Methodological differences between studies confound one-size-fits-all approaches to managing surface waterways for food and water safety.

Even though differences in methodology (e.g., sample volume and detection method) have been shown to affect observed microbial water quality, multiple sampling and laboratory protocols continue to be used for water quality monitoring. Research is needed to determine how these differences impact the comparability of findings to generate best management practices and the ability to perform meta-analyses. This study addresses this knowledge gap by compiling and analyzing a data set representing 2,429,990 unique data points on at least one microbial water quality target (e.g., Salmonella presence and Escherichia coli concentration). Variance partitioning analysis was used to quantify the variance in likelihood of detecting each pathogenic target that was uniquely and jointly attributable to non-methodological versus methodological factors. The strength of the association between microbial water quality and select methodological and non-methodological factors was quantified using conditional forest and regression analysis. Fecal indicator bacteria concentrations were more strongly associated with non-methodological factors than methodological factors based on conditional forest analysis. Variance partitioning analysis could not disentangle non-methodological and methodological signals for pathogenic Escherichia coli, Salmonella, and Listeria. This suggests our current perceptions of foodborne pathogen ecology in water systems are confounded by methodological differences between studies. For example, 31% of total variance in likelihood of Salmonella detection was explained by methodological and/or non-methodological factors, 18% was jointly attributable to both methodological and non-methodological factors. Only 13% of total variance was uniquely attributable to non-methodological factors for Salmonella, highlighting the need for standardization of methods for microbiological water quality testing for comparison across studies.IMPORTANCEThe microbial ecology of water is already complex, without the added complications of methodological differences between studies. This study highlights the difficulty in comparing water quality data from projects that used different sampling or laboratory methods. These findings have direct implications for end users as there is no clear way to generalize findings in order to characterize broad-scale ecological phenomenon and develop science-based guidance. To best support development of risk assessments and guidance for monitoring and managing waters, data collection and methods need to be standardized across studies. A minimum set of data attributes that all studies should collect and report in a standardized way is needed. Given the diversity of methods used within applied and environmental microbiology, similar studies are needed for other microbiology subfields to ensure that guidance and policy are based on a robust interpretation of the literature.

Salmonella Prevalence Is Strongly Associated with Spatial Factors while Listeria monocytogenes Prevalence Is Strongly Associated with Temporal Factors on Virginia Produce Farms.

The heterogeneity of produce production environments complicates the development of universal strategies for managing preharvest produce safety risks. Understanding pathogen ecology in different produce-growing regions is important for developing targeted mitigation strategies. This study aimed to identify environmental and spatiotemporal factors associated with isolating Salmonella and Listeria from environmental samples collected from 10 Virginia produce farms. Soil (n = 400), drag swab (n = 400), and irrigation water (n = 120) samples were tested for Salmonella and Listeria, and results were confirmed by PCR. Salmonella serovar and Listeria species were identified by the Kauffmann-White-Le Minor scheme and partial sigB sequencing, respectively. Conditional forest analysis and Bayesian mixed models were used to characterize associations between environmental factors and the likelihood of isolating Salmonella, Listeria monocytogenes (LM), and other targets (e.g., Listeria spp. and Salmonella enterica serovar Newport). Surrogate trees were used to visualize hierarchical associations identified by the forest analyses. Salmonella and LM prevalence was 5.3% (49/920) and 2.3% (21/920), respectively. The likelihood of isolating Salmonella was highest in water samples collected from the Eastern Shore of Virginia with a dew point of >9.4°C. The likelihood of isolating LM was highest in water samples collected in winter from sites where <36% of the land use within 122 m was forest wetland cover. Conditional forest results were consistent with the mixed models, which also found that the likelihood of detecting Salmonella and LM differed between sample type, region, and season. These findings identified factors that increased the likelihood of isolating Salmonella- and LM-positive samples in produce production environments and support preharvest mitigation strategies on a regional scale. IMPORTANCE This study sought to examine different growing regions across the state of Virginia and to determine how factors associated with pathogen prevalence may differ between regions. Spatial and temporal data were modeled to identify factors associated with an increased pathogen likelihood in various on-farm sources. The findings of the study show that prevalence of Salmonella and L. monocytogenes is low overall in the produce preharvest environment but does vary by space (e.g., region in Virginia) and time (e.g., season), and the likelihood of pathogen-positive samples is influenced by different spatial and temporal factors. Therefore, the results support regional or scale-dependent food safety standards and guidance documents for controlling hazards to minimize risk. This study also suggests that water source assessments are important tools for developing monitoring programs and mitigation measures, as spatiotemporal factors differ on a regional scale.

