Progress 09/01/08 to 08/31/13
Outputs
Target Audience: Target audiences reached during this reporting period included fresh produce growers and processors, as well as grower/shipper organizations, and academic professionals who conduct extension and research on fresh produce. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project provided training opportunities for graduate students and postdoctoral fellows accross multiple academic institutions and states. Professional development activites came in the form of presentation at scientific conferences, and symposia. How have the results been disseminated to communities of interest? Results of the project have been disseminated over the life of the project to various target audiences comprised of interested stakeholders that produce fresh produce. Such audences included growers and processors of freshproduce and vegetables including cantaloupes, tomatoes, leafy greens, onions, and potatoes. The ojective of these presentations was to effect a change in the knowledge of the target audiences with respect to acceptance the new knowledge obtained fm this project. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have developed and evaluateda suite of methods that can be used concentrate microorganisms (bacteria and viruses) from large volumes of water used in fresh produce production. We have also developed several novel methods to detect foodborne pathogens and indicator microorganisms. Models that have evaluated the best parameters (indicator microorganisms, environmental parameters) to use to estimate the presence of Salmonella species in the fresh produce growing environment have been deveoped.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria
monocytogenes from Large Volumes of Agricultural Water. Bisha, B., Adkins, J. A., Jokerst, J.
C., Chandler, J. C., Perez-Mendez, A., Coleman, S. M., Sbodio, A. O., Suslow, T. V., Danyluk,M. D., Henry, C. S., Goodridge, L. D. Journal of Visualized Experiments. In Press.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Predicting Salmonella populations from biological, chemical, and physical indicators in Florida
surface waters. McEgan, R., Mootian, G., Goodridge, L.D., Schaffner, D.W., and Danyluk, M.D. Applied and Environmental Microbiology. 79:4094-4105.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Evaluation of a Simple and Cost Effective Filter Paper-Based Shipping and Storage Medium for
Environmental Sampling of F-RNA Coliphages. P�rez-M�ndez, A., Chandler, J. C., Bisha, B., Coleman, S. M., Zhanqiang, S., Gang, Y., Goodridge, L. D. Journal of Virological Methods. 194:60-66.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Evaluation of Molecular Alternatives to Traditional Serotyping for Salmonella enterica subs.
Enterica. S. Coleman, McEgan, R., Chandler, J., Bisha, B., Perez-Mendez, A., Manley, W.,
Probasco, K., Marshall, D., Danyluk, M, and Goodridge, L. 2013. International Association for Food Protection 99th Annual Meeting, Charlotte, North Carolina.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Evaluation of Several Drag Sampling Techniques for Isolation of Salmonella enterica from
Agricultural Environments. B. Bisha., Chandler, J., Perez?Mendez, A., Coleman, S., Probasco,
K., Marshall, D., Manley, W., and Goodridge, L. 2013. International Association for Food Protection 99th Annual Meeting, Charlotte, North Carolina.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Characterization and Rapid Detection of Cantaloupe?associated Listeria monocytogenes. J.
Chandler, Manley, W., Bisha, B., Adkins, J., Perez-Mendez, A., Coleman, S., Henry, C., and
Goodridge, L. 2013. International Association for Food Protection 99th Annual Meeting, Charlotte, North Carolina.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: During the past year, activities including experiments and outreach, continued from previous year and centered around the rationship between indicator and index organisms and the presence of foodbore pathogens. The experiments led to results relating to the understanding of the relationship between foodborne pathogens and physical and biological indicators, and will facilitate the development of methods to more accurately assess the presence of foodbore pathogens in the fresh produce growing environment. These activities enabled the PIs to mentor several graduate (MS and PhD) students, and post doctoral fellows. Audiences targeted were stakeholders within the fresh produce industry, food safety risk assessors, policy makers and regulators, public health authorities, and consumers. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: During the project period, results obtained from this project were presented to various target audiences comprised of interested stakeholders that produce fresh produce. Such audences included growers and processors offres produce and vegetables including cantaloupes, tomatoes, leafy greens, onions, and potatoes. The objective of these presentations was to effect a change in the knowledge of the target audiences with respect to acceptance of the new knowledge obtained fm this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A change in knowledge occurred as a result of the activities described above. Physical and biologcal indicators and index organisms were shown to be weakly correlated with Salmonella spp in tomato growing environments. Weak linear relationships existed between biological indicators (E. coli/coliforms) and Salmonella levels and between physicochemical indicators and Salmonella levels. The average rainfall (previous day, week and month) before sampling did not correlate well with bacterial levels. Logistic regression analysis showed that the E. coli concentration could predict the probability of enumerating Salmonella. The lack of good correlations between biological indicators with Salmonella levels and between physicochemical indicators and Salmonella levels shows that the relationship between pathogens and indicators is complex. Escherichia coli provide a fair ability to predict Salmonella levels in Central Florida surface water. This information demonstrates that the current practice of testing agricultural water for the presence of E. coli as an indication of te safety of the water for using during growth andharvest of fresh produce, may not be valid.
