Temporal and Agricultural Factors Influence Escherichia coli Survival in Soil and Transfer to Cucumbers.

Biological soil amendments of animal origin (BSAAO) increase nutrient levels in soils to support the production of fruits and vegetables. BSAAOs may introduce or extend the survival of bacterial pathogens which can be transferred to fruits and vegetables to cause foodborne illness. Escherichia coli survival over 120 days in soil plots (3 m2) covered with (mulched) or without plastic mulch (not mulched), amended with either poultry litter, composted poultry litter, heat-treated poultry pellets, or chemical fertilizer, and transfer to cucumbers in 2 years (2018 and 2019) were evaluated. Plots were inoculated with E. coli (8.5 log CFU/m2) and planted with cucumber seedlings (Supremo). The number of days needed to reduce E. coli levels by 4 log CFU (dpi4log) was determined using a sigmoidal decline model. Random forest regression and one-way analysis of variance (ANOVA; P < 0.05) identified predictors (soil properties, nutrients, and weather factors) of dpi4log of E. coli and transfer to cucumbers. The combination of year, amendment, and mulch (25.0% increase in the mean square error [IncMSE]) and year (9.75% IncMSE) were the most prominent predictors of dpi4log and transfer to cucumbers, respectively. Nitrate levels at 30 days and soil moisture at 40 days were also impactful predictors of dpi4log. Differing rainfall amounts in 2018 (24.9 in.) and 2019 (12.6 in.) affected E. coli survival in soils and transfer to cucumbers. Salmonella spp. were recovered sporadically from various plots but were not recovered from cucumbers in either year. Greater transfer of E. coli to cucumbers was also shown to be partially dependent on dpi4log of E. coli in plots containing BSAAO.IMPORTANCE Poultry litter and other biological soil amendments are commonly used fertilizers in fruit and vegetable production and can introduce enteric pathogens such as Escherichia coli O157:H7 or Salmonella previously associated with outbreaks of illness linked to contaminated produce. E. coli survival duration in soils covered with plastic mulch or uncovered and containing poultry litter or heat-treated poultry litter pellets were evaluated. Nitrate levels on day 30 and moisture content in soils on day 40 on specific days were good predictors of E. coli survival in soils; however, the combination of year, amendment, and mulch type was a better predictor. Different cumulative rainfall totals from year to year most likely affected the transfer of E. coli from soils to cucumbers and survival durations in soil. E. coli survival in soils can be extended by the addition of several poultry litter-based soil amendments commonly used in organic production of fruits and vegetables and is highly dependent on temporal variation in rainfall.

Enteric Viruses and Pepper Mild Mottle Virus Show Significant Correlation in Select Mid-Atlantic Agricultural Waters.

Enteric viruses (EVs) are the largest contributors to foodborne illnesses and outbreaks globally. Their ability to persist in the environment, coupled with the challenges experienced in environmental monitoring, creates a critical aperture through which agricultural crops may become contaminated. This study involved a 17-month investigation of select human EVs and viral indicators in nontraditional irrigation water sources (surface and reclaimed waters) in the Mid-Atlantic region of the United States. Real-time quantitative PCR was used for detection of Aichi virus, hepatitis A virus, and norovirus genotypes I and II (GI and GII, respectively). Pepper mild mottle virus (PMMoV), a common viral indicator of human fecal contamination, was also evaluated, along with atmospheric (air and water temperature, cloud cover, and precipitation 24 h, 7 days, and 14 days prior to sample collection) and physicochemical (dissolved oxygen, pH, salinity, and turbidity) data, to determine whether there were any associations between EVs and measured parameters. EVs were detected more frequently in reclaimed waters (32% [n = 22]) than in surface waters (4% [n = 49]), similar to PMMoV detection frequency in surface (33% [n = 42]) and reclaimed (67% [n = 21]) waters. Our data show a significant correlation between EV and PMMoV (R2 = 0.628, P < 0.05) detection levels in reclaimed water samples but not in surface water samples (R2 = 0.476, P = 0.78). Water salinity significantly affected the detection of both EVs and PMMoV (P < 0.05), as demonstrated by logistic regression analyses. These results provide relevant insights into the extent and degree of association between human (pathogenic) EVs and water quality data in Mid-Atlantic surface and reclaimed waters, as potential sources for agricultural irrigation. IMPORTANCE Microbiological analysis of agricultural waters is fundamental to ensure microbial food safety. The highly variable nature of nontraditional sources of irrigation water makes them particularly difficult to test for the presence of viruses. Multiple characteristics influence viral persistence in a water source, as well as affecting the recovery and detection methods that are employed. Testing for a suite of viruses in water samples is often too costly and labor-intensive, making identification of suitable indicators for viral pathogen contamination necessary. The results from this study address two critical data gaps, namely, EV prevalence in surface and reclaimed waters of the Mid-Atlantic region of the United States and subsequent evaluation of physicochemical and atmospheric parameters used to inform the potential for the use of indicators of viral contamination.

