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





- Patel, Jitu
- Sharma, Manan
- Millner, Patricia
- Luo, Yaguang - Sunny
- Nou, Xiangwu
- 2021
- 2020
- 2019
- 2018
- 2017
- 2016
- Publications
- Food Safety, (Animal and Plant Products) (108)

Research Project: Characterization and Mitigation of Bacterial Pathogens in the Fresh Produce Production and Processing Continuum **Location:
                            Environmental Microbial & Food Safety Laboratory** 2020 Annual Report **Objectives**
                                

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. **Approach**
                                

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. **Progress Report**
                                

Progress was made on all objectives and their sub-objectives, which fall under National Program 108, Component 1, Foodborne Contaminants. Activities of this project focus on Problem 1, Population Systems, and Problem 5, Intervention and Control Strategies.

Under Objective 1, the prevalence data for bacterial foodborne pathogens and virulence genes for Shiga-toxigenic Escherichia coli in irrigation water (six sites at Conococheague creek, PA) were collected. Populations of generic E. coli were significantly lower in water samples during winter season. Salmonella were recovered in 7% of water samples in early fall. Two genes responsible for causing illness, stx1 and stx2, were prevalent in 16 and 7% water samples, respectively. Similarly, prevalence data for the survival of E. coli in manure-amended soils in the Northeast and Mid-Atlantic regions were collected.

Progress was made in development of new procedures for rapid detection of foodborne pathogens in irrigation water. The type of water, the target pathogen, and the specific methods used affected the accuracy and reliability of procedures used to provide pathogen testing results for irrigation water. To minimize contamination of fresh produce via irrigation water, cost-effective solutions to improve the microbial quality of irrigation water are being investigated through the development of a zero-valent iron (ZVI) filtration system. This system will provide small-scale fruit and vegetable growers with a mechanism to improve irrigation water from various sources during growing seasons. Under Objective 2, growth potential of Listeria monocytogenes was examined on whole and fresh-cut fruits and vegetables under normal (generally practiced by industry) and temperature abuse storage or retail display conditions. The growth rate of L. monocytogenes was significantly influenced by the pH of fresh produce. Role of micronutrient, antibacterial substance, and produce microbiome on L. monocytogenes growth is being explored. Biofilm formation of Shiga-toxigenic E. coli (STEC) and L. monocytogenes was examined using minimum biofilm eradication concentration assay. The biofilms formed by these bacterial pathogens varied with initial inoculum levels and bacterial strains. Further, STEC serotype O145 was shown as a weak biofilm former than O26, O45, and O121 serotypes. Under Objective 4, ARS researchers in collaboration with Rutgers University, 3D-printed produce models with well-defined dimensions and surface physicochemical properties to simulate fresh-cut produce. The surfaces of varying densities of these objects were inoculated with bacteria and modelled to study the effect of flow dynamics on bacterial removal. Significant progress was made in evaluating the potential application of our patented in-flight washer (IFW) in removing organic matter (released from fresh-cut produce that depletes sanitizer rapidly) before the product is introduced to the flume for washing, thereby improving food safety and quality after packaging and during storage. Numerous trials were conducted using two IFW prototypes with various configurations, fresh-cut produce (shredded lettuce, diced tomato, and diced cabbage), and produce throughput levels (representative of 25-150% of typical levels for commercial processing) to test the organic removal capacity of IFW. The IFW with larger diameter and multiple water/air layers removed 50-60% of organic materials released from shredded lettuce at a throughput of 3,600 lb./hr and water flow rate of 10 gal/min. 
ARS researchers collaborated with researcher at Rutgers University researcher to investigate the inactivation efficacy of Plasma-activated water (PAW) sanitization methods on fresh lettuce contaminated with human pathogens. The PAW spray treatment on chopped romaine lettuce pieces reduced the inoculated pathogen population by over 0.5 log CFU/g. Follow-up studies will be focused on the scale-up application of PAW on fresh and fresh-cut produce through our patented in-flight washer and commercial cutter with customer-designed nozzles installed. **Accomplishments**
                                