Pre-heated blades for harvesting baby-leaves reduce the risk of Escherichia coli internalization in leaves.

BACKGROUND: Pathogenic enterobacteria can travel through the plant vascular bundles by penetrating from cuts and persisting into ready-to-eat leafy greens. Because the cutting site is the main point of entrance and uptake, we tested how different cutting strategies can reduce bacterial internalization in leaves. Horizontal cuts at the base of the leaves were performed with two different types of tools: the first with a scalpel (by pulling the blade) and the second with a scissor-action that has blades that cuts by gliding against a thicker blade. Scissor-action generally makes closer border cuts. Blades of both types of tools have worked at 25 °C and 200 °C. The present study aimed to determine how these different types of cuts and temperatures affected bacterial uptake in leaves. Experiments were repeated on different plant genotypes and at different wilting stages. RESULTS: Our findings showed that cutting baby-leaves with a scissor action at 200 °C significantly reduced the bacterial uptake compared to the not heated (which simulates a mechanized lettuce harvester). The most effective cutting treatments for reducing bacterial uptake were in the order: scissor 200 °C > scissor 25 °C > scalpel 200 °C > scalpel 25 °C. The scissor heated at 200 °C also prevented bacterial uptake on wilted baby-leaves. CONCLUSION: The findings of the present study could provide a further contribution in terms of safety during harvest and suggest that a pre-heated blade supports safety during harvest of leafy greens. © 2022 Society of Chemical Industry.

A Validated Preharvest Sampling Simulation Shows that Sampling Plans with a Larger Number of Randomly Located Samples Perform Better than Typical Sampling Plans in Detecting Representative Point-Source and Widespread Hazards in Leafy Green Fields.

Commercial leafy greens customers often require a negative preharvest pathogen test, typically by compositing 60 produce sample grabs of 150 to 375 g total mass from lots of various acreages. This study developed a preharvest sampling Monte Carlo simulation, validated it against literature and experimental trials, and used it to suggest improvements to sampling plans. The simulation was validated by outputting six simulated ranges of positive samples that contained the experimental number of positive samples (range, 2 to 139 positives) recovered from six field trials with point source, systematic, and sporadic contamination. We then evaluated the relative performance between simple random, stratified random, or systematic sampling in a 1-acre field to detect point sources of contamination present at 0.3% to 1.7% prevalence. Randomized sampling was optimal because of lower variability in probability of acceptance. Optimized sampling was applied to detect an industry-relevant point source [3 log(CFU/g) over 0.3% of the field] and widespread contamination [-1 to -4 log(CFU/g) over the whole field] by taking 60 to 1,200 sample grabs of 3 g. More samples increased the power of detecting point source contamination, as the median probability of acceptance decreased from 85% with 60 samples to 5% with 1,200 samples. Sampling plans with larger total composite sample mass increased power to detect low-level, widespread contamination, as the median probability of acceptance with -3 log(CFU/g) contamination decreased from 85% with a 150-g total mass to 30% with a 1,200-g total mass. Therefore, preharvest sampling power increases by taking more, smaller samples with randomization, up to the constraints of total grabs and mass feasible or required for a food safety objective. IMPORTANCE This study addresses a need for improved preharvest sampling plans for pathogen detection in leafy green fields by developing and validating a preharvest sampling simulation model, avoiding the expensive task of physical sampling in many fields. Validated preharvest sampling simulations were used to develop guidance for preharvest sampling protocols. Sampling simulations predicted that sampling plans with randomization are less variable in their power to detect low-prevalence point source contamination in a 1-acre field. Collecting larger total sample masses improved the power of sampling plans in detecting widespread contamination in 1-acre fields. Hence, the power of typical sampling plans that collect 150 to 375 g per composite sample can be improved by taking more, randomized smaller samples for larger total sample mass. The improved sampling plans are subject to feasibility constraints or to meet a particular food safety objective.

Efficacy of commercial overhead washing and waxing systems on the microbiological quality of fresh peaches.

