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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

2019 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 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 Salmonella enterica and Listeria monocytogenes in irrigation water sources (rivers, creeks, ponds, reclaimed water) in the Mid-Atlantic region were collected. Further analysis of data will help regulators in decision-making if non-microbiological indicators of water quality can be associated with pathogen prevalence in irrigation water. Data collected previously on the survival of E. coli in manure-amended soils in the Northeast United States are being analyzed to help growers in the Northeast U.S. determine if their manure application for fruit and vegetable production complies with proposed Food Safety Modernization Act (FSMA) rules. Under Objective 2, bacterial species that strongly affected the growth and biofilm formation by Listeria monocytogenes were isolated from fresh produce processing facilities. One strain (Brevundimonas naejangsanensis spp.) greatly enhanced the growth rate and biofilm formation of Listeria monocytogenes. Further characterization of the inter-species interactions could shed lights on the ability of L. monocytogenes to persist in food processing environment. Other strain (Bacillus amyloliquefaciens spp.) strongly inhibited the growth and biofilm formation of L. monocytogenes. The potential of this strain as a L. monocytogenes antagonist in food processing environment is being explored. Under Objective 3, Benzyl isothiocyanate (BIT), a compound present in cruciferous vegetables such as broccoli, significantly killed Salmonella on alfalfa seeds following treatment with 15 min. -The germination rate of alfalfa seeds treated with BIT was not different from untreated seeds. Under Objective 4, ARS researchers invented and patented a novel system that washes produce vertically (all existing systems wash produce horizontally). The system is effective in removal of organic matter from cut produce with improved process control and wash efficacy. "Single-pass" washing, in which water contacts the produce once and is not recirculated, is a recent industry development designed to reduce pathogen cross-contamination risk. The ARS team collaborated with an industry partner to compare produce quality and shelf life, and water and chemical usage between single-pass and flume systems. Results were shared with the industry to aid in developing and optimizing new washing processes. Lactic acid bacteria isolated from canine feces were used as a biocontrol to spray on strawberries previously contaminated with Salmonella or Listeria monocytogenes. Populations of these pathogens were reduced by 3-4 log CFU/g on strawberries after 7 days of storage at 4 or 10°C. Nano-emulsion of carvacrol, an essential oil reduced 3-5 log CFU/cm2 of E. coli O157:H7 on spinach and lettuce stored for 14 days at 4°C. Similarly, fruit extracts of lemon, yuzu and grape reduced Salmonella on cucumbers stored at refrigerated or room temperatures for 7 days.

4. Accomplishments
1. Sanitizer concentration is critical during produce wash to reduce food-safety risk. Improper washing can spread bacteria and increase health-risk associated with consumption of fresh produce. ARS researchers at Beltsville, Maryland, identified key operating conditions (including sanitizer concentration) affecting bacterial survival and transference during washing. An industry and a multi-agency taskforce used our findings to develop "Guidelines to Validate Control of Cross-Contamination during Washing of Fresh-Cut Leafy Vegetables". Our findings were also cited by the FDA as a scientific basis for the newly released “Draft Guidance for Industry: Guide to Minimize Food Safety Hazards of Fresh-cut Produce” to implement science- and risk-based food safety policies in support of Food Safety Modernization Act (FSMA). This research will help food processors in controlling bacteria that cause human illnesses due to consumption of contaminated fresh produce.

2. Standard operating procedures are required to control contamination on tomatoes during wash process. Tomato contamination with pathogens such as Salmonella can lead to costly food-borne illness outbreaks. ARS researchers at Beltsville, Maryland, in collaboration with Florida tomato growers and packers identified and quantified key operational parameters to prevent Salmonella survival and cross-contamination during tomato dump tank wash process. The findings were used by the industry to develop “Commodity Specific Food Safety Guidelines for the Fresh Tomato Supply Chain,” a critical food safety standard. The information will be helpful to processors in controlling disease-causing bacteria on tomato and subsequent human illnesses.

3. Location, season, and manure type affect survival of pathogens in manure-amended soils. The Produce Safety Rule of the Food Safety Modernization Act (FSMA) states that untreated manure must be applied 90 or 120 days prior to the harvest of edible produce crops to minimize contamination from pathogens potentially present in untreated manure. However, this interval was not scientifically validated. Over twelve separate field trials conducted in the mid-Atlantic U.S. over four years, ARS researchers at Beltsville, Maryland, in collaboration with university scientists showed that spatiotemporal factors (site, year, and season) affect survival durations of E. coli in manure-amended soils more than agricultural factors (manure type, organic or conventional management of soils, and depth of application) or weather effects. The results provide critical information to growers on potential risk of produce contamination with specific raw animal manure application. The Food and Drug Administration (FDA) will use these data to develop food safety standards for controlling bacterial contamination of fresh produce from soil.

