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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Environmental Microbial & Food Safety Laboratory » Research » Research Project #430368

Research Project: Characterization and Mitigation of Bacterial Pathogens in the Fresh Produce Production and Processing Continuum

Location: Environmental Microbial & Food Safety Laboratory

2017 Annual Report

1a. Objectives (from AD-416):
Objective 1: Investigate the mechanism(s) of introduction, transference, and survival of enterohemorrhagic Escherichia coli (EHEC), Salmonella, and Listeria to fresh produce at the farm level. Sub-objective 1a. Investigate the population dynamics of non-pathogenic E. coli and non-O157 EHEC in soils amended with biological soil amendments (BSA). Sub-objective 1b. Determine factors affecting persistence of EHEC, Salmonella and Listeria in soils amended with BSA. Objective 2: Determine the effects of multispecies biofilm formation on the survival, persistence, and dissemination of pathogenic bacteria in fresh produce processing environments and on contamination of fresh produce. Sub-objective 2a. Assess the biofilm formation capacity of foodborne bacterial pathogens in fresh produce processing environments and on fresh produce surfaces; identify environmental bacterial strains or species that promote multispecies biofilm formation on fresh produce or in processing environments. Sub-objective 2b. Elucidate factors controlling foodborne bacterial pathogen interactions in multispecies biofilms on fresh produce or in processing environments. Sub-objective 2c. Determine biofilm formation of non-O157 shiga-toxigenic E. coli (STEC) on abiotic and biotic surfaces. Objective 3: Investigate intervention strategies to minimize contamination of EHEC, Salmonella and Listeria on fresh produce at the farm level. Sub-objective 3a. Determine the role of Brassica vegetables in controlling enteric pathogens in soil. Sub-objective 3b. Develop pre-harvest interventions to control Listeria and Salmonella in cantaloupe. Objective 4: Develop effective intervention technologies to reduce pathogen survival and growth during processing and retail operations. Sub-objective 4a. Identify and validate food safety preventive controls for water application during fresh-cut processing. Sub-objective 4b. Investigate novel antimicrobials to control enteric pathogens on herbs. Objective 5: Assessment of microbial safety of fresh produce grown under non-conventional farming practices. Sub-objective 5a. Determine the effect of reclaim water on microbial safety of fresh produce grown in urban farming.

1b. Approach (from AD-416):
Mechanisms of introduction and transfer of pathogens on fresh produce (lettuce, spinach, leafy greens, fresh herbs) at the farm level will be investigated. Population dynamics of non-O157 Enterohemorrhagic E. coli (EHEC) and non-pathogenic E. coli in soils amended with biological soil amendments (BSA: manure, compost) will be investigated. Factors affecting growth and survival patterns of EHEC, Salmonella and Listeria in soils amended with BSA will be determined. The role of stress response genes on the survival of enteric pathogens in manure or manure-amended soils will be evaluated. Bacterial analysis will include the use of microbial culture and molecular methods to detect target pathogens in samples. Biofilm formation capacity of EHEC and Listeria monocytogenes will be assessed under conditions partially simulating produce production and processing environments. Bridge bacteria that promote the incorporation of pathogen in multispecies biofilms will be isolated and identified. Confocal microscopy, mass spectrometry, and metagenomic sequencing will be used to decipher the complexity of the multispecies biofilms. Intervention strategies will be investigated to minimize pathogen contamination at the farm level. Field studies will be conducted to determine the role of Brassica vegetables in killing EHEC, Salmonella, and Listeria in soil. Biological controls such as lactic acid bacteria will be evaluated at the farm level to control Listeria contamination on cantaloupe. Food safety preventive controls during fresh-cut processing operations will be identified and validated to reduce pathogen survival and growth on fresh produce. Validation of free chlorine concentration, role of produce particulates, and pathogen inactivation kinetics will be investigated to minimize pathogen cross-contamination. Fresh produce will be irrigated with reclaimed water to assess its microbial safety. Microbial risk assessment models will be used to determine microbial safety of fresh produce.

