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United States Department of Agriculture

Agricultural Research Service

Research Project: Microbial Ecology and Safety of Fresh Produce

Location: Environmental Microbial and Food Safety Laboratory

2013 Annual Report


1a.Objectives (from AD-416):
Objective 1. Investigate the mechanism(s) of introduction and transference of Shiga toxigenic E. coli (STEC) and Salmonella to fresh produce during growing, harvest, and postharvest handlings: (1a) Evaluate the growth and survival potential of Enterohemorrhagic E. coli (EHEC) and Salmonella in compost manure applied to leafy green fields and validate cross-contamination and temperature control steps in a Hazard Analysis Critical Control Point (HACCP) system for compost, (1b) Develop a science-based proximity assessment index for airborne bioaerosol emissions for tomato and fresh produce (leafy greens -LG) fields, (1c) Determine the influence of biocontrols and insect amplification and transmission of EHEC and Salmonella in tomato and leafy green crops, and (1d) Evaluate the role of cultivars and fertilization on leafy-green damage during processing and its potential for pathogen contamination.

Objective 2. Determine the persistence and survival of pathogens on fresh and fresh-cut leafy green produce: (2a) Determine the persistence of pathogens introduced at different levels on spinach and lettuce plants to verify the adequacy of the California Leafy Greens Marketing Agreement (LGMA), (2b) Identify and evaluate suitable surrogate microorganism for generic E. coli in irrigation water, and (2c) Develop a field-scale-zero valent or slow sand filtration column to inactivate E. coli O157:H12 (surrogate strain) in a field study.

Objective 3. Determine the effects of pathogen attachment, biofilm formation, and internalization on the extent of contamination of fresh produce: (3a) Determine the role of various virulence and stress factors on the ability of EHEC to persist on leafy green surfaces, and (3b) Determine the effect of biofilm formation on survival of STEC and Salmonella on fresh produce.

Objective 4: Develop effective intervention technologies during processing to inactivate pathogens, and/or to reduce their survival and growth while maintaining produce quality and shelf-life: (4a) Minimizing pathogen transference and infiltration during produce harvesting and post-harvest handling, (4b) Optimizing fresh-cut produce wash system configurations and operations to improve pathogen inactivation and minimize cross-contamination, and (4c) Effect of temperature and packaging atmospheric conditions on survival, growth, and virulence of EHEC.


1b.Approach (from AD-416):
Mechanisms of introduction and transfer of pathogens on fresh produce (lettuce, spinach, tomatoes, leafy greens) at the farm level will be investigated. Growth and survival patterns of avirulent strains of Enterohemorrhagic E. coli (EHEC) and Salmonella will be evaluated in composted manure with different pile size and configuration. Deposition of airborne-microbes onto fresh produce will be evaluated during different times of the year to determine proximity distance between fields and suspected nearby source of contamination. The role of insect vectors in transmission of pathogens to fresh produce will be studied and biocontrols will be used as a potential deterrent to insects. Bacterial analysis will include the use of microbial culture and molecular methods to detect target pathogens in samples. Persistence of EHEC and Salmonella on fresh produce will be determined when these pathogens are introduced at different levels via irrigation water. Clostridium perfringens and coliphages will be evaluated as suitable indicators for fecal contamination of irrigation water. Zero-valent iron (ZVI) columns will be evaluated as an intervention for removing pathogens from irrigation water. The role of specific virulence and stress factors on the ability of EHEC to attach and persist on fresh produce will be determined. Wild-type and curli- and cellulose-deficient strains of EHEC and Salmonella will be evaluated for their attachment and biofilm formation on fresh produce; biofilm formation on foliar surfaces will be determined by confocal laser scanning microscopy (CLSM). Existing and novel new antimicrobial wash treatments which remove biofilm from foliar surfaces will be evaluated. The effect of tomato dump tank management parameters on the probability and extent of Salmonella infiltration will be determined; the infiltration pattern will be determined by CLSM. The effect of the wash-cut sequence on pathogen cross-contamination during cutting and washing of fresh produce will be investigated. To minimize the chlorine degradation of wash water used for fresh produce wash, chlorine stabilizer will be used in wash water to evaluate its effect on chlorine stability of wash water and additional pathogen reduction on fresh produce. Pathogen growth and virulence as impacted by temperature abuse at a retail level and modified atmosphere packaging (MAP) of fresh produce will be evaluated.


