Source: UNIV OF SOUTH FLORIDA submitted to
RAPID CONCENTRATION/DETECTION OF FOODBORNE PATHOGENS FROM WASH WATER FOR ENHANCED SAFETY OF FRESH FRUITS AND VEGETABLES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1005362
Grant No.
2015-67017-23114
Project No.
FLAW-2014-05806
Proposal No.
2014-05806
Multistate No.
(N/A)
Program Code
A1331
Project Start Date
Feb 15, 2015
Project End Date
Feb 14, 2017
Grant Year
2015
Project Director
Lim, D.
Recipient Organization
UNIV OF SOUTH FLORIDA
(N/A)
TAMPA,FL 33620
Performing Department
Cell Biol,Microbiol & Mol Biol
Non Technical Summary
Foodborne outbreaks associated with fresh-cut fruits and vegetables have continued unabated despite widespread use of chemical sanitizers in commercial flume systems. However, these commercial washing practices do not guarantee a pathogen-free product; bacterial pathogens such as Escherichia coli O157:H7 and Listeria monocytogenes can be easily transferred from a small amount of contaminated product to previously uncontaminated product during fresh-cut processing. The small amount of processed product generally sampled for contamination relative to the large volume processed is a serious limitation of current monitoring efforts. A faster and more reliable way to overcome this limitation is to sample larger, more representative volumes of the produce wash water. This project utilizes a novel dead-end ultrafiltration, or DEUF, sampling method in combination with qPCR to detect foodborne pathogens E. coli O157:H7 and L. monocytogenes in produce wash water in a fresh-cut leafy green pilot-scale production facility and a commercial-scale leafy green processing facility. The innovative approach of using DEUF for concentrating pathogens from produce wash water will enable more reliable detection of pathogens in a sample matrix that better represents contamination in processed product. This research is expected to introduce a new method that the industry can use for enhanced detection of pathogens in fresh-cut produce, making it possible to more accurately screen finished product for contamination, potentially reducing hold times, product recalls and the incidence of illnesses associated with fresh-cut fruits and vegetables.
Animal Health Component
0%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71272991100100%
Goals / Objectives
The major goal of this project is to demonstrate improved detection of bacterial pathogens in processed lettuce using a novel DEUF sampling procedure that rapidly collects, concentrates and recovers pathogens from large volumes of produce wash water in combination with an AOAC-approved qPCR rapid detection method. This procedure will be used to detect E. coli O157:H7 and L. monocytogenes in produce wash water in a fresh-cut leafy green pilot-scale production facility and a commercial-scale leafy green processing facility.The objectives to achieve this goal are:1) evaluation of the treatment of leafy green wash water with various enzymes that degrade plant cell wall structure to permit faster filtration of larger volumes of wash water by DEUF,2) comparison of the DEUF concentration method with the standard FDA BAM sampling method to detect E. coli O157:H7 and L. monocytogenes in flume water at Michigan State University's fresh-cut leafy green pilot-scale production facility, and3) assessment of the DEUF sampling method to detect E. coli O157:H7 and L. monocytogenes at a commercial-scale leafy green processing facility.
Project Methods
The methods for the project will include training and direct involvement of academic, field, and industry scientists in project studies, including use of the DEUF concentration method to collect large produce wash water samples to detect E. coli O157:H7 and Listeria monocytogenes. These studies will be performed in the actual environments in which these scientists work. The success of the project will be measured by the ability of the DEUF concentration method to more reliably and accurately detect E. coli O157:H7 and Listeria monocytogenes in produce wash water compared to standard grab samples. Cultural isolation and qPCR detection probabilities of E. coli O157:H7 and L. monocytogenes will be determined in enriched, non-concentrated and DEUF concentrated lettuce wash samples as defined by AOAC. Besides determining detection probability per method, cultural isolation will be used to determine viable detection whereas qPCR will be used to confirm recovery of cells after DEUF concentration. Binary logistic regression will be used to compare the positive qPCR results (Ct values will be analyzed as 1 for any positive samples and 0 for any negative samples) from the concentrated and unconcentrated lettuce wash water samples. Nagelkerke R square, which can range from 0.0 to 1.0, denotes the effect size (the strength of the relationship); stronger associations have values closer to 1.0. Relationships will be considered significant when the P value for the model chi square is <0.05 and the confidence interval for the odds ratio does not include 1.0. Greater odds ratios indicate a higher probability of change in the dependent variable with a change in the independent variable. Statistical significance for all data will be accepted at the 95% confidence level (a < 0.05).