3. Progress Report:
Progress was made on all five objectives and their sub-objectives, which fall under National Program 108. The third season of a three-season field study was performed at the University of Vermont (objective 1). Declines of E. coli in manure-amended soils over 60 days were similar in the three seasons, but Listeria sp. survival profiles were different between season 2 and 3, indicating E. coli and Listeria may survive differently under the same conditions. Survival studies of enteric pathogens in soils containing various biological soil amendments (manure or heat-treated manure products) revealed that E. coli could survive for up to 205 days in soils amended with heat-treated poultry pellets (HTPP). Other study in growth chambers showed that regular irrigation of HTPP-amended soils increased Salmonella population in soil. Under objective 2, produce and environmental samples were collected from a major fresh produce processing plant to assess the biofilm formation and interactions with foodborne pathogens by environmental microorganism. The dynamic shifts of microbial community on baby spinach during processing and temperature-abused storage conditions, and in the processing environments were analyzed using metagenomic procedures. Nonpathogenic bacterial strains with strong biofilm formation, as well as those that enhanced biofilm formation by foodborne pathogenic strains were isolated. Previously we reported that Ralstonia insidiosa could induce precipitation by L. monocytogenes, a necessary step of biofilm formation. The transmission electron microscopy procedure revealed that L. monocytogenes cells were attracted to cores formed by R. insidiosa cells (objective 2). However, further analyses of R. insidiosa-L. monocytogenes biofilm formation on stainless steel coupons using scanning electron microscopy failed to generate consistent information. The transcriptomic analysis is being used to characterize the interaction of R. insidiosa with L. monocytogenes in dual culture growth and in biofilm formation. Under objective 3, significant progress was made on persistence of non-O157 shiga toxin-producing E. coli (STEC) on various equipment surfaces and fresh produce leaves. Recovery of attached STEC was 2-3 log CFU/g lower from their initial populations (~6.5 log CFU/g) when fresh produce was incubated for 48 h. Significant reductions in STEC populations were observed on spinach samples incubated for 48 h at 22°C. E. coli O26:H11 strain 5711 was recovered at significantly higher level than E. coli O121:H19 strain 5705 on cabbage and Romaine lettuce following 48 h incubation at 4°C. Under objective 4, progress was made on field-testing the installation of doors on open refrigerated display cases to support the implementation and compliance of U.S. Food Code on temperature control. Enabled by strong support from the retail industry, temperature loggers in major grocery stores in multiple locations were installed. Preliminary data showed significant reduction in temperature in cases with doors. Plans for testing for energy consumption, product quality and safety are in place. Major outcomes are expected to reduce pathogen growth during retail display and improve produce safety. The associated energy reduction will also provide incentive for the retailers to adopt this technology. The microbial quality of alternative water including reclaimed water, roof-harvest rainwater, and creek water was evaluated (objective 5). Persistence of indicator bacteria on spinach irrigated with alternative water was determined. Salmonella and E. coli O157:H7 were recovered from reclaimed water; whereas Listeria monocytogenes was recovered from roof-harvest water. Results suggested that reclaimed water required remediation treatment to reduce bacterial populations prior to use for irrigation of fresh produce. The creek water and roof-harvest water quality met Food Safety Modernization Act (FSMA) standards for irrigation; however, presence of pathogens in this water could contaminate fresh produce and cause foodborne illnesses. Repeat irrigation of spinach with reclaimed and roof-harvest water resulted in increased persistence of enterobacteriaceae and fecal coliform on spinach leaves. Presence of enterohemorrhagic E. coli, Salmonella spp, and L. monocytogenes in various types of non-traditional irrigation waters in the Mid-Atlantic region were surveilled regularly as a part of NIFA -funded project. From all water samples, 65% and 46% were positive for Salmonella spp. and L. monocytogens, respectively, and 53% contained E. coli populations greater than the level stated for irrigation water in the Produce Safety Rule of the FSMA.

