Fresh-cut Leafy Greens Research Articles

Growth models for Salmonella, E. coli O157:H7 and L. monocytogenes give different predictions for pathogen growth in cut leafy greens transportation, but are consistent in identifying higher risk conditions

Leafy greens are frequently implicated in foodborne disease outbreaks and cut-leafy greens are a food that requires time and temperature control for safety. Predictive microbiology uses mathematical models to predict the growth of bacteria based on environmental conditions. The objective of our study was to compare published square root growth models for Salmonella (n = 6), pathogenic E. coli (n = 6) and Listeria monocytogenes (n = 4) using real world transport temperature data. Data from trucks transporting fresh-cut leafy greens during cross-country shipments were used as temperature inputs to the models. Bacterial growth was computed using the temperatures from each probe in every truck over the duration of transit, which resulted in 12–18 growth predictions per truck for each model. Each model generally gave significantly different predictions than other models for the same organism. The exception was for the two Salmonella models predicting the least growth and the two Salmonella models predicting the most growth which gave predictions that were not significantly different. Although different models tended to give different predictions, their ability to rank risk by truck was generally consistent across models. While absolute risk might be dependent upon choice of model, relative risk is independent of model choice.

Persistence of Silver Nanoparticles Sorbed on Fresh-Cut Lettuce during Flume Washing and Centrifugal Drying

Increased agricultural use of silver nanoparticles (Ag NPs) may potentially lead to residual levels on fresh produce, raising food safety and public health concerns. However, the ability of typical washing practices to remove Ag NPs from fresh produce is poorly understood. This study investigated the removal of Ag NPs from Ag NP-contaminated lettuce during bench-top and pilot-scale washing and drying. Ag NP removal was first assessed by washing lettuce leaves in a 4-L carboy batch system using water containing chlorine (100 mg/L) or peroxyacetic acid (80 mg/L) with and without a 2.5% organic load and water alone as the control. Overall, these treatments removed only 3–7% of the sorbed Ag from the lettuce. Thereafter, Ag NP-contaminated lettuce leaves were flume-washed for 90 s in a pilot-scale processing line using ∼600 L of recirculating water with or without a chlorine-based sanitizer (100 mg/L) and then centrifugally dried. After processing, only 0.3–3% of the sorbed Ag was removed, probably due to the strong binding of Ag with plant organic materials. Centrifugation only removed a minor amount of Ag as compared to flume washing. However, the Ag concentration in the ∼750 mL of centrifugation water was much higher as compared to the flume water, suggesting that the centrifugation water would be preferred when assessing fresh-cut leafy greens for Ag contamination. These findings indicate that Ag NPs may persist on contaminated leafy greens with commercial flume washing systems unable to substantially reduce Ag NP levels.

Evaluation of DNA barcode abiotic surrogate as a predictor for inactivation of E. coli O157:H7 during spinach washing

Fresh-cut leafy greens are washed in water with antimicrobials such as free chlorine to mitigate risk of pathogen cross-contamination. Validation of the process in a commercial setting poses challenges, including the identification of effective surrogates that can be used directly on the processing line during commercial operation. The objective of this study was to evaluate the use of an abiotic, DNA-based indicator as a potential surrogate for Escherichia coli O157:H7 during the washing of spinach. E. coli O157:H7 and DBAS (DNA barcode abiotic surrogate) were inoculated onto spinach leaves and washed in water with free chlorine. E. coli O157:H7 and DBAS populations were enumerated by selective plate counting or qPCR, respectively, and by laser scanning confocal microscopy. DBAS displayed high sensitivity to free chlorine at concentrations ≥12.5 mg/L, similar to concentrations commonly used in leafy green washing. Linear regression analysis indicated that inactivation of E. coli O157:H7 and DBAS had comparable inactivation responses (R2 = 0.76). Microscopy analysis also showed similar rates of decontamination between the two analytes from the surface of spinach. These results show that DBAS is a promising abiotic surrogate for onsite validation of antimicrobial processes targeting foodborne pathogens such as E. coli O157:H7 on fresh produce.

