Cultivating Food Safety Together: Insights About the Future of Produce Safety in the U.S. Controlled Environment Agriculture Sector.

Controlled environment agriculture (CEA) is a rapidly growing sector that presents unique challenges and opportunities in ensuring food safety. This manuscript highlights critical gaps and needs to promote food safety in CEA systems as identified by stakeholders (n=47) at the Strategizing to Advance Future Extension andResearch (S.A.F.E.R.) CEA conference held in April 2023 at The Ohio State University's Ohio CEA Research Center. Feedback collected at the conference was analyzed using an emergent thematic analysis approach to determine key areas of focus. Research-based guidance is specific to the type of commodity, production system type, and size. Themes include the need for improved supply chain control, cleaning, and sanitization practices, pathogen preventive controls and mitigation methods and training and education. Discussions surrounding supply chain control underscored the significance of the need for approaches to mitigate foodborne pathogen contamination. Effective cleaning and sanitization practices are vital to maintaining a safe production environment, with considerations such as establishing standard operating procedures, accounting for hygienic equipment design, and managing the microbial communities within the system. Data analysis further highlights the need for risk assessments, validated pathogen detection methods, and evidence-based guidance in microbial reduction. In addition, training and education were identified as crucial in promoting a culture of food safety within CEA. The development of partnerships between industry, regulatory, and research institutions are needed to advance data-driven guidance and practices across the diverse range of CEA operations and deemed essential for addressing challenges and advancing food safety practices in CEA. Considering these factors, the CEA industry can enhance food safety practices, foster consumer trust, and support its long-term sustainability.

Use of a silver-based sanitizer to accelerate Escherichia coli die-off on fresh-cut lettuce and maintain produce quality during cold storage: Laboratory and pilot-plant scale tests.

Outbreaks and product recalls involving romaine and iceberg lettuce are frequently reported in the United States. Novel technologies are needed to inactivate pathogens without compromising product quality and shelf life. In this study, the effects of a process aid composed of silver dihydrogen citrate, glycerin, and lactic acid (SGL) on Escherichia coli and Listeria monocytogenes concentrations on lettuce immediately after washing and during cold storage were evaluated. Sensory and quality attributes of fresh-cut iceberg lettuce were also evaluated. Laboratory results indicated that application of SGL solution for 30 s as a first step in the washing process resulted in a 3.15 log reduction in E. coli O157:H7 immediately after washing. For E. coli O157:H7 a significant difference between SGL treatment and all other treatments was maintained until day 7. On day zero, SGL led to a 2.94 log reduction of L. monocytogenes. However, there was no significant difference between treatments with or without SGL regardless of storage time. Pilot-plant results showed that samples receiving SGL spray followed by chlorinated flume wash exhibited a greater reduction (1.48 log) in nonpathogenic E. coli populations at the end of shelf life than other treatments (p < 0.05). Additional pilot plant tests were conducted to investigate the hypothesis that SGL residues could continue to impact microbial survival on the final washed lettuce. Results show that pathogens introduced subsequent to flume washing of lettuce pretreated with SGL solution were not affected by antimicrobial residues. The final quality and shelf life of flume washed lettuce were also unaffected by pretreatment with SGL. In conclusion, the results of this study demonstrate that this new technology has the potential to accelerate E. coli die-off on fresh-cut lettuce during cold storage and improve product safety, while not affecting quality throughout the shelf life of the finished products.

Differential Survival of Escherichia coli and Listeria spp. in Northeastern U.S. Soils Amended with Dairy Manure Compost, Poultry Litter Compost, and Heat-Treated Poultry Pellets and Fate in Raw Edible Radish Crops.

ABSTRACT: Composted or heat-treated biological soil amendments of animal origin (BSAAOs) can be added to soils to provide nutrients for fresh produce. These products lower the risk of pathogen contamination of fresh produce compared with the use of untreated BSAAOs; however, meteorological conditions, geographic location, and soil properties can influence the presence of pathogenic bacteria or their indicators (e.g., generic Escherichia coli) and allow potential for produce contamination. Replicated field plots of loamy or sandy soils were tilled and amended with dairy manure compost (DMC), poultry litter compost (PLC), or no compost (NoC) over two field seasons and noncomposted heat-treated poultry pellets (HTPPs) during the second field season. Plots were inoculated with a three-strain cocktail of rifampin-resistant E. coli (rE. coli) at levels of 8.7 log CFU/m2. Direct plating and most-probable-number methods measured the persistence of rE. coli and Listeria spp. in plots through 104 days postinoculation. Greater survival of rE. coli was observed in PLC plots in comparison to DMC plots and NoC plots during year 1 (P < 0.05). Similar trends were observed for year 2, when rE. coli survival was also greater in HTPP-amended plots (P < 0.05). Survival of rE. coli depended on soil type, and water potential and temperature were significant covariables. Listeria spp. were found in NoC plots, but not in plots amended with HTPPs, PLC, or DMC. Radish data demonstrate that PLC treatment promoted the greatest level of rE. coli translocation compared with DMC and NoC treatments (P < 0.05). These results are consistent with findings from studies conducted in other regions of the United States, and they inform northeast produce growers that composted and noncomposted poultry-based BSAAOs support greater survival of rE. coli in field soils. This result has the potential to affect the food safety risk of edible produce grown in BSAAO-amended soils as a result of pathogen contamination.

