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United States Department of Agriculture

Agricultural Research Service


Location: Environmental Microbial & Food Safety Laboratory

2012 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:
An evaluation of non-pathogenic E. coli and attenuated E. coli O157:H7 in various manures (dairy, poultry litter, dairy manure liquids, horse) applied to fields was conducted at two different field sites in Maryland. The seasonality, type of manure, and soil-type affected the survival of E. coli in field environments. Results show that the persistence of non-pathogenic E. coli may be a good indicator for the persistence of E. coli O157:H7 in raw manure applied to fields. A field-scale system was utilized to evaluate the effectiveness of a biosand filter (S), and a biosand filter incorporated with zero valent iron (ZVI) in decontaminating irrigation water. Filtered waters were subsequently overhead irrigated to spinach plants. ZVI-filtered water contained significantly less E. coli O157:H12 than S-filtered water, and spinach irrigated with ZVI-irrigated water also contained less bacteria than that irrigated with sand-filtered water. Zero-valent iron filtration may provide a low cost, efficient mitigation treatment for irrigation water to be used by small-scale farmers. Lytic bacteriophages specific for E. coli O157:H7 were able to prevent cross-contamination of fresh-cut lettuce. Lytic bacteriophages were sprayed with or immersed into lytic bacteriophages solutions, and then subsequently exposed to E. coli O157:H7. E. coli O157:H7 declined more rapidly on the bacteriophage-treated lettuce than the control (no bacteriophages) lettuce stored at 4°C for 7 days. These results indicate that lytic bacteriophages can help reduce E. coli O157:H7 contamination on fresh cut lettuce during refrigerated storage. Essential oils were evaluated as a potential produce wash treatment to reduce pathogens on romaine and iceberg lettuce. Cinnamaldehyde and Sporan significantly reduced E. coli O157:H7 and Salmonella on iceberg and romaine lettuce. 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. The revised Food and Drug Administration (FDA) Food Code now includes cut leafy greens among foods that require time and temperature control for safety (TCS) at or below 41°F (5°C). ARS scientists examined the display case operational parameters and retained store product handling schemes (thermostat settings, defrost schedules, produce locations within each shelf, stock rotations, type of case shelving) on temperature maintenance and produce quality and safety, and also determined that the commonly-used infrared thermometer technology is not an appropriate method for determining the product temperature of bagged leafy greens. Findings provide critical information to the industry for improving compliance with the FDA food code concerning TCS foods.

4. Accomplishments
1. Role of curli and cellulose expression in adherence of shiga-toxigenic E. coli to spinach leaves. Cellular appendages, such as curli fibers, and cellulose, a constituent of extracellular matrix, have been suggested to be involved in E. coli attachment and persistence in fresh produce. ARS researchers in Beltsville documented that curli-expressing E. coli O157:H7 strains developed stronger association with spinach leaf surfaces, whereas curli-deficient mutants attached to spinach at significantly (P < 0.01) lower numbers. Attachment of cellulose-impaired mutants to spinach leaves was not significantly different from that of curliated strains. The relative attachment strength of E. coli O157:H7 to spinach increased with incubation time for the curli-expressing strains. Scanning confocal microscopy (LSCM) analysis of inoculated leaves revealed that curli-expressing E. coli O157:H7 were surrounded by extracellular structures strongly immunostained with anti-curli antibodies. Production of cellulose was not required to develop strong attachment to spinach leaf. These results indicate that curli fibers are essential for strong attachment of E. coli O157:H7 to spinach where as cellulose is dispensable.

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 alternatives to chemicals. The antimicrobial effect of apple, hibiscus, olive, and lemongrass extract was evaluated against Salmonella on organic romaine and iceberg lettuce, and on spinach. Olive extract exhibited significant antimicrobial effect, resulting in up to 3 log CFU/g Salmonella reduction followed by lemongrass and apple extract. The antimicrobial effect of hibiscus extract was marginal. The antimicrobial effect of these extracts increased with exposure/contact time. Plant extracts were more effective in reducing Salmonella on romaine and iceberg lettuce than on spinach. This study demonstrates potential use of natural plant extract to reduce Salmonella on organic leafy greens.

