Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT 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. 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. This is the final report for the project 8042-32000-006-00D which terminated in March 2021. 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, data continue to be collected and analyzed examining the survival of E. coli in manure-amended soils in the Northeast and Mid- Atlantic United States. Heat-treated poultry litter pellets supported sustained persistence of E. coli compared to gradual decline that occurred in soil amended with compost. At 123 days post application of soil amendments, very low numbers of E. coli were recovered from baby spinach, but all radish samples regardless of compost or pellet amendment were positive for E. coli. Sediment samples collected from three different sites at Conococheague creek, Pennsylvania, were analyzed for indicator and pathogenic bacteria. E. coli populations recovered from sediments were affected by the season and were lower in winter compared to summer season. Salmonella, and E. coli O17:H7 were recovered from 4% of the sediment samples. The rapid methods for the detection of foodborne pathogens in irrigation water are being developed. Methods to recover antibiotic-resistant Salmonella enterica from surface waters are currently being evaluated for the environmental working group of the National Antimicrobial Resistance Monitoring and Surveillance (NARMS) program. Under objective 2, multi-species biofilm formation of E. coli O157:H7 was investigated in combination with promotor bacteria-Ralstonia insidiosa at various hydrodynamic shear stresses. The biofilms formed by E. coli O157:H7 varied with shear stress, type of equipment surfaces, and presence of promotor bacteria. The growth potential and kinetics of Listeria monocytogenes on 14 different whole and fresh-cut fruits and vegetables under standard industry practices and temperature abuse storage or retail display conditions was investigated. ARS scientists also assessed factors determining L. monocytogenes growth on fresh produce, including produce physiochemical characteristics and microbiome. Under objective 3, probiotic lactic acid bacteria (LAB) were used to control non-pathogenic surrogate bacteria on lettuce at the organic farm in Chambersburg, Pennsylvania. Field-grown lettuce cultivars -Green star and New Red Fire were sprayed with Listeria innocua or E. coli O157:H12 followed by LAB spray and then harvested periodically. The LAB significantly killed target surrogate bacteria. The antimicrobial effect of LAB was prominent on ⿿New Red Fire⿿ lettuce than on ⿿Green star⿿ lettuce cultivar leaves. Under objective 4, the efficacy of carvacrol nanoemulsion was evaluated as a washing treatment to reduce E. coli O157:H7 on fresh produce. E. coli O157:7 was significantly reduced on lettuce and spinach leaves following treatment with carvacrol emulsion and during storage at 10°C. The nanoemulsion did not affect the color of spinach or lettuce leaves after 14 days of storage. ARS scientists in collaboration with scientists at Howard University, D.C. and Volcani Institute, Israel evaluated the antimicrobial potential and mechanism of a novel sanitizer formula (gallic acid, hydrogen peroxide, and lactic acid) on E. coli O157:H7 and Listeria monocytogenes inactivation on baby spinach. While each component showed microbial reduction, a significant synergistic effect was found with the combination. The combination also accelerated E. coli O157:H7 die off on baby spinach during cold storage. Microarray based rapid method is being developed to detect Salmonella in fresh produce. The method detected all fresh produce samples spiked at 5 CFU per 25 g following enrichment for 3 hours and concentration by centrifugation or concentrator® pipette. ACCOMPLISHMENTS 01 Rainfall and soil amendment type affect pathogen survival and transfer to cucumbers in fields. Poultry litter and other biological soil amendments used as fertilizers in fresh produce production can introduce enteric pathogens to soil and contaminate fresh produce. ARS scientists evaluated E. coli survival duration in soils covered with plastic mulch or uncovered and containing poultry litter, heat-treated poultry litter pellets. Nitrate levels on day 30 and soil moisture content on day 40 were good predictors of E. coli survival in soils; however, knowledge of the combination of year, amendment, and mulch type was a better predictor of E. coli survival. Cumulative rainfall totals and pattern of rainfall events most likely affected the transfer of E. coli from soils to cucumbers and survival durations in soil. The results provide organic farmers specific knowledge about the potential effect of soil amendments on the microbial safety of fresh produce. 02 Irrigation water quality and leaf topography affect the persistence of non-pathogenic surrogate bacteria on lettuce. Lack of irrigation water due to drought, climate change, and increasing urbanization is a severe issue of agriculture production. Alternative water such as secondary- treated wastewater and roof-harvest rainwater may be used to overcome water scarcity while maintaining food security and food safety. ARS scientists irrigated lettuce grown at the farm (Chambersburg, Pennsylvania ) with these waters containing non-pathogenic E. coli O157:H12 or E. coli K12 bacteria. Lettuce samples were harvested at specific days post-irrigation and analyzed for populations of inoculated surrogate bacteria. Irrigation with roof-harvest rain water resulted in higher recovery of these surrogates on lettuce leaves. The difference in leaf characteristics of lettuce cultivars influenced the persistence of these surrogates on lettuce leaves. The findings provide small farmers alternative sources of irrigation water during water scarcity, and knowledge about how the leaf structure associated with cultivar influence the microbial safety of fresh produce. 03 Zero-valent iron filtration removes pathogens from irrigation water. Bacterial pathogens in irrigation waters are responsible for several outbreaks and recalls associated with vegetables. Small scale farmers require cost-effective mitigation such as zero-valent iron filtration to reduce levels of pathogenic bacteria in irrigation water intended for fruits and vegetables. ARS scientists collected pond water samples at the research farm, contaminated with non-pathogenic E. coli, and then filtered through either a sand or Zero-valent iron (ZVI) filter. Results showed that ZVI reduced more E. coli than sand filtration in water, and spinach plants irrigated with ZVI-filtered water had less E. coli than when irrigated with sand-filtered water. This work benefits farmers and growers by designing and evaluating cost-effective filtration strategies to reduce E. coli in irrigation waters.
