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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
2008 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
A study was conducted to determine: (1) microbial content of seasonal produce grown by farmers in the Mid-Atlantic region, (2) Persistence of E. coli O157:H7 on spinach and soil, (3) the effect of green manure on survival of E. coli O157:H7 in soil, (4) the effect of leaf age and radiation exposure on survival of E. coli O157:H7 on spinach, and (5) the microbial assessment of compost fertilizer used by farmers. Locally grown produce (tomatoes, leafy greens, strawberries, green onions, and herbs) did not contain either E. coli O157:H7 or Salmonella spp. A study was initiated to determine persistence of E. coli O157:H7 on spinach leaves and in soil. Populations of E. coli O157:H7 survived for a shorter duration on spinach shoots than in soil. The rise in popularity of leafy green produce commodities has promoted the use of green manure because not all parts of the harvested commodity are packaged in the pre-washed bagged products. The application of green manure at the field level is practical because it reduces the waste at processing facilities and lowers transportation costs from the field. E. coli O157:H7 populations detected with cabbage manure at 35 days were significantly lower than the E. coli O157:H7 populations detected in spinach or daikon manures. An ongoing study is being conducted to determine the effect of green manure on other foodborne pathogens. Survival of E. coli O157:H7 on spinach leaves declined gradually and varied with the age of leaf. After 28 days, older leaves partially shaded and sometimes in contact with moist soil had greater populations than upper (intermediate age) or crown (youngest age) leaves. A study was conducted to characterize the microbial quality of finished, marketable compost prepared from a wide range of organic residuals from 15 U.S. commercial facilities. Prevalence of toxigenic E. coli in commercial composts was very low despite the relatively high number of samples that contained E. coli. Research is being carried out to determine the effect of essential oils on survival of E. coli O157:H7 and Salmonella spp. in soil. The efficacy of essential oil supplements for control of pathogens in compost tea is being investigated. The effectiveness of a bacteriophage, viruses that can kill pathogenic foodborne bacteria, was examined to kill E. coli O157:H7 on fresh produce. The bacteriophage treatment reduced populations of E. coli O157:H7 immediately upon application to lettuce, and reduced populations on cantaloupes occurred over 7 days. The suitability of bacteriophage treatment to kill E. coli O157:H7 in biofilm is being studied. The growth pattern of three strains of Escherichia albertii on fresh-cut iceberg lettuce revealed that E. albertii 9194 showed marginal growth when stored at 5 deg C, indicating that this strain of E. albertii may be more persistent at low temperatures than other strains. These projects address the National Program 108 Action Plan 1 (Pathogens, Toxins and Chemical Contaminants, Pre-harvest) by developing a better understanding of introduction, persistence and survival of foodborne pathogens on fresh produce.


4.Accomplishments
1. Longitudinal Microbiological Survey of Fresh Produce Grown by Farmers in the Mid-Atlantic Region - Limited data on production practices relative to microbial/pathogen content of fresh produce are currently available for either organic or conventional farms in the Mid-Atlantic region, some of which market directly to consumers. A study was conducted to evaluate the microbial content of seasonal produce grown during 2006-07 relative to various production practices and inputs. In addition to produce, soil and water samples used in the production systems were analyzed for the content of E. coli (generic and pathogenic), Salmonella spp., and Listeria spp. Produce (tomatoes, leafy greens, strawberries, green onions, and herbs) were collected from matched pairs of organic and conventional farms. E. coli were confirmed by biochemical, fatty acid analysis profiles, and multiplex PCR (utilized for confirmation of E. coli O157:H7). Farm practices at each site were obtained by interviews, on-site observations, and written responses from participants. Confirmed isolates of E. coli included 34 and 46 soil, and 5 and 15 commodity samples in 2006 and 2007, respectively. No EHEC strains were determined from PCR screening, and no salmonellae were confirmed. Pathogenic Listeria spp. were recovered from strawberry and soil samples on one farm by enrichments in 2007. E. coli in excess of drinking water quality limits were recovered from seven of 17, and 11 of 28 samples in 2006 and 2007 respectively. (National Program 108 Component 1.1 Pathogens, Toxins and Chemical Contaminants Pre-harvest; Problem Area 1.1.3 – Ecology, Host Pathogen and Chemical Contaminants Relationships)