Bacterial Community Dynamics Distinguish Poultry Compost from Dairy Compost and Non-Amended Soils Planted with Spinach.

The aim of this study was to determine whether and how poultry litter compost and dairy manure compost alter the microbial communities within field soils planted with spinach. In three successive years, separate experimental plots on two fields received randomly assigned compost treatments varying in animal origin: dairy manure (DMC), poultry litter (PLC), or neither (NoC). The composition and function of bacterial and fungal communities were characterized by the amplicon sequencing of marker genes and by the ecoenzyme activity, respectively. The temporal autocorrelation within and among years was adjusted by principal response curves (PRC) to analyze the effect of compost on community composition among treatments. Bacteria in the phylum Bacteriodetes, classes Flavobacteriia and Spingobacteriales (Fluviicola, Flavobacteriia, and Pedobacter), were two to four times more abundant in soils amended with PLC than DMC or NoC consistently among fields and years. Fungi in the phylum Ascomycota were relatively abundant, but their composition was field-specific and without treatment differences. The ecoenzyme data verify that the effects of PLC and DMC on soil communities are based on their microbial composition and not a response to the C source or nutrient content of the compost.

A Predictive Model for Survival of Escherichia coli O157:H7 and Generic E. coli in Soil Amended with Untreated Animal Manure.

This study aimed at developing a predictive model that captures the influences of a variety of agricultural and environmental variables and is able to predict the concentrations of enteric bacteria in soil amended with untreated Biological Soil Amendments of Animal Origin (BSAAO) under dynamic conditions. We developed and validated a Random Forest model using data from a longitudinal field study conducted in mid-Atlantic United States investigating the survival of Escherichia coli O157:H7 and generic E. coli in soils amended with untreated dairy manure, horse manure, or poultry litter. Amendment type, days of rain since the previous sampling day, and soil moisture content were identified as the most influential agricultural and environmental variables impacting concentrations of viable E. coli O157:H7 and generic E. coli recovered from amended soils. Our model results also indicated that E. coli O157:H7 and generic E. coli declined at similar rates in amended soils under dynamic field conditions.The Random Forest model accurately predicted changes in viable E. coli concentrations over time under different agricultural and environmental conditions. Our model also accurately characterized the variability of E. coli concentration in amended soil over time by providing upper and lower prediction bound estimates. Cross-validation results indicated that our model can be potentially generalized to other geographic regions and incorporated into a risk assessment for evaluating the risks associated with application of untreated BSAAO. Our model can be validated for other regions and predictive performance also can be enhanced when data sets from additional geographic regions become available.

Salmonella enterica in Soils Amended with Heat-Treated Poultry Pellets Survived Longer than Bacteria in Unamended Soils and More Readily Transferred to and Persisted on Spinach.

