Progress 04/01/11 to 03/16/16
Outputs
Progress Report 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. 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. In this final year of the five-year project, significant progress was made on all objectives to be addressed in FY16. A naturally-occurring epiphytic bacillus strain was highly effective as biocontrol in reducing Salmonella Newport to whole tomatoes, leaves, and blossoms of tomato plants (objective 1). Significant reductions of Salmonella were observed on leaves and blossoms inoculated with biocontrol strain. Experiments performed for objective 1 demonstrated attachment mechanism(s) of enteric pathogens on fresh produce. The persistence of E. coli O157:H7 on spinach leaves was dependent on spinach cultivars. Curli-expressing E. coli O157:H7 persisted at significantly higher level on spinach leaves of Waitiki cultivars. E. coli O157:H7 persistence was influenced by roughness and stomata density of spinach leaves. For objective 3 on biofilm, our study revealed that cellulose formation by E. coli O157:H7 was not required for stronger attachment to spinach leaf. Biofilm formation varied with type of fresh produce; and cut surface of fresh produce facilitated increased biofilm formation of E. coli O157:H7. Role of virulence factors was elucidated (objective 3) where E. coli O157:H7 strains lacking the intimin (eae) virulence factor declined to lower numbers on microgreens than the wild-type strain, but the level of expression of eae did not increase during attachment to microgreen tissue. To improve the microbial safety of fresh produce, the sanitizer efficiency was increased using chlorine stabilizer (objective 4). The washing aid T128 significantly increased the efficacy of chlorine wash against bacterial cross contamination while maintaining the quality of leafy green vegetables under real world fresh-cut processing conditions. Experiments for objective 4 demonstrated that packaging conditions could affect the expression of E. coli O157:H7 virulence factors on shredded lettuce. Conditions representing commercial packaging conditions and refrigerated temperatures did not promote the expression of virulence factors of E. coli O157:H7, but abusive temperatures and ambient conditions led to increased expression of these factors. This is the final report for the project 8042-32420-005-00D terminating September 2016. Key findings of the project are as follows. Avian pathogenic E. coli isolated from poultry broilers and manure survived for longer durations on basil, spinach and lettuce compared to E. coli O157:H7 when simultaneously inoculated on to these leafy green commodities. Salmonella isolated from fresh produce persisted at significantly higher numbers on spinach plants when sprayed with contaminated water compared to Salmonella isolated from poultry. Zero- valent iron (ZVI) filtration of irrigation water contaminated with E. coli O157:H7 was effective in reducing and killing bacteria in the water. Cellular appendages, such as curli fibers were involved in E. coli attachment and persistence in fresh produce. Natural antimicrobials such as cinnamaldehyde and sporan were good alternative to chemical sanitizers as a produce wash to kill bacterial pathogens on organic fresh produce. The sanitizing effect of free chlorine in removing Salmonella biofilms was enhanced significantly when chlorine was combined with T128 stabilizer. The results of the study have been used by the regulatory agencies U.S. Food and Drug Administration (FDA) and California Leafy Green Marketing Association (LGMA) to update GAPs of commodity specific guidelines at the pre-harvest level. Fresh-cut produce industry has adopted new washing aid T28 to improve the chlorine sanitizer efficiency. Accomplishments 01 Turkey manure dust particles extend Salmonella persistence on spinach. The survival of Salmonella in turkey manure dust (TMD) alone and in manure dust on spinach plants was assessed. Results showed TMD protected Salmonella on spinach leaves from inactivation from ultraviolet light more than water, allowing Salmonella to survive for longer periods. Salmonella also survived for longer durations (291 days) in manure dust, with the greatest survival in dust with the smallest particle size and the lowest moisture content. This work is helpful to growers in selecting buffer zones between farm and animal rearing facilities to minimize airborne contamination of Salmonella to proximate leafy green farms.
Impacts
(N/A)
Publications
- Sharma, M., Ingram, D., Graham, L. 2016. Foodborne outbreaks and potential routes of contamination in fresh and fresh-cut fruits and vegetables. Foodborne Diseases. p. 19-36.