3.Progress Report:
The persistence of Salmonella isolated from poultry or produce was compared following irrigation of spinach plants. The contamination source, biofilm formation and inoculation level influenced the persistence of Salmonella on growing spinach. Produce isolates formed stronger biofilm on abiotic surfaces and persisted at significantly higher numbers on spinach leaves. Results show that the source of Salmonella contamination will affect its persistence on produce surfaces. The persistence of E. coli O157:H7 on spinach leaves varied with spinach cultivars and surface appendages of E. coli O157: H7 strains. Cultivar Waitiki with highest leaf roughness supported significantly higher E. coli O157:H7 populations on its leaves compared to other cultivars- Emilia, Lazio and Space. Leafy green cultivars that don’t support pathogen growth and persistence could be used by the growers to minimize produce associated outbreaks and recall. E. coli O157:H7 could internalize into hydroponically grown intact spinach plants through the root system and move to the stem and leaf level. Wounding of the root system in hydroponically grown spinach increased the incidence of E. coli O157:H7 internalization and translocation to the edible portions of the plant. E. coli O157:H7 internalization was observed in greater numbers when plants were grown in soil then in those grown hydroponically. Curli expression by E. coli O157:H7 and spinach cultivar did not affect its root uptake by spinach plants. The finished compost as a cover over static and windrow piles to inactivate pathogens during composting process was evaluated. Weed seed germination as an indication of finished compost was evaluated. E. coli O157:H12 and Salmonella reduced rapidly in compost piles covered with 30 cm finished compost compared to that in non-covered control piles. Further, the rate of reduction was substantially more rapid in the windrow than in the static piles. Germination of 'broadleaf dock' seeds after 28 days in static and non-covered piles was consequent to recovery of E. coli O157:H7 from these piles. Compost processors can use finished compost as a cover to rapidly reduce pathogens during composting process that will help reduce cross contamination of fresh produce via soil. Internalization of pathogenic bacteria in tomatoes during washing dump tanks is a serious concern for the tomato industry and for consumer safety. The impact of storage temperature and duration on the survival and growth of pathogens and the correlation between decay development and pathogen presence in tomatoes were also investigated. These studies demonstrated that pathogens can infiltrate into tomatoes during dump tank handling even under conditions of elevated dump tank water temperature and brief immersion time. Delaying the time between stem-removal and immersion in dump tank water significantly reduced both incidence and severity of pathogen internalization as did reducing immersion time. This research provides critical information to the FDA and the tomato industry for developing science-based tomato food safety practices against pathogen internalization.


4.Accomplishments
1. Efficacy of fresh-cut produce wash system in controlling cross-contamination of fresh produce. The fresh-cut produce industry currently does not have the capacity to maintain stable sanitizer concentration during commercial operation. Washing produce without effective sanitizer concentration could drastically spread disease-causing bacteria leading to significant increases in human illness from consumption of contaminated produce. Working closely with the industry, USDA-ARS scientists at Beltsville, MD conducted a comprehensive analysis of the relationship between sanitizer loss in produce wash operation and the spread of bacteria. They demonstrated that the sanitizer concentration in the industry’s food safety program is insufficient for preventing bacteria survival and spread. Additionally, they proved that re-washing contaminated produce is not an effective corrective action as it does not eliminate the presence of bacteria. These studies revealed the clear risks in current industry practices, and further provided the quantitative data for the FDA to develop science-based food safety regulations for preventive controls against bacterial contamination. Industry is currently using the findings to revise wash operational procedures. The information is also used by the Homeland Security Department to develop counter bioterrorism programs.

