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.

A comparison of methods to detect low levels of Salmonella enterica in surface waters to support antimicrobial resistance surveillance efforts performed in multiple laboratories.

Developing effective and sensitive detection methods for antimicrobial resistant Salmonella enterica from surface water is a goal of the National Antimicrobial Resistance Monitoring System (NARMS). There are no specified methods for recovery of S. enterica in surface waters in the U.S. A multi-laboratory evaluation of four methods - bulk water enrichment (BW), vertical Modified Moore Swab (VMMS), modified Standard Method 9260.B2 (SM), and dead-end ultrafiltration (DEUF) - was undertaken to recover S. enterica from surface water. In Phase 1, one-liter volumes of water were collected from the same site on five different dates. Water was shipped and analyzed at four different laboratory locations (A, B, C, and D) for recovery of 1) inoculated fluorescent S. Typhimurium strain (ca. 30 CFU/L) and 2) Salmonella present in the water sampled. At each location, BW, VMMS, or SM recovery was performed on five separate 1 L water samples. Twenty 1 L water samples were subjected to each recovery method, and overall, sixty 1 L samples were assayed for Salmonella. Inoculated, fluorescent Salmonella Typhimurium and environmental Salmonella spp. were recovered from 65 % (39/60) and 45 % (27/60) of water samples, respectively. BW, VMMS, and SM recovered fluorescent S. Typhimurium from 60 %, 60 %, and 75 % of inoculated samples, respectively. Analysis by Chi-squared test determined laboratory location had a significant (p < 0.05) effect on fluorescent S. Typhimurium recovery compared to method or date of water collection. In Phase 2, recovery of inoculated fluorescent S. Typhimurium from 1 L samples by SM and DEUF was compared at laboratory locations B and D. SM and DEUF recovered fluorescent S. Typhimurium from 100 % (20/20) and 95 % (19/20) of inoculated water samples, respectively; laboratory location (p > 0.05) did not affect Salmonella recovery. Uniform laboratory methodology and training should be prioritized in conducting Salmonella recovery from surface water in laboratories.

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.

The effect of a first flush rainwater harvesting and subsurface irrigation system on E. coli and pathogen concentrations in irrigation water, soil, and produce.

Climate change is stressing irrigation water sources, necessitating the evaluation of alternative waters such as harvested rainwater to determine if they meet water quality and food safety standards. We collected water, soil, and produce samples between June and August 2019 from two vegetable rain garden (VRG) sites in Frederick, Maryland that harvest rainwater using a first flush system, and deliver this water to produce through subsurface irrigation. The raised VRG beds include layers of gravel, sand, and soil that act as filters. We recorded the average surface soil moisture in each bed as well as antecedent precipitation. All water (n = 29), soil (n = 55), and produce (n = 57) samples were tested for generic E. coli using standard membrane filtration, and water samples were also tested for Salmonella spp. and Listeria monocytogenes by selective enrichment. No Salmonella spp. or L. monocytogenes isolates were detected in any water samples throughout the study period. Average E. coli levels from all harvested rainwater samples at both sites (1.2 and 24.4 CFU/100 mL) were well below the Good Agricultural Practices food safety guidelines. Only 7 % (3/44) of produce samples from beds irrigated with harvested rainwater were positive for E. coli. E. coli levels in soil samples were positively associated with average surface soil moisture (r2 = 0.13, p = 0.007). Additionally, E. coli presence in water samples was marginally associated with a shorter length of antecedent dry period (fewer days since preceding rainfall) (p = 0.058). Our results suggest that harvested rainwater collected through a first flush system and applied to produce through subsurface irrigation meets current food safety standards. Soil moisture monitoring could further reduce E. coli contamination risks from harvested rainwater-irrigated produce. First flush and subsurface irrigation systems could help mitigate climate change-related water challenges while protecting food safety and security.

Levels of Salmonella enterica and Listeria monocytogenes in Alternative Irrigation Water Vary Based on Water Source on the Eastern Shore of Maryland.

