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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
2011 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
The persistence of Salmonella strains, isolated from produce vs. poultry, on spinach leaves was evaluated. Salmonella were spray inoculated onto spinach plants and analyzed weekly. Salmonella were undetectable throughout the study when irrigation water was contaminated with low levels; however, Salmonella were recovered every week from spinach leaves when inoculated at high levels. Salmonella persisted at significantly higher numbers when contaminated with produce isolates. Total Organic carbon (TOC) levels were evaluated for the ability to support the growth and survival of E. coli O157:H7 on spinach leaves at temperatures similar to those encountered in a typical growing cycle. At 0 ppm TOC levels, low and high populations of E. coli O157:H7 survived for 6 and 13 days, respectively; while at 12 and 120 ppm TOC, low populations reached 7-8 log CFU/ml by 6 days, while high populations reached 7-8 log CFU/ml by day 3. TOC levels did not affect the survival of E. coli O157:H7 when introduced to foliar surfaces. The survival, persistence, and dispersal of E. coli O157:H7, Salmonella, and Listeria in frass of hornworm larvae, that had ingested a one-time dose of 100, 1000 or 10000 bacteria, was determined. All three bacteria survived the gut of Manduca without causing harm to the larvae. Bacterial survival and growth was dose-dependent and generally persisted in frass at least three days after initial ingestion. The survival of Salmonella Newport on tomatoes co-inoculated with a naturally-occurring epiphytic bacillus strain TS-15 was evaluated. S. newport was significantly lower on tomatoes, leaves and blossoms after TS-15 application. Essential oils were evaluated as a potential produce wash treatment to reduce pathogens on fresh-cult produce. Sporan alone or in combination with acetic acid significantly reduced Salmonella and E. coli O157:H7 on spinach and fresh-cut lettuce without affecting color or texture. Caprylic acid (a GRAS ingredient) also reduced E. coli O157:H7 and Salmonella on fresh produce without affecting the produce quality.

Modified Atmospheric Packaging (MAP) conditions increased the expression of specific virulence factors in E. coli O157:H7 after three days of storage at 15°C. In summary, over the past 5 years of this project, we determined that contamination of fresh produce via soil was reduced by soil treatment with essential oils; biosand filters and zero valent Iron columns were effective in reducing Escherichia coli O157:H7 from irrigation water; E. coli O157:H7 and Salmonella persisted on fresh produce for longer duration when repeatedly contaminated at higher levels; E. coli O157:H7 and Salmonella attached to produce surfaces within 5 min of contact time, although attachment varied with strain, produce type, produce surface, and contact time; E. coli O157:H7 strains expressing curli fibers attached more strongly to spinach leaves at significantly higher numbers than curli deficient strains; natural antimicrobials (Sporan, Cinnamaldehyde, caprylic acid) and bacteriophages significantly reduced pathogen contamination from fresh produce surfaces.


4.Accomplishments
1. Survival and dispersion of E. coli O157:H7, Salmonella, and Listeria from hornworms on tomato. Insects are suspected of vectoring enteric pathogens on fresh produce. Larvae of Manduca sexta L. (hornworms) are common, well-camouflaged pests of tomato that chew the leaves and deposit their frass onto leaves and fruits of the plants. In laboratory studies with laboratory-reared hornworms, we investigated the survival, persistence, and dispersal of E. coli O157:H7, Salmonella, and Listeria in frass produced by hornworm larvae that ingested a one-time dose of 100, 1000 or 10000 bacteria. Results of multiple trials show that all three bacteria not only survived the gut of Manduca, but also grew without harm to the larvae. Bacterial survival and growth was dose-dependent and generally persisted in frass at least three days after initial ingestion of the dosed tomato leaves. Amplification and dispersion of the bacteria from larvae and frass onto tomato foliage and fruits was visualized with a fluorescent tracer, before and after simulated rainfall. This study provides clear evidence of the role of hornworm larval feeding and frass in amplifying and dispersing E. coli, Salmonella, and Listeria onto tomato plants and fruits.

2. Epiphytic Bacillus as a biocontrol for Salmonella newport on whole tomato plants. Tomatoes have been implicated in foodborne outbreaks of Salmonella newport and other Salmonella serotypes. Long-term intervention measures to reduce Salmonella prevalence on tomatoes in pre- and post-harvest environments are needed. In this study we tested in situ a previously isolated and characterized naturally-occurring epiphytic bacillus strain, TS-15, as a biocontrol for Salmonella newport using an attenuated strain inoculated onto whole tomatoes, leaves, and blossoms of tomato plants in an insect-screened high tunnel with a split-plot design. Survival of Salmonella after challenge was measured on samples with and without antagonist at days 0, 1, 2, 3, and 5 for blossoms and 6 for tomatoes and leaves, respectively. After the antagonist was applied onto the tomatoes, leaves, and blossoms of tomato plants, the concentration of S. Newport was significantly lower compared with controls. Few plants (less than 10 percent) had detectable levels of Salmonella in blossoms by day 5. The rate of decrease in bacterial concentration was significantly greater on leaves and blossoms inoculated with the antagonist than on those without the antagonist. However, no statistically significant difference was found in the mortality rate of Salmonella on tomato fruits in our study. This study provides evidence that the antagonist TS-15 is highly effective in reducing Salmonella newport on tomato plants and has potential for reducing Salmonella in field production of tomatoes.