Improving temperature management and retaining quality of fresh-cut leafy greens by retrofitting open refrigerated retail display cases with doors

Abstract Open refrigerated display cases in supermarkets are prevalent in the United States despite being prone to temperature fluctuations. We collaborated with a chain retailer in Florida to retrofit existing open vegetable display cases with doors, and monitored the display temperature profile over 5 months. Quality evaluations were performed on baby spinach, spring mix, and chopped romaine lettuce after storage under simulated open and closed case conditions. Doors improved spatial and temporal temperature uniformity in whole and fresh-cut vegetable cases. Optimal storage temperature for ready-to-eat produce (≤5 °C) was recorded over 98% of the time after door installation versus 49–88% before retrofitting. An overall temperature decrease in the fresh-cut produce case from 3.62 to 0.66 °C necessitated a thermostat setting adjustment of +1.1 °C to avoid freezing of salad product. Elevated headspace CO2 concentrations and higher populations of aerobic bacteria (6.6 log CFU g−1), yeasts and molds (4.6 log CFU g−1) were found under open display conditions for one or more salad products tested. Baby spinach leaves from the open case environment were lighter green and more yellowish in color. Electrolyte leakage and ascorbic acid content of the three salad types were similar between the open and closed door displays. Superior overall visual quality and stronger purchase intent were found for baby spinach and spring mix samples from the closed display case. These products had less severe off-odor and discoloration, and crispier texture than those held in the open case. The decreased overall temperature and improved temperature uniformity in closed display cases, along with better produce quality and potential energy savings, may encourage retailers to consider retrofitting open displays with doors or purchasing closed displays.

Efficacy of Acetic Acid or Chitosan for Reducing the Prevalence of Salmonella- and Escherichia coli O157:H7–Contaminated Leafy Green Plants in Field Systems

Outbreaks associated with fresh-cut leafy greens continue to occur despite efforts to implement horticultural practices that minimize introduction of enteric pathogens to the crop. The experimental trials in this study were designed to examine the efficacy of an acetic acid (AA)- and chitosan-based spray treatment, applied 1 day prior to harvest, for reducing the prevalence of Escherichia coli O157:H7 (O157) and Salmonella in field-grown leafy greens contaminated at levels detectable only through enrichment culture. Responses to the treatment solution were variable and depended on the type of leafy green (leafy lettuce, spinach, or cabbage), cultivar, pathogen, and AA concentration (0.3 to 0.7%). No significant differences in E. coli O157 prevalence were found for untreated and treated cabbage heads and spinach plants (P > 0.05). In contrast, treatment significantly affected Salmonella on 'Bravo F1' green cabbage and '7-Green' spinach (P < 0.05), with odds ratios of 2.2 and 3.3 for finding the pathogen on untreated versus treated greens, respectively. Salmonella was also 7.1 times more likely to be found on an untreated lettuce plant than on a lettuce plant sprayed with a 0.7% AA treatment solution (95% confidence interval [CI], 4.1 to 12.2; P < 0.0001). In studies addressing the efficacy of chitosan (0.1 or 0.3%), this chemical failed to reduce the prevalence of either pathogen on lettuce (P > 0.05). Similarly, spraying with 0.3% AA did not affect the prevalence of Salmonella on lettuce plants (P > 0.05); however, treatment solutions with 0.4% AA reduced the likelihood of detecting Salmonella in treated versus untreated plants by 6.6 times (95% CI, 2.1 to 20.9; P = 0.0007). After the lettuce was harvested and hand washed, consumers failed to distinguish either visually or organoleptically between untreated lettuce and lettuce sprayed with an acetic acid solution (P > 0.05). These results indicate that acetic acid could be used to reduce the microbiological risk of preharvest leafy greens.