Publications
- Detection of Salmonella spp. from large volumes of water by modified Moore swabs and tangential flow filtration. McEgan, R., Rodrigues, C.A., Sbodio, A., Suslow, T.V., Goodridge, L. D., and Danyluk M. D. Letters in Applied Microbiology. 2012. in press
- Framework for Developing Research Protocols: Evaluating Microbial Hazards and Controls during Production that Pertain to the Quality of Agricultural Water contacting Produce that may be consumed Raw. Harris, L. J., Bihn, E. A., Bender, J., Blessington, T.,Danyluk, M. D., Delaquis, P., Goodridge, L., Ibekwe, A. M., Ilic, S., Kniel, K., LeJeune, J. T., Schaffner, D. W., Stoeckel, D., and Suslow, T. V. Journal of Food Protection. 2012. 75:2251-2273.
- Development of a Paper-Based Analytical Device for Colorimetric Detection of Select Foodborne Pathogens. Jokerst, J. C., Adkins, J. A., Bisha, B., Mentele, M. M., Goodridge, L. D., and Henry, C. S. Analytical Chemistry. 2012. 84:2900−2907.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: During this project period, experiments were conducted that relate to the concentration of produce related pathogens (E. coli O157:H7 and Salmonella spp.) from large volumes of water used in produce production. An ongoing and a major focus of this project is the evaluation of the Modified Moore Swab (MMS) as a method to concentrate bacterial pathogens in water. It was hypothesized that the use of the MMS would allow for significant concentration of the target pathogens to easily detectable levels from large volumes of water in a reasonable amount of time. The objective was to evaluate the efficacy of the MMS method for concentration of E. coli O157:H7 and Salmonella spp. in (irrigation) water used in produce production, as a low tech way of concentrating bacteria, and then to compare the MMS to a high tech (continuous centrifugation) way of concentrating. In one series of experiments, water samples were spiked at levels of 10(1), 10(2), 10(3), and 10(4) CFU/100 ml with three-strain cocktails of either E. coli O157:H7 or Salmonella serovars, which had been previously transformed with a plasmid to express resistance to ampicillin as well as green, red, or cyan fluorescent proteins. Plating was performed before and after concentration on tryptic soy agar supplemented with ampicillin in order to quantitate the concentration efficiencies of each method. The two lowest spiking levels were also enriched in low volumes of tryptic soy broth supplemented with ampicillin followed by testing via lateral flow devices. Significant (P < 0.05) concentrations of initial levels of E. coli O157:H7 in the range of 0.7 to 1.0 and 1.2 to 1.4 log were achieved within approximately 35 min of processing time via MMS and CFC, respectively. Similarly, significant (P < 0.05) concentrations were also achieved for Salmonella with 0.9 to 1.2 and 1.2 to 1.4 log concentration for MMS and CFC, respectively. There were no statistical differences (P > 0.05) between the two concentration methods in their ability to concentrate either of the two target bacteria. Significantly (P > 0.05) more spiked samples were detected by lateral flow devices following concentration and enrichment than for nonconcentrated, enriched samples. From these experiments it is concluded that both MMS and CFC have potential to be used to enhance the sensitivity of downstream bacterial detection methods used to test irrigation water for the presence of foodborne pathogens. Due to cost effectiveness and the low tech construction of the modified moore swabs, it was decided to evaluate these devices for use in routine water sampling of water used during produce production. The devices are currently being evaluated at three sites by collaboratoers on the study in the states of Florida, Michigan, and Ohio. PARTICIPANTS: Memebers of the research team who worked on this project include: Colorado State University: Lawrence Goodridge, Bledar Bisha, Alma Perez-Mendez, University of California Davis: Trevor Suslow, University of Florida: Michelle Danyluk, Rached McEgan, Ohio State University: Jeff LeJeune, Rutgers University: Don Schaffner, Gabriel Mootian, University of Guelph: Manel Griffiths, Yongheng Yang, In addition, an opportunity for training arose from this project, in the form of a visit from Rachel McEgan at the University of Florida to Lawrence Goodridge's lab at Colorado State University to learn how to conduct Pulsed Field Gel Electrophoresis which she is using to subtype Salmonella isolates obtained during this project. TARGET AUDIENCES: During this project period the results of the ongoing work were communicated to three separate groups including academics (the S-1033 USDA multistate group on pre and post harvest food safety) during the annual meeting for this group (of which several PIs on this project are members), the Florida Association for Food Protection, and the Arkansas Associaation for Food protection. These last two gruops are comprised mainly of food industry professionals. Therefore, the dissemination of research results allowed for extension and outreach to many members of the food production arena including industry and academics. