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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Environmental Microbial & Food Safety Laboratory » Research » Research Project #430368

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

Location: Environmental Microbial & Food Safety Laboratory

2018 Annual Report

1a. Objectives (from AD-416):
Objective 1: Investigate the mechanism(s) of introduction, transference, and survival of enterohemorrhagic Escherichia coli (EHEC), Salmonella, and Listeria to fresh produce at the farm level. Sub-objective 1a. Investigate the population dynamics of non-pathogenic E. coli and non-O157 EHEC in soils amended with biological soil amendments (BSA). Sub-objective 1b. Determine factors affecting persistence of EHEC, Salmonella and Listeria in soils amended with BSA. Objective 2: Determine the effects of multispecies biofilm formation on the survival, persistence, and dissemination of pathogenic bacteria in fresh produce processing environments and on contamination of fresh produce. Sub-objective 2a. Assess the biofilm formation capacity of foodborne bacterial pathogens in fresh produce processing environments and on fresh produce surfaces; identify environmental bacterial strains or species that promote multispecies biofilm formation on fresh produce or in processing environments. Sub-objective 2b. Elucidate factors controlling foodborne bacterial pathogen interactions in multispecies biofilms on fresh produce or in processing environments. Sub-objective 2c. Determine biofilm formation of non-O157 shiga-toxigenic E. coli (STEC) on abiotic and biotic surfaces. Objective 3: Investigate intervention strategies to minimize contamination of EHEC, Salmonella and Listeria on fresh produce at the farm level. Sub-objective 3a. Determine the role of Brassica vegetables in controlling enteric pathogens in soil. Sub-objective 3b. Develop pre-harvest interventions to control Listeria and Salmonella in cantaloupe. Objective 4: Develop effective intervention technologies to reduce pathogen survival and growth during processing and retail operations. Sub-objective 4a. Identify and validate food safety preventive controls for water application during fresh-cut processing. Sub-objective 4b. Investigate novel antimicrobials to control enteric pathogens on herbs. Objective 5: Assessment of microbial safety of fresh produce grown under non-conventional farming practices. Sub-objective 5a. Determine the effect of reclaim water on microbial safety of fresh produce grown in urban farming.

1b. Approach (from AD-416):
Mechanisms of introduction and transfer of pathogens on fresh produce (lettuce, spinach, leafy greens, fresh herbs) at the farm level will be investigated. Population dynamics of non-O157 Enterohemorrhagic E. coli (EHEC) and non-pathogenic E. coli in soils amended with biological soil amendments (BSA: manure, compost) will be investigated. Factors affecting growth and survival patterns of EHEC, Salmonella and Listeria in soils amended with BSA will be determined. The role of stress response genes on the survival of enteric pathogens in manure or manure-amended soils will be evaluated. Bacterial analysis will include the use of microbial culture and molecular methods to detect target pathogens in samples. Biofilm formation capacity of EHEC and Listeria monocytogenes will be assessed under conditions partially simulating produce production and processing environments. Bridge bacteria that promote the incorporation of pathogen in multispecies biofilms will be isolated and identified. Confocal microscopy, mass spectrometry, and metagenomic sequencing will be used to decipher the complexity of the multispecies biofilms. Intervention strategies will be investigated to minimize pathogen contamination at the farm level. Field studies will be conducted to determine the role of Brassica vegetables in killing EHEC, Salmonella, and Listeria in soil. Biological controls such as lactic acid bacteria will be evaluated at the farm level to control Listeria contamination on cantaloupe. Food safety preventive controls during fresh-cut processing operations will be identified and validated to reduce pathogen survival and growth on fresh produce. Validation of free chlorine concentration, role of produce particulates, and pathogen inactivation kinetics will be investigated to minimize pathogen cross-contamination. Fresh produce will be irrigated with reclaimed water to assess its microbial safety. Microbial risk assessment models will be used to determine microbial safety of fresh produce.

3. Progress Report:
Progress was made on all 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.

4. Accomplishments
1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.

Review 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.
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.
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. 125:133-141.