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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL ECOLOGY AND SAFETY OF FRESH ON-FARM ORGANICALLY GROWN PRODUCE
2010 Annual Report


1a.Objectives (from AD-416)
1. Determine the prevalence, diversity, and quantity of bacteria associated with organic as compared to conventional fresh produce in Maryland and Virginia. 2. Determine colonization and survival rates of bacterial pathogens associated with selected organic and conventional fresh produce. 3. Investigate the mechanism(s) of introduction and transference of E. coli O157:H7 to lettuce and leafy greens during growing, harvest, postharvest handlings and processing operations. 4. Determine the persistence and survival of Escherichia coli O157:H7 on fresh and fresh-cut leafy green produce. 5. Compare the extent of enteric pathogen transmission by a chewing insect, Colorado Potato Beetle, in organically- and conventionally-grown produce.


1b.Approach (from AD-416)
A farm-based investigation will be conducted to assess the prevalence, diversity, and quantity of epi- and endophytic saprophytes and gastroenteric bacteria associated with organic as compared to conventional fresh produce. Bacterial analyses will include use of microbial cultural and molecular methods to characterize the enteric and saprophytic microflora on fresh market produce types most commonly reported in foodborne illness outbreaks: tomatoes, salad greens: lettuce, arugula, mesculin, spinach, beets; herbs: basil, cilantro, parsley; strawberries; cucurbits: cucumbers and melons. In addition, practices at each farm will be identified and characterized to associate analytical results with microbial quality of farm inputs and operations. Bacterial analysis will include total aerobic heterotrophic bacteria, enteric bacteria (coliforms), Aeromonas spp., Bacillus cereus, C. perfringens, E. coli, Enterococcus spp., L. monocytogenes, and Salmonella. Microbial community analysis of epi- and endophytic bacteria by DGGE will be used for tomato fruit and salad greens from organic and conventional farms. Growth chamber and field plot experiments will be conducted to establish the pathogen concentrations required to establish populations that survive on different plants (e.g., tomatoes, greena, basil, scallions, carrots, and strawberries). Survival of pathogens (Aeromonas, EHEC, L. monocytogenes, and Salmonella) on/in plants grown in organic and conventional soils with composted or aged manure (spike with pathogens) and irrigated with contaminated river water or sprayed with compost tea will be determined by enrichment and enumeration of rhizosphere, cortical root tissue, and/or on the edible portion of plants. Organic practices that can prevent pathogen growth in compost tea and internal plant tissues will be evaluated in growth chamber and field studies with tomato. Finally, the potential for a chewing insect, Colorado Potato Beetle, to transfer bacterial pathogens to the surface and interior tissue of tomato plants and initiate systemic colonization will be investigated for organic and conventional production methods.


3.Progress Report
Studies were conducted to determine the antimicrobial properties of essential oils in controlling E. coli O157:H7 in organic soil. Cinnamaldehyde, ecotrol and vinegars significantly reduced E. coli O157:H7 populations in soil, whereas eugenol was the least effective oil in controlling E. coli O157:H7. Commercial products and crop residue treatments can be used with clear plastic film as a soil biofumigation to eliminate fecal contamination of E. coli O157:H7 and Salmonella in soil.

The role of insects as a vector in pathogen transmission was determined. Results suggested that the insect frass from hornworms feeding on contaminated tomato foliage can spread E. coli O157:H7 throughout a plant canopy and onto fruits.

Spinach plants were spray irrigated with Salmonella every two weeks to determine the adequacy of the California Leafy Green Marketing Agreement (LGMA) criteria established for irrigation water. Salmonella were not detected after 1 day following irrigation of water contaminated with as high as 4 log CFU Salmonella/100 ml. The research indicates that LGMA standards for irrigation water are too stringent and may create undue burden to farmers, especially in Mid-Atlantic area where surface water used for irrigation has higher generic E. coli populations.

Persistence of E. coli O157:H7 on Romaine lettuce varied with the strain. In general, most strains persisted on lettuce leaves for at least 14 days when inoculated with 6 log cfu/ leaf. Environmental stress factors might be responsible for rapid decline of the pathogen on produce surfaces.

Biosand filters (BSF) were evaluated as an intervention to reduce pathogens in irrigation water. E. coli O157:H7 were not detected from the BSF 15 days post-introduction of 20 L water containing 6 log cfu/ml of E. coli O157:H7. These results provide the basis for field testing of biosand filters as a treatment for contaminated irrigation water intended for fresh produce.

Organic materials from fresh-cut produce can rapidly inactivate free chlorine in washing solution. A chlorine stabilizer was evaluated for the potential of increasing chlorine efficacy during sanitizing washing of fresh-cut lettuce. While the treatment did not influence significant reduction of bacterial pathogen, it helped prevent cross-contamination of fresh produce in the presence of organic load.