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A change in knowledge occurred as a result of the activities described above. Modified Moore Swabs were shown to be a cost effective alternative to the use of continuous centrifugation (an accepted way to concentrate bacteria from water), and it was also shown that this method is neccesary to increase the sensitivity of test methods for bacterial pathogens. When coupled with the results from 2010, which indicated that MMS was essential for more efficiently screening multiple pallets of incoming raw material in totes and packaged salads for contamination, it is clear that the evaluation and optimization of this technology has led to findings and results that can be used to influence behaviors leading to increased food safety production practices during fresh produce production.
Publications
- Marisa Bunning , Daniel Woo, Martha B. Stone, Bledar Blisha, Lawrence D. Goodridge. 2011. Microbial quality of mixed salad greens purchased from farmers' market vendors and a retail grocer. Institute of Food Technologists Annual Meeting and Food Expo, New Orleans, LA.
- Bisha, B., Perez-Mendez, A., Danyluk, M. D., and Goodridge, L. D. 2011. Evaluation of Modified Moore Swabs and Continuous Flow Centrifugation for Concentration of Salmonella and Escherichia coli O157:H7 from Large Volumes of Water. Journal of Food Protection 74:1934-1937.
- Willford, J. D., Bisha, B., Bolenbaugh, K.E., and Goodridge, L.D. 2011. Luminescence based enzyme labeled phage (Phazyme) assays for rapid detection of Shiga toxin producing Escherichia coli serogroups. Bacteriophage 1(2): 101-110.
- Rachel McEgan, Lawrence D. Goodridge and Michelle D. Danyluk. 2011. Isolation of Salmonella spp. from Surface Waters in Florida over a Five-month Period. International Association of Food Protection Annual Meeting, Milwaukee, WI. Abstract P1-134
- Bronte Roberts, Bledar Bisha, Kristofer Bruun, Katie Fialko and Lawrence D. Goodridge. 2011. Evaluation of a Portable, Recycled Vertical Flow Constructed Wetland as a Low Cost Treatment System for Greywater Reuse in Food Production. International Association of Food Protection Annual Meeting, Milwaukee, WI. Abstract P2-61
- Lawrence Goodridge. 2011 Bacteriophage: Friends, Foes or a Little of Both International Association of Food Protection Annual Meeting, Milwaukee, WI. Symposium Abstract S7
- Lawrence Goodridge 2011. Enzyme-based assays for rapid detection of Listeria monoctytogenes. Institute of Food Technologists Annual Meeting and Food Expo, New Orleans, LA.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: During this project period, several activities were conducted in the form of experiments that relate to the concentration of produce related pathogens (E. coli O157:H7 and Salmonella spp.) and indicators of fecal contamination from water used in produce production. The Disruptor Filter was used to concentrate F specific phages from artificially contaminated water samples. The volume of water samples used in these studies was 10 L. Sterile distilled water was inoculated with MS2 phage and each water sample was filtered through a single layer Disruptor Filter. Following capture, a variety of eluants were tested for their ability to release the entrapped phage from the filter. The presence of phage was enumerated by the Double Layer Agar method. In addition to concentration of indicators of fecal contamination, a major focus of this project is the evaluation of the Modified Moore Swab (MMS) as a method to concentrate bacterial pathogens in water. It was hypothesized that the use of the MMS would allow for significant concentration of the target pathogens to easily detectable levels from large volumes of water in a reasonable amount of time. The objective was to evaluate the efficacy of the MMS method for concentration of E. coli O157:H7 and Salmonella spp. in water used in produce production. In one series of experiments, three independent experimental setups were conducted to evaluate the capture sensitivity of MMS by inoculating with and without 1.0 g of a clay‐loam soil from 10 L of nanopure water. The target concentration of generic E. coli was adjusted from a standardized suspension in 0.1% BPW that had been held for 24h at 2.5 C. The water turbidity level after addition of the clay‐loam soil was measured to be 14 FAU, typical of