2. Effect of Leaf Age and Radiation Exposure on Survival of E. coli O157:H7 on Spinach - A study was conducted in an organically managed High Tunnel system to determine the survival of E. coli O157:H7 on 'Whale' cultivar of spinach relative to leaf age and exposure to radiation. High tunnels are polyethylene film-covered growing structures that provide a semi-protected growing environment for certain fruits and vegetables. The population of E. coli declined from initial inoculated concentrations of approximately 175,000 colony-forming units per leaf to an average of 71 most probable number (mpn) per leaf in 7 days. Populations of E. coli continued to decline to very low to undetectable numbers per leaf up through 28 days. On average, older leaves partially shaded and sometimes in contact with moist soil had greater populations, 48 mpn per leaf, than upper (intermediate age) or crown (youngest age) leaves, less than 1 mpn per leaf by day 28. These results show a rapid population die-off of E. coli on upright leaves (upper and crown leaves which became the upper leaves as plants grew) within the high tunnel environment. (National Program 108 Component 1.1 Pathogens, Toxins and Chemical Contaminants Pre-harvest; Problem Area 1.1.3 – Ecology, Host Pathogen and Chemical Contaminants Relationships)

3. Persistence of Enterohemorrhagic and Non-Pathogenic E. coli on Spinach Leaves and in the Rhizosphere - No studies have examined how long E. coli O157:H7 can survive under field conditions on spinach leaves. Survival of E. coli O157:H7 and non-pathogenic E. coli was evaluated on spinach plants and in organic soil in a growth chamber that simulates field conditions. Populations of E. coli O157:H7 survived for a shorter duration on spinach shoots than in soil. Non-pathogenic E. coli were detected intermittently on spinach up to 28 days. These populations were not affected by the addition of fertilizer close to day 14 of the study. Results suggest survival of these E. coli O157:H7 and non-pathogenic strains were similar on leaves and in rhizosphere soil. (National Program 108 Component 1.1 Pathogens, Toxins and Chemical Contaminants Pre-harvest; Problem Area 1.1.3 – Ecology, Host Pathogen and Chemical Contaminants Relationships)

4. Effect of Green Manure on Survival of E. coli O157:H7 in Soil - Extracts of fresh leaf spinach are known to support growth of E. coli O157:H7, but it is not known to what extent this organism will grow or survive on spinach leaves that remain in the field after harvest of the crop and soil tillage. A growth chamber study was conducted for 35 days to determine the survival of E. coli O157:H7 in soil containing organic residue of spinach leaves with and without organic residue of cabbage leaves or daikon radish root. These crops were included because they are known to release a group of biologically active anti-bacterial isothiocyanic esters, and could be potential biofumigants. Results showed a rapid decline in E. coli populations on spinach leaves in all soils. There was a trend toward greater declines in E. coli populations over time in soils with spinach leaves and cabbage leaf residue than in soil with spinach leaves and daikon root or no additional crop residue. The results show that some crop residues can influence survival and die-off of E. coli O157:H7 populations in soils on a short-term basis and may have potential use as biofumigants to accelerate decline and persistence of this organism in some cropping systems. (National Program 108 Component 1.1 Pathogens, Toxins and Chemical Contaminants Pre-harvest; Problem Area 1.1.4 – Intervention strategies)

5. Prevalence of Pathogenic and Spoilage Microorganisms in Organic and Conventional Tomato Plants - Tomato petiole and stem print samples were obtained from a single conventional production system over 3 years using this sap collection method. No E. coli, salmonellae, or Listeria were recovered from tomato petiole sap or stem prints from this single field study or from petiole sap samples from organic and conventional fields where compost, creek/pond water, and other field practices were used. Pseduomonas, Xanthomonas, Chryseomonas, Curtobacterium, and Microbacterium spp. were isolated from samples. The few Enterobacteriaceae isolates obtained from petiole sap and stem prints included: a single isolate of Enterobacteri intermedius, two isolates of Erwinia chrysanthemi, and several isolates of Pantoea agglomerans. The numbers and types of bacteria on roots were significantly greater than those obtained from petioles of these same plants. Bacterial populations on calyx and stem scars of fruits from these plants were significantly greater than those from petiole sap. Results indicate that culturable bacteria are commonly present inside plant sap although at relatively low levels in comparison with roots and calyx/stem areas of tomato fruits. The stem area of tomato fruits is a site of potential survival and growth for bacteria and subsequent entry into fruits. Biofilms can easily develop in this part of the fruit when water collects there during fruit maturation. (National Program 108 Component 1.1 Pathogens, Toxins and Chemical Contaminants Pre-harvest; Problem Area 1.1.3 – Ecology, Host Pathogen and Chemical Contaminants Relationships)