1. Novel antimicrobial to control seed decontamination for improving sprout safety. Contaminated sprouts have caused 50 foodborne outbreaks resulting in more than 2500 illnesses. The current standard of chlorine treatment for seed decontamination is not effective. Also, the use of high chlorine concentrations may pose an occupational health hazard. ARS scientists in Beltsville, Maryland, used a naturally occurring compound in green vegetables to kill Salmonella on alfalfa seeds. An industry partner is working with ARS scientists to scale-up the use of the green vegetable extract for controlling Salmonella on alfalfa seeds and improving sprout safety. 2. Control of foodborne pathogens on cantaloupe at the farm level. Cantaloupes contaminated with Listeria monocytogenes have been implicated in several foodborne outbreaks in the United States. Current post-harvest wash treatment of cantaloupe with chlorine has limited effectiveness. ARS scientists in Beltsville, Maryland, used Lactic acid bacteria (LAB) as a biocontrol spray application on cantaloupes inoculated with surrogate bacteria L. innocua on the farm. The population of L. innocua was reduced significantly on cantaloupes harvested at 5 and 7 days after LAB treatment. LAB was also effective in reducing L. monocytogenes on cantaloupes as post-harvest application. The information is useful for cantaloupe growers and packers to minimize the risk of pathogen contamination and improve food safety. 3. Reduce food safety risks of imported papayas. Contaminated papayas imported from Mexico have caused several salmonellosis outbreaks. Following an urgent request from the produce industry, ARS scientists in Beltsville, Maryland, investigated the effect of simulated packing house operation conditions on the cross-contamination of Salmonella on papayas. Results indicated that disinfectants such as chlorine and peracetic acid can reduce but not eliminate the presence of Salmonella on papayas. These results are used by the industry in developing “Food Safety Best Practices for the Growing and Handling of Mexican Papaya” guidelines. 4. Improve the food safety of fresh-cut produce during cold storage. Pathogen contamination on fresh-cut produce has led to several foodborne outbreaks. Safety of fresh-cut produce can be improved by accelerating bacterial die-off on fresh produce during storage. In partnership with the fresh produce industry, ARS scientists in Beltsville, Maryland, investigated a novel technology for bacterial inactivation on fresh produce in simulated commercial fresh-cut processing conditions. Results indicated that silver citrate in combination with a process aid rapidly killed bacteria on romaine lettuce during cold storage. The fresh-cut industry partner used the results to improve safety of fresh-cut produce. 5. Non-traditional water sources for irrigation of fresh produce. Alternative irrigation water sources are required to meet increasing demands of food supply for growing human population and during water scarcity. In collaboration with university researchers, ARS scientists in Beltsville, Maryland, used secondary-treated waste water (STW) and rain water (RW) for spinach irrigation at a farm to determine its effect on microbial safety of spinach. Spinach irrigated with STW or RW were free from pathogens, and had similar or lower levels of generic E. coli. Produce growers can use such non-traditional irrigation waters during water scarcity, provided that these waters contain lower levels of indicator bacteria and free from pathogens. 6. Irrigation water source influence foodborne pathogen survival in the Mid-Atlantic U.S. The use of contaminated surface irrigation water can lead to pathogen contamination on fresh produce. ARS scientists in Beltsville, Maryland, in collaboration with university researchers, analysed rivers, creeks, ponds and recycled water samples in Maryland and Delaware for foodborne pathogens. Salmonella and L. monocytogenes were found in 50% and 31% of water samples, respectively. These pathogens were frequently recovered from rivers and creeks than from pond or reclaimed waters. Farmers, growers and regulators can use the results to improve the microbial quality of irrigation water and produce safety. 