2. Antimicrobial activity of plant-derived compounds against Salmonella on organic leafy greens. Organic foods are produced without the use of any chemicals, and hence, natural plant compounds may be good alternative to chemicals. The antimicrobial effect of cinnamaldehyde, Sporan, and oregano oil was evaluated against foodborne pathogens on fresh produce. Oregano oil as low as 0.1% concentration reduced Salmonella by 1.5-3 log per g on lettuce and spinach following 2 min treatment. Up to 3 log reductions in E. coli O157:H7 and Salmonella populations was observed when lettuce was treated with 800 ppm cinnamaldehyde or 1000 ppm Sporan. The texture and the color of iceberg and romaine lettuce treated with essential oils were not different from the control lettuce after 14 days. This study demonstrates the potential of Sporan and cinnamaldehyde as effective lettuce washes that do not affect lettuce color and texture.

3. Lytic bacteriophages to control contamination of pathgens in fresh produce. Fresh-cut produce may be contaminated at any stage during farm-to-fork continuum. Contaminated fresh produce may cause outbreak of human illnesses associated with its consumption. Lytic baceriophages, viruses that specifically infect and kill bacteria, have been proposed as an alternative disinfection technique for pathogenic bacteria. To evaluate efficacy, bacteriophages were applied to fresh cut lettuce inoculated with E. coli O157:H7. Results show that populations of pathogenic bacteria were reduced in studies simulating produce cross-contamination during refrigerated storage. These bacteriophages also worked synergistically with sodium hypochlorite to reduce E. coli O157:H7 populations on fresh cut lettuce more than the application of hypochlorite solutions alone, indicating effectiveness under current leafy green processing conditions. The study reveals the potential of bacteriophages in reducing cross-contamination of fresh produce.

4. Persistence of avian pathogenic E. coli on leafy greens. Avian pathogenic E. coli (APEC) could be an unrecognized source of human illness, if produce is contaminated through the use of poultry manure applied to agricultural fields. This study assessed the survival of avian pathogenic E. coli on lettuce and spinach, as compared to E. coli O157:H7 and E. coli O104:H4, both known human pathogens. APEC isolated from poultry broilers and manure were able to survive for longer durations on basil, spinach and lettuce compared to E. coli O157:H7 when simultaneously inoculated on to these leafy green commodities. A possible explanation of the increased duration of the avian pathogenic strains compared to the E. coli O157:H7 isolates is their better adaption to environmental stresses. Poultry manure used for soil fertility should be properly composted to minimize contamination of soil with avian pathogenic E. coli.


Review Publications
Yossa, N., Patel, J.R., Millner, P.D., Ravishankar, S., Martin Lo, Y. 2013. Antimicrobial activity of Cinnamaldehyde and Sporan against Escherichia coli O157:H7 and Salmonella on lettuce. Foodborne Pathogens and Disease. 10(1):87-96.

Yossa, N., Patel, J.R., Millner, P.D., Murphy, C.F., Bauchan, G.R., Lo, M. 2012. Antibacterial activity of cinnamaldehyde and Sporan against Escherichia coli O157:H7 and Salmonella. Journal of Food Processing and Preservation. DOI: 10.1111/jfpp.12026.

Moore-Neibel, K., Gerber, C., Patel, J.R., Friedman, M., Jaroni, D., Ravishankar, S. 2013. Antibacterial activity of oregano oil against antibiotic resistant Salmonella enterica on organic leafy greens at varying exposure times and storage temperatures. Food Microbiology. 34(1):123-129.

Shen, C., Luo, Y., Nou, X., Wang, Q., Millner, P.D. 2013. The effects of free chlorine concentration, organic load, and exposure time on the inactivation of Salmonella, Escherichia coli O157:H7 and non-O157 STEC. Journal of Food Protection. 76(3):386-393.

Luo, Y., Nou, X., Millner, P.D., Zhou, B., Shen, C., Yang, Y., Wu, Y., Wang, Q., Fen, H., Shelton, D.R. 2012. A pilot plant scale evaluation of a new process aid for enhancing chlorine efficacy against pathogen survival and cross-contamination during produce wash. International Journal of Food Microbiology. 158(2):133-139.

Shen, C., Luo, Y., Nou, X., Bauchan, G.R., Zhou, B., Wang, Q., Millner, P.D. 2012. Fresh produce washing aid, T-128, enhances inactivation of salmonella and pseudomonas biofilms on stainless steel in chlorinated wash solutions. Applied and Environmental Microbiology. 78(19):6789-98.

Last Modified: 12/21/2014
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