Transfer and Redistribution of Salmonella Typhimurium LT2 and Escherichia coli O157:H7 during Pilot-Scale Processing of Baby Spinach, Cilantro, and Romaine Lettuce

Several outbreaks of foodborne illness traced to leafy greens and culinary herbs have been hypothesized to involve cross-contamination during washing and processing. This study aimed to assess the redistribution of Salmonella Typhimurium LT2 during pilot-scale production of baby spinach and cilantro and redistribution of Escherichia coli O157:H7 during pilot-scale production of romaine lettuce. Four inoculated surrogate:uninoculated product weight ratios (10:100, 5:100, 1:100, and 0.5:100) and three inoculation levels (103, 101, and 10-1 CFU/g) were used for the three commodities. For each of three trials per condition, 5-kg batches containing uninoculated product and spot-inoculated surrogate products at each ratio and inoculation level were washed for 90 s in a 3.6-m-long flume tank through which 890 L of sanitizer-free, filtered tap water was circulated. After washing and removing the inoculated surrogate products, washed product (∼23, 225-g samples per trial) was analyzed for presence or absence of Salmonella Typhimurium or E. coli O157:H7 by using the GeneQuence Assay. For baby spinach, cilantro, and romaine lettuce, no significant differences ( P > 0.05) in the percentage of positive samples were observed at the same inoculation level and inoculated:uninoculated weight ratio. For each pathogen product evaluated (triplicate trials), inoculation level had a significant impact on the percentage of positive samples after processing, with the percentage of positive samples decreasing, as the initial surrogate inoculation level decreased. The weight ratio of contaminated:noncontaminated product plays an important role: positive samples ranged from 0% to 11.6% ± 2.05% and from 68.1% ± 33.6% to 100% among the four ratios at inoculation of 10-1 and 101 CFU/g, respectively. To our knowledge, this study is the first to assess the redistribution of low levels of pathogens from incoming product to leafy greens during processing and should provide important data for microbial risk assessments and other types of food safety analyses related to fresh-cut leafy greens.

Minimizing pathogen growth and quality deterioration of packaged leafy greens by maintaining optimum temperature in refrigerated display cases with doors

Retail display of packaged fresh-cut leafy greens is a critical stage of cold chain management and is prone to temperature fluctuations when produce is displayed in open cases, due to infiltration of ambient air into the case. Previous studies demonstrated that retrofitting open display cases with doors is the most effective solution to bring produce temperatures into compliance with FDA Food Code (<5 °C), retain product quality longer, and reduce operational energy costs. In this study, we evaluated changes in quality attributes and populations of inoculated bacterial pathogens (Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes) in packaged baby spinach, chopped romaine, and lettuce mix displayed in a refrigerated case retrofitted with doors. All products displayed in the case with doors maintained high freshness and attractiveness after 3-day display, and the quality was comparable to that of products stored at a constant temperature in a 4 °C cold room. No substantial changes in pathogen populations were observed during the display period. These results demonstrate that retrofitting display cases with doors is a practical means of reducing temperature fluctuations and damage for fresh-cut products.

Approaches toward Identification of Surrogates To Validate Antimicrobial Washes as Preventive Controls for Fresh-Cut Leafy Greens

In fresh-cut produce production, antimicrobials may be used during washing to control the risk of cross-contamination by microbial hazards. Surrogate microorganisms have long been used to validate processes, but none have been identified for validating the efficacy of antimicrobial washing of fresh-cut produce. The objective of this study was to develop procedures by which surrogates may be identified for use in validating the control of cross-contamination for fresh-cut lettuce operations. Four microbial characteristics, which may be important factors in cross-contamination events, were quantitatively evaluated in potential surrogate microorganisms for comparison to a reasonably foreseeable hazard, Escherichia coli O157:H7: sensitivity to chlorine in solution, sensitivity to chlorine on lettuce leaf surfaces, shedding from contaminated lettuce leaves into the water during washing, and cross-contamination from inoculated to uninoculated lettuce leaves during chorine washing. A procedure of practical quantitative experiments for comparing the characteristics reduced the original pool of 80 potential strains, which consisted of lactic acid bacteria, probiotics, and isolates obtained from lettuce enrichment cultures, to five strains: Lactobacillus plantarum, Pediococcus pentosaceus, probiotic 22C, and two lettuce enrichment isolates. These strains may be evaluated in additional studies involving comparisons to other reasonably foreseeable hazards and including other potential process variables that should be understood and controlled to prevent cross-contamination in fresh-cut lettuce operations.