Essential oils were evaluated as a potential produce wash treatment to reduce pathogens on fresh-cult produce. Sporan and cinnamaldehyde significantly reduced Salmonella and E. coli O157:H7 on fresh-cut spinach and lettuce leaves. Increased concentrations (> 800 ppm) of these oils adversely affected color and texture of spinach and lettuce leaves. The effect of temperature abuse during storage of fresh produce on virulence expression of E. coli O157:H7 was determined. The Modified Atmospheric Packaging (MAP) conditions increased the expression of specific virulence factors in E. coli O157:H7after three days of storage at 15°C. The results shows that fresh-cut lettuce stored under commercial MAP conditions can potentially increase the virulence of the pathogen.


4.Accomplishments
1. Antimicrobial properties of essential oils against E. coli O157:H7 in organic soil. Soil can be a significant source of preharvest contamination of produce by pathogens. Demand for natural pesticides such as essential oils for organic farming continues to increase. We examined the antimicrobial activity of several essential oils against E. coli O157:H7 in soil. E. coli O157:H7 populations in soil were reduced by up to 5 log cfu/g after 24 h incubation at room temperature with 2% cinnamanaldehyde, Ecotrol, Sporan or vinegar. Reduction in E. coli O157:H7 by eugenol was not significantly different from control. Overall, E. coli O157:H7 strain 4406 was the most sensitive of all the five strains tested and cinnamaldehyde was superior to other treatments in reducing E. coli O157:H7 in soil. This study shows the potential use of oils in organic produce environments to effectively reduce Salmonella populations in soil.

2. Soil biofumigation. The effect of biofumigation on reduction of E. coli and Salmonella in lettuce and spinach production soils was tested in a field-replicated trial for the second time in late summer 2009. Soil cassettes were artificially contaminated with nonpathogenic strains and buried prior to solarization treatments. Different types of plastic films as well as black and white-on-black plastic mulch (commonly used mulch types in vegetable production) were compared in the study. In addition, all mulch types were evaluated as treatments for biofumigation in previously planted leafy greens beds in organic high tunnels and in organic open field soils. Several commercial products and one crop residue treatment were tested in the high tunnel plots and nematode eradication was also evaluated in these plots. Results with single rate applications show significant promise for elimination of E. coli O157:H7 and Salmonella from fecal contamination in soil using clear plastic film biofumigation. Exceptionally heavy rainfall during the experimental period in 2009 inundated some of the open field plots. The experiment will be repeated in 2010 season to obtain a second year of the open field plot data.

3. Insect as a vector in transmission of pathogens. The growth and survival of E. coli O157:H7 in hornworm larvae (Manduca sexta) were evaluated in replicated, multi-dose level (1000 to 100,000 cfu per insect single dose) laboratory studies on tomato leaves. Insects were reared in the laboratory and 4th instar stages were selected for assays. Results indicate a clear dose response in terms of the frequency and amount of contamination in insect frass produced. Greatest amounts of contamination were obtained in frass 48 hr after initial single dose. Viable E. coli O157:H7 survived well on molted exoskeleton, but was rarely recovered on the 5th instar larvae. These results indicate that insect frass from hornworms feeding on contaminated tomato foliage can amplify and spread E. coli O157:H7 from localized contamination throughout a plant canopy and onto fruits developing on plants.

4. Assessment of recommended irrigation water standards for leafy greens. The California Leafy Green Marketing Agreement (LGMA) recommends that water containing less than 235 MPN (most probable number) E. coli / 100 ml be used in irrigating leafy greens in the field. However, no evaluation of these standards has been undertaken. The individual 3-week old spinach plants were spray inoculated every 2 week with ~ 5 ml water containing as high as 13,000 Salmonella per 100 ml. Salmonella were undetectable (less than 1.1 MPN/g) on spinach leaves after 1 day or after 6 weeks when irrigation water was contaminated at this level. Similar results were reported when low levels of E. coli O157:H7 and avian pathogenic E. coli were inoculated on spinach leaves. The effect of total organic carbon in irrigation water on E. coli O157:H7 persistence was determined. The carbon levels did not influence E. coli O157:H7 persistence on spinach leaves. Repeated irrigation of plants with water containing low levels of E. coli or Salmonella does not enhance survival on spinach leaves. Results may aid in understanding the role of irrigation water in Salmonella contamination on spinach leaves.