Overhead spray washing and waxing systems (WWS) are used commercially to reduce the risk of microbial contamination and improve the quality of fresh produce during packing. This study evaluated the microbiological quality of overhead spray water and spent peach wash water, as well as fresh peaches before and after they pass the WWS. Pre- and post-washed/waxed peach samples (n = 192) and overhead spray water and spent peach wash water samples (n = 54) were collected several times over the course of a processing day in three packing facilities located in the state of Georgia. Populations of total aerobes (TA), yeasts and molds (YM), and coliforms (TC) and the presence of thermotolerant coliforms (TTC) and enterococci (EC) were measured in collected samples. The average TA and TC counts and the incidences of TTC and EC were significantly higher (P < 0.05) on peach samples collected after the WWS compared to those collected before the WWS. Counts and incidences of TA, YM, and TC in spent peach wash water were significantly higher than in the overhead spray water where neither TTC nor EC was detected. Results suggest that the commercial washing and waxing systems had little effect in improving the microbiological quality of fresh peaches.

Shiga Toxin-Producing Escherichia coli Infections Associated With Romaine Lettuce-United States, 2018.

BACKGROUND: Produce-associated outbreaks of Shiga toxin-producing Escherichia coli (STEC) were first identified in 1991. In April 2018, New Jersey and Pennsylvania officials reported a cluster of STEC O157 infections associated with multiple locations of a restaurant chain. The Centers for Disease Control and Prevention (CDC) queried PulseNet, the national laboratory network for foodborne disease surveillance, for additional cases and began a national investigation. METHODS: A case was defined as an infection between 13 March and 22 August 2018 with 1 of the 22 identified outbreak-associated E. coli O157:H7 or E. coli O61 pulsed-field gel electrophoresis pattern combinations, or with a strain STEC O157 that was closely related to the main outbreak strain by whole-genome sequencing. We conducted epidemiologic and traceback investigations to identify illness subclusters and common sources. A US Food and Drug Administration-led environmental assessment, which tested water, soil, manure, compost, and scat samples, was conducted to evaluate potential sources of STEC contamination. RESULTS: We identified 240 case-patients from 37 states; 104 were hospitalized, 28 developed hemolytic uremic syndrome, and 5 died. Of 179 people who were interviewed, 152 (85%) reported consuming romaine lettuce in the week before illness onset. Twenty subclusters were identified. Product traceback from subcluster restaurants identified numerous romaine lettuce distributors and growers; all lettuce originated from the Yuma growing region. Water samples collected from an irrigation canal in the region yielded the outbreak strain of STEC O157. CONCLUSIONS: We report on the largest multistate leafy greens-linked STEC O157 outbreak in several decades. The investigation highlights the complexities associated with investigating outbreaks involving widespread environmental contamination.

Heavy metal content of produce grown in San Juan County (New Mexico, USA).

The Animas River Watershed has long received discharges of naturally occurring acid rock drainage; however, on August 5, 2015, three million gallons flowed into the agricultural region of Farmington, New Mexico and the Navajo Nation. Consumers and growers in the region were fearful that produce might absorb heavy metals from contaminated irrigation water originating from these rivers. Samples were collected from the region including corn (n = 30), pumpkin (n = 10), squash (n = 10), and cucumber (n = 10) then processed and tested using inductively coupled plasma-optical emission spectrometry (ICP-OES) for concentrations of nine metals of interest. These include toxic metals: Al, As, and Pb, which were compared to the World Health Organization limits, 18.29 mg d-1, 0.192 mg d-1, and 0.05 mg kg-1, respectively and essential metals: Cr, Fe, Mn, Zn, Ca, and Cu whose levels were compared to the National Academies' dietary references for tolerable upper intake levels. Results indicate that produce grown in the region contained significantly less metal than the allowable limits, except for Pb in two corn samples. This research is the first attempt to monitor and analyze heavy metal absorption of produce in the area.

Salmonella enterica recovery from river waters of the Maryland Eastern Shore reveals high serotype diversity and some multidrug resistance.

