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

ABSTRACT 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.

Download Full-text

Evaluation of Potential Impacts of Free Chlorine during Washing of Fresh‐Cut Leafy Greens on Escherichia coli O157:H7 Cross‐Contamination and Risk of Illness

Risk Analysis ◽

10.1111/risa.13818 ◽

2021 ◽

Author(s):

Amir Mokhtari ◽

Hao Pang ◽

Sofia Santillana Farakos ◽

Gordon R. Davidson ◽

Elizabeth Noelia Williams ◽

...

Keyword(s):

Escherichia Coli ◽

Free Chlorine ◽

Escherichia Coli O157 ◽

Cross Contamination ◽

Leafy Greens ◽

Fresh Cut

Download Full-text

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

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-058 ◽

2014 ◽

Vol 77 (9) ◽

pp. 1487-1494 ◽

Cited By ~ 11

Author(s):

ANNEMARIE L. BUCHHOLZ ◽

GORDON R. DAVIDSON ◽

BRADLEY P. MARKS ◽

EWEN C. D. TODD ◽

ELLIOT T. RYSER

Keyword(s):

Escherichia Coli ◽

Membrane Filtration ◽

Fluorescent Protein ◽

Pilot Scale ◽

Escherichia Coli O157 ◽

E Coli ◽

Leafy Greens ◽

Iceberg Lettuce ◽

Processing Line ◽

Fresh Cut

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.

Download Full-text

Modeling of Free Chlorine Consumption and Escherichia coli O157:H7 Cross‐Contamination During Fresh‐Cut Produce Wash Cycles

Journal of Food Science ◽

10.1111/1750-3841.14774 ◽

2019 ◽

Vol 84 (10) ◽

pp. 2736-2744 ◽

Cited By ~ 4

Author(s):

Mohammadreza Dehghan Abnavi ◽

Ali Alradaan ◽

Daniel Munther ◽

Chandrasekhar R. Kothapalli ◽

Parthasarathy Srinivasan

Keyword(s):

Escherichia Coli ◽

Free Chlorine ◽

Escherichia Coli O157 ◽

Cross Contamination ◽

Fresh Cut

Download Full-text

Effect of Modified Atmosphere Packaging on the Persistence and Expression of Virulence Factors of Escherichia coli O157:H7 on Shredded Iceberg Lettuce†

Journal of Food Protection ◽

10.4315/0362-028x.jfp-10-427 ◽

2011 ◽

Vol 74 (5) ◽

pp. 718-726 ◽

Cited By ~ 21

Author(s):

MANAN SHARMA ◽

SUDESNA LAKSHMAN ◽

SEAN FERGUSON ◽

DAVID T. INGRAM ◽

YAGUANG LUO ◽

...

Keyword(s):

Escherichia Coli ◽

Virulence Factors ◽

Modified Atmosphere Packaging ◽

Ambient Air ◽

Modified Atmosphere ◽

Escherichia Coli O157 ◽

Atmospheric Conditions ◽

E Coli ◽

Leafy Greens ◽

Fresh Cut

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.

Download Full-text

Determination of Free Chlorine Concentrations Needed To Prevent Escherichia coli O157:H7 Cross-Contamination during Fresh-Cut Produce Wash†

Journal of Food Protection ◽

10.4315/0362-028x.jfp-10-429 ◽

2011 ◽

Vol 74 (3) ◽

pp. 352-358 ◽

Cited By ~ 113

Author(s):

YAGUANG LUO ◽

XIANGWU NOU ◽

YANG YANG ◽

ISABEL ALEGRE ◽

ELLEN TURNER ◽

...

Keyword(s):