6. Microbiological Assessment of Commercially Available Composts - A study was conducted to characterize the microbial quality of finished, marketable compost prepared from a wide range of organic residuals from 15 U.S. commercial facilities. Whole cell FAME profiles of 261 E. coli isolates were used to select 183 isolates for virulence factor screening by multiplex PCR assays targeted to stx1, stx2 and eae genes were conducted on the selected isolates. Results show 53 percent and 6.7 percent of compost facilities had product that exceeded the USEPA 503 limit for Class A product: fecal coliforms greater than 1000 MPN per g and Salmonella greater than 3 MPN per 4g, respectively. One E. coli isolate (0.55 percent) positive for stx2 was recovered from sewage sludge compost that met the EPA503 standards. In total, 67 percent of samples were positive for E. coli and 78.7 percent of samples met the fecal coliform standard. Prevalence of toxigenic E. coli in commercial composts was very low despite the relatively high number of samples that contained E. coli. All facilities use outdoor compost systems in which most, but not all, particles are subjected to pathogen destructive thermal process time-temperatures. Data in this study show that commercial composts that meet the fecal coliform and Salmonella standards may still contain low levels of pathogenic E. coli. Pathogen content data are needed to help ensure selection/use of quality composts by fresh produce growers. (National Program 108 Component 1.1 Pathogens, Toxins and Chemical Contaminants Pre-harvest; Problem Area 1.1.3 – Ecology, Host Pathogen and Chemical Contaminants Relationships)

7. Biocontrol of Escherichia coli O157:H7 on Fresh-cut Lettuce and Cantaloupe by Treatment with Bacteriophage - The effectiveness of a mixture of bacteriophages, viruses that can kill pathogenic foodborne bacteria, was examined to kill E. coli O157:H7 on cut pieces of iceberg lettuce and cantaloupe stored at 4 deg C. The bacteriophage treatment reduced populations of E. coli O157:H7 immediately upon application to lettuce, and reduced populations on cantaloupes occurred over 7 days. A meeting of industry experts at the International Lettuce and Leafy Greens Research Conference indicated that a kill-step for fresh produce was of the highest priority of the industry. This study is the first to show the effectiveness of a bacteriophage mixture to kill E. coli O157:H7 on fresh-cut lettuce and cantaloupes at refrigerated temperatures. In another study in collaboration with North Carolina A&T University, procedures were developed to use a bacteriophage mixture to kill E. coli O157:H7 on the surface of lettuce leaves. A 2-log reduction was achieved on the surface of lettuce pieces. These methods will be standardized for the application of bacteriophage to other produce commodities. (National Program 108 Component 1.2. Pathogens, Toxins and Chemical Contaminants Postharvest; Problem Area 1.2.4 – Processing Intervention Strategies)

8. Survival and Growth of Escherichia albertii on Fresh-cut Lettuce and in Ground Meats (beef and turkey) Stored at Various Temperatures - Escherichia albertii is a lesser known pathogen but may have the ability to cause foodborne illness, and has been found in the rhizosphere of tomato plants. These strains had been previously evaluated for phenotypic properties related to pressure, acid and heat tolerance. The behavior of three strains of E. albertii on fresh-cut iceberg lettuce, ground turkey, and ground beef stored at different temperatures was evaluated. E. albertii 9194 showed marginal growth when stored at 5 deg C, indicating that this strain of E. albertii may be more persistent at low temperatures than other strains. As expected, all strains of E. albertii grew on all commodities at abusive temperatures. This is the first examination of E. albertii on fresh-cut produce or in ground meat products. This work was done in collaboration with University of Georgia and Texas A&M University. (National Program 108 Component 1.1 Pathogens, Toxins and Chemical Contaminants Pre-harvest; Problem Area 1.1.3 – Ecology, Host Pathogen and Chemical Contaminants Relationships)


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of Non-Peer Reviewed Presentations and Proceedings3
Number of Newspaper Articles and Other Presentations for Non-Science Audiences2

Review Publications
Sharma, M., Mudd, C.L., Eastridge, J.S. 2008. Effective household disinfection methods of kitchen sponges. Food Control. http://dx.doi.org/10.1016/j.foodcont.2008.05.020

Last Modified: 9/10/2014
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