The Delmarva Peninsula, a major agricultural and recreational region for the U.S. states of Delaware, Maryland and Virginia, experiences recurrent salmonellosis disease. Previous studies point to water environments as a potential persistent environmental reservoir of Salmonella enterica. To evaluate this hypothesis, water from the four main rivers of the Maryland Eastern Shore on the Delmarva Peninsula was tested for the presence of S. enterica, and recovered isolates were characterized for antimicrobial resistance. Sampling was performed in autumn and spring to evaluate temporal persistence at twenty four sites along the Choptank, Nanticoke, Pocomoke and Wicomico Rivers. Water (10 L) was filtered through sterile modified Moore swabs in situ. Swabs were selectively enriched for S. enterica and presumptive salmonellae were confirmed by PCR amplification of the Salmonella-specific invA and hilA genes. The serogroup of 402 isolates was determined, followed by serotype characterization for 157 isolates selected to represent all the identified serogroups across all samples. S. enterica was isolated from all the rivers in both seasons and was detected in 35/46 (65%) of surface water samples, with equivalent recovery in spring (70%) and fall (61%). The likelihood of isolating S. enterica was higher for the Nanticoke and Pocomoke Rivers, χ2 (3, N = 46) = 12.75, p < 0.01. In total, 18 serotypes of S. enterica were identified, and serotype diversity differed between the fall and spring samplings. Newport was the most frequently isolated serotype, both overall and in the fall, identified in 8/46 samples (17%). Typhimurium was the predominant serotype in spring. Some temporal and biogeographic patterns were observed in S. enterica recovery, but 6/18 serotypes were identified in both seasons. The majority (84%) of isolates were pan-susceptible, including all those tested from the Wicomico River (N = 19). Twenty five isolates (16%) from 9 samples were resistant to at least one antimicrobial, including serotypes Typhimurium, Newport, Litchfield, III 17:z10: e,n,x,z15, III 60:I and IV_40:z4: z32: -. Of the isolates that were resistant to a single antimicrobial (N = 12), resistance was to streptomycin or sulfisoxazole. Thirteen isolates were multidrug resistant, nine exhibiting resistance to ampicillin, sulfisoxazole, tetracycline, amoxicillin/clavulanic acid, cefoxitin and ceftriaxone, and four to sulfisoxazole and tetracycline. The widespread presence and diversity of S. enterica in Delmarva rivers are concerning given the frequent use of rivers and tributaries as a source of irrigation and for recreation. Future research should seek to determine specific point sources of S. enterica for surface river waters, and risks associated with acquisition of antimicrobial resistance traits.

Review of water quality criteria for water reuse and risk-based implications for irrigated produce under the FDA Food Safety Modernization Act, produce safety rule.

Questions related to the safety of alternative water sources, such as recycled water or reclaimed water (including grey water, produced water, return flows, and recycled wastewater), for produce production have been largely un-explored at the detail warranted for protection of public health. Additionally, recent outbreaks of Escherichia coli (E. coli) in fresh produce, in which agricultural water was suspected as the source, coupled with heightened media coverage, have elevated fruit and vegetable safety into the forefront of public attention. Exacerbating these concerns, new Federal regulations released by the U.S. Food and Drug Administration (FDA) as part of implementation of the FDA Food Safety Modernization Act (FSMA), require testing of agricultural water quality for generic E. coli. Here, we present a review of water quality criteria - including surface water, groundwater recreational water, and water reuse - in an attempt to better understand implications of new FDA regulations on irrigated produce. In addition, a Quantitative Microbial Risk Assessment (QMRA) was conducted to estimate risks from pathogen contamination of food crops eaten fresh under the context of FDA regulations to provide perspective on current water reuse regulations across the country. Results indicate that irrigation water containing 126 CFU/100 mL of E. coli correspond to a risk of GI illness (diarrhea) of 9 cases in 100,000,000 persons (a 0.000009% risk) for subsurface irrigation, 1.1 cases in 100,000 persons (a 0.0011% risk) for furrow irrigation, and 1.1 cases in 1000 persons (a 0.11% risk) for sprinkler irrigation of lettuce. In comparison to metrics in states that currently regulate the use of recycled water for irrigation of food crops eaten fresh, the FDA FSMA water quality metrics are less stringent and therefore the use of recycled water presents a reduced risk to consumers than the FDA regulations. These findings, while limited to a one-time exposure event of lettuce irrigated with water meeting FSMA water quality regulations, highlight the need for additional assessments to determine if the scientific-basis of the regulation is protective of public health.

Evaluation of post-contamination survival and persistence of applied attenuated E. coli O157:H7 and naturally-contaminating E. coli O157:H7 on spinach under field conditions and following postharvest handling.

