Soil Microclimate and Persistence of Foodborne Pathogens Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica Newport in Soil Affected by Mulch Type.

Mulching is a common agricultural practice that benefits crop production through soil moisture retention, weed suppression, and soil temperature regulation. However, little is known about the effect of mulch on foodborne pathogens present in soil. In this study, the influence of polyethylene plastic, biodegradable corn-based plastic, paper, and straw mulches on Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica Newport populations in soil was investigated. Silt loam soil in troughs was inoculated with a cocktail of the pathogens and covered with mulch or left bare, then incubated for 21 days, during which bacteria were enumerated and environmental parameters monitored. Bacterial counts declined in all treatments over time (p < 0.001) but persisted at 21 days at 0.8-0.95 log CFU/g. Pathogens also declined as a factor of mulch cover (p < 0.01). An exponential decay with asymptote model fit to the data revealed slower rates of decline in soil under mulches for all pathogens (p < 0.05) relative to bare soil. Compared to the average for all treatments, rates of decay in bare soil were 0.60 (p < 0.001), 0.45 (p < 0.05), and 0.63 (p < 0.001) log CFU/g/d for E. coli O157:H7, L. monocytogenes, and Salmonella, respectively. Linear multiple regression revealed that soil hydrological parameters were positively correlated (p < 0.05) with bacterial counts, while day soil temperatures were negatively correlated (p < 0.001), suggesting that higher day temperatures and lower moisture content of bare soil contributed to the faster decline of pathogens compared to mulched soil. A microcosm experiment using field soil from lettuce cultivation suggested no influence of prior mulch treatment on pathogens. In summary, pathogen decline in soil was modified by the soil microclimate created under mulch covers, but the effect appeared was restricted to the time of soil cover. Slower decline rates of pathogens in mulched soil may pose a risk for contamination of fresh market produce crops.

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.

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.

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.

Metagenomic analysis of bacterial and viral assemblages from a freshwater creek and irrigated field reveals temporal and spatial dynamics.

Lotic surface water sites (e.g. creeks) are important resources for localized agricultural irrigation. However, there is concern that microbial contaminants within untreated surface water may be transferred onto irrigated soil and crops. To evaluate this issue, water samples were collected between January 2017 and August 2018 from a freshwater creek used to irrigate kale and radish plants on a small farm in the Mid-Atlantic, United States. In addition, on one sampling date, a field survey was conducted in which additional water (creek source and point-of-use) and soil samples were collected to assess the viral and bacterial communities pre- and post- irrigation. All samples were processed for DNA extracts and shotgun sequenced on the Illumina HiSeq platform. The resulting metagenomic libraries were assembled de novo and taxonomic and functional features were assigned at the contig and peptide level. From these data, we observed that Betaproteobacteria (e.g. Variovorax) dominated the water, both at the source and point-of-use, and Alphaproteobacteria (e.g. Streptomyces) dominated both pre- and post-irrigated soil. Additionally, in the creek source water there were variations in the abundance of the dominant bacterial genera and functional annotations associated with seasonal characteristics (e.g. water temperature). Antibiotic resistance genes and virulence factors were also identified in the creek water and soil, with the majority specific to their respective habitat. Moreover, an analysis of clustered regularly interspaced short palindromic repeat (CRISPR) arrays showed the persistence of certain spacers through time in the creek water, as well as specific interactions between creek bacteriophages and their hosts. Overall, these findings provide a more holistic picture of bacterial and viral composition, dynamics, and interactions within a freshwater creek that can be utilized to further our knowledge on its suitability and safety for irrigation.

Waxing and cultivar affect Salmonella enterica persistence on cucumber (Cucumis sativus L.) fruit.