Impacts
(N/A)
Publications
- Solaiman, S., Allard, S.M., Callahan, M., Jian, C., Handy, E.T., East, C.L. , Haymaker, J., Bui, A., Craddock, H., Murray, R., Kulkurni, P., Anderson- Coughlin, B., Craighead, S., Gartley, S., Vanore, A., Duncan, R., Foust, D. , Taabodi, M., Sapkota, A., May, E., Hashem, F., Parveen, S., Kniel, K., Sharma, M., Sapkota, A.R., Micalef, S.A. 2020. A longitudinal assessment of Escherichia coli, total coliforms, Enterococcus and Aeromonas spp. dynamics in alternative irrigation water sources: A CONSERVE study. Applied and Environmental Microbiology. https://doi.org/10.1128/AEM.00342- 20.
- Zhen, J., Luo, Y., Wang, D., Dinh, Q., Lin, S., Sharma, A., Block, E.M., Yang, M., Gu, T., Pearlstein, A.J., Yu, H., Zhang, B. 2021. Nondestructive multiplex detection of foodborne pathogens with background microflora and symbiosis using a paper chromogenic array and advanced neural network. Biosensors and Bioelectronics. 183:113209. https://doi.org/10.1016/j.bios. 2021.113209.
- Mei, L., Zhang, F., Zhang, J., Li, Y., Liu, Y., Luo, Y., Wang, Q. 2020. Alkynyl silver modified chitosan as a novel antimicrobial coating material for potential food applications. Carbohydrate Polymers. 254:117416. https:/ /doi.org/10.1016/j.carbpol.2020.117416.
- Teng, Z., Luo, Y., Zhou, B., Wang, Q., Hapeman, C.J. 2021. Characterization and mitigation of chemical oxygen demand and chlorine demand from fresh produce wash water. Food Control. 127:1008112. https:// doi.org/10.1016/j.foodcont.2021.108112.
- Brecht, J., Xie, Y., Abrahan, C., Bornhorst, E., Luo, Y., Monge-Brenes, A., Vorst, K., Brown, W. 2020. Improving temperature management and retaining quality of freshcut leafy greens by retrofitting open refrigerated retail display cases with doors. Journal of Food Engineering. https://doi.org/10. 1016/j.jfoodeng.2020.110271.
- Liu, X., Yang, M., Luo, Y., Wang, S., Zhou, B., Teng, Z., Dillow, H., Gu, T., Reed, K., Sharm, A., Jia, Z., Yu, H., Zhang, B. 2021. Machine learning- enabled non-destructive paper chromogenic array detection of multiplexed viable pathogens on food. Nature Food. 2:110-117. https://www.x-mol.com/ paperRedirect/1362513694001762304.
- Zhou, B., Luo, Y., Teng, Z., Millner, P.D., Pearlstein, A. 2020. A novel in-flight washing system on bacterial reduction and quality of fresh-cut lettuce. Food Control. https://doi.org/10.1016/j.foodcont.2020.107538.
- Zhou, B., Luo, Y., Teng, Z., Nou, X., Millner, P.D. 2022. Factors impacting water quality and microbiota during simulated dump tank wash of grape tomatoes. Journal of Food Protection. 84:695-703. https://doi.org/10. 4315/JFP-20-343.
