, lettuce becomes more bitter, has a tendency to bolt (premature flower stalk formation), and generates loose, fluffy heads. At temperatures near freezing, young plants are not damaged but growth is slowed, whereas the outer leaves of mature lettuce plants may be damaged. (43)
In the United States, the bulk of lettuce production (including romaine lettuce) occurs in two states, California and Arizona, and together, they provide a year-round supply of lettuce for domestic consumption. Other states where commercial acreage is devoted to seasonal lettuce production is much less and includes Colorado (July to September), Florida, (December to April), New Mexico (April to May), and Washington (June to September). (40)Within these areas, soils used for cultivation should be well-drained and rich in organic matter. The majority of soils meeting those criteria within California are classified as silt loams or sandy soils. Heavy clay soils may also be used to grow lettuce as long as they have good soil structure that provides for adequate drainage. (43)
Within the major growing areas of the U.S., romaine lettuce is typically cultivated on raised beds (10 to 25 cm [4 to 10 in]) that may be either narrow (102 to 107 cm [40 to 42 in]) or wide (203 to 213 cm [80 to 84 in]). On these beds, pelleted seed is deposited just below the surface in two or three (narrow beds) or five or six (wide beds) parallel rows using a precision planter. (40,43) Transplants may also be set into the beds by hand or with the assistance of a waterwheel. These transplants have been typically seeded into cell plug trays in the greenhouse four to six weeks prior to going to the field. (50) In the major production areas, transplanting may be selected at the start of the season or at midseason when there is a need to get a crop in to meet production schedules.(43) In minor production areas, on the other hand, transplants may be preferred due to the short growing season. Regardless, transplanting provides for more accurate spacing and final plant population and reduced exposure to insect damage, drought or early season stresses. (50)
Several types of irrigation (sprinkler, drip, and furrow) are used to provide continuous moisture for maximum yields and quality. Initially, spray irrigation (6.1 to 12.2 cm [2 to 4 in]) is used prior to seeding to soften the soil for seedbed preparation. Then every two to three days, it is applied to seeded soil or transplants until seeds emerge or transplants have been established (usually 6 to 10 days after transplanting). Irrigation continues for the remainder of the growth cycle with the highest use of water occurring during the last month of the crop. During this time, surface drip irrigation with drip tape is often installed and utilized as it permits growers to water frequently during the rapid vegetative growth phase. This type of irrigation is also advantageous as romaine lettuce is shallow-rooted and drip irrigation, rather than spray-overhead irrigation, is more effective at delivering water directly to the roots and hence, less water is required (36 to 55 cm [12 to 18 in] for drip compared to 54 to 73 cm [18 to 24 in] for spray irrigation). Drip irrigation also facilitates weekly fertigation with low rates of fertilizer as opposed to having to apply side-dressings of nitrogen close to the romaine lettuce roots (applied typically 2-3 weeks after seeding and thinning has taken place). When utilized, drip tape is installed between rows and typically retrieved before harvesting so that it may be reused for subsequent crops. Furrow irrigation, the least efficient in terms of water usage (73 to 91 cm [24 to 30 in]), is used primarily in Arizona and in areas where strong winds exist.(40)
Several other practices that have been applied during the cultivation of romaine lettuce are currently less common but could become more prevalent as good quality water sources become scarce. One is the use of plasticulture when used with drip irrigation. Advantages to use of plasticulture is controlling weeds, moderating soil temperatures and hence maximizing plant growth and minimizing water evaporation. (50) Another set of practices to extend the growing season for romaine lettuce is to grow the plants in high tunnels, greenhouses, or hydroponic systems. In Florida where this latter system has seen rapid growth, the predominant design consists of plastic or other lightweight channels, gutters or tubes holding multiple transplants over which a thin film of nutrient solution trickles over the bare roots of each plant. The whole system is at a sloped angle enabling catchment of the unused nutrient solution that is then filtered or aerated and then recycled back to a reservoir for reuse. (35)
Harvesting and postharvest handling practices
Historically, romaine lettuce has been hand harvested as whole plants; however, small growers selling bagged greens may choose to harvest the crop as individual leaves. Typically, the time for romaine lettuce to reach market maturity will depend on growing conditions but harvested heads should be ones that will just be starting to close as waiting too long can result in bolting. (50) Once the romaine head is cut at the base, the head is trimmed of loose, discolored, damaged, diseased, and soiled leaves. These heads may then be placed on a conveyor belt where they are sprayed with a chlorinated water solution (generally 200 ppm) before being packed into 24 count (head) cartons. (40)
Hand harvest of lettuce requires a large amount of human capital. For example, in an article detailing the Arizona lettuce growing and harvesting operations, it was acknowledged that more than 45,000 legal guest workers from Mexico commuted across the border into Yuma every day for the purposes of harvesting up to 1 million boxes of lettuce each day. (39) Automated mechanized romaine harvesters that utilize a water knife to cleanly cut the heads in the field have been developed and are now being used in some commercial operations. (45)