3. Evaluation of washing aid in inactivation of Salmonella in biofilms. The effect of the washing aid, T-128, on inactivation of Salmonella enterica serovars Thompson or Newport, or Pseudomonas fluorescens populations in biofilms on stainless steel was evaluated under conditions of increasing organic matter loads. For both Salmonella and Pseudomonas, the sanitizing effect of free chlorine (1.0-5.0 mg per L) was enhanced significantly when combined with T-128. Application of T-128 decreased the free chlorine depletion rate caused by increasing organic matter in wash waters, and significantly augmented inactivation of bacteria in biofilms compared to treatments without T-128. Results show T-128 can aid in sanitizing stainless steel contact surfaces during fresh-cut produce processing.

4. Chlorine and washing aid to reduce pathogens in biofilms on cantaloupe rind. The efficacy of chlorinated water (CW) solutions, with or without the washing aid, T-128, on inactivation of natural microbial flora and enteric pathogen biofilms on cantaloupe rinds was evaluated. With free chlorine (FC) at 500-2000 mg per L, the sanitizing effect on the natural microflora and on the inoculated strains were enhanced significantly by 1.0-2.0 log CFU per square cm when combined with T-128 in the washing solutions. An additional significant reduction of 0.7-1.0 log CFU per square cm for S. Poona and E. coli O157:H7 was observed in CW at 500-1000 mg per L containing T-128 when rinds were brush-scrubbed during the washing. These results indicate that T-128 can aid in reducing pathogen viability in biofilms on cantaloupe rinds, and thereby can aid in reducing food safety risks associated with fresh cantaloupes.

Review Publications
Macarisin, D., Patel, J.R., Bauchan, G.R., Giron, J., Sharma, V.K. 2012. Role of curli and cellulose expression by Escherichia coli O157:H7 on the cell’s ability to attach to spinach. Foodborne Pathogens and Disease. 9(2):160-167.

Moore-Niebel, K., Gerber, C., Patel, J.R., Friedman, M., Ravishankar, S. 2012. Antimicrobial activity of lemongrass oil against Salmonella enterica on organic leafy greens. Journal of Applied Microbiology. 112:485-492.

Xia, X., Luo, Y., Yang, Y., Schneider, K., Meng, J. 2012. Effects of tomato variety, temperature differential and post-stem removal time on internalization of Salmonella Thompson into tomatoes. Journal of Food Protection. 75(2):297-303.

Patel, J.R., Macarisin, D., Sangly, G., Murphy, C.A. 2012. Inactivation and injury of pathogens on intact beef treated with hydrodynamic pressure. Innovative Food Science and Emerging Technologies. 14:38-45.

Moore, K., Patel, J.R., Jaroni, D., Friedman, M., Ravishankar, S. 2011. Antimicrobial activity of apple, hibiscus, olive, and hydrogen peroxide formulations against Salmonella enterica on organic leafy greens. Journal of Food Protection. 74(10):1676-1683.

Ingram, D.T., Callahan, M.L., Ferguson, S.E., Hoover, D., Shelton, D.R., Millner, P.D., Patel, J.R., Kniel, K., Sharma, M. 2012. The use of Zero-valent iron biosand filters to reduce E. coli O157:H12 in irrigation water applied to spinach plants in a field setting. Journal of Applied Microbiology. 112(3):551-560.

Pimstein, A., Eitel, J.U., Long, D.S., Mufradi, I., Karnieli, A., Bonfil, D.J. 2009. A spectral index to monitor the head-emergence of wheat in semi-arid conditions. Field Crops Research. 111:218-225.

Zhou, B., Feng, H., Luo, Y., Millner, P.D. 2012. Improved Design and Ultrasound-assisted Sanitation of Lettuce Harvesting Knives for Minimizing Escherichia coli O157:H7 Contamination. Journal of Food Protection. 75(3):563-566.

Yang, Y., Luo, Y., Millner, P.D., Turner, E.R., Feng, H. 2012. Assessment of Escherichia coli O157:H7 transference from soil to Iceberg Lettuce via a contaminated harvesting knife. International Journal of Food Microbiology. 153(3):345-350.

Last Modified: 05/27/2017
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