Research Project: Intervention Strategies to Mitigate the Food Safety Risks Associated with the Fresh Produce Supply Chain

Location: Environmental Microbial & Food Safety Laboratory

2023 Annual Report

Objectives
Objective 1: Evaluate the microbial safety of alternative fresh produce production systems and the efficacy of preventive practices. Sub-objective 1.a: Investigate sources of pathogen contamination on fresh produce grown by aquaponics (APs). Sub-objective 1.b: Determine the relationship of AP design and operational practices on microbiome characteristics and efficacy of water and probiotic treatments in reducing pathogen risks on produce. Sub-objective 1.c: Develop mitigation strategies for controlling pathogens in microgreen and sprouts. Objective 2: Develop and evaluate novel mitigation strategies to control foodborne pathogens at pre- and post-harvest levels. Sub-objective 2.a. Develop pre-harvest interventions to control pathogens on leafy greens. Sub-objective 2.b. Develop intervention strategies to control pathogens on fresh-cut leafy greens while maintaining quality and shelf life. Sub-objective 2.c. Optimize fresh-cut process to reduce pathogen presence on tomatoes. Objective 3: Characterize pathogen interactions with multi-species biofilms and efficacy of interventions on abiotic surfaces. Sub-objective 3.a. Investigate single and multi-species biofilm formation by foodborne bacterial pathogens in fresh produce processing environments. Sub-objective 3.b. Develop mitigation strategies to control biofilm formation in fresh produce processing environment.

Approach
Sources of pathogen contamination of fresh produce grown using aquaponics production (AP) system will be investigated. The aquaponics system inputs (seeds, transplant media, water, fish, feed) will be analyzed for fecal coliforms, spoilage bacteria, and foodborne pathogens (Shiga-toxigenic E.coli, Salmonella spp., Listera monocytogenes). Fresh produce (basil, cilantro, brassica leafy greens, pepper, pak choi) grown by AP system will be evaluated for spoilange and pathogenic bacteria, and produce yield. The effect of probiotics in controlling surrogate pathogens (avirulent E. coli, L. innocua, Salmonella spp.) on fresh produce seeds/seedlings and APs water will be determined. Nanobubble, UV light, and plasma functionalized water will be evaluated for seed germination. plant growth promotion, and system disinfection. Changes in fish and plant microbiome due to antimicrobial treatments will be evaluated. Sprout seeds (alfalfa, broccoli, clover, mung bean, radish) treated with glucosonilate compounds will be evaluated for rate of seed germination, inhibition of foodborne pathogens, and potential transfer of inoculated pathogens to sprouts. Seeds of microgreens contaminated with pathogens will be grown in different growing media to determine the effect of growth media on pathogen survival, and yield and quality of microgreens. Biocontrols and plant-based nanoemulsions will be investigated as pre-harvest interventions to control pathogen surrogate E. coli O157:H12 at the farm level. The effect of size distribution of particulates generated during commercial fresh-cut vegetables (baby spinach, diced cabbage, shredded lettuce) operation on wash water turbidity and subsequent pathogen cross-contamination will be investigated. Te effect of wash-cut sequence on pathogen cross-contamination on tomatoes will be evaluated and fresh-cut processing of tomatoes will be optimized to minimize pathogen contamination. Multi-species biofilm formation of L. monocytogenes and E. coli O157:H7 with promotor strain Ralstonia insidiosa will be assessed on different equipment surfaces at variable flow rates. Biofilm studies will be simulated in pilot plant to identify potential hotspots of biofilm formation. Novel antimicrobials such as plant-based nanoemulsion, antagonistic bacteria; and new technologies (dry steam, nonflammable ethanol, surface coating) will be investigated for biofilm removal in laboratory and pilot plant studies, respectively.