Temperatures experienced by fresh-cut leafy greens during retail storage and display

Temperatures experienced by fresh-cut leafy greens during retail storage and display

Food safety management system (FSMS) adjusted to the characteristics of the leafy greens production chain context in Spain

The shift from local to global food markets as well as other food safety concerns such as the pressure of climate change has increased the demand on current horticultural production systems to guarantee the microbiological (e.g., control of Salmonella and pathogenic E. coli) and chemical (e.g., pesticide residues) safety of fresh produce. To deal with these concerns, food safety management systems (FSMS) that are tailored to specific company situations are needed. However, the context of the production, processing and trade chains influence the FSMS. The objective of this study was to gain insights into the food safety context of fresh produce production and processing companies located in southeastern Spain, a leading European horticultural area. A diagnostic tool was applied in 13 producer and 7 processing companies focused on the trade of fresh-cut leafy greens throughout the Eu. The diagnostic tool consisted of a questionnaire about the context of the company and its risk profile. Results showed that product and process riskiness were moderate to high for most of the companies surveyed. All the companies in this study complied with voluntary standards such as GLOBAL GAP, BRC and IFS. Multiple certification requirements are forcing these companies to develop a generic sector-based FSMS adapted to their situations.

Evaluation of food safety related behaviors of retail and food service employees while handling fresh and fresh-cut leafy greens

Foodborne illness outbreaks are often associated with fresh and fresh-cut produce (specifically leafy greens); however, effective training and methods of prevention have not been readily available for the retail and food service industry. The objective of this study was to assess and evaluate the current state of compliance and employee handling practices for fresh and fresh-cut produce, and to identify factors influencing employees' fresh and fresh-cut handling practices using an observational survey tool. A total of sixteen restaurants and fifteen grocery stores in Texas and Ohio were observed. The results revealed low compliance of employees' leafy green handling practices such as improper use of thermometers, lack of documentation, and poor employee hygiene. Statistical analysis results suggested that employees' food safety behaviors vary based on several factors such as operation size, cuisine, and operation type. The results of the study will provide practical guidelines for training employees handling leafy green produce at fresh and fresh-cut packing facilities, produce distribution centers, grocery stores, and restaurants.

Transit temperatures experienced by fresh-cut leafy greens during cross-country shipment

There have been very limited time and temperature profiles published for the United States indicating the conditions experienced by fresh-cut, bagged leafy greens during U.S. cold chain transport and distribution. The objective of this study was to monitor and fully characterize truck trailer temperatures during the transport of fresh-cut leafy greens shipped from Salinas, CA and Yuma, AZ, to distribution centers across the United States. A total of sixteen shipments were monitored for trailer temperature from June, 2010 to May, 2011, utilizing trailers provided by multiple major Western processor/shippers. Twenty-four or thirty temperature loggers, total, were placed on six pallets and along the sidewalls during the loading of each trailer. Sensors were placed on pallet stacks at the front, middle and rear of each trailer. Two pallet stacks positioned side-by-side were monitored at each location. Additional sensors were placed 4 feet above the floor on each sidewall adjacent to the monitored stacks. In five of the sixteen shipments, an additional sensor with a probe was placed in each monitored stack at the middle position and the probe was inserted 24 inches into the center of the stack. Data was analyzed for the effect of location within a trailer, on a sidewall, or on or within a pallet; and on a pass/fail basis where any sensor in a trailer recording temperatures above 5 °C subjected the entire load to rejection. Analyzing sensor data on a pass/fail basis yielded highly skewed results as the analysis indicated all trailers failed to maintain adequate temperature during transit. However, only 16.7% of the 431 sensors used in the study recorded temperatures greater than 5 °C and nearly twice as many (47) were found on sidewalls than pallets (25). The FDA standard for pre-cut leafy greens appears to overestimate high temperature abuse as it does not consider where the abuse is occurring in a trailer. Cold temperature abuse was less extensive with only 39 of 431 sensors recording temperatures below −0.17 °C. The data suggests that the product was also under-heated in fall as 22 of 39 sensors recording temperatures <−0.17 °C did so during this period. Though the interior was not greatly different in temperature than the exterior of pallet stacks (2.87 vs. 2.09 °C, respectively), the results indicate the importance of proper stacking and good air-flow around boxes of product in a trailer. For some runs, localized freezing of pre-cut greens may have occurred but, overall, the trailer refrigeration units appeared to be efficient though there was large variability in trailer performance. Microbial modeling utilizing Escherichia coli O157:H7 and Listeria monocytogenes should be conducted based on the cold–abuse profiles generated in this study to determine the potential for the growth of these microbes on pre-cut leafy greens during subsequent retail storage and display.