Irrigation water sources have been shown to harbor foodborne pathogens and could contribute to the outbreak of foodborne illness related to consumption of contaminated produce. Determining the probability of and the degree to which these irrigation water sources contain these pathogens is paramount. The purpose of this study was to determine the prevalence of Salmonella enterica and Listeria monocytogenes in alternative irrigation water sources. Water samples (n = 188) were collected over 2 years (2016 to 2018) from 2 reclaimed water plants, 3 nontidal freshwater rivers, and 1 tidal brackish river on Maryland's Eastern Shore (ESM). Samples were collected by filtration using modified Moore swabs (MMS) and analyzed by culture methods. Pathogen levels were quantified using a modified most probable number (MPN) procedure with three different volumes (10 liters, 1 liter, and 0.1 liter). Overall, 65% (122/188) and 40% (76/188) of water samples were positive for S. enterica and L. monocytogenes, respectively. For both pathogens, MPN values ranged from 0.015 to 11 MPN/liter. Pathogen levels (MPN/liter) were significantly (P < 0.05) greater for the nontidal freshwater river sites and the tidal brackish river site than the reclaimed water sites. L. monocytogenes levels in water varied based on season. Detection of S. enterica was more likely with 10-liter filtration compared to 0.1-liter filtration. The physicochemical factors measured attributed only 6.4% of the constrained variance to the levels of both pathogens. This study shows clear variations in S. enterica and L. monocytogenes levels in irrigation water sources on ESM. IMPORTANCE In the last several decades, Maryland's Eastern Shore has seen significant declines in groundwater levels. While this area is not currently experiencing drought conditions or water scarcity, this research represents a proactive approach. Efforts, to investigate the levels of pathogenic bacteria and the microbial quality of alternative irrigation water are important for sustainable irrigation practices into the future. This research will be used to determine the suitability of alternative irrigation water sources for use in fresh produce irrigation to conserve groundwater.

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.

Treatment of COVID-19 Patients with Convalescent Plasma in Houston, Texas.

BACKGROUND: COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for more than 100 years. METHODS: Patients (n=25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28 to April 14, 2020. Patients were transfused with convalescent plasma obtained from donors with confirmed SARS-CoV-2 infection and had been symptom free for 14 days. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 post-transfusion. Clinical improvement was assessed based on a modified World Health Organization 6-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. RESULTS: At baseline, all patients were receiving supportive care, including anti-inflammatory and anti-viral treatments, and all patients were on oxygen support. At day 7 post-transfusion with convalescent plasma, nine patients had at least a 1-point improvement in clinical scale, and seven of those were discharged. By day 14 post-transfusion, 19 (76%) patients had at least a 1-point improvement in clinical status and 11 were discharged. No adverse events as a result of plasma transfusion were observed. The whole genome sequencing data did not identify a strain genotype-disease severity correlation. CONCLUSIONS: The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease. Randomized, controlled trials are needed to determine its efficacy.

Treatment of Coronavirus Disease 2019 (COVID-19) Patients with Convalescent Plasma.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for >100 years. Patients (n = 25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28, 2020, to April 14, 2020. Patients were transfused with convalescent plasma, obtained from donors with confirmed severe acute respiratory syndrome coronavirus 2 infection who had recovered. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 after transfusion. Clinical improvement was assessed on the basis of a modified World Health Organization six-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. At day 7 after transfusion with convalescent plasma, nine patients had at least a one-point improvement in clinical scale, and seven of those were discharged. By day 14 after transfusion, 19 (76%) patients had at least a one-point improvement in clinical status, and 11 were discharged. No adverse events as a result of plasma transfusion were observed. Whole genome sequencing data did not identify a strain genotype-disease severity correlation. The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.

Prevalence of Salmonella and Listeria monocytogenes in non-traditional irrigation waters in the Mid-Atlantic United States is affected by water type, season, and recovery method.

Irrigation water contaminated with Salmonella enterica and Listeria monocytogenes may provide a route of contamination of raw or minimally processed fruits and vegetables. While previous work has surveyed specific and singular types of agricultural irrigation water for bacterial pathogens, few studies have simultaneously surveyed different water sources repeatedly over an extended period of time. This study quantified S. enterica and L. monocytogenes levels (MPN/L) at 6 sites, including river waters: tidal freshwater river (MA04, n = 34), non-tidal freshwater river, (MA05, n = 32), one reclaimed water holding pond (MA06, n = 25), two pond water sites (MA10, n = 35; MA11, n = 34), and one produce wash water site (MA12, n = 10) from September 2016-October 2018. Overall, 50% (84/168) and 31% (53/170) of sampling events recovered S. enterica and L. monocytogenes, respectively. Results showed that river waters supported significantly (p < 0.05) greater levels of S. enterica than pond or reclaimed waters. The non-tidal river water sites (MA05) with the lowest water temperature supported significantly greater level of L. monocytogenes compared to all other sites; L. monocytogenes levels were also lower in winter and spring compared to summer seasons. Filtering 10 L of water through a modified Moore swab (MMS) was 43.5 (Odds ratio, p < 0.001) and 25.5 (p < 0.001) times more likely to recover S. enterica than filtering 1 L and 0.1 L, respectively; filtering 10 L was 4.8 (p < 0.05) and 3.9 (p < 0.05) times more likely to recover L. monocytogenes than 1L and 0.1 L, respectively. Work presented here shows that S. enterica and L. monocytogenes levels are higher in river waters compared to pond or reclaimed waters in the Mid-Atlantic region of the U.S., and quantitatively shows that analyzing 10 L water is more likely recover pathogens than smaller samples of environmental waters.

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.

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

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

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.

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

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

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

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

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

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

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

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