- Kim, S., Bradshaw, R., Kulkarni, P., Allard, S., Chiu, P.C., Sapkota, A.R., Kniel, K.E., Newell, M.J., Handy, E.T., East, C.L., Sharma, M. 2020. Zero- valent iron-sand filtration reduces Escherichia coli in surface water and leafy green growing environments. Frontiers in Sustainable Food Systems. https://doi.org/10.3389/fsufs.2020.00112.
- Yin, H., Gupta, N., Chen, C., Pradhan, A., Patel, J.R., Boomer, A.M. 2020. Persistence o Escherichia coli O157:H12 and Escherichia coli K12 as non- pathogenic surrogates for O157 on lettuce cultivars irrigated with alternative waters in the field. Frontiers in Sustainable Food Systems. https://doi.org/10.3389/fsufs.2020.555459.
- Kumar, G., Patel, J.R., Ravishankar, S. 2020. Contamination of spinach at germination: A route to persistence and environmental reintroduction by Salmonella. International Journal of Food Microbiology. https://doi.org/10. 1016/j.ijfoodmicro.2020.108646.
- Thippareddi, H., Balamurugan, S., Patel, J.R., Manpreet, S., Brassard, J. 2020. Coronaviruses ⿿ potential human threat from foodborne transmission?. LWT - Food Science and Technology. https://doi.org/10.1016/j.lwt.2020. 110147.
- Yin, H., Boomer, A.M., Chen, C., Patel, J.R. 2020. Efficacy of benzyl isothiocyanate for controlling Salmonella on alfalfa seeds and sprouts. International Journal of Food Science and Technology. https://doi.org/10. 1111/ijfs.14520.
- Boomer, A., Hsin-Bai, Y., Patel, J.R. 2019. Antibiofilm efficacy of Peptide 108 against listeria monocytogenes and shiga toxigenic Escherichia coli on equipment surfaces. Journal of Food Protection. https://doi.org/10. 4315/0362-028X.JFP-19-168.
- Yin, H., Kumar, V.N., Macarisin, D., Patel, J.R. 2020. Biocontrol of Listeria on cantaloupes in the field with lactic acid bacteria. Journal of Food Protection. https://doi.org/10.1111/jfpp.14465.
- Yin, H., Chen, C., Kiaranth, S., Byun, S., Mayer, C., Harriger, D., Patel, J.R., Pradhan, A. 2020. Effect of cultivars and irrigation waters on persistence of indicator bacteria on lettuce grown in high tunnel. Journal of Food Safety. https://doi.org/10.1111/jfs.12795.
- Litt, P.K., Kelly, A., Omar, A., Johnson, G., Vinyard, B.T., Kniel, K.E., Sharma, M. 2021. Temporal and agricultural factors influence E. coli survival in soil and transfer to cucumbers. Applied and Environmental Microbiology. https://doi.org/10.1128/AEM.02418-20.
- Nong, W., Guan, W., Yin, Y., Lu, C., Wang, Q., Luo, Y., Zhang, B., Wu, J., Guan, Y. 2021. Photothermal metal-organic framework nano-generators for non-contact microorganism inactivation. Advanced Functional Materials. https://doi.org/10.1016/j.cej.2021.129874.
- Bolton, S., Gu, G., Gulbronson, C., Kramer, M.H., Luo, Y., Zografos, A., Nou, X. 2021. Evaluation of DNA barcode abiotic surrogate as a predictor for inactivation of E. coli O157:H7 during spinach washing. Journal of Food Science and Technology. https://doi.org/10.1016/j.lwt.2021.111321.
- Gu, G., Bolten, S., Mendes-Oliveira, G., Zhou, B., Teng, Z., Pearlstein, D. , Luo, Y., Millner, P.D., Nou, X. 2020. Salmonella inactivation and sponge/ microfiber mediated cross-contamination during papaya wash with chlorine or peracetic acid as sanitizer. Postharvest Biology and Technology. https:/ /doi.org/10.1016/j.fm.2020.103677.
- Wallis, A., Gu, G., Ramachandran, O., Reed, E., Ottesen, A., Nou, X., Cox, K. 2021. Endophytic bacterial communities in apple leaves are minimally impacted by streptomycin use for fire blight management. Phytobiomes Journal. https://doi.org/10.1094/PBIOMES-11-20-0081-R.
