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.

Urea Release by Intermittently Saturated Sediments from a Coastal Agricultural Landscape.

Urea-N is linked to harmful algal blooms in lakes and estuaries, and urea-N-based fertilizers have been implicated as a source. However, the export of urea-N-based fertilizers appears unlikely, as high concentrations of urea-N are most commonly found in surface waters outside periods of fertilization. To evaluate possible autochthonous production of urea-N, we monitored urea-N released from drainage ditch sediments using mesocosms. Sediments from a cleaned (recently dredged) drainage ditch, uncleaned ditch, forested ditch, riparian wetland, and an autoclaved sand control were isolated in mesocosms and flooded for 72 h to quantify urea-N, NH-N, and NO-N in the floodwater. Sediments were flooded with different N-amended solutions (distilled HO, 1.5 mg L NH-N, 3.0 mg L NH-N, 2.6 mg L NO-N, or 5.1 mg L NO-N) and incubated at three water temperatures (16, 21, and 27°C). Urea-N concentrations in mesocosms representing uncleaned and cleaned drainage ditches were significantly greater than nonagricultural sediments and controls. While flooding sediments with N-enriched solution had no clear effect on urea-N, warmer (27°C) temperatures resulted in significantly higher urea-N. Data collected from field ditches that were flooded by a summer rainstorm showed increases in urea-N that mirrored the mesocosm experiment. We postulate that concentrations of urea-N in ditches that greatly exceed environmental thresholds are mediated by biological production in sediments and release to stagnant surface water. Storm-driven urea-N export from ditches could elevate the risk of harmful algal blooms downstream in receiving waters despite the dilution effect.

Persistence and Surface Transport of Urea-Nitrogen: A Rainfall Simulation Study.

Studies of harmful algal blooms and associated urea concentrations in the Chesapeake Bay and in coastal areas around the globe strongly suggest that elevated urea concentrations are associated with harmful algal blooms. The observed increased frequency and toxicity of these blooms in recent decades has been correlated with increased agricultural use of N inputs and increased use of urea as a preferred form of commercial N. This rainfall simulation study sought to assess the potential for different N fertilizers and manures to contribute to urea in runoff from a Coastal Plain soil on the Eastern Shore of Maryland. Under worst-case conditions, ~1% of urea-N applied as commercial fertilizer and surface-applied poultry litter was lost in runoff in a simulated rainfall event, roughly equivalent to a 1-yr return period rain storm in the study area, 12 h after application. Cumulative urea-N losses, including four subsequent weekly rainfall events, approached 1.7% from urea-N fertilizer containing a urease inhibitor. Urea-N loss from incorporated poultry litter was negligible, and losses from dairy manure were intermediate. These losses are likely confined to hydrological contributing areas that extend several meters from a drainage ditch or stream for storms with frequent recurrence intervals. Cumulative dissolved N losses in runoff (urea-N + ammonium-N + nitrate-N) as a proportion of total applied plant-available N were <5%, suggesting that most of the applied N was lost by other pathways or was immobilized in soil. Results also highlight the potential for simple management options, such as shallow incorporation or timing, to greatly reduce urea runoff losses.

Bacterial diversity in agricultural drainage ditches shifts with increasing urea-N concentrations.

Urea-based fertilizers applied to crop fields can enter the surface waters of adjacent agricultural drainage ditches and contribute to the nitrogen (N) loading in nearby watersheds. Management practices applied in drainage ditches promote N removal by the bacterial communities, but little is known about the impacts of excess urea fertilizer from crop fields on the bacterial diversity in these ditches. In 2017, sediments from drainage ditches next to corn and soybean fields were sampled to determine if fertilizer application and high urea-N concentrations alters bacterial diversity and urease gene abundances. A mesocosm experiment was paired with a field study to determine which bacterial groups respond to high urea-N concentrations. The bacterial diversity in the ditch next to corn fields was significantly different from the other site. The bacterial orders of Rhizobiales, Bacteroidales, Acidobacteriales, Burkholderiales, and Anaerolineales were most abundant in the ditch next to corn and increased after the addition of urea-N (0.5 mg N L-1) during the mesocosm experiment. The results of our study suggests that urea-N concentrations >0.07 mg N L-1, which are higher than concentrations associated with downstream harmful algal blooms, can lead to shifts in the bacterial communities of agricultural drainage ditches.

Natural sources and controlling factors of urea-nitrogen concentrations in agricultural drainage ditches.

