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

Location: Environmental Microbial & Food Safety Laboratory

2021 Annual Report

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

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

Progress Report
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
1. 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.

2. 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.

3. 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.

Review 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.