The efficacy of zero valent iron-sand filtration on the reduction of Escherichia coli and Listeria monocytogenes in surface water for use in irrigation.

The use of surface and recycled water sources for irrigation can reduce demand on critical groundwater resources. Treatment or mitigation may be necessary for the use of these alternative water sources in order to reduce risk associated with microbial pathogens present in the water. In this study, the efficacy of a zero-valent iron (ZVI) sand filter was assessed for the reduction of Listeria monocytogenes and Escherichia coli in surface water. Water recovered from an agricultural pond was inoculated with E. coli TVS353 and an environmental L. monocytogenes isolate at 7 Log10 CFU/mL and horizontally filtered over a six-month period through a PVC pipe filter, filled with 35%:65% (volume:volume) ZVI:sand or sand alone. Filtered water was used to irrigate lettuce and bacterial persistence on lettuce leaves was determined for 7 days post-irrigation. Both ZVI:sand-filtered water and sand-filtered water contained significantly (p < 0.005) lower levels of E. coli and L. monocytogenes compared to initial unfiltered inoculated water. Population reductions of E. coli and L. monocytogenes were comparable after sand filtration. However, ZVI:sand filtration resulted in significantly greater population reductions of L. monocytogenes (P < 0.05) compared to E. coli. Populations of E. coli on leaves of lettuce plants irrigated with ZVI:sand-filtered water were not significantly lower than populations on plants irrigated with sand-filtered irrigation water over the 7-day period. However, populations of L. monocytogenes on lettuce leaves irrigated with ZVI-treated water were significantly lower than counts on plants irrigated with sand-filtered irrigation water on days 3 and 4 post irrigation (p = 0.052 and p = 0.042 for days 3 and 4, respectively. The differences observed in reductions of L. monocytogenes and E. coli by ZVI filtration is due to the differing effect that ZVI disruption has on Gram-positive and Gram-negative cell walls and membranes. ZVI- sand filters show promising results as an inexpensive on-farm technology for the mitigation of enteric foodborne bacterial populations in pond water over a six-month period.

Zerovalent iron-sand filtration can reduce the concentration of multiple antimicrobials in conventionally treated reclaimed water.

Irrigation with reclaimed water is increasing in areas that lack access to, and infrastructure for, high-level treatment and distribution. Antimicrobial residues are known to persist in conventionally treated reclaimed water, necessitating the investigation of reuse site-based mitigation options to further reduce these contaminants. We examined the effectiveness of a 50:50 volume/volume, particle matched, micro-scale zerovalent iron (ZVI)-sand filter in reducing concentrations of mixtures of antimicrobials present in pH-unadjusted conventionally treated reclaimed water. Twelve antimicrobials (azithromycin, ciprofloxacin, erythromycin, linezolid, oxacillin, oxolinic acid, penicillin G, pipemidic acid, sulfamethoxazole, triclocarban, tetracycline and vancomycin) were quantified using high performance-liquid chromatography-tandem mass spectrometry in reclaimed water, and ZVI-sand filtered reclaimed water, in a two-month long greenhouse-based experiment. Data were analyzed using a non-parametric rank-based approach. ZVI-sand filtration significantly reduced concentrations of azithromycin, ciprofloxacin, oxolinic acid, penicillin G, sulfamethoxazole, linezolid, pipemidic acid and vancomycin. Azithromycin, the antimicrobial with the highest median concentration (320 ng/L), was reduced to below the limit of detection after ZVI-sand filtration. Inorganic element (antimony, beryllium, cadmium, chromium, iron, lead, selenium and thallium) and water quality (free and total chlorine, nitrates, nitrites, pH and total dissolved solids) analyses showed that ZVI-sand filtered reclaimed water quality (nitrate, salinity, and inorganic elements) met the recommended guidelines for agricultural irrigation with reclaimed water. Based on our initial results, ZVI-sand filtration may be a promising basis for a point-of-use filtration system for reclaimed water irrigation on small-scale farms.