Methodology for modeling the disinfection efficiency of fresh-cut leafy vegetables wash water applied on peracetic acid combined with lactic acid

A methodology to i) assess the feasibility of water disinfection in fresh-cut leafy greens wash water and ii) to compare the disinfectant efficiency of water disinfectants was defined and applied for a combination of peracetic acid (PAA) and lactic acid (LA) and comparison with free chlorine was made. Standardized process water, a watery suspension of iceberg lettuce, was used for the experiments. First, the combination of PAA+LA was evaluated for water recycling. In this case disinfectant was added to standardized process water inoculated with Escherichia coli (E. coli) O157 (6logCFU/mL). Regression models were constructed based on the batch inactivation data and validated in industrial process water obtained from fresh-cut leafy green processing plants. The UV254(F) was the best indicator for PAA decay and as such for the E. coli O157 inactivation with PAA+LA. The disinfection efficiency of PAA+LA increased with decreasing pH. Furthermore, PAA+LA efficacy was assessed as a process water disinfectant to be used within the washing tank, using a dynamic washing process with continuous influx of E. coli O157 and organic matter in the washing tank. The process water contamination in the dynamic process was adequately estimated by the developed model that assumed that knowledge of the disinfectant residual was sufficient to estimate the microbial contamination, regardless the physicochemical load. Based on the obtained results, PAA+LA seems to be better suited than chlorine for disinfecting process wash water with a high organic load but a higher disinfectant residual is necessary due to the slower E. coli O157 inactivation kinetics when compared to chlorine.

Impact of Organic Load on Escherichia coli O157:H7 Survival during Pilot-Scale Processing of Iceberg Lettuce with Acidified Sodium Hypochlorite

Chemical sanitizers are routinely used during commercial flume washing of fresh-cut leafy greens to minimize cross-contamination from the water. This study assessed the efficacy of three chlorine treatments against Escherichia coli O157:H7 on iceberg lettuce, in wash water, and on surfaces of a pilot-scale processing line using flume water containing various organic loads. Iceberg lettuce (5.4 kg) was inoculated to contain 10(6) CFU/g of a 4-strain cocktail of nontoxigenic, green fluorescent protein-labeled, ampicillin-resistant E. coli O157:H7 and held for 24 h at 4°C before processing. Lettuce was shredded using a Urschel TransSlicer, step conveyed to a flume tank, washed for 90 s using water alone or one of three different sanitizing treatments (50 ppm of total chlorine either alone or acidified to pH 6.5 with citric acid or T-128) in water containing organic loads of 0, 2.5, 5, or 10% (wt/vol) blended iceberg lettuce, and then dried using a shaker table and centrifugal dryer. Next, three 5.4-kg batches of uninoculated iceberg lettuce were processed identically. Various product (25 g), water (50 ml), and equipment surface swab (100 cm(2)) samples were homogenized in neutralizing buffer, diluted appropriately, and plated on tryptic soy agar containing 0.6% (wt/vol) yeast extract and 100 ppm of ampicillin without prior 0.45- m m membrane filtration to quantify E. coli O157:H7. Organic load negatively impacted the efficacy of all three chlorine treatments (P < 0.05) at the end of processing, with typical E. coli O157:H7 reductions of >5 and 0.9 to 3.7 log CFU/ml for organic loads of 0 and 10%, respectively. Organic load rarely had a significant impact (P < 0.05) on the efficacy of chlorine, chlorine plus citric acid, or chlorine plus T-128 against E. coli O157:H7 on iceberg lettuce. Reduced sanitizer efficacy generally corresponded to changes in total solids, chemical oxygen demand, turbidity, and maximum filterable volume, indicating that these tests may be effective alternatives to the industry standard of oxygen/reduction potential.