4. Zero-valent iron filtration improves microbial and chemical irrigation water quality. Shiga-toxigenic E. coli (STEC) in irrigation water was responsible for several outbreaks associated with Romaine lettuce in 2018, highlighting the need for mitigation strategies to improve microbial water quality. ARS researchers at Beltsville, Maryland, designed and optimized a zero-valent iron (ZVI) filtration system which can be used by small farmers in their irrigation water systems. Results showed that different ZVI systems consistently reduced levels of antibiotics and bacterial pathogens (E. coli, Listeria monocytogenes) in surface and reclaimed wastewater compared to sand filtration. ZVI is a promising mitigation treatment for small farmers to reduce chemical and microbial contaminants in irrigation water.

5. Organic fertilizers can affect survival durations of bacterial pathogens in soils and on leafy greens. Bacterial pathogens like Salmonella spp. can be introduced to produce-growing environments through contaminated irrigation water, animal intrusions, or soil/ manure runoff. ARS researchers at Beltsville, Maryland, showed that Salmonella Newport can grow to high populations in soil runoff containing heat-treated poultry pellets (HTTP), a commonly used organic fertilizer in vegetable production. Soils amended with HTTP also supported longer survival durations of Salmonella Newport than unamended soils, and promoted more transfer of the pathogen from soils to leaves of spinach plants. These findings provide farmers with an improved understanding of specific factors that affect and promote pathogen survival in pre-harvest produce growing environments.

6. Shiga-toxigenic E. coli is present in potential irrigation water sources in the U.S. Contamination of irrigation water with Shiga-toxigenic Escherichia coli (STEC) was responsible for two outbreaks associated with leafy greens grown in California and Arizona in 2018. Little is known about the prevalence of STEC in other produce-growing regions of the U.S. ARS researchers at Beltsville, Maryland, in collaboration with university scientists analyzed more than 500 samples of surface non-tidal water, tidal brackish water, recycled water, and other potential irrigation water sources in the Mid-Atlantic U.S from twelve different sites. Approx. 2.4% (12/510) of samples were positive for STEC, compared to almost 11% of water samples from California were positive for STEC. Growers can use these results to use intervention strategies for controlling disease-causing bacteria in irrigation water.

7. Microbial ecology in food processing environments. Understanding microbial ecology of environmental surfaces is critical to improve sanitation practices in food processing. ARS researchers at Beltsville, Maryland, investigated the microbiome in a commercial fresh-cut produce processing facility. The identities and relative abundance of bacterial species on a variety of environmental surfaces were determined before and after routine sanitation. The study identified a core residential microbiota (a collection of bacterial species) abundantly presenting in the processing facility. The results benefit the research community for better understanding the microbial ecology in food processing environment, and fresh produce processors for knowing the microbiome in the processing facilities.

8. Microbiome changes on spinach irrigated with non-conventional water. Changes in microbial community of fresh produce irrigated with non-conventional water and its influence on pathogen persistence is unknown. ARS researchers at Beltsville, Maryland, compared the microbiomes in three different types of irrigation water, including ground water, reclaimed waste water, and roof-harvest rain water; and investigated the microbiome change on spinach in field before and after irrigation. The results indicated that the most abundant bacterial species on spinach retained dominant presence after irrigation with different types of irrigation water, while irrigation resulted in a transient increase of multiple bacterial species on spinach, including species of potential opportunistic pathogens. This study provided information to growers on the potential risks using non-conventional irrigation water on bacterial persistence on spinach.