This study determined the variability in population uniformity of an applied mixture of attenuated E. coli O157:H7 (attEcO157) on spinach leaves as impacted by sampling mass and detection technique over spatial and temporal conditions. Opportunistically, the survival and distribution of naturally contaminating pathogenic E. coli O157:H7 (EcO157), in a single packaged lot following commercial postharvest handling and washing, was also evaluated. From the main study outcomes, differences in the applied inoculum dose of 100-fold, resulted in indistinguishable population densities of approximately Log 1.1 CFU g-1 by 14 days post-inoculation (DPI). Composite leaf samples of 150 g and the inclusion of the spinach petiole resulted in the greatest numerical sensitivity of detection of attEcO157 when compared to 25 and 150 g samples without petioles (P < 0.05). Differences in population density and protected-site survival and potential leaf internalization were observed between growing seasons and locations in California (P < 0.05). A Double Weibull model best described and identified two distinct populations with different inactivation rates of the inoculated attEcO157. Linear die-off rates varied between 0.14 and 0.29 Log/Day irrespective of location. Detection of EcO157- stx1-negative and stx2-positive, resulting from a natural contamination event, was observed in 11 of 26 quarantined commercial units of washed spinach by applying the 150 g sample mass protocol. The capacity to detect EcO157 varied between commercial test kits and non-commercial qPCR. Our findings suggest the need for modifications to routine pathogen sampling protocols employed for lot acceptance of spinach and other leafy greens.

Meeting Report: Key Outcomes from a Collaborative Summit on Agricultural Water Standards for Fresh Produce.

On February 27 to 28, 2018, the Produce Safety Alliance convened a national water summit in Covington, KY to discuss the requirements of the United States Food and Drug Administration's (FDA) Food Safety Modernization Act Standards for the Growing, Harvesting, Packing, and Holding of Produce for Human Consumption (Produce Safety Rule [PSR]). The goals of the meeting were to better understand the challenges growers face in implementing the requirements in Subpart E-Agricultural Water and work collaboratively to develop practical solutions to meet fruit and vegetable production needs while protecting public health. To meet these goals, the summit engaged a diverse group of stakeholders including growers, researchers, extension educators, produce industry members, and regulatory personnel. Key outcomes included defining implementation barriers due to diversity in water sources, distribution systems, commodity types, climates, farm size, and production activities. There was an articulated need for science-based solutions, such as the use of agricultural water system assessments and sharing of federal, state, and regional water quality data, to ensure qualitative and quantitative standards reduce microbial risks. These identified challenges and needs resulted in significant debate about whether reopening the PSR-Subpart E for modification or attempting to address concerns through guidance would provide the best mechanism for alleviating concerns. In addition, training, outreach, and technical assistance were identified as vital priorities once the concerns are formally addressed by FDA. The water summit highlighted the critical need for transparency of FDA's progress on reevaluating the Subpart E requirements to help guide growers' decisions regarding the use of agricultural water.

Novel sanitization approach based on synergistic action of UV-A light and benzoic acid: Inactivation mechanism and a potential application in washing fresh produce.

Antimicrobial activity of the simultaneous UV-A light and benzoic acid (BA) treatment against stationary phase Escherichia coli O157:H7 was investigated. While 15 mM BA or UV-A light exposure for 30 min alone caused < 1 logarithmic reduction in the bacterial population, > 5 logarithmic reductions were induced by the simultaneous application of UV-A and 15 mM BA in 30 min, demonstrating a synergistic antimicrobial effect. Due to its ability to increase cell membrane permeability, addition of EDTA (1 mM) was able to decrease the required concentration of BA in the simultaneous treatment from 15 to 8 mM. Microbial inactivation was a result of simultaneous membrane damage, intracellular acidification, and intracellular oxidative stress. The simultaneous treatment was effective in the presence of organic load of up to 500 mg/L of chemical oxygen demand (COD) and was able to lower cross-contamination risk during simulated washing of spinach (Spinacia oleracea) without adversely affecting its color.

Occurrence of enteric viruses in reclaimed and surface irrigation water: relationship with microbiological and physicochemical indicators.