Tracking an Escherichia coli O157:H7–Contaminated Batch of Leafy Greens through a Pilot-Scale Fresh-Cut Processing Line

Cross-contamination of fresh-cut leafy greens with residual Escherichia coli O157:H7-contaminated product during commercial processing was likely a contributing factor in several recent multistate outbreaks. Consequently, radicchio was used as a visual marker to track the spread of the contaminated product to iceberg lettuce in a pilot-scale processing line that included a commercial shredder, step conveyor, flume tank, shaker table, and centrifugal dryer. Uninoculated iceberg lettuce (45 kg) was processed, followed by 9.1 kg of radicchio (dip inoculated to contain a four-strain, green fluorescent protein-labeled nontoxigenic E. coli O157:H7 cocktail at 10(6) CFU/g) and 907 kg (2,000 lb) of uninoculated iceberg lettuce. After collecting the lettuce and radicchio in about 40 bags (∼22.7 kg per bag) along with water and equipment surface samples, all visible shreds of radicchio were retrieved from the bags of shredded product, the equipment, and the floor. E. coli O157:H7 populations were quantified in the lettuce, water, and equipment samples by direct plating with or without prior membrane filtration on Trypticase soy agar containing 0.6% yeast extract and 100 ppm of ampicillin. Based on triplicate experiments, the weight of radicchio in the shredded lettuce averaged 614.9 g (93.6%), 6.9 g (1.3%), 5.0 g (0.8%), and 2.8 g (0.5%) for bags 1 to 10, 11 to 20, 21 to 30, and 31 to 40, respectively, with mean E. coli O157:H7 populations of 1.7, 1.2, 1.1, and 1.1 log CFU/g in radicchio-free lettuce. After processing, more radicchio remained on the conveyor (9.8 g; P < 0.05), compared with the shredder (8.3 g), flume tank (3.5 g), and shaker table (0.1 g), with similar E. coli O157:H7 populations (P > 0.05) recovered from all equipment surfaces after processing. These findings clearly demonstrate both the potential for the continuous spread of contaminated lettuce to multiple batches of product during processing and the need for improved equipment designs that minimize the buildup of residual product during processing.

Transfer of Escherichia coli O157:H7 from equipment surfaces to fresh-cut leafy greens during processing in a model pilot-plant production line with sanitizer-free water.

Escherichia coli O157:H7 contamination of fresh-cut leafy greens has become a public health concern as a result of several large outbreaks. The goal of this study was to generate baseline data for E. coli O157:H7 transfer from product-inoculated equipment surfaces to uninoculated lettuce during pilot-scale processing without a sanitizer. Uninoculated cored heads of iceberg and romaine lettuce (22.7 kg) were processed using a commercial shredder, step conveyor, 3.3-m flume tank with sanitizer-free tap water, shaker table, and centrifugal dryer, followed by 22.7 kg of product that had been dip inoculated to contain ∼10(6), 10(4), or 10(2) CFU/g of a four-strain avirulent, green fluorescent protein-labeled, ampicillin-resistant E. coli O157:H7 cocktail. After draining the flume tank and refilling the holding tank with tap water, 90.8 kg of uninoculated product was similarly processed and collected in ∼5-kg aliquots. After processing, 42 equipment surface samples and 46 iceberg or 36 romaine lettuce samples (25 g each) from the collection baskets were quantitatively examined for E. coli O157:H7 by direct plating or membrane filtration using tryptic soy agar containing 0.6% yeast extract and 100 ppm of ampicillin. Initially, the greatest E. coli O157:H7 transfer was seen from inoculated lettuce to the shredder and conveyor belt, with all equipment surface populations decreasing 90 to 99% after processing 90.8 kg of uncontaminated product. After processing lettuce containing 10(6) or 10(4) E. coli O157:H7 CFU/g followed by uninoculated lettuce, E. coli O157:H7 was quantifiable throughout the entire 90.8 kg of product. At an inoculation level of 10(2) CFU/g, E. coli O157:H7 was consistently detected in the first 21.2 kg of previously uninoculated lettuce at 2 to 3 log CFU/100 g and transferred to 78 kg of product. These baseline E. coli O157:H7 transfer results will help determine the degree of sanitizer efficacy required to better ensure the safety of fresh-cut leafy greens.