Romaine lettuce heads have moderate respiration rates but they are generally higher than rates for iceberg lettuce. As such, it is even more critical that the product is quickly cooled to extend its shelf-life. Consequently, the cartons of romaine lettuce will be shipped to a facility where field heat may be removed through vacuum cooling although forced air cooling is being used with some success. (50) Once these operations are performed, romaine typically has a storage life of two to three weeks if stored at the proper temperature (0 to 5°C) and relative humidity (95%). However, during storage, the lettuce should never be stored with products that give off ethylene, such as apples, pears, or cantaloupes, because ethylene damage on romaine appears as discolored spots on the midrib. Although low oxygen atmospheres will reduce respiration, atmospheres containing carbon dioxide are not generally beneficial to intact heads. In contrast, cut romaine lettuce, is commonly packaged in low oxygen (< 1%) and high carbon dioxide (7 to 10%) atmospheres because these conditions control browning on the cut surfaces. (5)
Pre-harvest sources and fate of contamination
As discussed in section four, the majority of romaine lettuce consumed worldwide is grown in open agricultural systems. In such systems, pathogen contamination may therefore arise from a number of contaminated sources including: animal manure-based soil amendments; irrigation water; run-off water from livestock operations; insects; aerosols; and wildlife intrusion and subsequent deposition of their fecal matter. (11) In turn, the extent of contamination of the crop will vary with the contamination source but may span the gamut from a localized event to the entire crop potentially being initially contaminated. In addition, even if the pathogen does not get deposited onto the plant’s surface, there is the potential for it to be later contaminated through indirect events, such as water splashing contaminated soil or wildlife scat onto the plant. (1,6,27)
When a pathogen comes into contact with the phyllosphere (aerial) tissue, distribution on the surface is highly variable and likely a reflection of variations in both the chemical compounds and physical topography at individual sites. For example, Kroupitsky et al. (2011) found that Salmonella tended to localize preferentially near the petiole (stem) as opposed to the end of the leaf blade. The pathogen also had a higher affinity for the abaxial (underside) compared to the adaxial (topside) portion of leaves. Internalization of both Salmonella and E. coli O157:H7 into the phyllosphere tissue has also occurred with the pathogen entering via the stomata (breathing apertures). When initial dosages are typical of those that could be encountered under field conditions, however, these internalized pathogens appear to be transient residents as they can no longer be detected 24 to 48 h after being internalized. (12) Similarly, a large portion of E. coli O157:H7 that is deposited on a plant’s surface is also quickly inactivated; however, there is a slower rate of decline of the surviving cells. (26) For example, when E. coli O157:H7 was deposited on the upper surface of romaine lettuce heads in the field, within 2 h after being exposed, the pathogen population had already been reduced by a thousand-fold. Four weeks after exposure, however, a low number of surviving cells could still be detected. (29)
Two main environmental factors are primarily responsible for the initially rapid pathogen inactivation that occurs on lettuce plants: ultraviolet radiation and desiccation. In addition, it is conjectured that surviving pathogen cells are ones that have been deposited into niches where exposure to the two environmental factors are minimized. Such protection then likely affords sufficient time for the cells to modify their metabolism and become more stress-resistant. (13)
A third component that has the potential to alter the fate of enteric pathogens on romaine lettuce is biological in nature. For example, the persistence of E. coli O157:H7 was enhanced on romaine lettuce when the plant pathogen responsible for downy mildew disease was also present. (41) The role of indigenous microflora on the survival of enteric pathogens, on the other hand, has exhibited positive, negative, as well as no effect in several studies. Such variability is likely a reflection of the diversity and complexity of the indigenous microflora that may exist on plant tissue as well as the target pathogen and the conditions under which each study was conducted. For example, the levels of culturable indigenous bacteria showed significant positive correlations to the titers of multiple surrogate enteric viruses on romaine lettuce (14), whereas there was no effect on the survival of E. coli O157:H7 when field-grown microflora was transferred onto romaine lettuce plants being cultivated in a growth chamber. (51) In contrast, indigenous microflora present in the rhizosphere (soil located in close proximity to the roots) is conjectured to impede the internalization of enteric pathogens through the roots of lettuce plants. (12) This conclusion was based on the observation that in field soils having large numbers of indigenous microflora, internalization into roots only occurred when enteric pathogen populations were artificially added to the soil to levels that would not occur naturally (i.e., more than a million cells per gram). In contrast, there have been two scenarios where internalization of enteric pathogens into roots has been observed at lower pathogen levels. In the one case, the plants had been growing in field soil that had been subjected to high heat to dramatically reduce the native microflora. In the other case, the plants were being cultivated hydroponically (10) and thus there would be much lower levels of microflora present in the water than would be present in soil.