4. Accomplishments
1. Environmental bacteria can enhance biofilm formation by foodborne pathogens. Ralstonia (R.) insidiosa, a bacterium found in the environment, is an opportunistic pathogen that often contaminates water supply systems. ARS scientists found that R. insidiosa isolated from produce packing facilities promoted the incorporation of the disease causing strain Escherichia (E.) coli O157:H7 into a dual species biofilm, which is adherence of bacteria to surfaces including those in packing plants. R. insidiosa also enhanced the formation of biofilm by other pathogenic E. coli strains, Salmonella, and Listeria (L.) monocytogenes strains in dual species cultures. The bacterium seems to play the role of “bridge bacteria” in multispecies biofilm formation. R. insidiosa induced aggregation, a key step in biofilm formation, by L. monocytogenes in mixed cultures. This information is useful for developing new antimicrobial wash treatments to improve the microbial safety of fresh produce.

2. Pathogenic bacteria are internalized in cantaloupes during post-harvest treatment. Postharvest practices such as cooling of cantaloupes in ice water (hydro-cooling) and washing reduce the contamination of fresh fruits by human pathogens, such as Listeria (L.) monocytogenes. Scientists at ARS in collaboration with the FDA investigated the potential for L. monocytogenes to be internalized into cantaloupes during dump tank washing and immersion-type hydro-cooling in water contaminated with L. monocytogenes. Water containing L. monocytogenes infiltrated both full slip (stem removed – stem scar) and clipped (residual stem) cantaloupes through the stem scars/stems during hydro-cooling and was then transferred to interior flesh of the fruit. The incidence and level of L. monocytogenes internalized in the flesh of the fruit were not significantly affected by water temperature or cantaloupe variety. The results reveal potential health safety risk during hydro-cooling of cantaloupe and emphasize need for pathogen control at both, farm level and at the packing facility.

3. Growth of pathogenic bacteria on cantaloupe varies with its contamination site. Recent outbreaks of foodborne illnesses associated with cantaloupe consumption require investigation of pathogen survival on cantaloupe. ARS researchers determined that Rocky Ford cantaloupes, which were implicated in a deadly listeriosis outbreak in 2011, were no more likely to support the survival of the pathogen Listeria (L.) monocytogenes than the Athena variety of cantaloupes. It was also demonstrated that the site of contamination on cantaloupes affected the survival and growth of L. monocytogenes more than growth temperature or variety. L. monocytogenes was able to survive and grow on the stem-scar area of intact melons, even under refrigeration temperatures, but not on the rind. On fresh-cut melons, the L. monocytogenes also grew even at refrigeration temperatures. The results show potential routes of contamination of cantaloupes with L. monocytogenes.

4. Seminal research used by FDA and industry to develop science- and risk-based food safety practices. Fresh produce processors traditionally have used a specific free-chlorine level [1 ppm (part per million)] as the “Control Limit” and a re-wash as the “Corrective Action” in Hazard Analysis and Critical Control Points (HACCP) programs. ARS scientists determined that this industry-standard "Control Limit" chlorine concentration does not prevent pathogen cross-contamination, and that re-washing of contaminated product is an ineffective "Corrective Action". The research clearly documented significant risk factors associated with “generally-considered-safe” operating practices. Follow up studies further demonstrated that a minimum of 10 ppm free-chlorine was required to effectively prevent pathogen cross-contamination during washing. Recommendations have been adopted by leading processors, and incorporated in the interagency and industry taskforce whitepaper entitled “Guidelines to Validate Control of Cross-Contamination during Washing of Fresh-Cut Leafy Vegetables”.