Prevalence of Shiga-toxigenic and atypical enteropathogenic Escherichia coli in untreated surface water and reclaimed water in the Mid-Atlantic U.S.

The microbial quality of irrigation water has increasingly become a concern as a source of contamination for fruits and vegetables. Non-traditional sources of water are being used by more and more growers in smaller, highly diversified farms in the Mid-Atlantic region of the U.S. Shiga-toxigenic E. coli (STEC) have been responsible for several outbreaks of infections associated with the consumption of leafy greens. Our study evaluated the prevalence of the "big seven" STEC serogroups and the associated enterohemorrhagic E. coli (EHEC) virulence factors (VF) genes in conventional and nontraditional irrigation waters in the Mid-Atlantic region of the U.S. Water samples (n = 510) from 170 sampling events were collected from eight untreated surface water sites, two wastewater reclamation facilities, and one vegetable processing plant, over a 12-month period. Ten liters of water were filtered through Modified Moore swabs (MMS); swabs were then enriched into Universal Pre-enrichment Broth (UPB), followed by enrichment into non-O157 STEC R&F broth and isolation on R & F non-O157 STEC chromogenic plating medium. Isolates (n = 2489) from enriched MMS from water samples were screened for frequently reported STEC serogroups that cause foodborne illness: O26, O45, O103, O111, O121, O145, and O157, along with VF genes stx1, stx2, eae, and ehxA. Through this screening process, STEC isolates were found in 2.35% (12/510) of water samples, while 9.0% (46/510) contained an atypical enteropathogenic E. coli (aEPEC) isolate. The eae gene (n = 88 isolates) was the most frequently detected EHEC VF of the isolates screened. The majority of STEC isolates (stx1 or stx2) genes mainly came from either a pond or reclamation pond water site on two specific dates, potentially indicating that these isolates were not spatially or temporally distributed among the sampling sites. STEC isolates at reclaimed water sites may have been introduced after wastewater treatment. None of the isolates containing eae were determined to be Escherichia albertii. Our work showed that STEC prevalence in Mid-Atlantic untreated surface waters over a 12-month period was lower than the prevalence of atypical EPEC.

Human Norovirus and Its Surrogates Induce Plant Immune Response in Arabidopsis thaliana and Lactuca sativa.

Human norovirus is the leading cause of foodborne illness worldwide with the majority of outbreaks linked to fresh produce and leafy greens. It is essential that we thoroughly understand the type of relationship and interactions that take place between plants and human norovirus to better utilize control strategies to reduce transmission of norovirus in the field onto plants harvested for human consumption. In this study the expression of gene markers for the salicylic acid (SA) and jasmonic acid (JA) plant defense pathways was measured and compared in romaine lettuce (Lactuca sativa) and Arabidopsis thaliana Col-0 plants that were inoculated with Murine Norovirus-1, Tulane Virus, human norovirus GII.4, or Hank's Balanced Salt Solution (control). Genes involving both the SA and JA pathways were expressed in both romaine lettuce and A. thaliana for all three viruses, as well as controls. Studies, including gene expression of SA- and JA-deficient A. thaliana mutant lines, suggest that the JA pathway is more likely involved in the plant immune response to human norovirus. This research provides the first pieces of information regarding how foodborne viruses interact with plants in the preharvest environment.

Survival and Transfer of Murine Norovirus within a Hydroponic System during Kale and Mustard Microgreen Harvesting.