AIMS: To assess the prevalence of enteric viruses in different irrigation water sources and in the irrigated produce, and the possible links with microbiological and physicochemical water characteristics. METHODS AND RESULTS: The prevalence and levels of Escherichia coli, Norovirus (NoV) genogroup I (GI) and II (GII), as well as Hepatitis A virus were assessed in three types of water: surface water (surface-W), reclaimed water subjected to secondary treatment (secondary-W) and reclaimed water subjected to tertiary treatment (tertiary-W), as well as in zucchini irrigated with these irrigation water sources. Chemical oxygen demand (COD), turbidity, total suspended solids, alkalinity and maximum filterable volume (MFV) were also measured in the water. Higher prevalence of NoV in secondary-W (GI 100%, GII 55·6%) and tertiary-W (GI 91·7%, GII 66·7%) compared with surface-W (GI 58·4%, GII 22·2%) was observed. Nov GI showed positive correlation with E. coli (Spearman's correlation coefficient = 0·68, P < 0·01), and with some physicochemical parameters such as COD (0·52, P < 0·01), turbidity (0·52, P < 0·01) and MFV (0·54, P < 0·01). Escherichia coli and enteric viruses were not detected in zucchini. CONCLUSION: There is a potential risk of contamination of crops with NoV when reclaimed water is used for irrigation. SIGNIFICANCE AND IMPACT OF THE STUDY: Increase the knowledge on the prevalence of enteric viruses in different irrigation water sources, and its consequences for fresh produce safety.

Effect of the irrigation regime on the susceptibility of pepper and tomato to post-harvest proliferation of Salmonella enterica.

Raw produce is increasingly recognized as a vehicle of human gastroenteritis. Non-typhoidal Salmonella, pathogenic Escherichia coli, and other human pathogens have been isolated from fruits and vegetables in the field and in the marketplace, which led to the hypothesis that these microbes can use plants as alternate hosts. However, environmental and physiological factors that facilitate persistence of these bacteria in the crop production environment and make produce more vulnerable to post-harvest contamination have not been fully delineated. This study tested the effect of irrigation regimes on the susceptibility of peppers and tomatoes to post-harvest proliferation of Salmonella. The experiments were carried out over three experimental seasons in two locations using seven strains of Salmonella. The irrigation regime per se did not affect susceptibility of tomatoes and peppers to post-harvest proliferation of Salmonella; however, in some of the seasons, irrigation regime-dependent differences were observed. Red peppers and tomatoes were more conducive to proliferation of Salmonella than green fruit in all seasons. Inter-seasonal differences were the strongest factors affecting proliferation of Salmonella in peppers.

Effects of nitrogen and potassium fertilization on the susceptibility of tomatoes to post-harvest proliferation of Salmonella enterica.

Fresh fruits and vegetables are increasingly recognized as vehicles of salmonellosis. Pre- and post-harvest environmental conditions, and physiological, and genetic factors are thought to contribute to the ability of human pathogens to persist in the production environment, attach to, colonize and proliferate in and on raw produce. How field production conditions affect the post-harvest food safety outcomes is not entirely understood. This study tested how varying nitrogen and potassium fertilization levels affected the "susceptibility" of tomatoes to Salmonella infections following the harvest of fruits. Two tomato varieties grown over three seasons under high, medium, and low levels of nitrogen and potassium fertilization in two locations were inoculated with seven strains of Salmonella. Even though the main effects of nitrogen and potassium fertilization on the susceptibility of tomatoes to infections with Salmonella enterica were not statistically significant overall, differences in nitrogen concentrations in plant tissues correlated with the susceptibility of partially ripe tomatoes (cv. Solar Fire) to Salmonella. Tomato maturity and the season in which tomatoes were produced had the strongest effect on the ability of Salmonella to multiply in tomatoes. Tomato phenolics, accumulation of which is known to correlate with rates of the N fertilization, did not inhibit growth of Salmonella in vitro.

Southern Region Produce Safety Alliance Grower Training: Using Pre- and Post-Training Knowledge Assessments to Understand Training Effectiveness.

The Produce Safety Alliance (PSA) grower training was introduced in 2016 as the standardized curriculum to meet the training requirements of the Food and Drug Administration's (FDA) Food Safety Modernization Act's (FSMA) Produce Safety Rule (PSR). The PSR states that at least one supervisor or responsible party from each farm must have successfully completed this food safety training or one equivalent to the standardized curriculum, as recognized by the FDA. This study evaluated the effectiveness of PSA trainings conducted between 2017 and 2019 in the Southern United States by the Southern Regional Center for Food Safety Training, Outreach, and Technical Assistance by analyzing pre- and posttest assessments. Effectiveness was based on a 25-question knowledge assessment administered to participants before (n = 2494) and after (n = 2460) each training. The knowledge assessment indicated the overall effectiveness of the training, with average scores increasing significantly from pretest (15.9/25, 63.4%) to posttest (20.3/25, 81.3%) (P < 0.001). The greatest knowledge gains were seen in the Postharvest Handling and Sanitation, How to Develop a Farm Food Safety Plan, and Agricultural Water modules. Notably, these modules had lower posttest scores compared to the other modules, indicating that the amount of knowledge gained did not necessarily correspond with a sufficient understanding of the material. To ensure that participants understand all aspects of the PSR and best practices to minimize food safety risks, additional or advanced trainings may be needed. Additionally, the current testing instrument (pre-/posttest) used for PSA grower training, while validated, may not be optimal, thus alternative methods to assess the training effectiveness are likely needed.