Practices for prevention of contamination in the field and during harvest
Contamination of romaine lettuce may occur anywhere along the farm-to-fork continuum. Hence, it stands to reason that both management and regulatory guidelines (to avoid contamination) and interventions (to reduce cross-contamination and reduce contamination) have been developed for application throughout the continuum. Initially after it became apparent that produce was associated with multiple outbreaks, voluntary guidance in the form of Good Agricultural Practices (GAPs) and Good Manufacturing Practices (GMPs) began to be issued in the late 90’s by both national and international groups to address food safety concerns associated with various commodities consumed in the raw state. Examples of these documents included:
• GlobalGap’s ‘Control Points and Compliance Criteria. Integrated Farm Assurance. Fruits and Vegetables’;
• U.S. Food and Drug Administration’s ‘Guide to Minimize Microbial Food Safety Hazards of Leafy Greens’ and
• California Leafy Green Marketing Board’s ‘Commodity Specific Guidelines for the Production and Harvest of Lettuce and Leafy Greens’.
Along with these documents, many operations were being required to submit to third party audits that used these guidelines or had their own unique conformance requirements that needed to be met in order for the product to be harvested and sold to distributors. When deadly outbreaks continued to occur, the federal government authorized through the Food Safety Modernization Act (FSMA) the creation of a more comprehensive and restrictive food safety system in the United States that relied on the establishment of science-based prevention-oriented mandatory standards, some of which include numerical criteria. Produce is just one area addressed in FSMA but within this broad group of products, FSMA focuses on practices that may introduce or spread biological hazards via five routes:
1. agricultural water;
2. biological soil amendments;
3. domesticated and wild animals;
4. worker training and health and hygiene; and
5. equipment, tools, and buildings.
The standards included in the Produce section of FSMA apply to producers and packers of raw agricultural commodities, both domestic and imported. However, farms that have an average annual value of produce sold during the previous three-year period of $25,000 or less are excluded. Exclusion of such farms, that could include operations such as U-pick farms, and farmers selling at farmer’s markets or roadside stands, was based on: 1) the concern that compliance would increase their costs and potentially put them out of business; and 2) their relatively small scale of covered total acreage within the U.S. (1.5%) and in turn, little measurable public health impact. Hence, although some farmers’ markets have established food safety rules that vendors must comply with, for the most part, food safety assurance of romaine lettuce purchased from small-scale markets will be dependent on verification by the end-consumer. For more information on the FSMA Produce Safety Rule, see a summation of the key requirements (48) or the Federal Register which has the complete document. (49)
Post-harvest interventions to minimize contamination and cross-contamination
A common practice applied to romaine lettuce either as heads or as a component of pre-packaged salads is washing with water to remove soil and debris from its surface. Another practice employed by many retail grocery stores is to submerge leafy greens in water for 5 to 15 min, followed by refrigeration, which gives products a fresh look and crispy texture. (20) However, the problem with both of these practices is that submersion in water may dislodge enteric pathogens from contaminated product into the water and when no active sanitizers are present in the water, those pathogens may then transfer to uncontaminated lettuce (17,23) and processing equipment surfaces. (2) Consequently, to limit cross-contamination during washing operations, inclusion of sanitizers, such as chlorine (sodium hypochlorite) or peroxyaectic acid (Tsunami) in water has been shown to be effective. (8,23) In particular, due to its wide antimicrobial activity and low cost, chlorine is the most common sanitizer used for washing lettuce with concentrations of 50 to 200 ppm and contact times of 1 to 3 min being recommended. (7) However, one disadvantage to using chlorine is its high reactivity with organic matter that decreases the levels of active chlorine in the water that is capable of inactivating pathogens. (9) Although inclusion of a chlorine-stabilizer, T-128, has been shown to extend the efficacy of chlorine when high organic loads are present in the wash water (31), there is also public concern that trihalomethane by-products formed in the presence of organic matter during disinfection of drinking water would also be formed in wash water. (37) Hence, chlorine dioxide has been suggested as an alternative to sodium hypochlorite for a wash water sanitizer as the former possesses higher oxidation capacity and forms fewer halogenated by-products than the latter. (24,37)