Hydroponically grown microgreens are gaining in popularity, but there is a lack of information pertaining to their microbiological safety. The potential risks associated with virus contamination of crops within a hydroponic system have not been studied to date. Here a human norovirus (huNoV) surrogate (murine norovirus [MNV]) was evaluated for its ability to become internalized from roots to edible tissues of microgreens. Subsequently, virus survival in recirculated water without adequate disinfection was assessed. Kale and mustard seeds were grown on hydroponic pads (for 7 days with harvest at days 8 to 12), edible tissues (10 g) were cut 1 cm above the pads, and corresponding pieces (4 cm by 4 cm) of pads containing only roots were collected separately. Samples were collected from a newly contaminated system (recirculated water inoculated with ∼3 log PFU/ml MNV on day 8) and from a previously contaminated system. (A contaminated system without adequate disinfection or further inoculation was used for production of another set of microgreens.) Viral titers and RNA copies were quantified by plaque assay and real-time reverse transcription (RT)-PCR. The behaviors of MNV in kale and mustard microgreens were similar (P > 0.05). MNV was detected in edible tissues and roots after 2 h postinoculation, and the levels were generally stable during the first 12 h. Relatively low levels (∼2.5 to ∼1.5 log PFU/sample of both edible tissues and roots) of infectious viruses were found with a decreasing trend over time from harvest days 8 to 12. However, the levels of viral RNA present were higher and consistently stable (∼4.0 to ∼5.5 log copies/sample). Recirculated water maintained relatively high levels of infectious MNV over the period of harvest, from 3.54 to 2.73 log PFU/ml. Importantly, cross-contamination occurred easily; MNV remained infectious in previously contaminated hydroponic systems for up to 12 days (2.26 to 1.00 PFU/ml), and MNV was detected in both edible tissues and roots. Here we see that viruses can be recirculated in water, even after an initial contamination event is removed, taken up through the roots of microgreens, and transferred to edible tissues. The ease of product contamination shown here reinforces the need for proper sanitation.

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.

Application of Bacillus subtilis to the roots of leafy greens, in the presence of Listeria innocua and Salmonella Newport, induces closure of stomata.

Plant growth-promoting rhizobacterium Bacillus subtilis UD1022 has been shown to trigger an induced systemic response in Arabidopsis thaliana. This interaction causes plant stomata to close, protecting the plant from infection by plant pathogens and thereby increasing crop yield. The purpose of this study was to determine whether UD1022 applied to the roots of plants is able to induce stomata closure in leafy greens as well as influence the persistence of human pathogens (Listeria and Salmonella) on plants. UD1022 induced stomata closure in the presence of human pathogens on both lettuce and spinach 3 h post-inoculation (p<0.0001). Results were confirmed by root inoculation with heat-killed UD1022, which did not induce stomata closure. Presence of UD1022 on lettuce roots significantly reduced the persistence of Listeria on plants after 3 days post-inoculation (p=0.02) but had less of an effect on the persistence of Salmonella. The results of this study indicate that plant growth-promoting rhizobacterium B. subtilis UD1022 may be able to prevent contamination by some human pathogens. This is the first study to investigate the use of plant growth-promoting rhizobacteria to control the persistence of human pathogens on plants.

Effectiveness of calcium hypochlorite on viral and bacterial contamination of alfalfa seeds.

Alfalfa sprouts have been involved in numerous foodborne outbreaks, which has increased the awareness for seed and sprout safety. This study compared the effectiveness of calcium hypochlorite (Ca(OCl)2) on the inactivation of bacteria and viruses on alfalfa seeds and in the presence of a simulated organic load. Alfalfa seeds were inoculated with human norovirus (huNoV) genogroup II (GII), murine norovirus (MNV), Tulane virus (TV), Escherichia coli O104:H4, and Salmonella enterica serovar Agona. Seeds were treated with Ca(OCl)2 (2000 ppm or 20,000 ppm with the average of free chlorine 1388 ± 117 mg/L and 11,472 ± 1500 mg/L, respectively, pH adjusted to 7.00). The reduction of huNoV genomic copies indicated that huNoV was relatively resistant to Ca(OCl)2 regardless of concentrations. Significant reductions were observed in the order of TV < Salmonella Agona < MNV < E. coli O104:H4 at 20,000 ppm Ca(OCl)2. A similar trend was found at 2000 ppm Ca(OCl)2 in the order of TV, Salmonella Agona, MNV < E. coli O104:H4. Ca(OCl)2 at 20,000 ppm was more effective than 2000 ppm for all the organisms tested. This trend was also observed in samples containing an artificial organic material load. Ca(OCl)2 activity on virus inactivation decreased as the organic load increased. Reduction was greater in fetal bovine serum-containing samples compared to alfalfa seeds, indicating a close relationship between the organisms and alfalfa seeds. Ca(OCl)2 could not completely inactivate bacteria or viruses inoculated on seeds, and high levels of E. coli O104:H4 and Salmonella Agona were present on sprouts from sanitized seed samples following a 7-day germination period.