Untreated biological soil amendments of animal origin (BSAAO) are commonly used as biological fertilizers but can harbor foodborne pathogens like Salmonella enterica, leading to potential transfer from soils to fruits and vegetables intended for human consumption. Heat-treated poultry pellets (HTPP) can provide produce growers with a slow-release fertilizer with a minimized risk of pathogen contamination. Little is known about the impact of HTPP-amended soil on the survival of Salmonella enterica The contributions of RpoS and formation of viable but nonculturable cells to Salmonella survival in soils are also inadequately understood. We quantified the survival of Salmonella enterica subsp. enterica serovar Newport wild-type (WT) and rpoS-deficient (ΔrpoS mutant) strains in HTPP-amended and unamended soil with or without spinach plants over 91 days using culture and quantitative PCR methods with propidium monoazide (PMA-qPCR). Simulated "splash" transfer of S. Newport from soil to spinach was evaluated at 35 and 63 days postinoculation (dpi). The S. Newport WT and ΔrpoS mutant reached the limit of detection, 1.0 log CFU/g (dry weight), in unamended soil after 35 days, whereas 2 to 4 log CFU/g (dry weight) was observed for both WT and ΔrpoS mutant strains at 91 dpi in HTPP-amended soil. S. Newport levels in soils determined by PMA-qPCR and plate count methods were similar (P > 0.05). HTPP-amended soils supported higher levels of S. Newport transfer to and survival on spinach leaves for longer periods of time than did unamended soils (P < 0.05). Salmonella Newport introduced to HTPP-amended soils survived for longer periods and was more likely to transfer to and persist on spinach plants than was S. Newport introduced to unamended soils.IMPORTANCE Heat-treated poultry pellets (HTPP) often are used by fruit and vegetable growers as a slow-release fertilizer. However, contamination of soil on farms may occur through contaminated irrigation water or scat from wild animals. Here, we show that the presence of HTPP in soil led to increased S. Newport survival in soil and to greater likelihood of its transfer to and survival on spinach plants. There were no significant differences in survival durations of WT and ΔrpoS mutant isolates of S. Newport. The statistically similar populations recovered by plate count and estimated by PMA-qPCR for both strains in the amended and unamended soils in this study indicate that all viable populations of S. Newport in soils were culturable.

Survival and Growth of Wild-Type and rpoS-Deficient Salmonella Newport Strains in Soil Extracts Prepared with Heat-Treated Poultry Pellets.

Manure runoff can transfer pathogens to farmlands or to water sources, leading to subsequent contamination of produce. Untreated biological soil amendments, like manure, can be contaminated with foodborne pathogens, such as Salmonella Newport, which may lead to transfer of the pathogen to fruits or vegetables. Studies have reported the occurrence and survival of Salmonella in manure or manure slurries. However, data on the survival and growth of Salmonella Newport is lacking in matrices simulating runoff. We quantified the survival and growth of wild-type (WT) Salmonella Newport and rpoS-deficient (Δ rpoS) strains in sterile and nonsterile soil extracts prepared with (amended) or without (unamended) heat-treated poultry pellets at 25°C. Salmonella Newport WT and Δ rpoS populations reached a maximum cell density of 6 to 8 log CFU/mL in 24 to 30 h in amended and unamended soil extracts and remained in stationary phase for up to 4 days. Salmonella Newport in amended soil extracts exhibited a decreased lag phase (λ , 2.87 ± 1.01 h) and greater maximum cell densities ( Nmax, 6.84 ± 1.25 CFU/mL) compared with λ (20.10 ± 9.53 h) and Nmax (5.22 ± 0.82 CFU/mL) in unamended soil extracts. In amended soil extract, the Δ rpoS strain had no measurable λ , similar growth rates (µmax) compared with WT, and a lower Nmax compared with the WT strain. Unamended, nonsterile soil extracts did not support the growth of Salmonella Newport WT and led to a decline in populations for the Δ rpoS strain. Salmonella Newport had lower cell densities in nonsterile soil extracts (5.94 ± 0.95 CFU/mL) than it did in sterile soil extracts (6.66 ± 1.50 CFU/mL), potentially indicating competition for nutrients between indigenous microbes and Salmonella Newport. The most favorable growth conditions were provided by amended sterile and nonsterile soil extracts, followed by sterile, unamended soil extracts for both Salmonella Newport strains. Salmonella Newport may grow to greater densities in amended extracts, providing a route for increased Salmonella levels in the growing environments of produce.

Survival of Escherichia coli in Manure-Amended Soils Is Affected by Spatiotemporal, Agricultural, and Weather Factors in the Mid-Atlantic United States.