Escherichia Coli ◽

Free Chlorine ◽

Chlorine Concentration ◽

Wash Water ◽

Escherichia Coli O157 ◽

Cross Contamination ◽

E Coli ◽

Wash Solution ◽

Fresh Cut ◽

Experimental Trials

This study was conducted to investigate the effect of free chlorine concentrations in wash water on Escherichia coli O157:H7 reduction, survival, and transference during washing of fresh-cut lettuce. The effectiveness of rewashing for inactivation of E. coli O157:H7 on newly cross-contaminated produce previously washed with solutions containing an insufficient amount of chlorine also was assessed. Results indicate that solutions containing a minimum of 0.5 mg/liter free chlorine were effective for inactivating E. coli O157:H7 in suspension to below the detection level. However, the presence of 1 mg/liter free chlorine in the wash solution before washing was insufficient to prevent E. coli O157:H7 survival and transfer during washing because the introduction of cut lettuce to the wash system quickly depleted the free chlorine. Although no E. coli O157:H7 was detected in the wash solution containing 5 mg/liter free chlorine before washing a mix of inoculated and uninoculated lettuce, low numbers of E. coli O157:H7 cells were detected on uninoculated lettuce in four of the seven experimental trials. When the prewash free chlorine concentration was increased to 10 mg/liter or greater, no E. coli O157:H7 transfer was detected. Furthermore, although rewashing newly cross-contaminated lettuce in 50 mg/liter free chlorine for 30 s significantly reduced (P = 0.002) the E. coli O157:H7 populations, it failed to eliminate E. coli O157:H7 on lettuce. This finding suggests that rewashing is not an effective way to correct for process failure, and maintaining a sufficient free chlorine concentration in the wash solution is critical for preventing pathogen cross-contamination.

Download Full-text

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

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-558 ◽

2012 ◽

Vol 75 (11) ◽

pp. 1920-1929 ◽

Cited By ~ 35

Author(s):

ANNEMARIE L. BUCHHOLZ ◽

GORDON R. DAVIDSON ◽

BRADLEY P. MARKS ◽

EWEN C. D. TODD ◽

ELLIOT T. RYSER

Keyword(s):

Escherichia Coli ◽

Membrane Filtration ◽

Tap Water ◽

Plant Production ◽

Health Concern ◽

Escherichia Coli O157 ◽

Romaine Lettuce ◽

E Coli ◽

Leafy Greens ◽

Fresh Cut

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.

Download Full-text

Quantitative Transfer of Escherichia coli O157:H7 to Equipment during Small-Scale Production of Fresh-Cut Leafy Greens

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-489 ◽

2012 ◽

Vol 75 (7) ◽

pp. 1184-1197 ◽

Cited By ~ 51

Author(s):

ANNEMARIE L. BUCHHOLZ ◽

GORDON R. DAVIDSON ◽

BRADLEY P. MARKS ◽

EWEN C. D. TODD ◽

ELLIOT T. RYSER

Keyword(s):

Escherichia Coli ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Small Scale ◽

Escherichia Coli O157 ◽

E Coli ◽

Leafy Greens ◽

Iceberg Lettuce ◽

Fresh Cut ◽

Green Fluorescent

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.

Download Full-text

Inactivation of Escherichia coli O157:H7 on the Intact and Damaged Portions of Lettuce and Spinach Leaves by Using Allyl Isothiocyanate, Carvacrol, and Cinnamaldehyde in Vapor Phase

Journal of Food Protection ◽

10.4315/0362-028x-72.10.2046 ◽

2009 ◽

Vol 72 (10) ◽

pp. 2046-2055 ◽

Cited By ~ 27

Author(s):

MOHAMMAD M. OBAIDAT ◽

JOSEPH F. FRANK

Keyword(s):

Escherichia Coli ◽

Vapor Phase ◽

Allyl Isothiocyanate ◽

Escherichia Coli O157 ◽

Pathogen Population ◽

Tissue Samples ◽

E Coli ◽

Leafy Greens ◽

Leaf Surfaces ◽

Spinach Leaves

Antimicrobials in the vapor phase might be more effective in inactivating Escherichia coli O157:H7 cells attached to leafy greens than aqueous antimicrobials. We determined the activity of allyl isothiocyanate (AIT), cinnamaldehyde, and carvacrol against E. coli O157:H7 on intact and damaged lettuce and spinach tissue. Samples were treated with various concentrations of antimicrobial in the vapor phase at 0, 4, and 10°C in an enclosed container. On intact lettuce surface, the vapor of the lowest concentration of these antimicrobials inactivated >4 log of E. coli O157:H7 at 0 and 4°C in 4 days and at 10°C in 2 days. However, at the tissue damaged by cutting, the highest concentration reduced the population by 4 log at 0°C and 2 to 4 log at 4°C in 4 days. These concentrations also reduced the population of the pathogen by 1 to 3 log at 10°C in 2 days. The pathogen population on spinach surface was reduced by 1 log less than on lettuce surface. However, reduction of the pathogen within spinach tissue was 2 and 3 log less than within lettuce tissue at 0 and 4°C, respectively. Overall, greater inactivation occurred on lettuce than spinach leaves and on the leaf surfaces than at the damaged area. Using antimicrobials in the vapor phase may improve the safety of refrigerated leafy greens marketed in sealed packages.

Download Full-text