Review Publications
Gu, G., Yin, H., Ottesen, A., Bolten, S., Patel, J.R., Rideout, S., Nou, X. 2019. Microbiota in ground water and alternative irrigation water, and spinach microbiota impacted by irrigation with different type of water. Phytobiomes Journal. .
Shah, M.K., Bradshaw, R.N., Nyarko, E., Millner, P.D., Neher, D., Weicht, T., Bergholz, T.M., Sharma, M. 2019. Survival and growth of wild-type and rpoS-deficient Salmonella Newport strains in soil extracts amended with heat-treated poultry pellets. Journal of Food Protection.
Kulkarni, P., Raspanti, G., Bui, A., Bradshaw, R., Kniel, K., Chiu, P., Sharma, M., Sapkota, A., Sapkota, A.R. 2019. Zerovalent iron-sand filtration can reduce the concentration of multiple antimicrobials in conventionally treated reclaimed wastewater. Environmental Research.
Gu, G., Ottesen, A., Bolten, S., Wang, L., Luo, Y., Rideout, S., Lyu, S., Nou, X. 2019. Impact of routine sanitation on Zone 3 microbiome in a fresh-cut produce processing facility. International Journal of Food Microbiology. 294:31-41. .
Sharma, M., Millner, P.D., Hashem, F., Vinyard, B.T., East, C.L., Handy, E.T., White, K.E., Stonebraker, R., Cotton, C.P. 2019. Survival of Escherichia coli in manure-amended soils is affected by spatiotemporal, agricultural, and weather factors in the Mid-Atlantic United States. Applied and Environmental Microbiology. 85:e02392-18.
Bornhorst, E.R., Luo, Y., Park, E., Vinyard, B.T., Nou, X., Zhou, B., Turner, E.R., Millner, P.D. 2018. Immersion-free, single-pass, commercial fresh-cut produce washing system: an alternative to traditional flume processing. Postharvest Biology and Technology. 146:124-133.
Guan, Y., Teng, Z., Mei, L., Zhang, J., Wang, Q., Luo, Y. 2018. An entrapped metal-organic framework system for controlled release of ethylene. Journal of Colloid and Interface Science. 533:207-215.
Guan, Y., Luo, Y., Teng, Z., Zhou, B., Mei, L., Bauchan, G.R., Wang, Q. 2018. A novel sensing chip for probing chlorine permeation into simulated produce cracks. Advanced Materials Interfaces. 5(13):119-130.
Shah, M.K., Bradshaw, R., Nyarko, E., Handy, E.T., East, C.L., Millner, P.D., Bergholz, T.M., Sharma, M. 2019. Salmonella Newport in soils amended with heat-treated poultry pellets survived longer and more readily transferred to and persisted on spinach. Applied and Environmental Microbiology. 85:e02392-18.
Haymaker, J., Sharma, M., Parveen, S., Hashem, F., May, E.B., Handy, E.T., White, C., East, C.L., Bradshaw, R., Micallef, S., Callahan, M., Allard, S., Anderson, B., Craighead, S., Gartley, S., Vanore, A., Kniel, K.E., Solaiman, S., Bui, A., Murray, R.T., Craddock, H.A., Kulkarni, P., Foust, D., Duncan, R., Taabodi, M., Sapkota, A.R. 2019. Prevalence of shiga-toxigenic and atypical enteropathogenic Escherichia coli in untreated surface water and reclaimed water in the Mid-Atlantic U.S. Environmental Research. 172:630-636.
Panthi, S., Sapkota, A.R., Raspannti, G., Allard, S., Bui, A., Craddock, H., Murrary, R., Zhu, L., East, C.L., Handy, E.T., Callahan, M., Haymaker, J., Kulkarni, P., Anderson, B., Craighead, S., Gartley, S., Vanore, A., Betancourt, W.Q., Duncan, R., Foust, D., Sharma, M., Micallef, S.A., Gerba, C., Parveen, S., Hashem, F., May, E., Kalmia, K., Pop, M., Ravishankar, S., Sapkota, A. 2019. Herbicides, antibiotics, stimulants, and disinfectants in agricultural water sources. Environmental Research. 174:1-8.
Marik, C.M., Anderson, B., Gartley, S., Craighead, S., Bradshaw, R., Kulkarni, P., Sharma, M., Kniel, K.E. 2019. The efficacy of zero valent iron filtration on the reduction of Escherichia coli and Listeria monocytogenes in surface water for use in irrigation. Environmental Research. 173:33-39.
Teng, Z., Van Haute, S., Zhou, B., Hapeman, C.J., Millner, P.D., Wamg, Q., Luo, Y. 2018. Impacts and interactions of organic compounds with chlorine sanitizer in recirculated and reused produce processing water. PLoS One. 13(12):1-15.
Xiao, Z., Rausch, S.R., Luo, Y., Sun, J., Yu, L., Wang, Q., Chen, P., Yu, L., Stommel, J.R. 2018. Microgreens of Brassicaceae: Genetic diversity of phytochemical concentrations and antioxidant capacities. LWT - Food Science and Technology. 101:731-737.
Zhang, B., Luo, Y., Kanyuck, K., Saenz, N., Zavalij, P., Mowery, J.D., Bauchan, G.R. 2018. Facile and template-free solvothermal synthesis of mesoporous/macroporous metal-organic framework nanosheets. RSC Advances. 8(58):33059-33064.