Daniel R. Shelton

Dehalogenation: A Novel Pathway for the Anaerobic Biodegradation of Haloaromatic Compounds

Microorganisms of lake sediment and sewage sludge anaerobically metabolized halobenzoates by a novel pathway. The primary degradative event was loss of the aryl halide without the alteration of the aromatic ring. Dehalogenation required strict anaerobic conditions and depended on the halogen and position, but not the number of halogen substituents. A stable methanogenic bacterial consortium was enriched from sludge and found capable of dehalogenating and often mineralizing a variety of halobenzoates to CH4 and CO2. The results suggest that reductive dehalogenation of aromatics could be important in removal of some chlorinated xenobiotics from the environment.

Escherichia Coli and Fecal Coliforms in Freshwater and Estuarine Sediments

It has been known for some time that substantial populations of fecal coliforms and E. coli are harbored in freshwater bottom sediments, bank soils, and beach sands. However, the relative importance of sediments as bacterial habitats and as a source of water-borne fecal coliforms and E. coli has not been recognized until recently, when a large number of publications have shown that in many cases the resuspension of sediment, rather then runoff from surrounding lands, can create elevated E. coli concentrations in water. This review is an attempt to develop the first comprehensive single source of existing information about fecal coliforms and E. coli in sediments and adjacent soils and to outline the knowledge gaps and research needs. The authors summarize available information on variability and environmental correlations of E. coli and FC concentrations in sediments, genetic diversity of E. coli in sediments, survival of E. coli and FC in sediments, release with resuspended sediment and settling of E. coli and FC, modeling of sediment effects on fate and transport of E. coli in surface waters, and implications for monitoring and management of microbiological water quality. The demonstrated role of pathogenic E. coli strains in food and water quality challenges reinforces the need in better understanding ecological and hydrological factors that affect functioning of sediments as E. coli reservoirs.

A handheld real time thermal cycler for bacterial pathogen detection.

The handheld advanced nucleic acid analyzer (HANAA) is a portable real time thermal cycler unit that weighs under 1 kg and uses silicon and platinum-based thermalcycler units to conduct rapid heating and cooling of plastic reaction tubes. Two light emitting diodes (LED) provide greater than 1 mW of electrical power at wavelengths of 490 nm (blue) and 525 nm (green), allowing detection of the dyes FAM and JOE/TAMRA. Results are displayed in real time as bar graphs, and up to three, 4-sample assays can be run on the charge of the 12 V portable battery pack. The HANAA was evaluated for detection of defined Escherichia coli strains, and wild-type colonies isolated from stream water, using PCR for the lac Z and Tir genes. PCR reactions using SYBR Green dye allowed detection of E. coli ATCC 11775 and E. coli O157:H7 cells in under 30 min of assay time; however, background fluorescence associated with dye binding to nonspecific PCR products was present. DNA extracted from three isolates of Bacillus anthracis Ames, linked to a bioterrorism incident in Washington DC in October 2001, were also successfully tested on the HANAA using primers for the vrrA and capA genes. Positive results were observed at 32 and 22 min of assay time, respectively. A TaqMan probe specific to the aroQ gene of Erwinia herbicola was tested on the HANAA and when 500 cells were used as template, positive results were observed after only 7 min of assay time. Background fluorescence associated with the use of the probe was negligible. The HANAA is unique in offering real time PCR in a handheld format suitable for field use; a commercial version of the instrument, offering six reaction chambers, is available as of Fall 2002.

Irrigation Waters as a Source of Pathogenic Microorganisms in Produce. A Review

There is increasing evidence that consumption of raw fresh produce is a major factor contributing to human gastrointestinal illness. A wide variety of pathogens contribute to food-borne illnesses, including bacteria (e.g., Salmonella, pathogenic Escherichia coli), protozoa (e.g., Cryptosporidium, Giardia), and viruses (e.g., noroviruses). Large-scale production of produce typically requires some form of irrigation during the growing season. There is a rapidly growing body of research documenting and elucidating the pathways of produce contamination by water-borne pathogens. However, many gaps still exist in our knowledge and understanding. The purpose of this review is to provide a comprehensive approach to the issue, including the most recent research. Topics covered include: temporal and spatial variability, and regional differences, in pathogen and indicator organism concentrations in water; direct and circumstantial evidence for contaminated water as a source of food-borne pathogens; fate and transport of pathogens and indicator organisms in irrigation systems, and the role of environmental microbial reservoirs; and current standards for irrigation water quality, and risk assessment. A concerted effort by researchers and practitioners is needed to maintain food safety of fresh produce in an increasingly intensive food production system and limited and declining irrigation water resources.