Progress Report
Progress was made on all objectives and their sub-objectives, which fall under National Program 108, Component 1, Foodborne Contaminants. Activities of this project focus on Problem 1, Population Systems, and Problem 5, Intervention and Control Strategies. Under Objective 1, studies were conducted on aquaponic production of leafy greens (lettuce, basil) to evaluate survival of inoculated surrogate bacteria Listeria innocua and Escherichia coli and to identify physicochemical parameters associated with bacterial persistence in aquaponics systems for up to 180 days after primary plant harvest. Listeria and E. coli populations declined significantly within 24 h post-inoculation and were undetectable by day 14. At harvest, Listeria and E. coli were recovered from lettuce roots and rockwool, but not from plant leaves or water samples. No E. coli was detected in fish feces or in lettuce crown tissue at day 180 after primary plant harvest. Foodborne pathogen survival on microgreens and growth media was investigated. Salmonella persistence on microgreens varied with growth media and source of contamination. Pathogen populations recovered from microgreens grown in compost-amended soil were significantly lower than those recovered when grown in environmentally sustainable growth media. Salmonella and E. coli O157:H7 were detected on microgreens when contaminated seed were used. There was a die-off of these pathogens; recovery was lower at the harvest (14 days) compared to 7 days. Further, pathogen persistence varied with among broccoli, mustard, red cabbage, and daikon microgreens used in the study. Under Objective 2, new oil-in-water benzyl isothiocyanate (BIT) nanoemulsion was formulated. The droplet size of this nanoemulsion remained stable for 56 days when stored at 4°C. The BIT nanoemulsion exerted significantly higher antimicrobial activity against E. coli O157:H7 strains resulting in inhibition zones of 13-18 mm dia. BIT nanoemulsion stored at 4°C remained effective against EHEC and up to 15 mm of inhibition zones were measured on day 56. In collaboration with commercial romaine producers/processors, research characterizing the forward processing practices (shipping raw commodity from production area in California and Arizona to distant facilities) and potential risks on the survival of Enterohemorrhagic E. coli as well as its infectivity was conducted. Samples of romaine lettuce collected in Salinas, California, (origin) and subjected to forward and source processing in Maryland and in California were analyzed for microbial load, microbiome shift, and product quality. Key operational parameters during packing, transport, and processing were determined. Under Objective 3, E. coli O157:H7 and L. monocytogenes biofilm removal from different equipment surfaces was studied using sanitizers. The extent of biofilm growth on equipment surfaces was influenced by the surface’s topographical features, hydrodynamic shear stress, and bacterial species. These pathogens in multispecies biofilms survived at higher rate following chlorine treatment of 1 or 4 min. In general, pathogen removal from biofilm on rubber surfaces was lower compared to stainless steel surfaces. The biofilm formation of Enterohemorrhagic E. coli isolates from a series of outbreaks implicating romaine lettuce as the source was investigated. Preliminary data suggested that these outbreak strains had stronger potential for biofilm formation. Genomic comparison with closely related reference strains is being conducted. In collaboration with researchers at University of Maryland, College Park, Maryland, transcriptomic characterization under the conditions of forward processing is being conducted.

Accomplishments
1. Biocontrol to minimize pathogen contamination on leafy greens at farm. Foodborne illnesses linked to contaminated leafy greens underline the need for effective natural approaches to improve produce safety at farm level. ARS scientists in Beltsville, Maryland, used lactic acid bacteria (LAB) as a biocontrol spray to control pathogens on lettuce and spinach at farms prior to harvest. LAB treatment significantly reduced non-pathogenic surrogate bacteria Listeria innocua and E. coli O157:H12 on lettuce and spinach at the farm level. The bactericidal effect of LAB varied with spinach cultivars. These findings will benefit growers in controlling pathogens on leafy greens at farm level prior to harvest for processing and packing.

2. Machine learning for foodborne Salmonella outbreaks prediction. Correlation between Salmonella outbreaks and weather trends has been documented; however, the relationship with genomic characteristics is not identified. In collaboration with University of Maryland researchers, ARS scientists in Beltsville, Maryland, used machine learning and count-based modeling methods to analyze the effect of differential gene expression and meteorological factors on Salmonella outbreaks. The final multi-variable Poisson regression model identified 127 significant predictor terms comprising 45 gene-only predictors, average temperature, average precipitation, and snow cover, and 79 gene-meteorological interaction terms. Regulatory agencies can use the findings to develop a more holistic model to predict disease outcome severity, which could extend to re-evaluating the risk to human health.

