Source: UNIVERSITY OF FLORIDA submitted to NRP
CONTROL OF FOOD-BORNE PATHOGENS IN PRE- AND POST-HARVEST ENVIRONMENTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0220803
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
S-1033
Project Start Date
Oct 1, 2007
Project End Date
Sep 30, 2012
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Citrus Research and Education Center, Lake Alfred
Non Technical Summary
An increasing number of gastrointestinal disease outbreaks have been linked to the consumption of fresh fruits and vegetables. The increase in the number of outbreaks caused by produce appears to be related to the increased demand for fresh fruits and vegetables. Between 1990 and 2001, contaminated fresh produce caused 148 outbreaks that account for approximately 9% of all food-borne outbreaks (Smith-DeWaal et al, 2002). Two of the most virulent foodborne pathogens, Salmonella and pathogenic Escherichia coli, have been responsible for many outbreaks associated with fresh fruits and vegetables. Fresh fruits and vegetables are considered high-risk foods because they are minimally processed and are susceptible to contamination at the farm.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7120999110020%
7121099110020%
7121199110020%
7121219110020%
7121499110020%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1499 - Vegetables, general/other; 1099 - Tropical/subtropical fruit, general/other; 0999 - Citrus, general/other; 1199 - Deciduous and small fruits, general/other; 1219 - Edible tree nuts, general/other;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The ovearall goal of this multistate project is to control foodborne pathogens in pre and post harvest environments. I will be involved in two of the specific objectives defined by the larger project: 1. Develop or improve methods for control or elimination of pathogens in pre-and post harvest environments including meat, poultry, seafood, fruits and vegetables and nutmeats. 2. Develop and validate mathematical modeling to gain understanding of pathogen behavior in macro and micro-environments. The US has seena dramatic increase in the number of Foodborne illnesses attributed to contaminated produce since the 1970s. The top five produce items, associated with 75% of produce outbreaks, are lettuce and leafy greens, melons, green onions and leafy herbs. Although all of these commodities are produced commercially in Florida, only Florida related tomatoes have been associated with foodborne outbreaks. Due to Florida's climate, many of these products are produced here when they can not be produced in other parts of the US, however the pre-harvest environment and food safety risks associated with it, are significantly different from other locations were produce is grown.
Project Methods
Common procedures will be developed between collaborators during these investigations. When ever possible the Bacteriological Analytical Manual (BAM, http://www.cfsan.fda.gov/~ebam/bam-toc.html) procedures will be used for enumeration and identification of the pathogens in the food products. Relevant equipment includes class II, type A biological safety hoods, microscopes, centrifuges, spiral-plater and scanner, autoclaves and other typical laboratory equipment necessary for these studies. Objective 1. Inoculation of produce: The produce samples will be spot inoculated with a cocktail of 5 E. coli O157:H7 strains and a cocktail of 5 Salmonella strains at different inoculation levels (103 to 106/g). Inoculation will be performed by diluting the respective cocktail mixture approximately 100-fold with sterile water to achieve a 107 CFU/g inoculum level. The produce samples will be spot inoculated with 10 - 100 ul of the inoculum,and allowed to dry for 30 minutes in a biosafety cabinet. A representative sample will be taken to determine the initial pathogen levels for each treatment as described below. To enumerate the E. coli O157:H7 and Salmonella, 10 g samples of each treatment will be homogenized with 90 ml of sterile 0.1% peptone and further 10 fold dilutions will be made using sterile 9 ml 0.1% peptone water blanks. The dilutions will be plated onto selective media. Total plate counts using non selective media, for the produce samples will also be determined to establish if any reduction of the normal sprout microflora occurs due to the various treatments. All experiments will be plated in duplicate for each. For samples that fall below detection limits (10 CFU/g), 25 g samples will then be analyzed for Salmonella using enrichment procedures according to the US FDA Bacteriological Analytical Manual (BAM) procedure. Decontamination Treatments: The response of several foodborne bacteriato UV, ozone, and hydrogen peroxide and other decontamination treatments will be examined. Bacterial foodborne pathogens to be used for the inoculated studies will include pathogenic E. coli strains associated with produce outbreaks and Salmonella spp. strains as described above. As a control for no treatment solution, sterile distilled water will be used at the same volume and treatment time for each produce item.