Contrary to the role of chemical agents being used as sanitizers to prevent cross-contamination during washing operations, a wide assortment of antimicrobials, including organic acids, ozone, plant essential oils, as well as the sanitizers discussed above, have been examined for their efficacy in reducing field-acquired contamination on lettuce. (32) For the majority of these antimicrobials, the efficacy has been relatively low resulting in only a 10 to 1000-fold reduction of the pathogen per gram of romaine lettuce. (28,53) Varying success for pathogen inactivation has also been achieved with different physical (ultraviolet light, ultrasound, irradiation, cold atmospheric plasma) and biological (antagonistic bacteria, bacteriophages, bacteriocins) interventions and the review by Olaimat and Holley (32) provides more detailed information on the studies in which they have been examined. Although the majority of those studies have not sought to differentiate the response of pathogens residing at different locations, internalized pathogens would likely be unaffected by aqueous antimicrobial treatments compared to physical interventions. (30) However, with any intervention that shows promise for inactivating pathogens on romaine lettuce, it is critical that before commercial implementation, verification be made as to whether the intervention treatment(s) affects the product’s quality either initially or during storage. In any event, at this point in time, these interventions are not being adopted by the industry for the purposes of reducing pathogen loads primarily due to a concern that any increased cost associated with the treatment would likely be transferred to the consumer who in turn would find it unacceptable.
As noted earlier in this review, romaine lettuce, whether as heads or in a packaged salad mix, should ideally be held at refrigeration times (0 to 5°C) during commercial transport, retail sale, and storage by the end user. Not only is this precaution necessary to reduce spoilage, but if pathogens are present, it minimizes their growth and decreases the possibility that there would be sufficient cells to make the individual consuming the product ill. For example, improper storage led to ca. 1000-fold increase in pathogens (L. monocytogenes, Salmonella, E. coli O157:H7) when shredded romaine lettuce was subjected to 25°C for 3 days and these increases occurred regardless of the composition of the atmosphere within the bag. (34) Even when a fresh-cut romaine salad mix was subjected to 5 different time-temperature profiles that would be considered standard for the industry, levels of both E. coli O157:H7 and L. monocytogenes increased ca. 1000-fold. (54) Such growth increases are therefore cause for concern as contrary to many consumers’ expectations that spoilage precedes any food safety risks, another study with packaged fresh-cut salad containing romaine and iceberg letter demonstrated that E. coli O157:H7 growth occurred before the product’s visual quality became unacceptable. (25)
Romaine lettuce is a hardy vegetable with a rich history dating back to at least the third millennium BCE. (43) In Ancient Egypt romaine lettuce was touted as an aphrodisiac, and was the sacred food of Min, the Egyptian god of fertility. (43) Romaine lettuce is used in the Maror plate during the Jewish ritual of Passover Seder. The dish symbolizes the bitterness and harshness of slavery the Jews endured in Ancient Egypt. In current times romaine lettuce is a common ingredient across the world. With its crisp texture and bitter herby taste, it pairs well with just about any ingredient. The lettuce is predominantly used in salads but can also be cooked, by for example grilled or sautéed. One of the most famous romaine lettuce recipes is the Caesar salad, in which the lettuce makes up the bulk of the dish. The Caesar salad is enjoyed across the globe, with each culture adding their own twist to the recipe. Romaine lettuce is sometimes used as an edible scoop for tabbouleh-like foods in Middle Eastern cuisine. In French cuisine grilled Romaine Hearts is a popular fresh vegetable dish. In Australia people enjoy their romaine lettuce tossed with fresh citrus fruits. Recently romaine lettuce has also made its way into space. In 2015 NASA astronauts on the International Space Station grew red romaine lettuce from the veggie plant growth system on the station’s orbiting laboratory.
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by Marilyn Erickson, PhD
Center for Food Safety
University of Georgia