3. 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 cinnamaldehyde and Sporan to reduce pathogens on spinach leaves. E.coli O157:H7 was more sensitive to antimicrobials than Salmonella. Sporan (800 ppm) alone or in combination with 200 ppm acetic acid significantly reduced Salmonella and E. coli O157:H7 on spinach leaves. Higher concentration of cinnamaldehyde (1000 ppm) was required to significantly reduce E. coli O157:H7. Populations of these pathogens reduced during storage of 14 days at 4°C. The color and texture quality of Sporan treated spinach was similar (P> 0.05) to control (water treated) leaves. Results show that Sporan, a natural GRAS antimicrobial could be used as an intervention to control pathogens in fresh-cut produce.

4. Caprylic acid to control pathogen on fresh produce. The potential use of caprylic acid (CA) for reducing E. coli O157:H7 and Salmonella on spinach and lettuce was evaluated. Fresh produce (spinach, iceberg and romaine lettuce) samples were inoculated with a cocktail of five E.coli O157:H7 or Salmonella strains, air dried for 30 min, and then treated with sodium caprylate (10, 25, and 50 ppm) or chlorine (50 ppm) for 1 min. Caprylic acid at 10 ppm reduced E. coli O157:H7 and Salmonella by 3.5 and 2.2 log cfu/g, respectively on Iceberg lettuce. The antimicrobial activity of caprylic acid was not significant on spinach leaves when washed with 10 ppm caprylic acid. Higher concentrations (25 and 50 ppm) of caprylic acid reduced these pathogens to undetectable level; however, the produce color was adversely affected at higher concentrations. A 5 ppm caprylic acid reduced E. coli O157:H7 and Salmonella by 2 and 1 log cfu/g, respectively, without affecting the color and texture of fresh produce. Caprylic acid, a GRAS ingredient can be used in conjunction with chlorine to enhance the pathogen reduction on fresh produce.

5. Bacteriophage to reduce pathogens on fresh produce. Lettuce sprayed with bacteriophages specific for E. coli O157:H7 (3.09 log PFU/cm2) and then inoculated with E. coli O157:H7 contained less E. coli O157:H7 than lettuce sprayed with phosphate buffered saline (PBS) and inoculated with the pathogen. E. coli O157:H7 counts were reduced by 1.3 log CFU/cm2 after 1 day at 4oC. Bacteriophage-treated lettuce had consistently lower E. coli O157:H7 populations than the control-treated lettuce over 7 days stored at 4oC. This work indicates that bacteriophage treatment may provide cut lettuce some protection against cross contamination with E. coli O157:H7.


Review Publications
Shelton, D.R., Karns, J.S., Coppock, C.R., Patel, J.R., Sharma, M., Pachepsky, Y.A. 2011. Comparison of generic E. coli vs. pathogenic E. coli virulence factors in an agricultural watershed: implications for irrigation water standards and leafy green commodities. Journal of Food Protection. 74:18-23.

Yossa, N., Patel, J.R., Millner, P.D., Lo, M. 2011. Inactivation of Salmonella in Organic Soil by Cinnamaldehyde, Eugenol, Ecotrol, and Sporan. Foodborne Pathogens and Disease. 8(2):311-317.

Patel, J.R., Sharma, M., Ravishankar, S. 2011. Effect of curli expression and hydrophobicity of E. coli O157:H7 on attachment to fresh produce surfaces. Journal of Applied Microbiology. 110:737-745.

Ingram, D.T., Patel, J.R., Sharma, M. 2011. The Effect of Repeated Irrigation with Water Containing Varying Levels of Total Organic Carbon on the Persistence of Escherichia coli O157:H7 on Baby Spinach. Journal of Food Protection. 74(5):709-717.

Mattson, T., Johny, A., Amalaradjou, M., More, K., Schreiber, D., Patel, J.R., Venkitanarayanan, K. 2011. Inactivation of Salmonella spp. on tomatoes by plant molecules. International Journal of Food Microbiology. 44(3):464-468.

Sharma, M., Lakshman, S., Ferguson, S.E., Ingram, D.T., Luo, Y., Patel, J.R. 2011. The effect of modified atmosphere packaging on the persistence and expression of virulence factors of Escherichia coli O157:H7 on shredded iceberg lettuce. Journal of Food Protection. 74(5):718-726.

Last Modified: 10/23/2014
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