Progress 10/01/11 to 09/30/12

Outputs
Target Audience: Produce growers, packers, processors and researchers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Attended S1033 Annual Meeting How have the results been disseminated to communities of interest? Presentations at extension and research meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? After 7d at 4°C, Salmonella was most effectively reduced by molybdate, at an average of 3.23 log CFU/ml. Solubor and manganese achieved a 1.4 log CFU/ml and 2.2 log CFU/ml, respectively, compared to the control of 1.4 log reduction. During 15°C incubation, Salmonella was most effectively reduced by manganese, at an average of 3.0 log CFU/ml. Solubor and molybdate achieved a 2.3 log CFU/ml and 1.4 log CFU/ml, respectively, compared to the control of 1.4 log reduction. During 30°C incubation, Salmonella was most effectively reduced by Solubor, at an average of 4.0 log CFU/ml. Molybdate achieved a 3.9 log CFU/ml reduction compared to the control of 3.1 log CFU/ml. Pathogen survival in copper sulfate, ferrous sulfate, and calcium nitrate did not occur under all three storage temperatures within 24 h, and in zinc sulfate within 72 h. These results demonstrate that Salmonella can survive in fertilizers and can be a source for pathogen contamination in foliar application in produce fields. Bivariate scatter plots of all indicator parameters and Salmonella levels were done. There were weak correlations (r2≈0.1) between E. coli and/or coliforms levels with Salmonella levels, suggesting that these biological indicators could not predict Salmonella levels. Similarly, chemical indicators could not predict (r2<0.1) Salmonella levels. Scatter plots between biological and chemical indicators and biological and physical indicators did not indicate significant relationships. The average rainfall (previous month) before sampling did not correlate well with bacterial levels. Contour plots showed that most observations of Salmonella, E. coli and coliforms occurred at around 1, 2, and 4 log MPN/100ml respectively. The role of index microorganisms and chemical indicators in determining the prevalence of Salmonella in at least some surface water may be limited. In the absence of a rapid indicator, direct screening of surface water for Salmonella may be necessary where Salmonella levels are high and where waters are applied close to harvest.

Publications

  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Teplitski, M., J.T. Noel, A. Alagely, and M.D. Danyluk. 2012. Functional genomics studies shed light on the nutrition and gene expression of non-typhoidal Salmonella and enterovirulent E. coli in produce. Food Research International. 45:576-586.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Kimber, M., H. Kaur, L. Wang, M.D. Danyluk, and L.J. Harris. 2012. Survival of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes on inoculated almonds and pistachios stored at -19, 4, and 24�C. Journal of Food Protection. 75:1394-1403.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Cevallos-Cevallos, J.M., E.D. Akins, L,M. Friedrich, M.D. Danyluk, and A.H. Simonne. 2012. Growth of Clostridium perfringens in refried beans. Journal of Food Protection. 75:1783-1790


Progress 10/01/07 to 09/30/12

Outputs
Target Audience: Produce growers, packers, processors, and researchres Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? All accomplishments are previously reported.