Evaluation of White Rot Fungus to Control Growth of Escherichia coli in Cattle Manure.

Biological soil amendments of animal origin, such as aqueous dairy manure, may be contaminated with microbial pathogens that can subsequently result in contaminated soil, water runoff, and crops. Multiple mitigation strategies are being evaluated to reduce these risks. Inclusion of filamentous fungus in a biofiltration system to inactivate pathogenic bacteria in aqueous dairy manure prior to land application is explored in this study as a preharvest preventative method. Of the microbes used to remediate biologically contaminated sites, ligninolytic white-rot fungi have been previously studied for their ability to degrade a wide variety of toxic or persistent environmental contaminants. Reduction of two E. coli strains (E. coli TVS355 and E. coli O157:H7 4407) was evaluated in aqueous dairy manure and PBS and in the presence of white-rot fungi Pleurotus ostreatus on three different nutrient sources (woodchips (WC), spent mushroom compost (SMC), and reticulated polyurethane foam (RPF)). Overall, E. coli TVS355 was more persistent in aqueous dairy manure and PBS, surviving for 50 days in the presence of P. ostreatus, with a final concentration of 4 log CFU/g in aqueous manure and 7 log CFU/g in PBS. However, greater (p < 0.0001) reduction of E. coli O157:H7 was observed, surviving for 50 days at an average of 4 log CFU/g in aqueous dairy manure and an average of 3 log CFU/g in PBS. Therefore, P. ostreatus has the potential to result in bacterial decay, with potential reduction observed in E. coli O157:H7. The RPF matrix showed positive results as a potential model for a nutrient limiting resource for P. ostreatus and could be the key to increased bacterial reductions if resulting in ligninolytic activity in order to seek other nutrient sources.

Simulated microgravity facilitates stomatal ingression by Salmonella in lettuce and suppresses a biocontrol agent.

As human spaceflight increases in duration, cultivation of crops in spaceflight is crucial to protecting human health under microgravity and elevated oxidative stress. Foodborne pathogens (e.g., Salmonella enterica) carried by leafy green vegetables are a significant cause of human disease. Our previous work showed that Salmonella enterica serovar Typhimurium suppresses defensive closure of foliar stomata in lettuce (Lactuca sativa L.) to ingress interior tissues of leaves. While there are no reported occurrences of foodborne disease in spaceflight to date, known foodborne pathogens persist aboard the International Space Station and space-grown lettuce has been colonized by a diverse microbiome including bacterial genera known to contain human pathogens. Interactions between leafy green vegetables and human bacterial pathogens under microgravity conditions present in spaceflight are unknown. Additionally, stomatal dynamics under microgravity conditions need further elucidation. Here, we employ a slow-rotating 2-D clinostat to simulate microgravity upon in-vitro lettuce plants following a foliar inoculation with S. enterica Typhimurium and use confocal microscopy to measure stomatal width in fixed leaf tissue. Our results reveal significant differences in average stomatal aperture width between an unrotated vertical control, plants rotated at 2 revolutions per minute (2 RPM), and 4 RPM, with and without the presence of S. typhimurium. Interestingly, we found stomatal aperture width in the presence of S. typhimurium to be increased under rotation as compared to unrotated inoculated plants. Using confocal Z-stacking, we observed greater average depth of stomatal ingression by S. typhimurium in lettuce under rotation at 4 RPM compared to unrotated and inoculated plants, along with greater in planta populations of S. typhimurium in lettuce rotated at 4 RPM using serial dilution plating of homogenized surface sterilized leaves. Given these findings, we tested the ability of the plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis strain UD1022 to transiently restrict stomatal apertures of lettuce both alone and co-inoculated with S. typhimurium under rotated and unrotated conditions as a means of potentially reducing stomatal ingression by S. typhimurium under simulated microgravity. Surprisingly, rotation at 4 RPM strongly inhibited the ability of UD1022 alone to restrict stomatal apertures and attenuated its efficacy as a biocontrol following co-inoculation with S. typhimurium. Our results highlight potential spaceflight food safety issues unique to production of crops in microgravity conditions and suggest microgravity may dramatically reduce the ability of PGPRs to restrict stomatal apertures.