- Salazar-Llorente, E., Morales, M., Sornoza, I., Mariduena-Zavala, M., Gu, G., Nou, X., Ortiz, J., Maldonado-Alvarado, P., Manual Cevallos, J. 2020. Microbiological quality of high-demand foods from three major cities in Ecuador. International Journal of Food Microbiology. https://doi.org/10. 4315/JFP-20-271.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report 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. 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. 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 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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. 08 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.
Impacts
(N/A)
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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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. 08 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.
Impacts
(N/A)
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.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report 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. 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. Progress was made on all five 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, data were collected for a soil amendment field study conducted at 3 different sites with twelve different trials in the Mid- Atlantic region. Six different statistical models have been developed to analyze those data to determine the contributions of agricultural factors including manure type, management (organic vs conventional), depth (surface vs tilled) and spatio-temporal factors (weather, location) on E. coli survival in manure-amended soils. Data have been shared with FDA for risk assessment purposes. Data from a three-season soil amendment field study performed at University of Vermont is currently being analyzed. Declines of E. coli and Listeria spp. in manure-amended soils over 60 days were similar in the three seasons, but Listeria spp. survival profiles were different between season 2 and 3, indicating E. coli and Listeria may survive differently under the same conditions. Surveillance of bacterial pathogens in non-traditional irrigation waters collected from the Mid-Atlantic regions revealed very low prevalence of shiga- toxigenic E. coli in these waters. Under Objective 2, we have obtained a large collection of environmental bacterial isolates from fresh produce and the processing environments. Screening of these isolates obtained strains that enhanced or inhibited biofilm formation by foodborne pathogens including Listeria monocytogenes. These isolates have been identified to species level by 16S rDNA sequencing. Whole genome sequencing is being used to further characterize the interactions with foodborne pathogens. Previously, we reported that R. insidiosa could induce L. monocytogenes aggregation, which require direct cell-cell contact. Transcriptomic analyses showed differential transcription of several L. monocytogenes genes in co-cultures with R. insidiosa. Several of the genes that are most affected by co-culturing have previously been shown to be associated with L. monocytogenes biofilm formations. The significance of the differentially expressed genes in co- culture biofilm formation is being further corroborated by proteomic analyses. Under Objective 2, novel antimicrobial peptides were used to remove bacterial biofilms formed by Listeria monocytogenes and shiga- toxigenic E. coli on various equipment surfaces. The extent of removal of bacterial biofilm was dependent on the concentration of peptide, bacterial strain, and type of equipment surface. Up to 4.3 log CFU/cm2 reduction of these bacterial pathogens was observed after treatment with 10-50 microgram/ml peptide. Under Objective 3, we made significant progress on controlling Listeria on cantaloupes at the farm level. Lactic acid bacteria isolated from canine feces were used as a biocontrol to spray on cantaloupes previously contaminated with Listeria in the field. Listeria were killed by 1.5-2 log CFU/cm2 on cantaloupes harvested 5-7 days after biocontrol application. Under Objective 4, ARS researchers in Beltsville, Maryland, developed a new sanitizer in-line mixing system to control chlorine during fresh-cut produce wash operation. The control system will be especially useful for researchers to run experiments with different concentrations of sanitizers, and for food processors who require dosing and monitoring of free chlorine at higher concentrations with space constraints. The system will be very important in technological advancement of non-immersive fresh-cut wash processing which minimizes water-mediated bacterial transfer caused by reuse of water. Benzyl isothiocyanate, a compound commonly found in Brassica family vegetables was a highly effective antimicrobial against Salmonella and E. coli O157:H7. At very low concentration, it inhibited motility and shiga toxin synthesis, which are required for bacteria to colonize and cause human infections. A field study was conducted to determine microbial quality of organic spinach irrigated with secondary-treated wastewater and rainwater. The microbial quality of spinach was not affected by these non-traditional irrigation waters when these waters contained low bacterial populations. The persistence of coliform bacteria was influenced by the growing season; bacterial die-off rate was higher on winter-grown spinach. Accomplishments 01 Spinach microbiota shifts following chlorine wash and storage at compliant and abusive temperatures. The microbial communities on fresh produce and in the processing environments could have profound effects on the growth and persistence of foodborne pathogens. Spinach samples were taken from a commercial fresh-cut processing facility and the microbiota compared before and after commercial washing in chlorinated water. Microbiota were also compared after storage in compliant (4 degrees Celsius) and abusive (10 and 15 degrees Celsius) temperatures. These analyses provided information regarding the dynamics of microbial populations during fresh-cut produce processing and storage. The information on the changes of the microbiota composition during chlorine washing and on microbiota restoration during storage is useful for developing antimicrobial intervention strategies and understanding the consequences of noncompliance with processing and storage standards. 