Untreated biological soil amendments of animal origin (BSAAO), such as manure, are commonly used to fertilize soils for growing fruit and vegetable crops and can contain enteric bacterial foodborne pathogens. Little is known about the comparative longitudinal survival of pathogens in agricultural fields containing different types of BSAAO, and field data may be useful to determine intervals between manure application and harvest of produce intended for human consumption to minimize foodborne illness. This study generated 324 survival profiles from 12 different field trials at three different sites (UMES, PA, and BARC) in the Mid-Atlantic United States from 2011 to 2015 of inoculated nonpathogenic Escherichia coli (gEc) and attenuated O157 E. coli (attO157) in soils which were unamended (UN) or amended with untreated poultry litter (PL), horse manure (HM), or dairy manure solids (DMS) or liquids (DML). Site, season, inoculum level (low/high), amendment type, management (organic/conventional), and depth (surface/tilled) all significantly (P < 0.0001) influenced survival duration (dpi100mort). Spatiotemporal factors (site, year, and season) in which the field trial was conducted influenced survival durations of gEc and attO157 to a greater extent than weather effects (average daily temperature and rainfall). Initial soil moisture content was the individual factor that accounted for the greatest percentage of variability in survival duration. PL supported greater survival durations of gEc and attO157, followed by HM, UN, and DMS in amended soils. The majority of survival profiles for gEc and attO157 which survived for more than 90 days came from a specific year (i.e., 2013). The effect of management and depth on dpi100mort were dependent on the amendment type evaluated.IMPORTANCE Current language in the Food Safety Modernization Act Produce Safety Rule states no objection to a 90- or 120-day interval between application of untreated BSAAO and harvest of crops to minimize transfer of pathogens to produce intended for human consumption with the intent to limit potential cases of foodborne illness. This regional multiple season, multiple location field trial determined survival durations of Escherichia coli in soils amended with manure to determine whether this interval is appropriate. Spatiotemporal factors influence survival durations of E. coli more than amendment type, total amount of E. coli present, organic or conventional soil management, and depth of manure application. Overall, these data show poultry litter may support extended survival of E. coli compared to horse manure or dairy manure, but spatiotemporal factors like site and season may have more influence than manure type in supporting survival of E. coli beyond 90 days in amended soils in the Mid-Atlantic United States.

Impacts and interactions of organic compounds with chlorine sanitizer in recirculated and reused produce processing water.

Water conservation and economics dictate that fresh produce processors reuse/recirculate the process water. However, the ensuing accumulation of organic matter in water depletes the chlorine sanitizer required for food safety. In this study, we comprehensively investigated chemical compounds that are responsible for water quality in relation to chemical oxygen demand (COD) and chlorine demand (CLD), the two most critical factors associated with water treatment and chlorine replenishment. Simulating commercial fresh-cut wash operations, multiple batches of diced cabbage (0.3 x 0.3 cm2) were washed in the same tank of water. The major components were isolated from the wash water and analyzed by HPLC. Sugars were the predominant compounds (82.7% dry weight) and the major contributor to COD (81.6%), followed by proteins/peptides (7.3% dry weight, 5.3% COD), organic acids (6.2% dry weight, 3.6% COD), and phenolics (0.5% dry weight, 0.5% COD). By repeated time course measures, the effect of these chemicals on CLD are dependent on the chemical structure, concentration in the wash water, and their rate of reaction. Proteins/peptides accounted for about 50% of the total CLD over a 120-min period and phenolics was 21% at 5 min, but diminished with time. The contribution by organic acids and sugars increased continuously, reaching 22% and 16% of total CLD at 120 min of chlorination, respectively. Collectively, these compounds represented 86% of the CLD in cabbage wash water at 5 min and greater than 94% CLD afterwards. This is the first systematic report on the source of COD and CLD during fresh produce washing. It provides essential information for the produce processors to develop safe, effective, and economical wash water treatment/reuse and chlorine replenishment strategies.

Presence of organohalide-respiring bacteria in and around a permeable reactive barrier at a trichloroethylene-contaminated Superfund site.

Trichloroethylene (TCE) is one of the most common groundwater contaminants in the United States; however clean-up efforts are a challenge due to its physical and chemical properties. TCE and several of its degradation products were detected in the groundwater of the Beaver Dam Road Landfill site (Beltsville, MD) at concentrations above accepted maximum contaminant levels. A permeable reactive barrier (i.e., biowall) was installed to remediate the groundwater. Microbial infiltration and colonization of the biowall with native site bacteria was expected to occur. An array of molecular biological tools was applied to survey the microbial community for presence of organohalide-respiring microorganisms at the site. Microorganisms belonging to methanogens, acetogens, sulfate-reducing bacteria, and chlorinated aliphatic hydrocarbon-metabolizing bacteria were identified, thus making way for the application of the microbial populations in the biowall bioaugmentation efforts. In concomitant laboratory studies, molecular approaches were used to monitor continuously-fed column reactors containing saturated biowall material spiked with a commercially-available, Dehalococcoides-containing culture (SDC-9), with or without zero-valent iron (ZVI) shavings. The column without ZVI had the highest abundance of Dehalococcoides spp. (2.7 × 106 cells g-1 material, S.D. = 3.8 × 105 cells g-1 material), while the addition of ZVI did not affect the overall population. Although the addition of ZVI and biostimulation did change ratios of the Dehalococcoides strains, the results suggests that if ZVI would be applied as a biowall material amendment, biostimulation would not be required to maintain a Dehalococcoides population. These experimental results will be utilized in future remediation and/or biowall expansion plans to utilize the natural resources most effectively at the biowall site.

Using a high-organic matter biowall to treat a trichloroethylene plume at the Beaver Dam Road landfill.