Detection of Salmonella enterica and Listeria monocytogenes in alternative irrigation water by culture and qPCR-based methods in the Mid-Atlantic U.S.

Alternative irrigation waters (rivers, ponds, and reclaimed water) can harbor bacterial foodborne pathogens like Salmonella enterica and Listeria monocytogenes, potentially contaminating fruit and vegetable commodities. Detecting foodborne pathogens using qPCR-based methods may accelerate testing methods and procedures compared to culture-based methods. This study compared detection of S. enterica and L. monocytogenes by qPCR (real-time PCR) and culture methods in irrigation waters to determine the influence of water type (river, pond, and reclaimed water), season (winter, spring, summer, and fall), or volume (0.1, 1, and 10 L) on sensitivity, accuracy, specificity, and positive (PPV), and negative (NPV) predictive values of these methods. Water samples were collected by filtration through modified Moore swabs (MMS) over a 2-year period at 11 sites in the Mid-Atlantic U.S. on a bi-weekly or monthly schedule. For qPCR, bacterial DNA from culture-enriched samples (n = 1,990) was analyzed by multiplex qPCR specific for S. enterica and L. monocytogenes. For culture detection, enriched samples were selectively enriched, isolated, and PCR confirmed. PPVs for qPCR detection of S. enterica and L. monocytogenes were 68% and 67%, respectively. The NPV were 87% (S. enterica) and 85% (L. monocytogenes). Higher levels of qPCR/culture agreement were observed in spring and summer compared to fall and winter for S. enterica; for L. monocytogenes, lower levels of agreement were observed in winter compared to spring, summer, and fall. Reclaimed and pond water supported higher levels of qPCR/culture agreement compared to river water for both S. enterica and L. monocytogenes, indicating that water type may influence the agreement of these results. IMPORTANCE: Detecting foodborne pathogens in irrigation water can inform interventions and management strategies to reduce risk of contamination and illness associated with fresh and fresh-cut fruits and vegetables. The use of non-culture methods like qPCR has the potential to accelerate the testing process. Results indicated that pond and reclaimed water showed higher levels of agreement between culture and qPCR methods than river water, perhaps due to specific physiochemical characteristics of the water. These findings also show that season and sample volume affect the agreement of qPCR and culture results. Overall, qPCR methods could be more confidently utilized to determine the absence of Salmonella enterica and Listeria monocytogenes in irrigation water samples examined in this study.

Survival of murine norovirus, Tulane virus, and hepatitis A virus on alfalfa seeds and sprouts during storage and germination.

Human norovirus (huNoV) and hepatitis A virus (HAV) have been involved in several produce-associated outbreaks and identified as major food-borne viral etiologies. In this study, the survival of huNoV surrogates (murine norovirus [MNV] and Tulane virus [TV]) and HAV was investigated on alfalfa seeds during storage and postgermination. Alfalfa seeds were inoculated with MNV, TV, or HAV with titers of 6.46 ± 0.06 log PFU/g, 3.87 ± 0.38 log PFU/g, or 7.01 ± 0.07 log 50% tissue culture infectious doses (TCID50)/g, respectively. Inoculated seeds were stored for up to 50 days at 22°C and sampled during that storage period on days 0, 2, 5, 10, and 15. Following storage, virus presence was monitored over a 1-week germination period. Viruses remained infectious after 50 days, with titers of 1.61 ± 0.19 log PFU/g, 0.85 ± 0.21 log PFU/g, and 3.43 ± 0.21 log TCID50/g for MNV, TV, and HAV, respectively. HAV demonstrated greater persistence than MNV and TV, without a statistically significant reduction over 20 days (<1 log TCID50/g); however, relatively high levels of genomic copies of all viruses persisted over the testing time period. Low titers of viruses were found on sprouts and were located in all tissues as well as in sprout-spent water sampled on days 1, 3, and 6 following seed planting. Results revealed the persistence of viruses in seeds for a prolonged period of time, and perhaps of greater importance these data suggest the ease of which virus may transfer from seeds to sprouts and spent water during germination. These findings highlight the importance of sanitation and prevention procedures before and during germination.