02 Microbiological quality of spinach irrigated with reclaimed waste water and roof-harvest water. Water scarcity is a serious issue and alternative water, such as reclaimed (reused) wastewater and roof- harvest water, may help overcome the scarcity of water while maintaining food security and food safety. Spinach grown in a controlled environment chamber was irrigated with alternative water for four weeks, and then spinach samples were collected weekly and analyzed for bacterial populations. A single irrigation with alternative water containing higher populations of total and fecal coliform bacteria did not necessarily result in higher populations of the coliform bacteria on spinach leaves; however, repeated irrigation with reclaimed wastewater resulted in higher numbers of E. coli positive spinach samples. Pathogens were not detected from any water or spinach samples under this investigation. Irrigation waters containing higher populations of total and fecal coliforms did not necessary result in higher populations of these bacteria on the spinach leaves. Repeated irrigation with reclaimed wastewater resulted in higher numbers of (nonpathogenic) E. coli positive spinach samples. Roof-harvest water had higher microbial quality than the reclaimed wastewater. Roof- harvest water irrigation did not increase the populations of fecal bacterial indicators on the irrigated spinach plants. The results show the potential use of roof-harvest water for irrigation of spinach without affecting the microbiological quality of the spinach. 03 The microbial quality of alternative irrigation waters. The availability of water for crop irrigation is decreasing due to droughts, population growth, and pollution. Implementation of the Food Safety and Modernization Act governing irrigation water standards discourages growers from using poor microbial quality water for produce crop irrigation. A method was evaluated to determine the microbial quality of wastewater, rainwater, and creek water in comparison to the membrane filtration method. No significant differences were observed concerning bacterial populations and pathogens. Recovery of fecal coliform bacteria in wastewater was lower than that found in filtered water samples. The study provides the microbial quality of non-traditional irrigation waters. 04 Proteomic of Staphylococcus aureus exposed to plant-derived antimicrobials. Consumers� preference for less chemicals in food has led researchers to explore natural antimicrobials to control foodborne bacteria. ARS scientists in Beltsville, Maryland, determined that punicalgins (plant derived antimicrobial substances) disrupted multiple bacterial cellular functions and inhibited bacterial growth of Staphylococcus aureus bacteria. This information is useful for understanding the functions of natural antimicrobials on potential foodborne pathogens. 05 Safe and effective water reuse. Reusing and reducing fresh-cut vegetable wash water is needed for sustained industry growth and a reduced environmental footprint. However, organic matter accumulated in reused wash water can lead to a loss of antimicrobial efficacy for chlorine disinfectant thus compromising the quality and safety of the washed products. ARS scientists in Beltsville, Maryland, identified that proteins and peptides are the major contributors to the loss of chlorine efficacy, and that sugars are important for developing effective wash water treatment and recycling programs. These findings will help vegetable processors develop safe, effective, and economical chlorine replenishment strategies and wash water reuse programs. 06 Essential oils control bacterial pathogens on fresh herbs. The demand for fresh herbs has increased in recent years due to health benefits and their distinct aroma in prepared food. Fresh herbs contaminated with shiga-toxigenic Escherichia coli and Salmonella bacterial species are associated with foodborne illnesses. Plant-based essential oils were evaluated on fresh herbs (basil, cilantro, dill, parsley, and tarragon) for their antimicrobial activities against Salmonella and E. coli. Treatments with specific concentrations of carvacrol or cinnamaldehyde killed E. coli and Salmonella on fresh herb leaves. There was no visual difference in herbs treated at lower concentrations of cinnamaldehyde or carvacrol. Results indicate use of novel, natural antimicrobials to kill E. coli and Salmonella without affecting the color attributes of fresh herbs. 07 Contamination of Listeria monocytogenes on cantaloupes at the packinghouse. Listeria monocytogenes is a bacterial pathogen which caused a large outbreak in 2011 was associated with contaminated cantaloupes. Research showed that packing house surfaces and equipment (nylon brushes, conveyor belts, foam pads) with cantaloupe juice on their surfaces supported increased survival of L. monocytogenes. Surfaces were also assessed for their ability to contaminate multiple cantaloupes. Foam surfaces contaminated more cantaloupes than conveyor belt surfaces (polyvinyl chloride, polyurethane, nitrile rubber). The study highlights the importance of plant sanitation and equipment design in controlling L. monocytogenes. 08 Rapid method to determine free chlorine levels in wash water during commercial-scale washing of fresh-cut produce. Substances released into the water during washing of the cut produce with the chlorine, sanitizer and reduce its efficacy. Maintenance of the level of free chlorine, a form that can interact with contaminants, is critical to avoid cross-contamination from bacteria during commercial-scale washing of fresh-cut produce. Controlling the sanitizer levels needed in fresh- cut produce wash water could improve if the chlorine demand is known in real-time. ARS scientists in Beltsville, Maryland, developed a rapid method using ultraviolet light absorbance to estimate chlorine demand for produce wash conditions. Ultraviolet light absorbance of the wash water was measured at two wavelengths. Based on these measurements, a predictive model for chlorine demand was developed and tested. The method shows promise for real-time application during commercial-scale washing of fresh-cut produce.