Trichloroethylene (TCE) is a highly effective industrial degreasing agent and known carcinogen. It was frequently buried improperly in landfills and has subsequently become one of the most common groundwater and soil contaminants in the USA. A common strategy to remediate TCE-contaminated sites and to prevent movement of the TCE plumes into waterways is to construct biowalls which consist of biomaterials and amendments to enhance biodegradation. This approach was chosen to contain a TCE plume emanating from a closed landfill in Maryland. However, predicting the effectiveness of biowalls is often site specific. Therefore, we conducted an extensive series of batch reactor studies at 12 °C as opposed to the typical room temperature to examine biowall fill-material combinations including the effects of zero-valent iron (ZVI) and glycerol amendments. No detectable TCE was observed after several months in the laboratory study when using the unamended 4:3 mulch-to-compost combination. In the constructed biowall, this mixture reduced the upstream TCE concentration by approximately 90% and generated ethylene downstream, an indication of successful reductive dechlorination. However, the more toxic degradation product vinyl chloride (VC) was also detected downstream at levels approximately ten times greater than the maximum contaminant level. This indicates that incomplete degradation also occurred. In the laboratory, ZVI reduced VC formation. A hazard quotient was calculated for the landfill site with and without the biowall. The addition of the biowall decreased the hazard quotient by 88%.

Association between bacterial survival and free chlorine concentration during commercial fresh-cut produce wash operation.

Determining the minimal effective free chlorine (FC) concentration for preventing pathogen survival and cross-contamination during produce washing is critical for developing science- and risk-based food safety practices. The correlation between dynamic FC concentrations and bacterial survival was investigated during commercial washing of chopped Romaine lettuce, shredded Iceberg lettuce, and diced cabbage as pathogen inoculation study during commercial operation is not feasible. Wash water was sampled every 30 min and assayed for organic loading, FC, and total aerobic mesophilic bacteria after chlorine neutralization. Water turbidity, chemical oxygen demand, and total dissolved solids increased significantly over time, with more rapid increases in diced cabbage water. Combined chlorine increased consistently while FC fluctuated in response to rates of chlorine dosing, product loading, and water replenishment. Total bacterial survival showed a strong correlation with real-time FC concentration. Under approximately 10 mg/L, increasing FC significantly reduced the frequency and population of surviving bacteria detected. Increasing FC further resulted in the reduction of the aerobic plate count to below the detection limit (50 CFU/100 mL), except for a few sporadic positive samples with low cell counts. This study confirms that maintaining at least 10 mg/L FC in wash water strongly reduced the likelihood of bacterial survival and thus potential cross contamination of washed produce.

Ammonia and Nitrous Oxide Emissions from Broiler Houses with Downtime Windrowed Litter.

An emerging poultry manure management practice is in-house windrowing to disinfect the litter. However, this practice is likely to increase emissions of ammonia (NH) and nitrous oxide (NO) from the windrowed litter. The objective of this study was to quantitatively compare NH and NO emissions from broiler houses with and without in-house windrowing. Two broiler houses at a commercial farm were used to compare the NH and NO emissions. Gas emission measurements were conducted continuously and simultaneously for both the control house (without windrowing) and the house with windrowing during the same production periods. The house emission rates were calculated by multiplying the hourly mean gas concentrations and the ventilation rates. The windrowed litter temperature was significantly higher than that of the control litter. The impact of downtime (the time lapse between flocks, during which the bird house is empty) windrowing litter on pathogen reduction was inconclusive because of very low or no recovery of both and spp. from control or windrowed litter samples, respectively. The windrowing house NH emissions were 26.2 and 16.6 kg d house, whereas for the control house, they were 14.6 and 12.8 kg d house in 2012 and 2013, respectively. The NO emissions from the windrowing house were also higher than those from the control house. The total NH and NO emissions from broiler houses practicing windrowing litter management were estimated to be 35.0 and 4.43 g bird, respectively, compared with 31.9 and 3.89 g bird for the control house, respectively.

Survival of Salmonella Newport on Whole and Fresh-Cut Cucumbers Treated with Lytic Bacteriophages.

