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

2014 ◽  
Vol 77 (9) ◽  
pp. 1487-1494 ◽  
Author(s):  
ANNEMARIE L. BUCHHOLZ ◽  
GORDON R. DAVIDSON ◽  
BRADLEY P. MARKS ◽  
EWEN C. D. TODD ◽  
ELLIOT T. RYSER

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 106 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.


2013 ◽  
Vol 76 (11) ◽  
pp. 1838-1845 ◽  
Author(s):  
GORDON R. DAVIDSON ◽  
ANNEMARIE L. BUCHHOLZ ◽  
ELLIOT T. RYSER

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 five commercial sanitizer treatments against Escherichia coli O157:H7 on iceberg lettuce, in wash water, and on equipment during simulated commercial production in a pilot-scale processing line. Iceberg lettuce (5.4 kg) was inoculated to contain 106 CFU/g of a four-strain cocktail of nontoxigenic, green fluorescent protein–labeled, ampicillin-resistant E. coli O157:H7 and processed after 1 h of draining at ~22°C. Lettuce was shredded using a commercial slicer, step-conveyed to a flume tank, washed for 90 s using six different treatments (water alone, 50 ppm of peroxyacetic acid, 50 ppm of mixed peracid, or 50 ppm of available chlorine either alone or acidified to pH 6.5 with citric acid [CA] or T-128), and then dried using a shaker table and centrifugal dryer. Various product (25-g) and water (50-ml) samples collected during processing along with equipment surface samples (100 cm2) from the flume tank, shaker table, and centrifugal dryer were homogenized in neutralizing buffer and plated on tryptic soy agar. During and after iceberg lettuce processing, none of the sanitizers were significantly more effective (P ≤ 0.05) than water alone at reducing E. coli O157:H7 populations on lettuce, with reductions ranging from 0.75 to 1.4 log CFU/g. Regardless of the sanitizer treatment used, the centrifugal dryer surfaces yielded E. coli O157:H7 populations of 3.49 to 4.98 log CFU/100 cm2. Chlorine, chlorine plus CA, and chlorine plus T-128 were generally more effective (P ≤ 0.05) than the other treatments, with reductions of 3.79, 5.47, and 5.37 log CFU/ml after 90 s of processing, respectively. This indicates that chlorine-based sanitizers will likely prevent wash water containing low organic loads from becoming a vehicle for cross-contamination.



2012 ◽  
Vol 75 (7) ◽  
pp. 1184-1197 ◽  
Author(s):  
ANNEMARIE L. BUCHHOLZ ◽  
GORDON R. DAVIDSON ◽  
BRADLEY P. MARKS ◽  
EWEN C. D. TODD ◽  
ELLIOT T. RYSER

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, ampicillin-resistant E. coli O157:H7 to contain ∼106, 104, and 102 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 cm2) 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-μm 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.



2014 ◽  
Vol 77 (10) ◽  
pp. 1669-1681 ◽  
Author(s):  
GORDON R. DAVIDSON ◽  
CHELSEA N. KAMINSKI ◽  
ELLIOT T. RYSER

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 106 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 cm2) 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-μm 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.



2012 ◽  
Vol 75 (11) ◽  
pp. 1920-1929 ◽  
Author(s):  
ANNEMARIE L. BUCHHOLZ ◽  
GORDON R. DAVIDSON ◽  
BRADLEY P. MARKS ◽  
EWEN C. D. TODD ◽  
ELLIOT T. RYSER

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 ~106, 104, or 102 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 106 or 104 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 102 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.



2011 ◽  
Vol 74 (5) ◽  
pp. 718-726 ◽  
Author(s):  
MANAN SHARMA ◽  
SUDESNA LAKSHMAN ◽  
SEAN FERGUSON ◽  
DAVID T. INGRAM ◽  
YAGUANG LUO ◽  
...  

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 N2), treatment B (near–ambient air atmospheric conditions in a gas-permeable film with microperforations), and treatment C (high-CO2 and low-O2 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 stx2, 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 stx2, 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.



2020 ◽  
Vol 83 (8) ◽  
pp. 1444-1462 ◽  
Author(s):  
GENEVIÈVE COULOMBE ◽  
ANGELA CATFORD ◽  
AMALIA MARTINEZ-PEREZ ◽  
ENRICO BUENAVENTURA

ABSTRACT Foodborne diseases are a major cause of illness in Canada. One of the main pathogens causing cases and outbreaks of foodborne illness in Canada is Escherichia coli O157:H7. From 2008 to 2018, 11 outbreaks of E. coli O157:H7 infection in Canada were linked to leafy greens, including 7 (63.6%) linked to romaine lettuce, 2 (18.2%) linked to iceberg lettuce, and 2 (18.2%) linked to other or unspecified types of leafy greens. The consumption of lettuce in Canada, the behavior of E. coli O157:H7 on lettuce leaves, and the production practices used for romaine and iceberg lettuce do not seem to explain why a higher number of outbreaks of E. coli O157:H7 infection were linked to romaine than to iceberg lettuce. However, the difference in the shape of iceberg and romaine lettuce heads could be an important factor. Among the seven outbreaks linked to romaine lettuce in Canada between 2008 and 2018, an eastern distribution of cases was observed. Cases from western provinces were reported only twice. The consumption of romaine and iceberg lettuce by the Canadian population does not seem to explain the eastern distribution of cases observed, but the commercial distribution, travel distances, and the storage practices used for lettuce may be important factors. In the past 10 years, the majority of the outbreaks of E. coli O157:H7 infection linked to romaine lettuce occurred during the spring (March to June) and fall (September to December). The timing of these outbreaks may be explained by the availability of lettuce in Canada, the growing region transition periods in the United States, and the seasonality in the prevalence of E. coli O157:H7. The consumption of romaine lettuce by the Canadian population does not explain the timing of the outbreaks observed. HIGHLIGHTS



