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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

2016 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:
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.

4. Accomplishments
1. 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.

2. 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.

3. 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.

4. 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 .

5. 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.

5. Significant Activities that Support Special Target Populations:

Review 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.