Agricultural drainage ditches accumulate high urea-nitrogen (N) concentrations even in the absence of urea fertilizer applications to adjacent crop fields. The accumulated urea, and other bioavailable forms of dissolved organic nitrogen (DON), can be flushed downstream during substantial rainfall events altering downstream water quality and phytoplankton communities. Sources of urea-N supporting its accumulation in agricultural drainage ditches are poorly understood. A ditch flooding event was simulated using mesocosms with N treatment solutions and monitored for changes in N concentrations, physicochemical properties, dissolved organic matter (DOM) composition, and N cycling enzymes. N concentrations were also monitored in field ditches after two rainfall events. Urea-N concentrations were higher with DON enrichment, but the treatment effects were temporary. The DOM released from the mesocosm sediments was dominated by terrestrial-derived, high molecular weight material. The lack of microbial-derived DOM and evidence from the bacterial gene abundances in the mesocosms suggests that urea-N accumulation after rainfall may not be associated with fresh biological inputs. The urea-N concentrations after spring rainfall and flooding with DON substrates indicated the urea from fertilizers may only temporarily affect urea-N concentrations in drainage ditches. Because urea-N concentrations increased with a high degree of DOM humification, sources of urea may derive from the slow decomposition of complex DOM structures. This study provides further insights of sources contributing to high urea-N concentrations and the types of DOM released from drainage ditches to nearby surface waters after hydrological events.

Short-term infection of striped bass Morone saxatilis with Mycobacterium marinum.

Striped bass Morone saxatilis were studied in order to characterize their immune responses over the short term following challenge with Mycobacterium marinum. The expression of immunity-related genes (IL-1beta, TNF-alpha, Nramp and TGF-beta) quickly increased following infection with M. marinum, but these genes were subsequently down-regulated despite the fact that bacterial counts remained high. The number of monocytes and neutrophils also initially increased at 1 d postinfection. This confirms the importance of these types of cells in initial inflammation and mycobacterial infection in striped bass. The phagocytic index of splenic leukocytes over these same time frames did not change significantly following infection. The discrete window in which inflammatory mechanisms were stimulated in striped bass may be related to the intracellular nature of this pathogen.

Seasonal and temporal factors leading to urea-nitrogen accumulation in surface waters of agricultural drainage ditches.

Urea-nitrogen (N) is commonly applied to crop fields, yet it is not routinely monitored despite its association with reduced water quality and its ability to increase toxicity of certain phytoplankton species. The purpose of this work was to characterize temporal fluctuations in urea-N concentrations and associated environmental conditions to infer sources of urea-N in agricultural drainage ditches. Physicochemical properties and N forms in ditch waters were measured weekly during the growing seasons of 2015-2018. Fertilizer application was only associated with spring peaks of urea-N concentrations in ditches next to cornfields, whereas summer peaks in ditches adjacent to corn (Zea mays L.) and soybean [Glycine max (L.) Merr.] fields were not associated with fertilizer applications. Environmental conditions of warmer temperatures, lower dissolved oxygen concentrations, and lower redox potentials were correlated with higher urea-N concentrations. In 2018, peaks of urea-N and ammonium-N during the summer co-occurred with peaks of dissolved organic N and total dissolved N, suggesting they might be associated with the breakdown of organic matter and with the turnover of the organic N pool. Although the highest urea-N concentrations occurred when ditch surface waters were hydrologically disconnected from nearby streams, heavy rainfalls can potentially flush accumulated urea-N into coastal waters, where it may affect algal bloom toxicity. Therefore, implementation of available drainage ditch management practices is recommended, but these strategies need to be optimized for targeting periods with high rainfall that coincide with fertilizer additions as well as for periods with low rainfall that promote stagnant water conditions.

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.

Aeromonas hydrophila, Bacillus thuringiensis, Escherichia coli and Pseudomonas aeruginosa utilization of Ammonium-N, Nitrate-N and Urea-N in culture.