3. Microarray-based system for rapid pathogen detection in fresh produce. Rapid detection of bacterial pathogens in fresh produce is required before it reaches to retail market or consumers to minimize illnesses. ARS scientists in Beltsville, Maryland, in collaboration with industry partner, optimized microarray based bacterial detection method to detect low contamination of Salmonella (~6 CFU/25 g) in fresh produce. After 6 h of enrichment, low level Salmonella artificial contamination on 100%, 98%, 90%, and 82% of Romaine lettuce, Iceberg lettuce, kale, and spinach samples were successfully detected. The overall analysis time of this methodology was between 8-11 h, in contrast to 2-5 days required for FDA standard method. This novel method will be helpful to processors for rapid Salmonella detection in fresh produce.

4. New antimicrobial sanitizer for disinfection of fresh produce. Baby spinach is nutrient-rich and in high demand by consumers owing to its convenience and freshness. However, contamination with foodborne human pathogens has led to public health concerns. ARS scientists in Beltsville evaluated a novel formulation of antimicrobial solution consisting of only food grade materials for pathogen reduction. The combination of gallic acid, hydrogen peroxide, and lactic acid significantly reduced the survival of pathogenic E. coli O157:H7, Listeria monocytogenes, and Salmonella spp. on spinach leaves while maintaining quality and shelf life of the treated produce. This finding can be the much-needed alternative to chlorine as sanitizer for fresh produce washing and disinfection operations especially for organic produce when the application of chlorine is restricted.

5. Evaluation of a new washing formulation for diced tomato. Antimicrobial washing of diced tomatoes is challenging due to the large amount of juices released at dicing. ARS scientists in Beltsville, Maryland, evaluated the impact of different sanitizers, including free chlorine, peracetic acid, and a new formulation containing peracetic acid, sulfuric acid surfactant, and copper against Salmonella and its effect on indigenous microbiota during washing and storage. New formulation efficiently reduced Salmonella on inoculated diced tomatoes and the transfer to non-inoculated tomato dice after washing. The treatment also inhibited the proliferation of most dominant bacteria on diced tomatoes during storage, such as Erwiniaceae, Curtobacterium, Pantoea, Erwinia and Enterobacterales spp., which may benefit product quality and safety. Tomato processing facilities can use the information to improve safety and quality of diced tomatoes.

6. Characterization of Listeria monocytogenes growth. L. monocytogenes is a foodborne pathogen ubiquitously present in agriculture environments and an increasing concern for various fresh produce commodity industries. A wide variety of sterile fresh produce juice were used to examine the ability of L. monocytogenes growth on corresponding fresh produce under optimal conditions (abundant nutrients without competing microorganisms). The results indicated that, consistent with current regulatory policies, product acidity was the primary determinant of L. monocytogenes growth, as the pathogen showed significant growth in juice extracted from produce with high pH (>5.6) and failure to grow in juice extracted from fruits with low pH (<4.1), irrespective of factors such as sugar content. Some of the fresh produce did not support L. monocytogenes growth potentially due to presence of antilisterial compounds. The information is useful for various commodity groups in assessing risks of L. monocytogenes growth.