Norovirus surrogate survival on spinach during preharvest growth.

Produce can become contaminated with human viral pathogens in the field through soil, feces, or water used for irrigation; through application of manure, biosolids, pesticides, and fertilizers; and through dust, insects, and animals. The objective of this study was to assess the survival and stability of human noroviruses and norovirus surrogates (Murine norovirus [MNV] and Tulane virus [TV]) on foliar surfaces of spinach plants in preharvest growth conditions. Spinach plants were housed in a biocontrol chamber at optimal conditions for up to 7 days and infectivity was determined by plaque assay. Virus inoculation location had the largest impact on virus survival as viruses present on adaxial leaf surfaces had lower decimal reduction time (D values) than viruses present on abaxial leaf surfaces. Under certain conditions, spinach type impacted virus survival, with greater D values observed from survival on semi-savoy spinach leaves. Additional UVA and UVB exposure to mimic sunlight affected virus survival on adaxial surfaces for both semi-savoy and smooth spinach plants for both viruses. Human GII norovirus inoculated onto semi-savoy spinach had an average D value that was not statistically significant from MNV and TV, suggesting that these surrogates may have similar survival on spinach leaves compared with human noroviruses. An understanding of the behavior of enteric viruses on spinach leaves can be used to enhance growers' guidelines and for risk assessment with certain growing conditions.

Pathogenic psychrotolerant sporeformers: an emerging challenge for low-temperature storage of minimally processed foods.

Sporeforming bacteria are a significant problem in the food industry as they are ubiquitous in nature and capable of resisting inactivation by heat and chemical treatments designed to inactivate them. Beyond spoilage issues, psychrotolerant sporeformers are becoming increasingly recognized as a potential hazard given the ever-expanding demand for refrigerated processed foods with extended shelf-life. In these products, the sporeforming pathogens of concern are Bacillus cereus, Bacillus weihenstephanensis, and Clostridium botulinum type E. This review article examines the foods, conditions, and organisms responsible for the food safety issue caused by the germination and outgrowth of psychrotolerant sporeforming pathogens in minimally processed refrigerated foods.

Microgravity and evasion of plant innate immunity by human bacterial pathogens.

Spaceflight microgravity and modeled-microgravity analogs (MMA) broadly alter gene expression and physiology in both pathogens and plants. Research elucidating plant and bacterial responses to normal gravity or microgravity has shown the involvement of both physiological and molecular mechanisms. Under true and simulated microgravity, plants display differential expression of pathogen-defense genes while human bacterial pathogens exhibit increased virulence, antibiotic resistance, stress tolerance, and reduced LD50 in animal hosts. Human bacterial pathogens including Salmonella enterica and E. coli act as cross-kingdom foodborne pathogens by evading and suppressing the innate immunity of plants for colonization of intracellular spaces. It is unknown if evasion and colonization of plants by human pathogens occurs under microgravity and if there is increased infection capability as demonstrated using animal hosts. Understanding the relationship between microgravity, plant immunity, and human pathogens could prevent potentially deadly outbreaks of foodborne disease during spaceflight. This review will summarize (1) alterations to the virulency of human pathogens under microgravity and MMA, (2) alterations to plant physiology and gene expression under microgravity and MMA, (3) suppression and evasion of plant immunity by human pathogens under normal gravity, (4) studies of plant-microbe interactions under microgravity and MMA. A conclusion suggests future study of interactions between plants and human pathogens under microgravity is beneficial to human safety, and an investment in humanity's long and short-term space travel goals.