A dearth of knowledge exists on the pathogen-commodity association of Salmonella enterica on cucumber, despite cucumbers being implicated in multiple salmonellosis outbreaks in recent years in the U.S. Data are lacking on cultivar susceptibility to Salmonella colonization in relation to heterogeneity in fruit surface morphology. Further, fruit waxing is a common practice in wholesale cucumber to preserve the water content of fruit and prolong shelf-life, but its impact on epiphytic microbiota is not well studied. This study investigated the survival of Salmonella Newport and S. Javiana on the surface of six cucumber cultivars of varying surface morphology and the effect of fruit waxing on the persistence of S. Newport. S. Newport and S. Javiana were spot inoculated onto fruit of cultivars 'Marketmore 97', 'Patio Snacker' and 'Corinto' (varieties with trichomes or spines), and 'Bella', 'Pepinex' and 'Summer Dance' (glabrous or smooth varieties). Cucumbers were held at room temperature for 24 h before inoculated sections of exocarp were excised for enteropathogen enumeration. S. Javiana persisted at higher populations than S. Newport (1.0 and 1.5 log CFU g-1 exocarp decline, respectively) after 24 h (P < 0.05). Fruit waxing lessened S. Newport population decline after 24 h on all cultivars tested (P < 0.01). While there was a ~2 log CFU g-1 exocarp decline in populations on unwaxed cucumbers, the reduction on waxed cucumbers was ~1 log CFU g-1 exocarp. A cultivar effect was also observed; 'Summer Dance' supported consistently higher and 'Corinto' and 'Marketmore 97' (though for S. Javiana only) consistently lower Salmonella levels. Cultivars with trichomes were associated with lower populations than glabrous cultivars (P < 0.05). Our data showed that Salmonella persistence on fruit differed for the two serotypes tested and that cultivar differences play a role in determining cucumber surface favourability for pathogen colonization. Additionally, fruit waxing may elevate the risk of Salmonella persistence on cucumber.

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.

Quenching by sodium thiosulfate does not influence 16S rRNA gene sequencing profiles of reclaimed water from three sites in the Mid-Atlantic, United States.

A quenching agent is commonly added to chlorinated, reclaimed water during sample collection to prevent chlorine-mediated die-off of viable microbiota. However, the effect of quenching on downstream 16S rRNA-based bacterial community analyses is unclear. We conducted a side-by-side comparison of 16S rRNA sequencing data from reclaimed water samples quenched with sodium thiosulfate and non-quenched samples. Our data showed that 16 S rRNA processing and sequencing methods, and resulting bacterial profiles, were not negatively impacted by quenching.

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.

Creek to Table: Tracking fecal indicator bacteria, bacterial pathogens, and total bacterial communities from irrigation water to kale and radish crops.

The impact of microbially contaminated irrigation water on risks to produce safety and public health is a complex issue that is not well understood. This study tracked fecal indicators, pathogenic bacteria, and total bacterial communities from a creek water irrigation source to irrigated produce to assess the impact of irrigation events on soil and produce-associated microbiota. Kale and radishes were drip-irrigated using Mid-Atlantic creek water in October 2017. Plant and soil samples were collected immediately before and after irrigation, and for 3 consecutive days thereafter. All samples (n = 134), including irrigation water, were tested for generic Escherichia coli and total coliforms (TC) using standard membrane filtration or direct plating, and for Salmonella enterica and Listeria monocytogenes by selective enrichment. DNA extracted from all samples was PCR-amplified for the V3-V4 region of the 16S rRNA gene for bacterial community profiling. In soil, TC levels were significantly higher immediately and 3 days post-irrigation compared to pre-irrigation (p < 0.01). E. coli levels in soil increased after irrigation, but the difference was not significant (p = 0.31), and die-off was not observed. No E. coli were detected on kale leaves. TC increased over the study period on radish roots (p < 0.01) but not kale leaves (p = 0.43). Although target pathogens were detected in irrigation water, S. enterica was detected from only one post-irrigation kale sample and L. monocytogenes was not detected in the field. The 16S rRNA gene sequencing data revealed differences in bacterial community structure and composition across sample types and showed that radish soil and root surface bacterial communities were more strongly influenced by irrigation compared to kale samples. This study provides insights into the impact of irrigation water on fresh produce microbiota, revealing that, although irrigation did influence crop-associated microbiota (especially below ground) in the field, bacterial pathogens were not likely transferred to the crop.

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

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

Soil Type, Soil Moisture, and Field Slope Influence the Horizontal Movement of Salmonella enterica and Citrobacter freundii from Floodwater through Soil.