- De Frias, J.A., Luo, Y., Kou, L., Zhou, B., Wang, Q. 2015. Improving spinach quality and reducing energy costs by retrofitting retail open refrigerated cases with doors. Postharvest Biology and Technology. 31(8) :114-120.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report 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. 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. Field studies were conducted to investigate novel pre-harvest interventions for control of pathogens on melons. Cantaloupes were grown at Beltsville Agricultural Research Center (BARC) high tunnels using organic farming practices. Cantaloupes were inoculated at the farm with L. innocua and E. coli O157:H12 and then sprayed with low concentration of octenidine dihydrochloride (OH). Results on surviving populations of these surrogate pathogens on cantaloupes harvested during 28 days revealed that 0.2% OH was effective in killing both E. coli O157: H12 and L. innocua to non-detectable levels. Greenhouse studies showed the E. coli survived at significantly higher populations in soils amended with poultry litter compared to either unamended soils or soils amended with liquid dairy manure. Greenhouse studies also showed that E. coli survival was influenced by different factors when studies were conducted in large pots versus small pots. In large pots, E. coli survival was significantly higher in poultry litter amended soils compared to unamended and liquid dairy manure amended soils but was not influenced by soil type. However, E. coli populations in small pots was influenced both by manure and soil type. Most attention on E. coli in biological soil amendments has focused on the presence of enterohemorrhagic E. coli (EHEC) but extraintestinal pathogenic E. coli (ExPEC) are a subset of E. coli capable of causing disease outside of the human gut and include uropathogenic (UPEC) and avian pathogenic E. coli (APEC). Of the 75 E. coli isolates recovered from finished compost, a thermally-treated nutrient-stabilized material commonly used as fertilizer in agricultural setting, 23% of isolates were determined to be APEC, 40% of E. coli isolates contained two APEC virulence factors, while no isolates were determined to be EHEC. This work indicates pathotypes of E. coli other than EHEC may be present in compost. The rpoS gene in the bacterial pathogens E. coli O157:H7 and Salmonella typhimurium may allow these organisms to survive for extended periods on spinach. Our study comparatively evaluated Salmonella and E. coli O157:H7 strains which do and do not possess the rpoS gene on spinach plants, along with the survival of murine norovirus (MNV) and Tulane Virus (TV), a surrogate for the viral pathogen norovirus. Salmonella and E. coli O157:H7 populations declined rapidly on foliar surfaces of spinach plants, regardless of rpoS status. Wild-type S. typhimurium populations declined less than other isolates co-inoculated on the plant surface over 4 days. MNV and TV titers remained relatively stable over the same period of time. With an increased incidence of foodborne illness outbreaks associated with fresh produce, the development of natural alternative sanitizers is gaining importance. The antimicrobial activity of clove bud oil in combination with grape seed or olive extract was evaluated against multi- drug resistant S. enterica on organic leafy greens. Antimicrobial wash treatment significantly reduced Salmonella on organic iceberg and romaine lettuce, and on spinach during storage at 4�C. The combination of clove bud oil and olive extract was more effective than the combination with grape seed extract. The results of this study demonstrate the potential use of combination treatments as alternative sanitizers for the organic produce industry to achieve effective antimicrobial activity. Accomplishments 01 Physical covering to kill enteric pathogens during composting process. Current composting practices provide minimal assurance that finished compost is free from enteric pathogen contamination. The effect of 30- cm covering of finished compost (FC) on survival of Escherichia coli O157:H7 and Salmonella in active, static and windrow composting systems was investigated. Salmonella were reduced significantly within 24 h in windrow piles and were below the detection limit after 3 and 7 days at internal locations of windrow and static piles containing FC covering, respectively. Likewise, E. coli O157:H7 were undetectable after one day in windrow piles covered with finished compost. Finished compost covering of compost piles aids in reduction of pathogens during the composting process. The research is currently used by leafy green trade associations. 02 Novel device to study pathogen inactivation kinetics with sanitizer. Scientists in Beltsville, Maryland, invented, fabricated, and tested a novel micro-fluidic mixer to accurately determine time-dose responses of pathogen inactivation with chlorine solutions from 0.1 second to 5 minute. This invention provides a valuable tool for researchers to determine pathogen inactivation kinetics in the sub-second time scale, filling the void of lab equipment needed to study pathogen inactivation kinetics in fractions of a second. The device is currently used by the Centers for Disease Control and Prevention (CDC) and the University of Maryland. 03 Comparative evaluation of E. coli O157:H7 detection procedures in compost. Two rapid immunomagnetic separation (IMS) protocols were evaluated by scientists in Beltsville, Maryland, to recover inoculated E. coli O157:H7 (1-2 log CFU/g) from 30 different commercial, finished compost samples. Both protocols detected E. coli O157:H7 in compost samples; however Polymerase Chain Reaction (PCR) techniques required the removal of inhibitors to reduce the possibility of a false negative result. Results showed that IMS protocols can be used to rapidly detect (within 8 h) E. coli O157:H7 in compost. The research will be helpful to compost industries, compost trade associations, and growers of the organic fresh produce. 04 Natural antimicrobials to reduce enteric pathogens on fresh produce. Caprylic acid is a GRAS (Generally Recognized As Safe) commonly found in coconut oil. Caprylic acid was evaluated as a produce wash to reduce E. coli O157:H7 and Salmonella on lettuce and spinach. Up to 5 log reductions in these pathogens was observed using 25 ppm caprylic acid. It was effective at low concentration (5 ppm) in reducing pathogens without affecting produce texture quality. The findings will help in exploring novel natural antimicrobials as a produce wash for organic fresh-cut produce.