7. Organic soil amendments (fertilizers) and irrigation affect Salmonella levels in soils. Salmonella Newport has caused several foodborne outbreaks. It has been found on fresh produce grown in the Mid-Atlantic U.S. where poultry litter and heat-treated poultry pellets are commonly used as biological soil amendments. ARS scientists in Beltsville, Maryland, along with university researchers, investigated survival pattern of S. Newport in these amendments and in fertilized soils. S. Newport survived at higher levels in soils fertilized with heat-treated poultry pellets. Irrigation event following soil contamination increased S. Newport levels in soils fertilized with these amendments. Farmers using poultry-based soil amendments for soil fertility can use this information for controlling pathogens in soil. 8. Farming practices and environmental factors affect survival of Escherichia coli in manure-amended soils. Fresh produce may be contaminated by pathgoens potentially present in untreated manure. The FDA Produce Safety Rule prohibits the use of untreated manure within 90 or 120 days prior to the harvest of edible produce crops. Data from 12 field trials over four years at three separate locations collected by ARS scientists in collaboration with the FDA were used to identify factors affecting E. coli survival in manure-amended soils. The poultry litter supported longer survival of E. coli than dairy or horse manure. Days of rainfall and soil moisture content affected E. coli survival in manure-amended soils. These results aid FDA in creating models determining the appropriate interval between application of raw manure and harvest of edible crops to minimize fresh produce contamination. **Review Publications**
                            *Patel, J.R., Yin, H., Bauchan, G.R., Mowery, J.D. 2020. Inhibition of Escherichia coli O157:H7 and Salmonella enterica virulence factors by benzyl isothiocyanate. Food Microbiology. https://doi.org/10.1016/j.fm.2019.103303.*
                                *Sharma, M., Handy, E.T., East, C.L., Kim, S., Jiang, C., Allard, S., Callahan, M.T., Micallef, S.A., Craighead, S., Anderson, B., Gartley, S., Vanore, A., Kniel, K., Haymaker, J.R., Duncan, R., Foust, D., White, C., Taabodi, M., Hashem, F., Parveen, S., May, E., Bui, A., Craddock, H., Kulkarni, P., Murray, R.T., Sapkota, A.R. 2020. Prevalence of Salmonella spp. and Listeria monocytogenes in non-traditional irrigation waters in the Mid-Atlantic United States is affected by water type, season, and recovery volume. PLoS One. 15(3):e0229365.*
                                *Bolten, S., Gu, G., Luo, Y., Van Haute, S., Zhou, B., Millner, P.D., Micallef, S.A., Nou, X. 2019. Salmonella inactivation and cross-contamination on cherry and grape tomatoes during washing in simulated commercial wash water. Food Microbiology. 87:103359. https://doi.org/10.1016/j.fm.2019.103359.*
                                *De Frias, A., Luo, Y., Zhou, B., Zhang, B., Ingram, D., Vorst, K., Brecht, J., Stommel, J.R. 2019. Effects of door opening pattern of an enclosed refrigerated display case on product temperature and energy consumption. Food Control. https://doi.org/10.1016/j.foodcont.2019.107044.*
                                *Gu, L., Chen, Q., Guo, A., Ruan, Y., Zhang, X., Nou, X. 2020. Differential effects of growth medium salinity on biofilm formation of Salmonella enterica svs. Enteritidis and Newport. Journal of Food Protection. 83(2):196-203. https://doi.org/10.4315/0362-028X.JFP-19-418.*
                                *Gu, G., Bolten, S., Mowery, J., Mowery, J.D., Luo, Y., Nou, X. 2020. Susceptibility of foodborne pathogens to sanitizers in produce rinse water and potential induction of viable but non-culturable state. Food Control. 112:107138.*
                                *Li, J., Teng, Z., Weng, S., Srinivasan, P., Zhou, B., Turner, E.R., Luo, Y. 2019. Dynamic changes in the physicochemical properties of fresh-cut produce wash water as impacted by commodity type and processing conditions. PLoS One. https://doi.org/10.1371/journal.pone.0222174.*
                                *Vorst, K., Brown, W., Steinmaus, S., Brecht, J.K., Xie, Y., Luo, Y., Bornhorst, E.R., Zhou, B., Shaw, A., Monge-Brenes, A. 2020. Temperature profiling of open- and closed-doored produce cases in retail grocery stores. Food Control. https://doi.org/10.1016/j.foodcont.2020.107158.*
                                *Pang, H., Mokhtari, A., Chen, Y., Oryang, D., Ingram, D.T., Sharma, M., Millner, P.D., Van Doren, J.M. 2020. A predictive model for survival of Escherichia coli O157:H7 and generic E. coli in soil amended with animal manure. Risk Analysis.  https://doi.org/10.1111/risa.13491.*
                                *Turner, E.R., Buchanan, R., Luo, Y. 2020. Microgreen production, nutrition, safety, and shelf life: A review. Journal of Food Science. 85(4):870-882. https://doi.org/10.1111/1750-3841.15049.*
                                *Van Haute, Sam, Luo, Y., Bolten, S., Gu, G., Nou, X., Millner, P.D. 2020. Survival of Salmonella enterica and shifts in microbial community as impacted by tomato wash water particulate size and chlorine treatment. Food Control. 90:103470. https://doi.org/10.1016/j.fm.2020.103470.*
                                *Yang, M., Cousineau, A., Liu, X., Sun, D., Li, S., Gu, T., Luo, S., Luo, Y., Xu, M., Zhang, B. 2020. Direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing on Nanopore MinION - promising strategies for multiplex identification of viable pathogens in food. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2020.00514.*


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