Review Publications
Bolten, S., Mowery, J.D., Gu, G., Redding, M., Kroft, B., Luo, Y., Nou, X. 2023. Listeria monocytogenes loss of cultivability on carrot is associated with the formation of mesosome-like structures. International Journal of Food Microbiology. https://doi.org/10.1016/j.ijfoodmicro.2023.110121.
Gu, G., Zhou, B., Mendes Oliveira, G., Redding, M., Luo, Y., Millner, P.D., Nou, X. 2023. Impact of sanitizer application on salmonella mitigation and microbiome shift on diced tomato during washing and storage. Postharvest Biology and Technology. https://doi.org/10.1016/j.postharvbio.2023.112268.
Gu, G., Murphy, C., Hamilton, A., Zheng, J., Nou, X., Rideout, S., Strawn, L. 2023. Effects of bactericides on Salmonella survival on the surface of and in inoculated tomato leaves. Journal of Food Safety. https://doi.org/10.1111/jfs.13043.
Rao, A., Patel, J.R., Pradhan, A. 2022. Application of alternative sources of water in agricultural food production - current trends and future prospects. Current Opinion in Food Science. 47. Article 100877. https://doi.org/10.1016/j.cofs.2022.100877.
Karanth, S., Patel, J.R., Shirmohammadi, A., Pradhan, A. 2023. Machine learning to predict foodborne salmonellosis outbreaks based on genome characteristics and meteorological trends. Current Research in Food Science. https://doi.org/10.1016/j.crfs.2023.100525.
Lichtendwald, M., Bolten, S., Luo, Y., Micallef, S.A., Millner, P.D., Nou, X. 2022. Growth and inactivation of Listeria monocytogenes in sterile extracts of fruits and vegetables: Impact of the intrinsic factors pH, sugar and organic acid content. International Journal of Food Microbiology. https://doi.org/10.1016/j.ijfoodmicro.2022.110043.
Teng, Z., Luo, Y., Pearlstein, D.J., Zhou, B., Johnson, C.M., Wang, Q., Fonseca, J.M. 2022. Agarose hydrogel composite supports microgreen growth with continuous water supply under terrestrial and microgravitational conditions. International Journal of Biological Macromolecules. 220:135-146. https://doi.org/10.1016/j.ijbiomac.2022.08.046.
Teng, Z., Luo, Y., Pearlstein, D.J., Wheeler, R., Johnson, C., Wang, Q., Fonseca, J.M. 2022. Microgreens for home, commercial, and space farming – a comprehensive update of the most recent developments. Annual Review of Food Science & Technology. 14:539-562. https://doi.org/10.1146/annurev-food-060721-024636.
Yin, H., Chen, C., Katchman, B., Newland, C., May, M., Patel, J.R. 2022. Rapid detection of Salmonella enterica in fresh produce by a novel microarray-based PathogenDx system. Food Microbiology. 107. Aricle 104086. https://doi.org/10.1016/j.fm.2022.104086.
Yin, H., Chen, C., Gu, G., Nou, X., Patel, J.R. 2022. Pre-harvest biocontrol of Listeria and Escherichia coli O157 on spinach and lettuce by lactic acid bacteria. International Journal of Food Microbiology. https://doi.org/10.1016/j.ijfoodmicro.2022.110051.
Zhang, T., Luo, Y., Zhou, B., Teng, Z., Huang, C., Nou, X. 2022. Sequential application of peracetic acid and UV irradiation (PAAUV/PAA) for improved bacterial inactivation in fresh-cut produce wash water. ACS Environmental Science & Technology Water. 2(7):1247-1253. https://doi.org/10.1021/acsestwater.2c00087.
Yang, M., Luo, Y., Sharma, A., Jia, Z., Wang, S., Wang, D., Lin, S., Pereault, W., Purohit, S., Gu, T., Dillow, H., Liu, X., Yu, H., Zhang, B. 2022. Nondestructive and multiplex differentiation of pathogenic microorganisms from spoilage microflora on seafood using paper chromogenic array and neural network. Food Research International. https://doi.org/10.1016/j.foodres.2022.112052.
Zhou, B., Luo, Y., Nou, X., Mwangi, E., Poverenov, E., Demokritou, P., Fonseca, J.M. 2023. Effects of a novel combination of gallic acid, hydrogen peroxide and lactic acid on pathogen inactivation and shelf-life of baby spinach. Food Control. 143. Article 109284. https://doi.org/10.1016/j.foodcont.2022.109284.