Pathogens in soil are readily mobilized by infiltrating water to travel downward through the soil. However, limited data are available on the horizontal movement of pathogens across a field. This study used a model system to evaluate the influence of soil type, initial soil moisture content, and field slope on the movement of Salmonella enterica serovar Newport across a horizontal plane of soil under flooding conditions. Three soil types of varying clay content were moistened to 40, 60, or 80% of their maximum water-holding capacities and flooded with water containing 6 log CFU/ml Salmonella Newport and Citrobacter freundii , the latter being evaluated as a potential surrogate for S. enterica in future field trials. A two-phase linear regression was used to analyze the microbial populations recovered from soil with increasing distance from the flood. This model reflected the presence of lag distances followed by a quantifiable linear decrease in the population of bacteria as a function of the distance from the site of flooding. The magnitude of the lag distance was significantly affected by the soil type, but this was not attributable to the soil clay content. The rate of the linear decline with distance from the flood zone was affected by soil type, initial soil moisture content, and soil incline. As the initial soil moisture content increased, the rate of decline in recovery decreased, indicating greater bacterial transport through soils. When flooding was simulated at the bottom of the soil incline, the rate of decline in recovery was much greater than when flooding was simulated at the top of the incline. There was no significant difference in recovery between Salmonella Newport and C. freundii , indicating that C. freundii may be a suitable surrogate for Salmonella Newport in future field studies.

Drip Line Flushing with Chlorine May Not Be Effective in Reducing Bacterial Loads in Irrigation Water Distribution Systems.

Irrigation water distribution systems are used to supply water to produce crops, but the system may also provide a protected environment for the growth of human pathogens present in irrigation water. In this study, the effects of drip tape installation depth and sanitization on the microbial quality of irrigation groundwater were evaluated. Drip tape lines were installed on the soil surface or 5 or 10 cm below the soil surface. Water samples were collected from the irrigation source and the end of each drip line every 2 weeks over an 11-week period, and the levels of Escherichia coli, total coliforms, aerobic mesophilic bacteria, and enterococci were quantified. Half of the lines installed at each depth were flushed with sodium hypochlorite for 1 h during week 6 to achieve a residual of 10 ppm at the end of the line. There was a statistically significant (P = 0.01) effect of drip tape installation depth and sanitizer application on the recovery of E. coli, with increased levels measured at the 5-cm depth and in nonsanitized lines, although the levels were at the limit of detection, potentially confounding the results. There was no significant effect of drip tape depth on total coliforms, aerobic mesophiles, or enterococci. In contrast, a statistically significant increase (P < 0.01) in the recovery of total coliforms was recorded from the ends of lines that received chlorine. This may be indicative of shedding of cells owing to degradation of biofilms that formed on the inner walls of the lines. These findings emphasize the need to better understand conditions that may lead to corrosion and increases in bacterial loads inside drip lines during flushing. Recommendations to growers should suggest collecting groundwater samples for testing at the end of drip lines rather than at the source. Guidelines on flushing drip lines with chlorine may need to include water pH monitoring, a parameter that influences the corrosive properties of chlorine.

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

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

Occurrence and Dispersal of Indicator Bacteria on Cucumbers Grown Horizontally or Vertically on Various Mulch Types.

No data exist on the impact of cultivation practices on food safety risks associated with cucumber. Cucumbers are typically grown horizontally over a mulch cover, with fruit touching the ground, but this vining plant grows well in vertical systems. To assess whether production system affects bacterial dispersal onto plants, field trials were conducted over 2 years. Cucumber cultivar 'Marketmore 76' was grown horizontally on plastic, straw, or bare ground or vertically on trellises installed on bare ground in soil previously amended with raw dairy manure. Fruit, flower, leaf, and soil samples were collected to quantify Escherichia coli , thermotolerant coliforms, and enterococci by direct plating. E. coli isolates were characterized by BOX-PCR to evaluate relatedness among strains. Although thermotolerant coliforms and enterococci were significantly less abundant on fruit in year 1 (P < 0.05), this result was not seen in year 2 when more rain was recorded. Instead, fruit from straw-mulched beds had higher levels of enterococci compared with fruit grown on bare ground (P < 0.05). Leaves on bare ground occasionally had more bacteria than did leaves on plastic mulch beds (P < 0.05). Production system did not impact flower-associated bacterial levels. E. coli isolates (n =127) were genotyped, generating 21 distinct fingerprints. Vertical production did not appear to be a barrier for E. coli dispersal to the crop, as suggested by numerous related isolates from soil and flowers on bare ground, straw-mulched, and trellised beds (subcluster B1). None of the isolates from soil and flowers in this subcluster were related to isolates recovered from fruit, showing that flower colonization does not necessarily lead to fruit colonization. One cluster of isolates contained those from flowers and fruits but not soil, indicating a source other than manure-amended soil. Straw may be a source of E. coli ; a number of closely related E. coli isolates were retrieved from soil and fruits from straw-mulched beds. Our approach revealed E. coli dispersal patterns and could be used to assess bacterial transmission in other production systems.