Quantitative transfer of Escherichia coli O157:H7 to equipment during small-scale production of fresh-cut leafy greens.

Postharvest contamination and subsequent spread of Escherichia coli O157:H7 can occur during shredding, conveying, fluming, and dewatering of fresh-cut leafy greens. This study quantified E. coli O157:H7 transfer from leafy greens to equipment surfaces during simulated small-scale commercial processing. Three to five batches (22.7 kg) of baby spinach, iceberg lettuce, and romaine lettuce were dip inoculated with a four-strain cocktail of avirulent, green fluorescent protein-labeled, ampicillinresistant E. coli O157:H7 to contain ∼10(6), 10(4), and 10(2) CFU/g, and then were processed after 1 h of draining at ∼23°C or 24 h of storage at 4°C. Lettuce was shredded using an Urschel TransSlicer at two different blade and belt speeds to obtain normal (5 by 5 cm) and more finely shredded (0.5 by 5 cm) lettuce. Thereafter, the lettuce was step conveyed to a flume tank and was washed and then dried using a shaker table and centrifugal dryer. Product (25-g) and water (40-ml) samples were collected at various points during processing. After processing, product contact surfaces (100 cm(2)) on the shredder (n = 14), conveyer (n = 8), flume tank (n = 11), shaker table (n = 9), and centrifugal dryer (n = 8) were sampled using one-ply composite tissues. Sample homogenates diluted in phosphate or neutralizing buffer were plated, with or without prior 0.45- m m membrane filtration, on Trypticase soy agar containing 0.6% yeast extract supplemented with 100 ppm of ampicillin to quantify green fluorescent protein-labeled E. coli O157:H7 under UV light. During leafy green processing, ∼90% of the E. coli O157:H7 inoculum transferred to the wash water. After processing, E. coli O157:H7 populations were highest on the conveyor and shredder (P<0.05), followed by the centrifugal dryer, flume tank, and shaker table, with ∼29% of the remaining product inoculum lost during centrifugal drying. Overall, less (P<0.05) of the inoculum remained on the product after centrifugally drying iceberg lettuce that was held for 1 h (8.13%) as opposed to 24 h (42.18%) before processing, with shred size not affecting the rate of E. coli O157:H7 transfer.

Effect of modified atmosphere packaging on the persistence and expression of virulence factors of Escherichia coli O157:H7 on shredded iceberg lettuce.

Fresh-cut leafy greens contaminated with Escherichia coli O157:H7 have caused foodborne outbreaks. Packaging conditions, coupled with abusive storage temperatures of contaminated lettuce, were evaluated for their effect on the potential virulence of E. coli O157:H7. Shredded lettuce was inoculated with 5.58 and 3.98 log CFU E. coli O157:H7 per g and stored at 4 and 15°C, respectively, for up to 10 days. Lettuce was packaged under treatment A (modified atmosphere packaging conditions used for commercial fresh-cut produce, in gas-permeable film with N(2)), treatment B (near-ambient air atmospheric conditions in a gas-permeable film with microperforations), and treatment C (high-CO(2) and low-O(2) conditions in a gas-impermeable film). E. coli O157:H7 populations from each treatment were determined by enumeration of numbers on MacConkey agar containing nalidixic acid. RNA was extracted from packaged lettuce for analysis of expression of virulence factor genes stx(2), eae, ehxA, iha, and rfbE. E. coli O157:H7 populations on lettuce at 4°C under all treatments decreased, but most considerably so under treatment B over 10 days. At 15°C, E. coli O157:H7 populations increased by at least 2.76 log CFU/g under all treatments. At 15°C, expression of eae and iha was significantly greater under treatment B than it was under treatments A and C on day 3. Similarly, treatment B promoted significantly higher expression of stx(2), eae, ehxA, and rfbE genes on day 10, compared with treatments A and C at 15°C. Results indicate that storage under near-ambient air atmospheric conditions can promote higher expression levels of O157 virulence factors on lettuce, and could affect the severity of E. coli O157:H7 infections associated with leafy greens.