Publications


    Progress 10/01/10 to 09/30/11

    Outputs
    Target Audience: Produce growers, packers, processors and researchers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Attended 2011 S1033 meeting How have the results been disseminated to communities of interest? Extension and profesional meetings What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Salmonella and L. monocytogenes populations decreased over 7 days at 4ºC, under all conditions, by ca. 1 log CFU/g (0.1-0.2 log CFU/g/day reductions) and 1-2 log CFU/g (0.1-0.3 log CFU/g/day reductions), respectively,. Salmonella and L .monocytogenes populations remained stable at ca. 3.0 to 3.5 log CFU/g for 7 days at 12ºC under all conditions. All Salmonella populations increased by ca. 1.5-2 log CFU/g over 48 h at 22ºC, under all conditions, with the greatest growth (2.1 log CFU/g) observed on the cut surface of samples stored in polyethylene containers. L. monocytogenes populations remained stable ca. 3 log CFU/g for 48 h at 22ºC, under all conditions. When held under refrigerated temperatures, Salmonella and L. monocytogenes do not grow on fresh-cut celery. Salmonella, but not L. monocytogenes, has the potential to grow on temperature-abused fresh-cut celery, especially if inoculated onto the cut surface. All water samples were positive for Salmonella following tangential flow filtration - real time PCR. Significant differences (P<0.05) exist between Salmonella MPN levels for the three counties sampled. Only one county showed a significant difference (P< 0.05) in Salmonella MPN levels between sampling months. The strongest correlations between Salmonella concentration and other parameters were air temperature and E. coli MPN, which were found to have correlation coefficients of 0.30 and 0.26, respectfully. When looking at just one county, a correlation coefficient of 0.43 was found for the air temperature. All other measured attributes showed correlation coefficients between 0.18 and -0.15. This work elucidates the continued presence of Salmonella in Central Florida surface waters, not strongly correlated to the standard coliform/E.coli testing commonly used to evaluate produce water safety. Populations of all three bacteria on peanut and pecan kernels decreased 1 to 2 logs during the initial 72 h drying. Reductions of all bacterial populations during 28 days of storage at -20, 4 and 23ºC on peanuts and pecans were not affected by inoculum level. When stored at -20ºC and 4ºC for 181 days, populations of Salmonella, L. monocytogenes, and E. coli O157:H7 remained stable at ca. 5 log CFU/g. At 23ºC, linear rates of decline were 0.32, 0.55, and 0.40 log CFU/month on peanuts and 0.21, 0.83, and 0.45 log CFU/month on pecans for Salmonella, L. monocytogenes, and E. coli O157:H7, respectively. Salmonella survives better than L. monocytogenes and E. coli O157:H7 on pecans and peanuts stored at ambient temperature, supporting its use as the target pathogen for nuts and nut products.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2011 Citation: 5. Danyluk, M.D., L.M. Friedrich, C. Bousquet, R.M. Goodrich-Schneider, M.E. Parish, and R. Rouseff. 2011. Prevalence, Amounts, Spoilage, and Mitigation of Alicyclobacillus spp. in Tropical and Subtropical Fruit Concentrates. Food Microbiology. 28:472-477.
    • Type: Journal Articles Status: Published Year Published: 2011 Citation: 4. Danyluk, M.D., and D.W. Schaffner. 2011. Quantitative microbial assessment for E. coli O157:H7 in leafy greens from farm to consumption using published data. Journal of Food Protection. 74:700-708
    • Type: Journal Articles Status: Published Year Published: 2011 Citation: 3. Cevallos-Cevallos, J.M., M.D. Danyluk, J. I. Reyes-De-Corcuera. 2011. GC-MS Based Metabolomics for rapid detection of Escherichia coli O157:H7 and Salmonella Typhimurium, Salmonella Muenchen and Salmonlla Hartford in beef and chicken. Journal of Food Science. 76: M238-M246.
    • Type: Journal Articles Status: Published Year Published: 2011 Citation: 2. Perez-Cacho, P.R., M.D. Danyluk, and R. Rouseff. 2011. GC-MS quantification and sensory thresholds of guaiacol in orange juice and the relationship between guaiacol and Alicyclobacillus. Food Chemistry. 129:45-50


    Progress 10/01/09 to 09/30/10

    Outputs
    Target Audience: Produce growers, packers and researchers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Attend the 2010 S1033 Annual Meeting How have the results been disseminated to communities of interest? Presentations at extension events and annual meetings What do you plan to do during the next reporting period to accomplish the goals? Further studies, to evaluate the influence of dirt and debris that may be present on tomato cartons and the fate of Salmonella in contact with tomatoes and tomato cartons is planned.