Agricultural drainage ditches represent a major source of nutrient pollution. Shifts in nitrogen source and use of animal manures have changed the bacterial composition both in species of bacteria and their abundance in agricultural ditches. This change affects how nitrogen is being cycled and potentially the final forms of available nutrients. In particular, animal manures often have bacteria such as Escherichia coli present, increasing the abundance of a bacterial species in ditches. Research has shown that the effect of different nitrogen sources is to change bacterial community composition (class, family). How this influences the role of an individual bacterial species is poorly understood. Thus, our question was how individual species would respond to different sources of nitrogen. We used Aeromonas hydrophila, Bacillus thuringiensis, Escherichia coli and Pseudomonas aeruginosa that are common in agricultural ditches and exposed them to different concentrations of nitrogen in cultures of 1 × 100 and 1 × 10-1 dilutions from a stock solution of bacteria. Nitrogen sources were ammonium chloride, sodium nitrate and urea. The results showed A. hydrophila and E. coli have strong similarities particularly with nitrate-N and urea-N utilization and the response was often correlated with the amount of nutrient added. P. aeruginosa while similar did not show any strong correlation with amount of nutrient added. B. thuringiensis was different from the other three bacteria in utilization or production. Research has provided insight into the role of some bacteria in nitrogen cycling and may be valuable in the future to developing management strategies to reduce nutrients.

Historical presence (1975-1985) of mycobacteriosis in Chesapeake Bay striped bass Morone saxatilis.

A retrospective analysis of archived tissue blocks has revealed that mycobacteriosis was apparent in Chesapeake Bay striped bass as early as 1984. Of 37 cases available from the years 1975 to 1985, 2 fish were found positive based on histopathology and genus-specific PCR. Multi-gene sequencing places the bacteria from the 2 positive cases (1984 and 1985) within the Mycobacterium tuberculosis clade with closest resemblance to the recently described fish pathogen M. pseudoshottsii. Our data confirms that mycobacteriosis is not a new disease of Chesapeake Bay striped bass and underscores the value of archived tissues in epidemiological examinations.

Tumor prevalence and biomarkers of exposure and response in brown bullhead (Ameiurus nebulosus) from the Anacostia River, Washington, DC and Tuckahoe River, Maryland, USA.

We evaluated liver and skin tumor prevalence and biomarkers of exposure and response in brown bullhead (Ameiurus nebulosus) from three locations in the Anacostia River (Washington, DC, USA), a Chesapeake Bay region of concern. The Tuckahoe River (Maryland, USA) served as a reference. Each river was sampled in fall 2000 and spring 2001. In the Anacostia, prevalence of liver tumors was 50 to 68%, and prevalence of skin tumors was 13 to 23% in large (> or = 260 mm, age > or = 3 years) bullheads. Liver and skin tumor prevalence was 10 to 17% and 0%, respectively, in small (150-225 mm, age 1-2 years) bullheads. Tuckahoe bullhead liver tumor prevalence was 0 to 3% (large) and 0% (small); none had skin tumors. Biliary polynuclear aromatic hydrocarbon (PAH)-like fluorescent metabolites and liver DNA adduct concentrations were elevated in large and small Anacostia bullheads. Mean adduct concentrations were 16 to 28 times higher than those in Tuckahoe fish. Chromatograms revealed a diagonal radioactive zone, indicating polycyclic aromatic compound (PAC)-DNA adducts. The biomarker data and the 10 to 17% liver tumor prevalence at ages 1 to 2 suggest that these year classes are likely to have a high prevalence as they reach age 3 and older. This study provides the strongest evidence to date of the role of PAHs in tumor development in Anacostia bullheads.

A protocol for conducting rainfall simulation to study soil runoff.

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The objective of this study was to characterize the effects of antecedent soil moisture content on the fate and transport of surface applied commercial urea, a common form of nitrogen (N) fertilizer, following a rainfall event that occurs within 24 hr after fertilizer application. Although urea is assumed to be readily hydrolyzed to ammonium and therefore not often available for transport, recent studies suggest that urea can be transported from agricultural soils to coastal waters where it is implicated in harmful algal blooms. A rainfall simulator was used to apply a consistent rate of uniform rainfall across packed soil boxes that had been prewetted to different soil moisture contents. By controlling rainfall and soil physical characteristics, the effects of antecedent soil moisture on urea loss were isolated. Wetter soils exhibited shorter time from rainfall initiation to runoff initiation, greater total volume of runoff, higher urea concentrations in runoff, and greater mass loadings of urea in runoff. These results also demonstrate the importance of controlling for antecedent soil moisture content in studies designed to isolate other variables, such as soil physical or chemical characteristics, slope, soil cover, management, or rainfall characteristics. Because rainfall simulators are designed to deliver raindrops of similar size and velocity as natural rainfall, studies conducted under a standardized protocol can yield valuable data that, in turn, can be used to develop models for predicting the fate and transport of pollutants in runoff.