Variations in Bacterial Communities and Antibiotic Resistance Genes Across Diverse Recycled and Surface Water Irrigation Sources in the Mid-Atlantic and Southwest United States: A CONSERVE Two-Year Field Study.

Reduced availability of agricultural water has spurred increased interest in using recycled irrigation water for U.S. food crop production. However, there are significant knowledge gaps concerning the microbiological quality of these water sources. To address these gaps, we used 16S rRNA gene and metagenomic sequencing to characterize taxonomic and functional variations (e.g., antimicrobial resistance) in bacterial communities across diverse recycled and surface water irrigation sources. We collected 1 L water samples (n = 410) between 2016 and 2018 from the Mid-Atlantic (12 sites) and Southwest (10 sites) U.S. Samples were filtered, and DNA was extracted. The V3-V4 regions of the 16S rRNA gene were then PCR amplified and sequenced. Metagenomic sequencing was also performed to characterize antibiotic, metal, and biocide resistance genes. Bacterial alpha and beta diversities were significantly different (p < 0.001) across water types and seasons. Pathogenic bacteria, such as Salmonella enterica, Staphylococcus aureus, and Aeromonas hydrophilia were observed across sample types. The most common antibiotic resistance genes identified coded against macrolides/lincosamides/streptogramins, aminoglycosides, rifampin and elfamycins, and their read counts fluctuated across seasons. We also observed multi-metal and multi-biocide resistance across all water types. To our knowledge, this is the most comprehensive longitudinal study to date of U.S. recycled water and surface water used for irrigation. Our findings improve understanding of the potential differences in the risk of exposure to bacterial pathogens and antibiotic resistance genes originating from diverse irrigation water sources across seasons and U.S. regions.

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.

Longitudinal Assessment of the Dynamics of Escherichia coli, Total Coliforms, Enterococcus spp., and Aeromonas spp. in Alternative Irrigation Water Sources: a CONSERVE Study.

As climate change continues to stress freshwater resources, we have a pressing need to identify alternative (nontraditional) sources of microbially safe water for irrigation of fresh produce. This study is part of the center CONSERVE, which aims to facilitate the adoption of adequate agricultural water sources. A 26-month longitudinal study was conducted at 11 sites to assess the prevalence of bacteria indicating water quality, fecal contamination, and crop contamination risk (Escherichia coli, total coliforms [TC], Enterococcus, and Aeromonas). Sites included nontidal freshwater rivers/creeks (NF), a tidal brackish river (TB), irrigation ponds (PW), and reclaimed water sites (RW). Water samples were filtered for bacterial quantification. E. coli, TC, enterococci (∼86%, 98%, and 90% positive, respectively; n = 333), and Aeromonas (∼98% positive; n = 133) were widespread in water samples tested. Highest E. coli counts were in rivers, TC counts in TB, and enterococci in rivers and ponds (P < 0.001 in all cases) compared to other water types. Aeromonas counts were consistent across sites. Seasonal dynamics were detected in NF and PW samples only. E. coli counts were higher in the vegetable crop-growing (May-October) than nongrowing (November-April) season in all water types (P < 0.05). Only one RW and both PW sites met the U.S. Food Safety Modernization Act water standards. However, implementation of recommended mitigation measures of allowing time for microbial die-off between irrigation and harvest would bring all other sites into compliance within 2 days. This study provides comprehensive microbial data on alternative irrigation water and serves as an important resource for food safety planning and policy setting.IMPORTANCE Increasing demands for fresh fruit and vegetables, a variable climate affecting agricultural water availability, and microbial food safety goals are pressing the need to identify new, safe, alternative sources of irrigation water. Our study generated microbial data collected over a 2-year period from potential sources of irrigation (rivers, ponds, and reclaimed water sites). Pond water was found to comply with Food Safety Modernization Act (FSMA) microbial standards for irrigation of fruit and vegetables. Bacterial counts in reclaimed water, a resource that is not universally allowed on fresh produce in the United States, generally met microbial standards or needed minimal mitigation. We detected the most seasonality and the highest microbial loads in river water, which emerged as the water type that would require the most mitigation to be compliant with established FSMA standards. This data set represents one of the most comprehensive, longitudinal analyses of alternative irrigation water sources in the United States.