Salmonella enterica associated with consumption of cucumbers ( Cucumis sativus ) has led to foodborne outbreaks in the United States. Whole and fresh-cut cucumbers are susceptible to S. enterica contamination during growing, harvesting, and postharvest handling. The application of lytic bacteriophages specific for S. enterica was evaluated to reduce Salmonella populations on cucumbers. Unwaxed cucumbers ('Lisboa' variety, or mini-cucumbers purchased at retail) were inoculated with Salmonella Newport (5 log CFU per cucumber) and were sprayed with 3.2 mL of phosphate-buffered saline (control) or 10 log PFU/ml of SalmoFresh, a Salmonella-specific bacteriophage preparation (phage), to deliver 4.76 × 107 PFU/cm2. Cucumbers were stored at 10 or 22°C for 7 days. Inoculated mini-cucumbers were sliced with a sterile knife to investigate Salmonella transfer to mesocarp, and cut pieces were stored at 4°C for 2 days. Populations (log CFU per cucumber) of Salmonella Newport on phage-treated whole cucumbers were significantly (P < 0.05) smaller (2.44 ± 0.94) than on control-treated cucumbers (4.27 ± 0.37) on day 0. Populations on phage-treated cucumbers stored at 10°C were 1.72 ± 0.77 and 1.56 ± 0.46, which were significantly lower than those on control-treated cucumbers (3.20 ± 0.48 and 2.33 ± 0.25) on days 1 and 4, respectively. Between days 0 and 1, populations on control-treated cucumbers stored at 10 and 22°C declined by 1.07 and 2.47 log CFU per cucumber, respectively. At 22°C, Salmonella Newport populations declined by 2.37 log CFU per cucumber between days 0 and 1. Phage application to whole cucumbers before slicing did not reduce the transfer of Salmonella Newport to fresh-cut slices. Lytic phage application may be a potential intervention to reduce Salmonella populations on whole cucumbers.

Survival and Growth of Listeria monocytogenes on Fresh-Cut "Athena" and "Rocky Ford" Cantaloupes During Storage at 4°C and 10°C.

Cantaloupes, marketed as "Rocky Ford," were implicated in the U.S. multistate outbreak of listeriosis in 2011, which caused multiple fatalities. Listeria monocytogenes can survive on whole cantaloupes and can be transferred to the flesh of melons. The growth of L. monocytogenes on fresh-cut "Athena" and "Rocky Ford" cantaloupe cultivars during refrigerated storage was evaluated. Fresh-cut cubes (16.4 cm3) from field-grown cantaloupes were each inoculated with 5 log10 CFU/mL of a multi-strain mixture of L. monocytogenes and stored at 4°C or 10°C. Inoculated fresh-cut cubes were also: (1) continuously stored at 4°C for 3 days; (2) temperature-abused (TA: 25°C for 4 h) on day 0; or (3) stored at 4°C for 24 h, exposed to TA on day 1, and subsequently stored at 4°C until day 3. L. monocytogenes populations on fresh-cut melons continuously stored at 4°C or 10°C were enumerated on selected days for up to 15 days and after each TA event. Brix values for each cantaloupe variety were determined. L. monocytogenes populations on fresh-cut cantaloupe cubes stored at 4°C increased by 1.0 and 3.0 log10 CFU/cube by day 7 and 15, respectively, whereas those stored at 10°C increased by 3.0 log10 CFU/cube by day 7. Populations of L. monocytogenes on fresh-cut cantaloupes stored at 10°C were significantly (p < 0.05) greater than those stored at 4°C during the study. L. monocytogenes showed similar growth on fresh-cut "Athena" and "Rocky Ford" cubes, even though "Athena" cubes had significantly higher Brix values than the "Rocky Ford" fruit. L. monocytogenes populations on fresh-cut cantaloupes exposed to TA on day 1 and then refrigerated were significantly greater (0.74 log10 CFU) than those stored continuously at 4°C for 3 days. Storage at 10°C or exposure to TA events promoted growth of L. monocytogenes on fresh-cut cantaloupe during refrigerated storage.

Survival and growth of Listeria monocytogenes on whole cantaloupes is dependent on site of contamination and storage temperature.

Whole cantaloupes (Cucumis melo L.), marketed as 'Rocky Ford', were implicated in a large multi-state outbreak of listeriosis in the United States in 2011; however, survival and growth of Listeria monocytogenes on whole cantaloupes remains relatively unexplored. The research presented here evaluated three different storage temperatures, two sites of contamination of cantaloupes, and two cantaloupe varieties to determine their effect on the survival of L. monocytogenes. 'Athena' and 'Rocky Ford' cantaloupe cultivars were grown in soil and harvested, and individual melons subsequently received a multi-strain inoculum of L. monocytogenes (6 log CFU/melon), which were then stored at 4°C, 10°C, and 25°C. Changes in L. monocytogenes populations on the rinds and stem scars of cantaloupes stored at each temperature were determined at selected times for up to 15days. An analysis of variance revealed that inoculation site and storage temperature significantly affected survival of L. monocytogenes on cantaloupes during storage (p<0.05), but cultivar did not influence L. monocytogenes (p>0.05). Populations of L. monocytogenes on stem scars of cantaloupes stored at 25°C increased by 1-2 log CFU/melon on day 1, and were significantly greater than those on cantaloupes stored at 4°C or 10°C (p<0.05), which remained constant or increased by approximately 0.3 log CFU/melon, respectively, over the same time period. A decrease of 2-5 log CFU/melon of L. monocytogenes occurred on the rinds of cantaloupes during storage by day 7, and were not significantly different at the three different storage temperatures (p>0.05). In trials performed in rind juice extracts, populations of L. monocytogenes decreased by 3 log CFU/mL when stored at 25°C by day 3, but grew by 3-4 log CFU/mL when stored at 4°C over 7days. Overall, site of contamination and storage temperature influenced the survival of L. monocytogenes on cantaloupes more than cantaloupe cultivar type.