5. Differential persistence of E. coli O157:H7 strains on romaine lettuce leaves. Limited information is available on ecological interactions influencing persistence of different E. coli O157:H7 strains on lettuce. We evaluated survival of six strains of E. coli O157:H7 isolated from produce and environmental samples on Romaine lettuce. Romaine lettuce (3 weeks old) grown in steam-pasteurized organic soil was inoculated with 6 log cfu of E. coli O157:H7 per leaf. Three of the six E. coli O157:H7 strains were non-detectable on lettuce leaves after 7 days. Five E. coli O157:H7 strains persisted on lettuce leaves after 14 days as determined by MPN, whereas only two isolates were recovered after 14 days by direct plating. Enrichment MPN assay is required to detect persistence of low numbers of E. coli O157:H7 on lettuce. The study shows rapid die off of E. coli O157:H7 once inoculated onto lettuce. This could be due to environmental stresses as well as foreign nature of foliar surface for enteric pathogens.

6. Mitigation of irrigation water using zero-valent iron treatment. Irrigation water has been implicated as a source of produce contamination with enteric pathogens. Mitigation strategies for irrigation water will help reduce contamination and subsequent outbreak. Commercial biosand filters (BSF) were evaluated to reduce pathogens such as E. coli O157:H7. Water containing 6 log CFU/ml of E. coli O157:H7 was filtered through the BSF column. E. coli O157:H7 were not detected from the column fifteen days after introduction of E. coli O157:H7. These results provide the basis for field testing of biosand filters and biosand filters incorporating zero-valent iron as a treatment for contaminated irrigation water intended for leafy greens.

7. Natural antimicrobials as a produce wash to reduce pathogens on fresh-cut produce. Customers’ preference for minimal chemicals have led the researchers to find alternative antimicrobials as a produce wash. We evaluated essential oils to reduce pathogens on fresh-cult spinach and lettuce leaves. Sporan and cinnamaldehyde at 800 ppm concentrations significantly reduced Salmonella and E. coli O157:H7 on fresh produce surface. Increased concentrations of these oils resulted in further reduction of Salmonella and E. coli O157:H7. However, the produce quality was poor when treated with the high conc. of oils. Texture and color of the treated leaves is being evaluated to determine adverse effect of these oil treatments, if any. Results show that oils could be used as an intervention to control pathogens in fresh-cut produce.

8. Evaluation of chlorine stabilizer to enhance killing efficacy of produce wash (chlorine). Organic materials from fresh-cut produce can rapidly inactivate free chlorine in washing solution. A chlorine stabilizer developed by the produce industry (T-128) was evaluated for the potential of increasing chlorine efficacy during sanitizing washing of fresh-cut lettuce. This product was found to improve chlorine free chlorine stability at the presence of high organic loads derived from lettuce juice, although the level of the improvement was marginal. However, the application of this product significantly reduced the survival of bacterial pathogens in the washing solution, even at high organic loads. This product did not significantly increase the inactivation of bacterial pathogens attached to lettuce surface. Nevertheless it significantly reduced the occurrence of cross contamination during produce washing.

9. Modified atmospheric packaging. Lettuce stored under commercial modified atmosphere packaging (MAP) was inoculated with E. coli O157:H7. Three different MAP conditions were used to determine if E. coli O157:H7 on lettuce under MAP and stored at abusive conditions may become more virulent than under non-MAP conditions. Lettuce contaminated with E. coli O157:H7 was stored at abusive temperatures (15°C) or at refrigered temperatures (4°C). Results indicated that specific MAP conditions influenced in the increased expression of specific virulence factors in E. coli O157:H7after three days of storage at 15°C. Expression of virulence factors at 4°C did not indicate any apparent differences. This work shows that fresh-cut lettuce stored under commercial MAP conditions can potentially increase the virulence of the pathogen.


5.Significant Activities that Support Special Target Populations
Provided collaboration and training to students from 1890 institutions (University of Maryland, Eastern Shore, MD; Alabama A&M University, Normal, AL)

Prepared two NIFA proposals with faculty from University of Maryland, Eastern Shore; North Carolina A & T, and Delaware State University, both proposals were funded.

Chesapeake Area Urban Farming Summit was held at BARC on June 18, 2010 (350 attendees including BARC's liason) at which food safety and health along with local production and access to fresh produce in inner-city areas in the region was a major focus.


Review Publications
Patel, J.R., Sharma, M. 2010. Differences in attachment of Salmonella enterica serovars to cabbage and lettuce leaves. International Journal of Food Microbiology. 139(1-2):41-47.

Yossa, N., Patel, J.R., Millner, P.D., Lo, M. 2010. Antimicrobial activity of essential oils against E. coli O157:H7 in organic soil. Food Control. 21(11):1458-1465.

Patel, J.R., Millner, P.D., Nou, X., Sharma, M. 2010. Persistence of enterohemorrhagic and non-pathogenic E. coli on spinach leaves and in rhizosphere soil. Journal of Applied Microbiology. 108:1789-1796.

Nou, X., Luo, Y. 2010. Whole-leaf sanitizing wash improves microbial reduction efficacy and prevents pathogen cross contamination during fresh-cut lettuce processing. Journal of Food Science. 75(5):M283-M290.

Last Modified: 12/20/2014
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