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.

Incidence of fecal indicator and pathogenic bacteria in reclaimed and return flow waters in Arizona, USA.

The quality of irrigation water used to cultivate produce that is consumed raw is an important issue with regard to food safety. In this study, the microbiological quality of potential irrigation water sources in Arizona was evaluated by testing for the presence of indicator and pathogenic bacteria. Reclaimed water samples were collected from two wastewater treatment plants and return flow samples were collected from two drainage canals and one return flow pond. Standard membrane filtration methods were used for detection of indicator bacteria. Water samples (n = 28) were filtered through cellulose ester membrane filters and bacterial populations were enumerated by placing the filters on selective agar. For detection of pathogens (Salmonella enterica, Listeria monocytogenes and Shiga toxin-producing E. coli (STEC)), water samples were filtered through Modified Moore swabs and enriched in Universal Pre-enrichment Broth, followed by selective enrichment broth for each pathogen. The enriched broth was streaked onto agar media selective for each pathogen. Presumptive colonies were confirmed by PCR/real-time PCR. Among the 14 reclaimed water samples from two sites, the ranges of recovered populations of E. coli, total coliforms, and enterococci were 0-1.3, 0.5-8.3 × 103, and 0-5.5 CFU/100 mL, respectively. No L. monocytogenes, Salmonella or STEC were found. In the 13 return flow water samples from 3 sites, the ranges of recovered populations of E. coli, total coliforms and enterococci were 1.9-5.3 × 102, 6.5 × 102-9.1 × 104, and 2.9-3.7× 103 CFU/100 mL, respectively. All samples were negative for L. monocytogenes. One (7.1%) of the return flow samples was positive for E. coli O145. Nine (64.3%) of the samples were positive for Salmonella. Both real-time PCR and culture-based methods were used for the detection of Salmonella and L. monocytogenes, and the results from the two methods were comparable. The findings of this study provide evidence that irrigation waters in Arizona, including reclaimed water and return flows, could be potential sources of bacterial contamination of produce. Additional work is needed to evaluate whether bacteria present in irrigation water sources transfer to the edible portion of irrigated plants and are capable of persisting through post-harvest activities.

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.

Pharmaceuticals, herbicides, and disinfectants in agricultural water sources.

Agricultural water withdrawals account for the largest proportion of global freshwater use. Increasing municipal water demands and droughts are straining agricultural water supplies. Therefore, alternative solutions to agricultural water crises are urgently needed, including the use of nontraditional water sources such as advanced treated wastewater or reclaimed water, brackish water, return flows, and effluent from produce processing facilities. However, it is critical to ensure that such usage does not compromise soil, crop, and public health. Here, we characterized five different nontraditional water types (n = 357 samples) for the presence of pharmaceuticals, herbicides, and disinfectants using ultra-high-pressure liquid chromatography tandem mass spectrometry based method (UPLC-MS/MS). We then evaluated whether the levels of these contaminants were influenced by season. The highest level of herbicides (atrazine) was detected in untreated pond water (median concentration 135.9 ng/L). Reclaimed water had the highest levels of antibiotics and stimulants including azithromycin (215 ng/L), sulfamethoxazole (232.1 ng/L), and caffeine (89.4 ng/L). Produce processing plant water also tended to have high levels of atrazine (102.7 ng/L) and ciprofloxacin (80.1 ng/L). In addition, we observed seasonal variability across water types, with the highest atrazine concentrations observed during summer months, while the highest median azithromycin concentrations were observed in reclaimed water during the winter season. Further studies are needed to evaluate if economically feasible on-farm water treatment technologies can effectively remove such contaminants from nontraditional irrigation water sources.

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.