Comparing the effectiveness of delivery style in produce safety training for growers.

The Produce Safety Alliance grower training has been offered since 2016. Prior to the pandemic, the course was offered exclusively in-person. During the pandemic, trainers were allowed to offer the course remotely. The effectiveness of in-person and remote delivery options was compared utilizing four methods: course evaluations completed at the training; a pre- and post-training knowledge assessment; a 1-year follow-up survey; and focus groups with course trainers. All methods, except the focus groups, were used as evaluation tools starting before and continuing during the pandemic. On the course evaluations, remote delivery and in-person participants rated their satisfaction with the training and their confidence in their ability to make changes at the same high rate. The knowledge assessment found remote delivery participants scored higher on the posttest than in-person participants when controlling for pretest score (p < 0.001); the effect size was between low and medium (ηp 2 = 0.025). On the follow-up survey, remote delivery participants reported making changes to food safety practices or infrastructure at a higher rate than in-person participants (68% vs. 53%, respectively, Χ2 (1, N = 700) = 6.372, p = 0.012, Cramer's V = 0.012 (very low)). There were demographic differences in educational level, job description, and number of years farming between the two populations. The focus group revealed advantages and disadvantages of both delivery methods, including internet availability, engagement activity, and course logistics and planning. Because no practical differences in outcome were measured between delivery methods and each had unique strengths, researchers recommend that educators should utilize both methods in the future. PRACTICAL APPLICATION: 1. When deciding between offering in-person or synchronous virtual training, trainers can feel confident that both delivery methods result in positive experiences from participants, learning, and behavior change. 2. PSA trainers may choose to offer the training remotely to increase accessibility for people who live in areas where there may not be enough growers to warrant holding an in-person training nearby, but should also consider that reliable high-speed internet access may not be available to all. 3. Remote delivery trainings can be smoother by hosting remote delivery participants at local extension or other partner offices where high-speed internet is available.

Irrigation Method Matters: Contamination and Die-off Rates of Escherichia coli on Dry Bulb Onions After Overhead and Drip Irrigation in Washington State (2022-2023).

Two U.S. outbreaks of salmonellosis in 2020 and 2021 were epidemiologically linked to red onions. The 2020 outbreak investigation implicated the production of agricultural water as a likely contamination source. Field trials were designed to investigate the prevalence and survival of Escherichia coli (surrogate for Salmonella) on dry bulb onions after the application of contaminated irrigation water at the end of the growing period. Irrigation water was inoculated at 3 log most probable number (MPN)/100 mL (2022 and 2023) or 5 log MPN/100 mL (2023, drip only) with a cocktail of rifampin-resistant E. coli and applied with the final irrigation (0.4 acre-inch/0.4 ha-cm) to onions. Onion bulbs (40 or 80) were sampled immediately after irrigation and throughout field curing (4 weeks) and E. coli was enumerated using an MPN method. For drip irrigation, at 3 log MPN/100 mL E. coli was detected on 13% of onions at 24 h but not detected at 0 h; at 5 log MPN/100 mL for drip irrigation applied to saturated soil, E. coli was detected in 63% of onions at 0 h. Prevalence significantly (P < 0.05), decreased after 7 d of curing with cell densities of 1-1,400 MPN/onion. At the end of field curing in 2023, 1/80 of onions had detectable E. coli (2.04 MPN/onion). E. coli was detected in a significantly smaller percentage of onions (2022: 13%; 2023: 68%) after a contaminated drip irrigation event compared to overhead irrigation (98-100%; P < 0.05). After overhead irrigation, E. coli was detected in onions (1-1,000 MPN/onion) on day 0. Prevalence decreased significantly (P < 0.05) after 7 d of field curing in both years (2022: 15%; 2023: 7%). E. coli was not detected on Calibra onions (80/year) at the end of field curing in either year but was detected at <12 MPN/onion in 2.5-3.75% of onions (n = 80) for other cultivars. These data confirm limited contamination risk associated with drip irrigation water quality and begin to quantify contamination risks associated with overhead irrigation of dry bulb onions.