Impacts
(N/A)
Publications
- Zhang, B., Luo, Y., Zhou, B., Millner, P.D. 2015. A novel micro fluidic mixer-based approach for determining inactivation kinetics of Escherichia coli O157:H7 in free chlorine solutions. Food Microbiology. 49: 152-160.
- Huang, J., Luo, Y., Nou, X. 2015. Growth of Salmonella and Listeria monocytogenes on fresh-cut cantaloupe under different temperature abuse scenarios. Journal of Food Protection. 78:1125-135.
- Lin, B., Luo, Y., Zhang, Z., Zhang, B., Zhou, B., Wang, Q. 2015. Development of silver/titanium dioxide/chitosan adipate nanocomposite as an antibacterial coating for fruit storage. LWT - Food Science and Technology. 63:1206-1213.
- Patel, J.R., Yossa, I., Macarisin, D., Millner, P.D. 2015. Physical Covering to control Escherichia coli O157:H7 and Salmonella in Static and Windrow Composting Process. Applied and Environmental Microbiology. 81:2063-2074.
- Patel, J.R., Keelara, S., Kumar, V.N. 2015. Reduction of Escherichia coli O157:H7 and Salmonella on fresh-cut produce by caprylic acid. Journal of Food Processing and Preservation. DOI:10.1111/jfpp.12468.
- Zhou, B., Luo, Y., Nou, X., Lyu, S., Wang, Q. 2015. Survival dynamics of Salmonella enterica, Listeria monocytogenes, and Escherichia coli in wash water during simulated chlorine depletion and replenishment processes. Food Microbiology. 50:88-96.
- Zhou, B., Luo, Y., Nou, X., Yang, Y., Wu, Y., Wang, Q. 2014. Effects of post-harvest handling conditions on internalization and growth of Salmonella enterica in tomatoes. Journal of Food Protection. 77:365-370.
- Zhang, B., Luo, Y., Aplin, J., Liu, Y., Bauchan, G.R., Payne, G.F., Wang, Q., Nou, X., Millner, P.D. 2015. Fabrication of biomimetically-patterned surfaces and their application to probing plant-bacteria interactions. ACS Applied Materials and Interfaces. 6(15):12467-12478.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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. An innovative Minimum Biofilm Eradication Concentration (MBEC) biofilm procedure was employed to investigate the efficacy of natural antimicrobials in removing biofilms formed by environmental isolates of Salmonella. Biofilm formation varied with strain; Salmonella reduction in biofilms was significantly influenced by natural antimicrobial concentrations. Cinnamaldehyde at 3000 ppm was comparable to 25 ppm chlorine in removing biofilms on polystyrene surfaces. Field studies were conducted to investigate antimicrobial activity of glucosinolate-derived compounds (GDC) from Brassica family plants (broccoli) in soil. Till-over of broccoli remnant following harvest served as antimicrobial in reducing inoculated E. coli O157:H12 in soil. Low concentration of Benzyl isothiocyanate (BIT, 0.039%) as a spray was superior to 1% natural plant antimicrobials in reducing E. coli O157:H12 populations in soil. The organism was undetectable after 42 days whereas it survived in untreated soil during 14 weeks of study. Understanding the interaction between plant surface and human pathogens is important to developing effective intervention technologies to remove and inactivate pathogens. A two-step replica molding method was developed for rapid fabrication of biomimetically patterned surfaces that mimic the surface topography of true plant leaves. The polydimethylsiloxane and agarose specimens reproduced leaf surface microstructures with unique chemical, physical, and biological features. The method facilitated reproducibility of experiments involving disinfection and attachment/ release of microbes from surfaces with microstructure identical to real produce surfaces. Accomplishments 01 Persistence of poultry Salmonella isolates on spinach plants. Poultry isolates of Salmonella could be a source of produce-borne illness if produce is contaminated through the use of poultry manure applied at the farm. Spinach grown in a controlled environment was spray-irrigated with water containing Salmonella isolated from poultry or produce and analyzed periodically for persistence on spinach leaves. 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. 02 Persistence of E. coli O157:H7 on leaves of different spinach cultivars. Pathogen attachment on spinach leaf surface could be influenced by the leaf structure of the cultivar. Four spinach cultivars (Emilia, Lazio, Space, and Waitiki) were grown in a controlled environment and spray-irrigated with water containing E. coli O157:H7. 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. 03 Role of plant cultivation conditions on E. coli internalization into spinach. The effect of hydroponic production of fresh produce on E. coli internalization requires evaluation since hydroponic farming practices have increased in recent years. Spinach grown in hydroponics and in soil were contaminated with E. coli O157:H7. Fractions of spinach plants (roots, stems, and leaves) were analyzed at specific intervals for E. coli internalization into spinach plant fractions. 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. Internalization was observed in greater numbers when plants were grown in soil as opposed to grown hydroponically. Curli expression by E. coli O157:H7 and spinach cultivar did not affect its root uptake by spinach plants.