Impacts
(N/A)
Publications
- Luo, Y., Zhou, B., Van Haute, S., Nou, X., Zhang, B., Teng, Z., Turner, E. R., Wang, Q., Millner, P.D. 2017. Association between bacterial survival and free chlorine concentration during commercial fresh-cut produce wash operation. Food Microbiology. 70:120-128.
- Park, E., Luo, Y., Marine, S.C., Everts, K.A., Micallef, S.A., Bolten, S.J. , Stommel, J.R. 2018. Consumer preference and physicochemical evaluation of organically grown melon. Postharvest Biology and Technology. 141:77-85.
- Mei, L., Teng, Z., Zhu, G., Liu, Y., Zhang, F., Li, Y., Guan, Y., Luo, Y., Chen, X., Wang, Q. 2017. Advanced materials interfaces. ACS Applied Materials and Interfaces. 9(40):3529-3530.
- De Frias, A.J., Luo, Y., Zhou, B., Turner, E.R., Millner, P.D., Nou, X. 2018. Minimizing pathogen growth and quality deterioration of packaged leafy greens by maintaining optimum temperature in refrigerated display cases with doors. Food Control. 92:488-495.
- Hsin-Bai, Y., Patel, J.R. 2018. Comparison of methods to determine the microbial quality of alternative irrigation waters. Agricultural Water Management. 201:38-45.
- Hsin-Bai, Y., Nou, X., Patel, J.R., Gu, G. 2018. Microbiological quality of spinach irrigated with reclaimed wastewater and roof-harvest water. Journal of Applied Microbiology.
- Lu, Y., Dong, W., Yang, T., Luo, Y., Wang, Q., Chen, P. 2017. Effect of preharvest CaCl2 spray and postharvest UV-B radiation on storage quality of broccoli microgreens, a richer source of glucosinolates. Journal of Food Composition and Analysis. 67(1):55-62.
- Gu, G., Ottesen, A., Bolten, S.J., Ramachandran, P., Reed, E., Rideout, S., Luo, Y., Patel, J.R., Brown, E., Nou, X. 2018. Shifts in spinach microbial communities after chlorine washing and storage at compliant and abusive temperatures. Food Microbiology. 73:73-84.
- Teng, Z., Luo, Y., Alborzi, S., Zhou, B., Chen, L., Zhang, J., Zhang, B., Millner, P.D., Wang, Q. 2017. Investigation on chlorine-based sanitization under stabilized conditions in the presence of organic load. International Journal of Food Microbiology. 67:150-157.
- Zhou, B., Luo, Y., Bauchan, G.R., Feng, H., Stommel, J.R. 2017. Visualizing pathogen internalization pathways in fresh tomatoes using MicroCT and confocal laser scanning microscopy. Food Control. 85:276-282.
- Cooper, B., Isalm, N., Xu, Y., Beard, H.S., Garrett, W.M., Gu, G., Nou, X. 2018. Quantitative proteomic analyses of Staphylococcus aureus treated with punicalagin, a natural antibiotic from pomegranate that disrupts iron homeostasis and induces SOS. Proteomics. 18:1700461.
- Patel, J.R., Keelara, S., Green, J.A. 2018. Inactivation of Escherichia coli O157:H7 and Salmonella on fresh herbs by plant essential oils. Foodborne Pathogens and Disease. 15(6):1-7.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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�. 05 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.