Survival and Persistence of Nonpathogenic Escherichia coli and Attenuated Escherichia coli O157:H7 in Soils Amended with Animal Manure in a Greenhouse Environment.

Animal manure provides benefits to agriculture but may contain pathogens that contaminate ready-to-eat produce. U.S. Department of Agriculture National Organic Program standards include 90- or 120-day intervals between application of manure and harvest of crop to minimize risks of pathogen contamination of fresh produce. Data on factors affecting survival of Escherichia coli in soils under greenhouse conditions are needed. Three separate studies were conducted to evaluate survival of nonpathogenic E. coli (gEc) and attenuated E. coli O157:H7 (attO157) inoculated at either low (4 log CFU/ml) or high (6 log CFU/ml) populations over 56 days. Studies involved two pot sizes (small, 398 cm(3); large, 89 liters), three soil types (sandy loam, SL; clay loam, CL; silt loam, SIL), and four amendments (poultry litter, PL; dairy manure liquids, DML; horse manure, HM; unamended). Amendments were applied to the surface of the soil in either small or large containers. Study 1, conducted in regularly irrigated small containers, showed that populations of gEc and attO157 (2.84 to 2.88 log CFU/g) in PL-amended soils were significantly (P < 0.05) greater than those in DML-amended (0.29 to 0.32 log CFU/g [dry weight] [gdw]) or unamended (0.25 to 0.28 log CFU/gdw) soils; soil type did not affect E. coli survival. Results from study 2, in large pots with CL and SIL, showed that PL-amended soils supported significantly higher attO157 and gEc populations compared with HM-amended or unamended soils. Study 3 compared results from small and large containers that received high inoculum simultaneously. Overall, in both small and large containers, PLamended soils supported higher gEc and attO157 populations compared with HM-amended and unamended soils. Populations of attO157 were significantly greater in small containers (1.83 log CFU/gdw) than in large containers (0.65 log CFU/gdw) at week 8, perhaps because small containers received more regular irrigation than large pots. Regular irrigation of small pots may have affected E. coli persistence in manure-amended soils. Overall, PL-amended soils in both small and large containers supported E. coli survival at higher populations compared with DML-, HM-, or unamended soils.

Metrics Proposed To Prevent the Harvest of Leafy Green Crops Exposed to Floodwater Contaminated with Escherichia coli.

UNLABELLED: The California Leafy Green Products Handler Marketing Agreement (LGMA) requires leafy green crops within 9 m of the edge of a flooded field not be harvested due to potential contamination (California Leafy Green Products Handler Marketing Board, Commodity Specific Flood Safety Guidelines for the Production and Harvest of Lettuce and Leafy Greens, 2012). Further, previously flooded soils should not be replanted for 60 days. In this study, the suitability of the LGMA metrics for farms in the Mid-Atlantic region of the United States was evaluated. The upper end of a spinach bed (in Beltsville, MD) established on a -5% grade was flooded with water containing 6 log CFU/ml Escherichia coli to model a worst-case scenario of bacterial movement through soil. Escherichia coli prevalence in soil and on foliar tissue was determined by most probable number (MPN) analysis at distances up to 9 m from the edge of the flood for 63 days. While E. coli was quickly detected at the 9-m distance within 1 day in the spring trial and within 3 days in the fall trial, no E. coli was detected on plants outside the flood zone after 14 days. On day 63 for the two trials, E. coli populations in the flood zone soil were higher in the fall than in the spring. Regression analysis predicted that the time required for a 3-log MPN/g (dry weight) decrease in E. coli populations inside the flood zone was within the 60-day LGMA guideline in the spring but would require 90 days in the fall. Overall, data suggest that the current guidelines should be revised to include considerations of field and weather conditions that may promote bacterial movement and survival. IMPORTANCE: This study tracked the movement of Escherichia coli from floodwater across a horizontal plane of soil and the potential for the contamination of distant leafy green produce. The purpose of this study was to address the validity of the California Leafy Green Products Handler Marketing Agreement recommendations for the harvest of leafy green crops after a flooding event. These recommendations were based on the turning radius of farming equipment but did not take into consideration the potential subsurface movement of pathogens in the water through soil. This research shows that further considerations of field slope, temperature, and additional rainfall events may be necessary to provide appropriate guidelines to prevent the harvest of leafy green crops contaminated by enteric pathogens in floodwaters. This study may be used to provide a framework for comprehensive recommendations to growers for good harvesting practices after a flooding event.