    Impacts
    What was accomplished under these goals? Salmonella populations, inoculated onto new and used cartons decreased over a 24 h drying period ca. 2.5 and 3 log units, respectively, while populations inoculated onto tomatoes decreased by ca. 1.5 log units under the same drying conditions. In general for new cartons, transfer coefficients are lower when the inoculum has dried for 24 h prior to transfer, and decrease with increasing contact times. This indicates the highest potential (worst case) for Salmonella transfer in new tomato cartons occurs under moist conditions and shorter contact times. In general, transfer coefficients were generally higher from used tomato cartons than from new tomato cartons. The highest potential (worst case) for Salmonella transfer in used tomato cartons occurs under wet conditions and longer contact times. An interesting observation is that Salmonella populations inoculated onto tomatoes in contact with cartons, and inoculated onto cartons in contact with tomatoes, following storage for 24 h and 7 days were often higher than populations on cartons or tomatoes alone. This phenomenon has resulted at times in transfer coefficients of greater than 1, indicating that more Salmonella than should have been present on the cardboard or tomato transferred.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2010 Citation: Strawn, L.K. and M.D. Danyluk. 2010. Fate of Escherichia coli O157:H7 and Salmonella spp. on Fresh and Frozen Cut Mangoes and Papayas. Int. J. Food Microbiol. 138:78-84.
    • Type: Journal Articles Status: Published Year Published: 2010 Citation: Strawn, L.K. and M.D. Danyluk. 2010. Fate of Escherichia coli O157:H7 and Salmonella spp. on Fresh and Frozen Cut Pineapples. J. Food Prot. 73:418-424.
    • Type: Journal Articles Status: Published Year Published: 2010 Citation: Dagulo, L., M.D. Danyluk, T.M. Spann, F.M. Valima, R.M Goodrich-Schneider, C.S Sims, and R. Rouseff. 2010. Chemical characterization of orange juice from trees infected with citrus greening (Huanglongbing). J. Food Sci. 75: C199-C207.


    Progress 10/01/08 to 09/30/09

    Outputs
    Target Audience: Produce researchers and growers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Attended 2009 S1033 Annual Meeting How have the results been disseminated to communities of interest? Workshops and presentations at international meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? E. coli O157:H7 and Salmonella spp. have the potential to grow on temperature abused fresh-cut mangoes and papayas held at 23°C and 12°C, and survive on fresh-cut pineapples. E. coli O157:H7 and Salmonella spp. can survive for extended periods of time on refrigerated (4°C) and frozen (-20°C) cut mangoes, papayas and pineapples. Our work indicates that both fresh and frozen cut mangoes, papayas and pineapples have the potential to be vectors for E. coli O157:H7 and Salmonella spp. transmission.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2009 Citation: Cevalos-Cevallos, J.M., J.I. Reyes-De-Corcuera, E. Exteberria, M.D. Danyluk, and G.E. Rodrick. 2009. The process of metabolomics analysis in food science. Trends Food Sci. 20:557-599.
    • Type: Journal Articles Status: Published Year Published: 2009 Citation: Friedrich, L.M., R. Goodrich-Schneider, M.E. Parish, and M.D. Danyluk. 2009. Mitigation of Alicyclobacillus spp. spores on food contact surfaces with aqueous chlorine dioxide and hypochlorite. Food Microbiol. 26:936-941.


    Progress 10/01/07 to 09/30/08

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Attending the 2008 S1033 multistate meeting. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? The results of the simulation indicate that even a small initial level of contamination at the point of harvest (<1 CFU/10 grams), stored under proper storage conditions (4 C) could result in a outbreak where more than 800 individuals would become ill, resulting in ~40 reported cases. Even mild temperature abuse (8 C for 7 days) would result in an outbreak of approximately the same size as the Fall 2006 US E. coli O157:H7 spinach outbreak.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2008 Citation: Amaratunga, K.S.P., M. D. Danyluk, L.J. Harris, and R.P. Singh. 2008. Effectiveness of Electrolyzed Water in Clean In Place Applications for Systems Fouled with Apple Juice. J. Food Manufacturing Efficiency. 2:15-22
    • Type: Journal Articles Status: Published Year Published: 2008 Citation: Danyluk, M.D., M. Nozawa-Inoue, K.R. Hristova, K.M. Scow, B. Lampinen and L.J. Harris. 2008. Survival and growth of Salmonella Enteritidis PT 30 in almond orchard soils. Journal of Applied Microbiology. 104: 1391-1399.
    • Type: Journal Articles Status: Published Year Published: 2008 Citation: Danyluk, M.D., M.T. Brandl, and L.J. Harris. 2008. Migration of Salmonella Enteritidis PT 30 through almond hulls and shells. Journal of Food Protection. 71:397-401.