Impacts
(N/A)
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.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report 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. 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. The persistence of E. coli and Listeria spp. in manure-amended soils under different management conditions was evaluated to determine survival mechanisms of foodborne pathogens (objective 1). E. coli and Listeria spp. survived for > 230 days in the Northeast, which was a longer duration than E. coli survival in the first season of this study (< 180 days). This work is concurrently evaluating Listeria spp. survival in manure- amended soils, and survival durations are similar to those of E. coli in the Northeast U.S. The methods for detecting non-O157 enterohemorrhagic E. coli (EHEC) from manure-amended soils and irrigation water were optimized to improve sensitivity and specificity of pathogen recovery (objective 1). Several different selective media and molecular methods were screened in the study. Two multiplex and one 11-plex PCR methods were selected as being efficient to detect the 'Big Six' serotypes of E. coli. Real-time multiplex PCR methods developed by an ARS laboratory (Clay Center) were identified and adapted to simultaneously screen manure-amended soils for Salmonella spp. and Listeria monocytogenes. Persistence of E. coli O157:H7 and Listeria monocytogenes on basil and cilantro grown using alternative farming practices (Bro-gro system) was evaluated to determine microbial safety of alternatively grown fresh produce (objective 5). Population of these pathogens declined on basil and cilantro leaves during 14-days; however, they were still detectable by a sensitive detection procedure. In general, L. monocytogenes were recovered at higher levels than E. coli O157:H7 on cilantro and basil leaves. There was no significant difference in persistence of E. coli O157:H7 and L. monocytogenes on conventionally and alternatively grown herb leaves. Conventional and organic fresh produce (n=142) purchased from local retailers were analyzed for presence of Salmonella, L. monocytogenes, and E. coli O157:H7 to assess their microbial safety (objective 5). Organic green chard was contaminated with E. coli O157:H7 and Salmonella where as red cabbage (n=2) were contaminated with E. coli O157:H7 and L. monocytogenes. There was no correlation between coliform populations and pathogen persistence on fresh produce. Total bacterial populations and coliform varied significantly with type of fresh produce. Role of biofilm formation on potential cross-contamination of fresh produce via equipment surfaces was determined (objective 2). Biofilm formation of non-O157 Enterohemorrhagic E. coli varied with material surface and strain. Most strains were recovered at significantly higher levels on PTFE and polycarbonate surfaces compared to populations recovered on stainless steel surfaces. E. coli O26 strain 3629 recovered from Stainless steel, polycarbonate and PTFE (7.06 - 7.44, log CFU/cm2) were significantly higher than E. coli O145 strain 3419 recovered from corresponding surfaces. In general, curli-expressing strains formed stronger biofilms on material surfaces. Challenges in maintaining adequate free chlorine levels in commercial produce wash systems were addressed by developing new technology (objective 4). Chlorine-dosing technology was developed to control chlorine in which chlorine is added according to an algorithm that predicts when the level would otherwise fall below the target. This technology will help in maintaining target chlorine levels throughout wash operations, and thus improving the safety of fresh-cut produce. Consumer awareness of fresh herbs and demand has increased in recent years due to health benefits and distinct aroma in prepared food. Natural antimicrobials can be used as a fresh produce was to replace inefficient chlorine wash (objective 4). Plant-based essential oils were evaluated on fresh herbs (Basil, Parsley, Tarragon, Cilantro, and Dill) for their antimicrobial properties against Salmonella and E. coli O157:H7. A treatment with 0.3% carvacrol and 0.5% cinnamaldehyde reduced these pathogens by 4 log CFU/g (P > 0.05%). E. coli O157:H7 and Salmonella populations were reduced further during storage of treated herbs. There were no color differences in herbs treated with cinnamaldehyde or 0.1% carvacrol from control samples. Understanding produce topography and plant-bacteria interaction on produce surfaces is critical in developing effective sanitizing treatments to remove pathogens (objective 4). Biomimetic plant surfaces that achieve high fidelity to the topography and microstructure of spinach leaf and cantaloupe rind surfaces were fabricated using either agar or polydimethylsiloxane. It facilitated reproducibility of experiments involving disinfection and attachment/release of microbes from surfaces having microstructure identical to real produce surfaces, but without any of the leaf-to-leaf or plant-to-plant variation. The technology will be of great interest in food safety related research aimed at understanding bacteria-host interaction and developing prospective control measures. Accomplishments 01 Poultry litter-amended soils enhance survival of E. coli. Role of contaminated manure on fresh produce contamination at farm level requires investigation to develop effective intervention strategies. ARS scientists at Beltsville, Maryland, investigated persistence of non- pathogenic surrogates in soils amended with different biological soil amendments (manure and compost) in greenhouses. Regardless of pot size evaluated, soils amended with poultry litter supported the survival of E. coli at higher populations than unamended soils or soils amended with horse manure or dairy manure. This work showed that greenhouse studies can be employed to collect practical data useful to FDA and those growers seeking variances and exceptions from FDA rules. 