Prevalence of STEC virulence markers and Salmonella as a function of abiotic factors in agricultural water in the southeastern United States.

Fresh produce can be contaminated by enteric pathogens throughout crop production, including through contact with contaminated agricultural water. The most common outbreaks and recalls in fresh produce are due to contamination by Salmonella enterica and Shiga toxin-producing E. coli (STEC). Thus, the objectives of this study were to investigate the prevalence of markers for STEC (wzy, hly, fliC, eaeA, rfbE, stx-I, stx-II) and Salmonella (invA) in surface water sources (n = 8) from produce farms in Southwest Georgia and to determine correlations among the prevalence of virulence markers for STEC, water nutrient profile, and environmental factors. Water samples (500 mL) from eight irrigation ponds were collected from February to December 2021 (n = 88). Polymerase chain reaction (PCR) was used to screen for Salmonella and STEC genes, and Salmonella samples were confirmed by culture-based methods. Positive samples for Salmonella were further serotyped. Particularly, Salmonella was detected in 6/88 (6.81%) water samples from all ponds, and the following 4 serotypes were detected: Saintpaul 3/6 (50%), Montevideo 1/6 (16.66%), Mississippi 1/6 (16.66%), and Bareilly 1/6 (16.66%). Salmonella isolates were only found in the summer months (May-Aug.). The most prevalent STEC genes were hly 77/88 (87.50%) and stx-I 75/88 (85.22%), followed by fliC 54/88 (61.63%), stx-II 41/88 (46.59%), rfbE 31/88 (35.22%), and eaeA 28/88 (31.81%). The wzy gene was not detected in any of the samples. Based on a logistic regression analysis, the odds of codetection for STEC virulence markers (stx-I, stx-II, and eaeA) were negatively correlated with calcium and relative humidity (p < 0.05). A conditional forest analysis was performed to assess predictive performance (AUC = 0.921), and the top predictors included humidity, nitrate, calcium, and solar radiation. Overall, information from this research adds to a growing body of knowledge regarding the risk that surface water sources pose to produce grown in subtropical environmental conditions and emphasizes the importance of understanding the use of abiotic factors as a holistic approach to understanding the microbial quality of water.

Escherichia coli Survival on Dry Bulb Onions Treated with Crop Protection Sprays Prepared using Contaminated Water in the Treasure Valley Growing Region.

Contaminated agricultural water has been implicated in produce-associated outbreaks, including dry bulb onions (Allium cepa). This study was designed to quantify risks associated with t contaminated water used to prepare crop protection sprays applied immediately before the onset of field curing of dry bulb onions. Laboratory experiments determining behavior of Salmonella and Escherichia coli in crop protection chemical solutions were performed to guide selection for field use. Field trials were conducted (2022, 2023) in eastern Oregon (Treasure Valley) using two onion cultivars ('Red Wing' and 'Cometa') inoculated with a rifampicin-resistant E. coli cocktail (3-4 log CFU/100 mL) suspended in fungicide solution or clay suspension, and applied with a backpack sprayer at the end of the growing season. Onions were sampled through the next 4 weeks of field curing and after 1 and 4-5 mos of postharvest storage. In 2022, onions were initially contaminated at a maximum cell density of 48 MPN/onion (Geometric mean (GM): 3.7 MPN/onion). At the end of curing, a single onion (out of 320) tested positive at 2 MPN/onion. In 2022, E. coli was not detected during postharvest storage (n = 160). In 2023, the application of contaminated sprays resulted in a maximum contamination of 275 MPN/onion (GM: 8.6 MPN/onion). At the end of the 2023 curing period, three out of 320 onions (0.9%) had detectable levels of E. coli (1-2 MPN/onion). Three 'Cometa' onions from the same plot that were treated with fungicide were positive for E. coli after 5 months of postharvest storage (2, 11, and 83 MPN/onion). These field trials indicate field curing conditions in the Treasure Valley help mitigate risks associated with contaminated water used for applying crop protection sprays. E. coli was detected on a small percentage of onions at low cell density after curing. The single onion with elevated E. coli populations after postharvest storage had internal damage characteristic of bacterial rot.