Progress 02/15/15 to 02/14/16

Outputs
Target Audience:The target audience for this project are academic and field scientists and students as well as industry executives interested in more reliable methods to collect representative samples of produce wash water to detect foodborne pathogens. Members of these groups were introduced during the first year of the project to the novel dead-end ultrafiltration (DEUF) sampling method used for collection of produce wash water. Changes/Problems:Some differences in culture and qPCR results were observed during the MSU pilot-scale production facility studies. These differences have been analyzed and minor protocol revisions have been made to minimize such differences in the June 2016 SmartWash Solutions Salinas studies. What opportunities for training and professional development has the project provided?Undergraduate and graduate students have been trained during this project reporting period on operation, maintenance, and use of the DEUF concentration method for collection of E. coli O157:H7 and L. monocytogenes in large volume flume water samples at Michigan State University's fresh-cut leafy green pilot-scale production facility. This training includes an extensive one week period in June 2015 when USF researchers Dr. Elizabeth Kearns and Sonia Castillo traveled to Michigan State University and trained MSU graduate student Ryann Gustafson and others how to properly operate and maintain the DEUF concentrator. Dr. Elizabeth Kearns, Sonia Castillo, Ryann Gustafson, Dr. Elliot Ryser, and Dr. Daniel Lim have exchanged numerous telephone calls and e-mails during this project period related to operation and maintenance of the DEUF concentrator and project experiments. How have the results been disseminated to communities of interest?A poster describing project goals and progress was presented at the July 24, 2015 USDA NIFA Project Directors Meeting in Portland, OR. A Food Safety Specialist and a Field Application Scientist from ThermoFisher Scientific visited USF in August 2015 and were introduced to the DEUF concentration method for collection of E. coli O157:H7 and L. monocytogenes in produce wash in combination with qPCR detection during an all-day training session. Dr. Elizabeth Kearns, Sonia Castillo, Ryann Gustafson, Dr. Elliot Ryser, and Dr. Daniel Lim have communicated with James Brennan and SmartWash Solutions staff regarding the protocol and plans for the June 2016 project studies are the SmartWash Solutions Salinas facility. What do you plan to do during the next reporting period to accomplish the goals?The DEUF sampling protocol to detect E. coli O157:H7 and L. monocytogenes will be assessed and compared with the standard FDA BAM sampling method in flume and centrifugation water at the SmartWash Solutions commercial-scale leafy green processing facility in Salinas, CA during a two week period in June 2016. A total of eight processing trials, each involving 2000 lbs (909 kg) of iceberg lettuce, will be performed. Iceberg lettuce will be inoculated at a level that generates approximately 1 viable E. coli O157:H7 per 125 ml and 1 viable L. monocytogenes per 25 ml of unconcentrated wash water for each of these processing trials. In addition to the flume water, these studies will also include analysis of spent centrifugation water after processing 2000 lbs. of iceberg lettuce with about 80 L of such water expected to be generated per trial. The trials will closely mimic current industry practices. Samples of flume and centrifugation water will be collected from each trial and sent to MSU for physicochemical analysis, including pH, oxidation/reduction potential (ORP), chemical oxygen demand (COD), total solids (TS), turbidity and Millipore maximum filterable volume (MMFV). In the same manner, subsamples of unconcentrated and DEUF concentrates will be collected, enriched and plated for bacteria. Aliquots of enriched samples will be shipped frozen to USF for qPCR analysis. Cultural isolation and qPCR detection probabilities of E. coli O157:H7 and L. monocytogenes will be determined in collected samples. Binary logistic regression will be used to compare the positive qPCR results (Ct values will be analysed as 1 for any positive samples and 0 for any negative samples) from the samples. Nagelkerke R square, which can range from 0.0 to 1.0, denotes the effect size (the strength of the relationship); stronger associations have values closer to 1.0. Relationships will be considered significant when the P value for the model chi square is <0.05 and the confidence interval for the odds ratio does not include 1.0. Greater odds ratios indicate a higher probability of change in the dependent variable with a change in the independent variable. Statistical significance for all data will be accepted at the 95% confidence level (p < 0.05). The results from these studies will be submitted as an abstract for presentation at the 2017 IAFP Meeting and/or submitted as a paper to a peer-reviewed scientific journal such as Journal of Food Protection.