02 Turkey manure dust particles extend Salmonella persistence on spinach. Investigation on airborne contamination of bacterial pathogen on fresh produce is required to establish microbial safety criteria at the farm level. The survival of Salmonella in turkey manure dust (TMD) alone and in manure dust on spinach plants was assessed. Results showed TMD protected Salmonella on spinach leaves from inactivation from ultraviolet light more than water, allowing Salmonella to survive for longer periods of time. Salmonella also survived for longer durations (291 days) in manure dust, with the greatest survival in dust with the smallest particle size and the lowest moisture content. This work is helpful to growers in selecting buffer zones between farm and animal rearing facilities to minimize airborne contamination of Salmonella to proximate leafy green farms. 03 Current metrics used by the California Leafy Greens Marketing Agreements (LGMA) requires reevaluation. Scientific validation is required to evaluate previously developed LGMA criteria for leafy greens and to ascertain microbial safety of fresh produce. ARS scientists at Beltsville, Maryland examined the 60-day interval between flooding of field and replanting of crops, and the 9 m (30 ft) �no harvest� zone from the edge of the flood that LGMA currently employ to prevent fecal contamination introduced to crops through flooding. In our intentionally flooded spinach field with a -5% slope, E. coli populations declined more slowly in fall trials than in spring trials, and E. coli in soils and on spinach plants were detected 9 m away from the edge of the flood. These results suggest that LGMA metrics should be revised to include considerations of field and weather conditions that may promote bacterial movement and survival. 04 Natural antimicrobials reduce Salmonella in biofilms. Bacteria in biofilms survive commonly used sanitizers and subsequently contaminate fresh produce at processing facilities. Salmonella isolates from conventional swine farm formed biofilm on Minimum Biofilm Eradication Concentration (MBEC) assay. Cinnamaldehyde and sporan at 1000 ppm significantly reduced Salmonella in biofilms. The bactericidal effect of these antimicrobials increased with their concentrations. Salmonella populations were reduced by 1 million from their initial populations of 10 million/cm2 when 2000 ppm conc. of these antimicrobials were used. Salmonella were undetectable when 3000 ppm of cinnamaldehyde or sporan was used. Organic fresh produce processors can use natural sanitizers to remove biofilm from equipment surfaces to minimize foodborne illnesses associated with consumption of fresh produce . 05 Novel antimicrobial controls enteric pathogens on fresh produce. With increased number of foodborne illnesses due to cantaloupe consumption, there is a need for effective antimicrobial wash to kill bacteria on cantaloupe. ARS scientists at Beltsville, Maryland, in collaboration with scientists at University of Connecticut, investigated the efficacy of a new disinfectant, octenidine dihydrochloride (OH) for killing bacteria such as Listeria monocytogenes, Salmonella spp., and Escherichia coli O157:H7 on cantaloupe surface. Cantaloupe rind plugs inoculated with theses pathogens were washed with OH or coated with chitosan containing OH. All OH wash treatments and OH coating significantly reduced L. monocytogenes, Salmonella spp. and E. coli O157:H7 on cantaloupe. The findings will help cantaloupe producers in exploring novel antimicrobials for cantaloupe to minimize potential human illnesses.
Impacts
(N/A)
Publications
- Rada, Z., Tood-Searle, J., Friedman, M., Patel, J.R., Jaroni, D., Ravishankar, S. 2016. Combining essential oils and olive extract for control of multi-drug resistant Salmonella enterica on organic leafy greens. Journal of Food Safety. 1(2):1-9.
- Whyte, C., Graham, L.P., Cotton, C.P., Hashem, F., Camp, M.J., Millner, P. D., Sharma, M. 2016. Survival and persistence of non-pathogenic Escherichia coli and attenuated Escherichia coli O157:H7 in soils amended with animal manure in a greenhouse environment. Journal of Food Protection. 79(6):913-921.
- Upadhyay, A., Chen, C., Yin, H., Upadhyay, I., Fancher, S., Liu, Y., Nair, M., Jankelunas, L., Patel, J.R., Venkitanarayanan, K. 2016. Inactivation of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 on cantaloupes by octenidine hydrochloride. Food Microbiology. 58(9):121-127.
- Wang, S., Luo, Y., Li, J., Zhou, B., Jacangelo, J., Schwab, K.J. 2015. Assessment and speciation of chlorine demand in fresh-cut produce wash water. Food Control. 60:543-551.
- Xiao, Z., Codling, E.E., Luo, Y., Nou, X., Lester, G.E., Wang, Q. 2016. Microgreens of brassicaceae: mineral composition and content of 30 varieties. Journal of Food Composition and Analysis. 49:87-93.
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