many irrigation reservoirs in the CA Central Coast. Inoculated water was filtered through the MMS unit by attaching the cassette to a peristaltic pump set at a flow rate of 1 liter per minute. Recovery was accomplished by extracting the MMS from the cassette and placing the saturated swab in a sterile Whirlpak Bag holding 100ml 2X TSB. The broth cultures were incubated without shaking at 37 C and duplicate 15ml sample aliquots extracted at 4 and 7 hours. Each 15ml enrichment subculture was filtered with an IsoGrid Membrane system and the grid placed on selective media and incubated overnight. Grids were inspected for fluorescence typical of E. coli GFP:kanr; at 18 and 36h. One event was conducted as part of this project. A symposia was sponsored at the International Association for Food Protection Annual Meeting, titled "Setting the Science-based Agenda for Co-management of Watershed Quality and Produce Safety". A website (www.testingproducesaety.com) has been developed that serves to introduce stakeholders to this project, and also to other produce safety related projects and websites. PARTICIPANTS: Lawrence D. Goodridge, Kendra K. Nightingale, Mansel Griffiths, Jeffrey LeJeune, Trevor V. Suslow, Michelle D. Danyluk, and Donald Schaffner. Partner organizations who contributed supplies and kits as part of the research incude Applied Biosystems (Carlsbad, CA) and Scientific Methods Inc. (Granger, IN). This project has provided the opportunity to train and mentor several graduate students and Post-doctoral fellows including: Bronte Roberts, Travis Steiner, Alma Perez-Mendez, Bledar Bisha, Adrian Sbodio, Sharyn Maeda, Mark Trent, Rachel McEgan and Yongheng Yang. TARGET AUDIENCES: The target audiences include stakeholders (industry, government, and academia) who attended the sponsored symposia. In addition, results from the project have been presented to date at more than 30 fruit and vegetable grower meetings, extension workshops, and educational/instructional venues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A change in knowledge occurred as a result of the activities described above. With respect to concentration of indicator organisms from large volumes of water, the results indicated that between 90% and 99.9% of the phage particles were retained in the filter. All of the elution buffers tested have been shown to have poor ability to release phage from the filter medium. The extent of release of phage by elution with 0.25M glycine of different pH was also investigated. Less than 1% of phage was recovered by 0.25 M glycine solution with pH over a range of 5.9 to 9.6. The ability of 3x Nutrient Broth (with pH from 7.4 to 9.5) was also investigated to release phage from the filter. The highest efficiency observed was for 3x Nutrient Broth adjusted to pH 8.0; when about 42% of the entrapped phage could be detected in the eluate. These results indicate that other elution buffers will be needed in order to effectively recover F specific phages from the concentration filter, before detection. Therefore, a variety of methods to improve elution of phage from filters will be examined. These include: i) physical disruption of the filter; ii) elution with buffers of different ionic strength; iii) elution with polar solvents and iv) direct extraction of nucleic acid from the filters. After concentration of bacterial pathogens by Modified Moore Swab (MMS), detection of low concentrations of inoculated pathogenic bacteria was possible across a wide range of environmental water quality (3 to > 900 FAU). As few as 6 CFU/10L were detected after 18h enrichment for both E. coli O157:H7 and Salmonella. This water was reduced to a turbidity range of 2‐18 FAU following passage through the MMS cartridge. The initial results led to a study evaluating the application of MMS filtration capture and enrichment in commercial settings. Over 200 preharvest water sources have been evaluated to date, using the MMS‐system in CA and AZ. Of these, 8 were positive for E.coli O157:H7 and 19 were positive for various Salmonella serotypes or serovars. Two samples were positive for both. Additional samples were positive for non‐O157 EHEC/STEC. Also, MMS capture‐detection was applied to assessments of postharvest re‐wash surveys of naturally contaminated leafy greens. Briefly, presumptive contaminated lots were re‐washed at ratios of 600g:1.85L of buffer and subjected to MMS for capture filtration followed by appropriate enrichment, qrtPCR, and selective and differential media for the target pathogen. MMS was essential for more efficiently screening multiple pallets of incoming raw material in totes and packaged salads for contamination. In one situation, MMS screening was used to detect 2/24 positive bags among pallets.