Impacts
(N/A)
Publications
- Patel, J.R., Singh, M., Macarisin, D., Sharma, M., Shelton, D.R. 2013. Differences in biofilm formation of produce and poultry Salmonella enterica isolates and their persistence on spinach plants. Food Microbiology. 36:388-394.
- Todd, J., Friedman, M., Patel, J.R., Jaroni, D., Ravishankar, S. 2013. The antimicrobial effects of cinnamon oil against multi-drug resistant Salmonella Newport on organic leafy greens. International Journal of Food Microbiology. 166:193-199.
- Landa-Salgado, P., Hernandez Anguiano, A.M., Vargas Hernandez, M., Eslava Campos, C., Chaidez Quiroz, C., Patel, J.R. 2013. Persistence of salmonella Typhimurium in Nopal. Revista Fitotecnia Mexicana. 36(2):147- 153.
- Zhou, B., Luo, Y., Nou, X., Millner, P.D. 2014. Development of a feed- forward sodium hypochlorite dosing scheme to maintain stable free chlorine concentration during simulated produce wash operations. Journal of Food Protection. 77:558-566.
- Zhou, B., Luo, Y., Turner, E.R., Yang, Y., Wang, Q., Schneider, K. 2014. Evaluation of current industry practices for maintaining tomato dump tank water quality during packinghouse operations. Journal of Food Processing and Preservation. DOI: 10.1111/jfpp.12200.
- Macarisin, D., Patel, J.R., Sharma, V.K. 2014. Role of curli and plant cultivation conditions on Escherichia coli O157:H7 internalization into organic spinach grown on hydroponics and in soil. International Journal of Food Microbiology. 173:68-53.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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.
Impacts
(N/A)
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.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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: strains developed stronger association with spinach leaf surfaces, where curli-deficient mutants attached to spinach at significantly (P < 0.01) lower numbers. Attachment of cellulose-impaired mutants to spinach leav was not significantly different from that of curliated strains. The relative attachment strength of E. coli O157:H7 to spinach increased wit 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 cellulo was not required to develop strong attachment to spinach leaf. These results indicate that curli fibers are essential for strong attachment o E. coli O157:H7 to spinach where as cellulose is dispensable. 02 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 t chemicals. The antimicrobial effect of apple, hibiscus, olive, and lemongrass extract was evaluated against Salmonella on organic romaine a iceberg lettuce, and on spinach. Olive extract exhibited significant antimicrobial effect, resulting in up to 3 log CFU/g Salmonella reductio followed by lemongrass and apple extract. The antimicrobial effect of hibiscus extract was marginal. The antimicrobial effect of these extract increased with exposure/contact time. Plant extracts were more effectiv 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. 03 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 sanitizin effect of free chlorine (1.0-5.0 mg per L) was enhanced significantly wh 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. 04 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 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 500-100 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 reduci food safety risks associated with fresh cantaloupes.
Impacts
(N/A)
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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. Persistence of coliphages, a potential indicator for fecal contamination of water, was evaluated on spinach. Coliphages (MS2 and phiX174) were inoculated on to spinach plants and recovery and enumeration techniques for phages were evaluated and optimized. The use of beef extract recovered slightly but significantly more phiX 174 phages than either sterile water or phosphate buffered saline from growing spinach plants after 20 days. When low levels of phage titers were applied to spinach plants, phiX 174 phages declined more rapidly on the surface of spinach plants than MS2 phage; however, at higher phage levels, MS2 phages declined more rapidly than phiX174 phages. The role of curli (a bacteria surface appendage) was investigated relative to its function in bacterial attachment to produce surfaces. The curli deficient mutants didn�t express curli at 20, 26, and 37�C, whereas wild strain and curli +ve strain developed a clear red phenotype on CR medium at 20 and 27�C. Curli deficient mutants attached to spinach leaves at significantly lower numbers than the wild strain. Lack of cellulose expression did not affect its attachment to spinach leaves. Confocal microscopy analysis of inoculated spinach revealed that bacteria firmly persisting on leaf surface were surrounded by an extracellular matrix recognized by rabbit anti-curli antibodies.
Impacts
(N/A)
Publications
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