5. Bacterial viruses reduce Salmonella contamination on cucumber. Cucumbers have been associated with recent outbreaks of salmonellosis, a GI disease that is caused by different strains of Salmonella bacteria. ARS researchers evaluated the survival of Salmonella (S.) Newport on cucumbers at different storage temperatures, and examined a novel antimicrobial approach for their control. S. Newport populations declined more quickly on whole cucumbers stored at temperatures that were higher than at the recommended post-harvest storage temperatures for cucumbers. They demonstrated that Salmonella could be transferred from the outside rind of the cucumber to the flesh. S. Newport did not grow on fresh-cut cucumbers stored at refrigerated temperatures. Bacterial viruses (bacteriophages) specific for Salmonella that do not affect humans reduced Salmonella populations on the cucumbers immediately after initial application. The results illustrated the extent of contamination routes during storage and the effectiveness of a novel antimicrobial for the killing of pathogens on cucumber.

Review Publications
Islam, N., Nagy, A., Garrett, W.M., Shelton, D.R., Cooper, B., Nou, X. 2016. Different cellular origins and functions of extracellular proteins from Escherichia coli O157:H7 and O104:H4 as determined by comparative proteomic analysis. Applied and Environmental Microbiology. doi: 10.1128/aem.00977-16.

Hernandez-Anguiano, A.M., Salgado, P.L., Eslava-Campos, C.A., Hernandez, M.V., Patel, J.R. 2016. Microbiological quality of fresh nopal juice. Microorganisms. 4(46):1-11.

Sharma, M., Reynnells, R. 2016. Importance of soil amendments: survival of bacterial pathogens in manure and compost used as organic fertizliers. Microbiology Spectrum. 4:1-13.

Nayarko, E., Kniel, K., Millner, P.D., Luo, Y., Handy, E.T., Reynnells, R., East, C.L., Sharma, M. 2016. Survival and growth of Listeria monocytogenes on whole cantaloupes is dependent on site of contamination and storage temperature. International Journal of Food Microbiology. 234:65-70.

Markland, S.M., Ingram, D.T., Kniel, K.E., Sharma, M. 2017. Water in agriculture. Microbiology Spectrum. (5)3.

Callahan, M., Micallef, S.A., Sharma, M., Millner, P.D., Buchanan, R.L. 2016. Investigating metrics proposed to prevent the harvest of leafy green crops contaminated by floodwater. Applied and Environmental Microbiology. 82:3746-3753.

Keelara, S., Patel, J.R., Siddhartha, T. 2016. Biofilm formation by environmental isolates of Salmonella and their sensitivity to natural antimicrobials. Foodborne Pathogens and Disease. 13(9):509-516.

Nyarko, E., Kniel, K., Reynnells, R., East, C.L., Handy, E.T., Luo, Y., Millner, P.D., Sharma, M. 2016. Survival and growth of Listeria monocytogenes on fresh-cut ‘Athena’ and ‘Rocky Ford’ cantaloupes during storage at 4 and 10°C. Foodborne Pathogens and Disease. 13:587-591.

Sharma, M., Dashiell, G., Handy, E.T., East, C.L., Reynnells, R., White, C., Nyarko, E., Hashem, F., Millner, P.D. 2017. Survival of Salmonella Newport on whole and fresh-cut cucumbers treated with lytic bacteriophages. Journal of Food Protection. 80:668-673.

Yan, S., Liu, H., Yang, T., Luo, Y., Chen, P. 2017. Dual effectiveness of ascorbic acid and ethanol combined treatment to inhibit browning and inactivate pathogens on fresh-cut apples. LWT - Food Science and Technology. 80:311-320.

Gombas, D., Luo, Y., Brennan, J., Shergill, G., Petran, R., Walsh, R., Hau, H., Khurana, K., Zomorodi, B., Rosen, J., Varley, R., Deng, K. 2017. Guidelines to validate control of cross-contamination during washing of fresh-cut leafy vegetables. Journal of Food Protection. 80(2):312-330.