Publications
- Abstracts Detection and Isolation of Salmonella from Surface Waters. R. McEgan, L. D. Goodridge, and Danyluk, M. D. Food Micro 2010, Copenhagen, Denmark, August 30th to September 3, 2010.
- Antimicrobial Incorporated Multi-angle Light Scattering Spectroscopy (ANIMALS) Facilitates Detection of Escherichia coli O157:H7 in Large Volumes of Irrigation Water. L. Goodridge, J. C. Leon, B. Bisha, M. Danyluk, M. Griffiths, J. LeJeune, D. Schaffner, and Suslow, T. HortScience 45(8) (Supplement)-2010 ASHS Annual Conference-August 2-5, 2010.
- Concentration of Large Volumes of Irrigation Water Facilitates Sensitive Detection of Foodborne Pathogens. L. Goodridge, B. Bisha, M. Danyluk, M. Griffiths, J. LeJeune, D. Schaffner, and Suslow, T. HortScience 45(8) (Supplement)-2010 ASHS Annual Conference-August 2-5, 2010.
- Rapid Detection of Viable Escherichia coli O157:H7 in Irrigation Water by Antimicrobial Incorporated Multi-Angle Light Scattering Spectroscopy. B. Bisha, J. C. Leon, S. Deshpande, and Goodridge, L. D. American Society for Microbiology, 110 General Meeting, San Diego, CA, May 23-27, 2010. Abstract P-2311/690.
- Evaluation of a rapid assay for concentration and detection of the FRNA bacteriophages as microbial indicators of Faecal Pollution. L. D. Goodridge, and Du Preez, M. Society for Applied Microbiology Summer Conference 2010. Abstract P18.
- Assessing the Performance of Modified Moore Swabs as a Low-cost Method of Large Volume Irrigation Source Surveys. A. Sbodio, S. Maeda, M. Trent and Suslow, T. V. International Association for Food Protection Annual Meeting, Anaheim, CA, August 1-4, 2010. Abstract P2-50.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: The main output completed during the reporting period was an activity. The activity was related to experiments conducted towards the development of rapid assays to detect Escherichia coli O157:H7, and fecal indicators in irrigation water. The assays were developed and have been evaluated using artificially contaminated river and agricultural runoff water. Other activities included analysis of water sources used for irrigation of fresh produce for the presence of Salmonella and E. coli O157:H7. Experiments have also begun with respect to development and optimization of a PCR serotyping method for Salmonella. One event was held during the reporting period. A program meeting between the PI, all of the coPIs and the Program Manager of the Specialty Crop Research Initiative was held at Colorado State University on August 18, 2009. During this meeting, the Program Manager was updated on the progress of the project thus far, and the PIs discussed the next steps in the research. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
During the reporting period, a change in knowledge occured through the development of rapid assays to detect E. coli O157:H7, and fecal indicators. The E. coli O157:H7 detection method combines the principles of light scattering and bacteriophage infection of its host in a new way to create a detection method not previously described. As such, the detection assay signifies an entirely new class of bacterial detection tests. The test for fecal indicators is based on detection of a class of indicators, called the FRNA bacteriophages. The assay is comprised of a resin based bacteriophage capture step, followed by reverse transcriptase polymerase chain reaction, or lateral flow-based, detection of the captured bacteriophages. Next, these tests will be used to gain knowledge regarding the presence of bacterial pathogens, and fecal (animal or human) contamination in fresh produce irrigation and process water, hopefully leading to a change in actions with respect to the information derived from the tests.
Publications
- Leon, J.C., Deshpande, S., and Goodridge, L. D. 2009. Rapid Detection of Viable Escherichia coli O157:H7 by Immunomagentic Separation and Light Scattering Spectroscopy. IAFP Program and Abstract Book Annual Meeting Grapevine, Texas(P2-114).
- Steiner, T., and Goodridge, L. D. 2009. Rapid Capture and Detection of Model Viruses from Large Volumes of Water. IAFP Program and Abstract Book Annual Meeting Grapevine, Texas(P2-34).
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