Human Norovirus Surrogates Persist in Nontraditional Sources of Irrigation Water in Excess of 100 Days.

Human norovirus (HuNoV) has been implicated as the leading cause of foodborne illness worldwide. The ability of HuNoV to persist in water can significantly impact food safety as agriculture and processing water could serve as vehicles of virus transmission. This study focused on the persistence and infectivity of the HuNoV surrogate viruses, murine norovirus (MNV), and Tulane virus (TV), after prolonged storage in diverse environmental water types currently used for agricultural irrigation. In this study, vegetable processing water (VW), brackish tidal surface water (SW), municipal reclaimed water (RW), and pond water (PW) were inoculated with each virus in a 1:10 v/v ratio containing virus at 3.5-4.5 logPFU/mL and stored at 16°C for 100 days. This time and temperature combination was chosen to mimic growing and harvest conditions in the mid-Atlantic area of the United States. Samples were then assayed for the presence of viral RNA using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approximately weekly throughout the study. Persistence of MNV and TV was not significantly different (p > 0.05) from one another in any water sample (n = 7) or the control (HBSS). However, there was variability observed in viral persistence across water samples with significant differences observed between several water samples. The presence of intact viral capsids enclosing the genomes of MNV and TV were evaluated by an RNase assay coupled with RT-qPCR on specific timepoints and determined to be intact up to and at 100 days after inoculation. TV was also shown to remain infectious in a cell culture assay (TCID50) up to 100 days of incubation. These findings are significant in that the potential for not only detection of enteric viruses can occur long after a contamination event occurs but these viruses may also remain infectious.

Temporal Stability of Salmonella enterica and Listeria monocytogenes in Surface Waters Used for Irrigation in the Mid-Atlantic United States.

Enteric bacterial pathogen levels can influence the suitability of irrigation water sources for fruits and vegetables. We hypothesize that stable spatial patterns of Salmonella enterica and Listeria monocytogenes levels may exist across surface water sources in the Mid-Atlantic U.S. Water samples were collected at four streams and two pond sites in the mid-Atlantic U.S. over 2 years, biweekly during the fruit and vegetable growing seasons, and once a month during nongrowing seasons. Two stream sites and one pond site had significantly different mean concentrations in growing and nongrowing seasons. Stable spatial patterns were determined for relative differences between the site concentrations and average concentration of both pathogens across the study area. Mean relative differences were significantly different from zero at four of the six sites for S. enterica and three of six sites for L. monocytogenes. There was a similarity between the mean relative difference distribution between sites over growing season, nongrowing season, and the entire observation period. Mean relative differences were determined for temperature, oxidation-reduction potential, specific electrical conductance, pH, dissolved oxygen, turbidity, and cumulative rainfall. A moderate-to-strong Spearman correlation (rs > 0.657) was found between spatial patterns of S. enterica and 7-day rainfall, and between relative difference patterns of L. monocytogenes and temperature (rs = 0.885) and dissolved oxygen (rs = -0.885). Persistence in ranking sampling sites by the concentrations of the two pathogens was also observed. Finding spatially stable patterns in pathogen concentrations highlights spatiotemporal dynamics of these microorganisms across the study area can facilitate the design of an effective microbial water quality monitoring program for surface irrigation water.

Human enteric pathogen internalization by root uptake into food crops.

With an increasing number of outbreaks and illnesses associated with produce contaminated before harvest, understanding the potential and mechanisms of produce contamination by enteric pathogens can aid in the development of preventative and post-harvest processing measures to reduce microbial populations. Enteric pathogens localized at subsurface sites on leafy green plant tissue prevent their removal during washing and inactivation by sanitizers. Root uptake of enteric pathogens and subsequent internalization has been a large area of research with results varying due to differences in experimental design, systems tested, and pathogens and crops used. The potential for uptake of foodborne pathogen, both bacterial and viral, through roots into food crops is reviewed. Various factors shown to affect the ability of human pathogens to internalize include growth substrate (soil vs. hydroponic solution), plant developmental stage, pathogen genus and/or strain, inoculum level, and plant species and cultivar. Several mechanisms of internalization ("active" vs. "passive") of bacteria to plant roots have also been hypothesized.