2001 ◽  
Vol 64 (3) ◽  
pp. 305-309 ◽  
Author(s):  
YUE LI ◽  
ROBERT E. BRACKETT ◽  
JINRU CHEN ◽  
LARRY R. BEUCHAT

This study determined the effects of mild heat and chlorine treatment followed by storage for up to 18 days at 5°C or 7 days at 15°C on the survival and growth of Escherichia coli O157:H7 inoculated onto fresh-cut iceberg lettuce. The efficacy of treatment with 20 ppm chlorine in killing the pathogen on lettuce at 50°C was determined. Treatment of lettuce with 20 ppm chlorine at either 20 or 50°C did not result in significantly greater reductions in populations of E. coli O157:H7 compared to respective treatments in water without chlorine. The pathogen steadily decreased in viability on treated lettuce throughout subsequent storage at 5°C for 18 days. The population increased by 2.3 to 3.2 log10 CFU/g within 2 days, then continued to increase at a slower rate through 7 days of storage at 15°C. At 4 and 7 days, significantly (α = 0.05) higher populations were reached on lettuce that had been treated at 50°C, compared to respective samples that had been treated at 20°C, regardless of the presence of 20 ppm chlorine in the treatment water. Treatment of lettuce with 20 ppm chlorine at 50 or 20°C before or after inoculation with E. coli O157:H7 did not have a marked influence on behavior of the pathogen during subsequent storage at 5 or 15°C.



2009 ◽  
Vol 72 (7) ◽  
pp. 1547-1552 ◽  
Author(s):  
J. L. TALLEY ◽  
A. C. WAYADANDE ◽  
L. P. WASALA ◽  
A. C. GERRY ◽  
J. FLETCHER ◽  
...  

The recent outbreak of Escherichia coli O157:H7 infection associated with contaminated spinach led to an investigation of the role of insects, which frequent fields of leafy greens and neighboring rangeland habitats, in produce contamination. Four leafy greens fields adjacent to cattle-occupied rangeland habitats were sampled using sweep nets and sticky traps. Agromyzid flies, anthomyiid flies, and leafhoppers were caught consistently in both rangeland and leafy greens production fields at all sites. An unexpected number of flies (n = 34) in the Muscidae and Calliphoridae families (known as filth flies because of their development in animal feces) were caught in one leafy greens field. A subset of these filth flies were positive (11 of 18 flies) for E. coli O157:H7 by PCR amplification using primers for the E. coli O157:H7–specific eae gene. Under laboratory conditions, house flies were confined on manure or agar medium containing E. coli O157:H7 tagged with green fluorescent protein (GFP) and then tested for their capacity to transfer the microbes to spinach plants. GFP-tagged bacteria were detected on surfaces of 50 to 100% of leaves examined by fluorescence microscopy and in 100% of samples tested by PCR. These results indicate that flies are capable of contaminating leafy greens under experimental conditions and confirm the importance of further investigation of the role of insects in contamination of fresh produce.



2010 ◽  
Vol 73 (3) ◽  
pp. 547-551 ◽  
Author(s):  
SANGHYUP JEONG ◽  
BRADLEY P. MARKS ◽  
ELLIOT T. RYSER ◽  
SCOTT R. MOOSEKIAN

Low-energy X-ray irradiation was assessed as a means of eliminating Escherichia coli O157:H7 on lettuce. Round-cut iceberg lettuce samples (2.54-cm diameter) were dip or spot inoculated with a three-strain cocktail of E. coli O157:H7, stored for 24 h at 4°C, and then irradiated at four dose levels up to 0.25 kGy using a prototype low-energy (70 kV) X-ray irradiator. E. coli O157:H7 survivors were quantified by plating on sorbitol MacConkey agar containing cefixime and tellurite. Dip inoculation yielded a D10-value of 0.040 ± 0.001 kGy, which is 3.4 times lower than a previously reported value of 0.136 kGy using gamma radiation. The D10-value for E. coli O157:H7 on spot-inoculated samples was 0.078 ± 0.008 kGy, which is about twice that of dip-inoculated samples. When 10 stacked leaves were irradiated from both sides, a dose of 0.2 kGy was achieved at the center of the stack with a surface dose of 1 kGy, corresponding to a ~5-log reduction of E. coli O157:H7 at the center of the stack. Based on these findings, low-energy X-ray irradiation appears to be a promising microbial inactivation strategy for leafy greens and potentially for other types of fresh produce.



2009 ◽  
Vol 72 (7) ◽  
pp. 1569-1575 ◽  
Author(s):  
MICHAEL A. JOHNSTON ◽  
MARK A. HARRISON ◽  
RUTH A. MORROW

Fresh-cut lettuce and spinach can become contaminated with pathogens at numerous points from the field to the retail market. Natural microflora present on fresh produce may help reduce the pathogen load. The objective of this study was to isolate natural microflora from fresh-cut iceberg lettuce and baby spinach and to determine whether these bacteria were antagonistic toward Escherichia coli O157:H7. Samples were collected under conditions that mimicked actual practices between production and retail sale. Evidence of naturally occurring microorganisms on fresh lettuce (295 isolates) and spinach (200 isolates) and of possible antagonistic activity toward E. coli O157:H7 was documented. Inhibitory activity by several isolates was due to either acid production or antimicrobial peptides. Bacteria with inhibitory activity were isolated from every step in the processing and handling of the fresh-cut iceberg lettuce and baby spinach.



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