Impacts
What was accomplished under these goals? Physical (e.g., pH, temperature) and chemical (e.g., enzymes, EDTA, surfactants) treatments of leafy green wash water that might degrade plant cell wall structures and reduce filter fouling, permitting faster filtration of larger volumes, were evaluated during this project reporting period as part of Objective 1. Results from these studies indicated that decreasing pH and increasing temperature increased flow rate of sieved lettuce wash up to 3.9X that of untreated sample. Viability of E. coli O157:H7 decreased after 30 min incubation at 40°C and pH 5. Listeria monocytogenes maintained viability for up to 45 min at pH 5-7 and temperature up to 50°C. Tested enzymatic treatments (e.g., pectinase, Viscozyme) showed flow rate improvement only when adjusted to a pH ≤5 and temperature ≥40°C. To more closely match the project test conditions (polysulfone ultrafilter and research facility temperature), the membrane filter was changed to polyethersulfone (PES) and a temperature of 4°C was included. The best improvement in filterability (1.4X the control) was observed in samples treated with 0.5% Tween 80 at pH 7 incubated at 40°C for 30 min. A novel dead-end ultrafiltration (DEUF) sampling method was used prior to qPCR or standard enrichment to determine presence/absence of E. coli O157:H7 and L. monocytogenes in wash water after pilot-scale processing of inoculated iceberg lettuce. The core of one head of iceberg lettuce was inoculated with an avirulent multi-strain cocktail of L. monocytogenes (J22F, J29H, M3) or E. coli O157:H7 (ATCC 43888, CV2b7, 6980-2, and 6982-2) to contain ~4.9 or 7.2 log CFU/g and then processed with 22.7 kg of uninoculated lettuce in a pilot-scale processing line by shredding, conveying, flume washing in a 3.3 m-long tank (90 s) followed by shaker table dewatering. Triplicate trials were conducted for each organism at inoculation levels of ~4.9 or 7.2 log CFU/g using 890 L of wash water with or without 50 ppm chlorine (XY-12 sanitizer), respectively, that contained 0, 2.5 or 5.0 % lettuce solids. After processing, duplicate 400 ml wash water samples were collected and examined for presence/absence of the target organism using standard enrichment protocols. In addition, duplicate 40 L samples were subjected to DEUF and then examined by both qPCR and standard enrichment. E. coli O157:H7 was grown by enriching 125 ml samples in 2x modified buffered peptone water with pyruvate (mBPWp) supplemented with acriflavin-cefsulodin-vancomycin, followed by isolation on sorbitol MacConkey containing cefixime and tellurite (SMAC-CT) and Rainbow O157 agars. L. monocytogenes was grown by enriching 25 ml samples in buffered Listeria enrichment broth containing acriflavine-cycloheximide-nalidixic acid followed by isolation on Modified Oxford agar (MOX). Two 750 ul aliquots from each enriched sample were screened for the presence of E. coli O157:H7 or L. monocytogenes using AOAC-approved confirmatory qPCR testing. Bacterial DNA was extracted using the PrepSEQ Rapid Spin Sample Preparation kit (Applied Biosystems, Life Technologies, Inc. Grand Island, NY) prior to amplification using the AOAC-approved MicroSEQ E. coli O157:H7 and L. monocytogenes Detection Kits (Applied Biosystems). Levels of assay inhibition were evaluated by assessing internal control reactions for each pathogen in the MicroSEQ kits. Enrichment and cultural isolation were performed at MSU with frozen aliquots shipped overnight to USF for qPCR analysis. Reductions in time to detection were evaluated for enriched samples containing very low levels of E. coli O157:H7 and L. monocytogenes. For selected processing runs in Task A containing a 0 and 5% organic load with sanitizer, aliquots were removed from the grab and DEUF concentrated samples of selected processing runs containing a 0 and 5% organic load with sanitizer at hourly intervals during enrichment starting at time 0 (addition of grab and DEUF samples into the enrichment media) and then frozen at -80°C for subsequent analysis using qPCR. Studies performed this past year compared the DEUF method with the standard FDA BAM sampling method at MSU's fresh-cut leafy green pilot-scale production facility. Eighteen complete trial sets were run with two grab samples and two DEUF retentate samples collected for E. coli O157:H7 and L. monocytogenes for each trial set. The grab samples and DEUF retentate samples were examined for bacteria by enrichment culture and by qPCR. Sixty-seven percent of E. coli and Listeria samples had the same results from DEUF qPCR and/or enrichment culture as from conventional FDA BAM sample qPCR and/or enrichment culture in trials containing 0%, 2.5%, and 5.0% organic loads with or without the use of a chlorine-based sanitizer. Thirty-three percent of E. coli and Listeria samples were positive by DEUF qPCR and/or enrichment culture, but negative by conventional FDA BAM sample qPCR and/or enrichment culture. These results suggest that the DEUF method with its larger, more representative size sample may be useful in increasing the probability of detecting E. coli and Listeria in leafy green processing water.

Publications

  • Type: Other Status: Other Year Published: 2015 Citation: USDA NIFA PD Meeting Poster