A novel microfluidic mixer-based approach for determining inactivation kinetics of Escherichia coli O157:H7 in chlorine solutions.

Determination of the minimum free chlorine concentration needed to prevent pathogen survival/cross-contamination during produce washing is essential for the development of science-based food safety regulations and practices. Although the trend of chlorine concentration-contact time on pathogen inactivation is generally understood, specific information on chlorine and the kinetics of pathogen inactivation at less than 1.00 s is urgently needed by the produce processing industry. However, conventional approaches to obtain this critical data have been unable to adequately measure very rapid responses. This paper reports our development, fabrication, and test of a novel microfluidic device, and its application to obtain the necessary data on pathogen inactivation by free chlorine in produce wash solution in times as short as 0.10 s. A novel microfluidic mixer with the capability to accurately determine the reaction time and control the chlorine concentration was designed with three inlets for bacterial, chlorine and dechlorinating solutions, and one outlet for effluent collection. The master mold was fabricated on a silicon wafer with microchannels via photopolymerization. Polydimethylsiloxane replicas with patterned microchannels were prototyped via soft lithography. The replicas were further assembled into the micromixer on glass via O2 plasma treatment, and the inlets were connected to a syringe pump for solution delivery. To determine the kinetics of free chlorine on pathogen inactivation, chlorine solutions of varying concentrations were first pumped into the micromixer, together with the addition of bacterial suspension of Escherichia coli O157:H7 through a separate inlet. This was followed by injection of dechlorinating solution to stop the chlorine-pathogen reaction. The effluent was collected and the surviving bacteria cells were enumerated using a modified 'Most Probable Number' method. Free chlorine concentration was determined using a standard colorimetric method. The contact time was experimentally set by adjusting the solution flow rate, and was estimated by computational fluid dynamics modeling. Results showed that 1) pathogen inactivation was significantly affected by free chlorine concentration (P < 0.0001) and subsecond reaction time (P < 0.0001) and their interactions (P < 0.0001); and 2) the current industry practice of using 1.0 mg/L free chlorine will require more than 1.00 s total contact to achieve a 5-log10 reduction in an E. coli O157:H7 population, whereas a 10.0 mg/L free chlorine solution will achieve 5-log10 reduction in as little as 0.25 s. Information obtained from this study will provide critical insight on kinetics of bacterial inactivation for a broad range of sanitizers and produce wash operational conditions, thus facilitating the development and implementation of science-based food safety regulations and practices for improving food safety.

Effect of proximity to a cattle feedlot on Escherichia coli O157:H7 contamination of leafy greens and evaluation of the potential for airborne transmission.

The impact of proximity to a beef cattle feedlot on Escherichia coli O157:H7 contamination of leafy greens was examined. In each of 2 years, leafy greens were planted in nine plots located 60, 120, and 180 m from a cattle feedlot (3 plots at each distance). Leafy greens (270) and feedlot manure samples (100) were collected six different times from June to September in each year. Both E. coli O157:H7 and total E. coli bacteria were recovered from leafy greens at all plot distances. E. coli O157:H7 was recovered from 3.5% of leafy green samples per plot at 60 m, which was higher (P < 0.05) than the 1.8% of positive samples per plot at 180 m, indicating a decrease in contamination as distance from the feedlot was increased. Although E. coli O157:H7 was not recovered from air samples at any distance, total E. coli was recovered from air samples at the feedlot edge and all plot distances, indicating that airborne transport of the pathogen can occur. Results suggest that risk for airborne transport of E. coli O157:H7 from cattle production is increased when cattle pen surfaces are very dry and when this situation is combined with cattle management or cattle behaviors that generate airborne dust. Current leafy green field distance guidelines of 120 m (400 feet) may not be adequate to limit the transmission of E. coli O157:H7 to produce crops planted near concentrated animal feeding operations. Additional research is needed to determine safe set-back distances between cattle feedlots and crop production that will reduce fresh produce contamination.