Denitrification and N mineralization from hairy vetch (Vicia villosa Roth) and rye (Secale cereale L.) cover crop monocultures and bicultures

N mineralization, N immobilization and denitrification were determined for vetch, rye and rye-vetch cover crops using large packed soil cores. Plants were grown to maturity from seed in cores. Cores were periodically leached, allowing for quantification of NO3− and NH4+ production, and denitrification incubations were conducted before and after cover crop kill. Gas permeable tubing was buried at two depths in cores allowing for quantification of N2O in the soil profile. Cover crops assimilated most soil N prior to kill. After kill, relative rates of N mineralization were vetch > rye-vetch mixture > fallow > rye. After correcting for N mineralization from fallow cores, net N mineralization was observed in vetch and rye-vetch cores, while net N immobilization was observed in rye cores. Denitrification incubations were conducted 5, 15 and 55 days after kill, with adjustment of cores to 75% water filled pore space (WFPS). The highest denitrification was observed in vetch cores 5 days after kill, when soil NO3− and respiration rates were high. Substantially lower denitrification was observed on subsequent measurement dates and in other treatments probably due to either limited NO3− or organic carbon in the soil. On day 5, 3%, 23%, 31% and 31% of the N2O was recovered in the headspace of fallow, vetch, rye and rye-vetch cores, respectively. The rest was stored in the soil profile. In a field study using intact soil cores, denitrification rates also peaked 1 week after cover crop kill and decreased significantly thereafter. Results suggest greater potential N losses from vetch than rye or rye-vetch cover crops due to rapid N-mineralization in conjunction with denitrification and potential leaching, prior to significant crop N-assimilation.

Survival of manure-borne E. coli in streambed sediment: effects of temperature and sediment properties.

Escherichia coli bacteria are commonly used as indicator organisms to designate of impaired surface waters and to guide the design of management practices to prevent fecal contamination of water. Stream sediments are known to serve as a reservoir and potential source of fecal bacteria (E. coli) for stream water. In agricultural watersheds, substantial numbers of E. coli may reach surface waters, and subsequently be deposited into sediments, along with fecal material in runoff from land-applied manures, grazing lands, or wildlife excreta. The objectives of this work were (a) to test the hypothesis that E. coli survival in streambed sediment in the presence of manure material will be affected by sediment texture and organic carbon content and (b) to evaluate applicability of the exponential die-off equation to the E. coli survival data in the presence of manure material. Experiments were conducted at three temperatures (4 degrees C, 14 degrees C, and 24 degrees C) in flow-through chambers using sediment from three locations at the Beaverdam Creek Tributary in Beltsville, Maryland mixed with dairy manure slurry in the proportion of 1000:1. Indigenous E. coli populations in sediments ranged from ca. 10(1) to 10(3)MPNg(-1) while approx 10(3) manure-borne E. coli MPNg(-1) were added. E. coli survived in sediments much longer than in the overlaying water. The exponential inactivation model gave an excellent approximation of data after 6-16 days from the beginning of the experiment. Slower inactivation was observed with the increase in organic carbon content in sediments with identical granulometric composition. The increase in the content of fine particles and organic carbon in sediments led not only to the slower inactivation but also to lower sensitivity of the inactivation to temperature. Streambed sediment properties have to be documented to better evaluate the role of sediments as reservoirs of E. coli that can affect microbiological stream water quality during high flow events.

Effect of Bovine Manure on Fecal Coliform Attachment to Soil and Soil Particles of Different Sizes

ABSTRACT Manure-borne bacteria can be transported in runoff as free cells, cells attached to soil particles, and cells attached to manure particles. The objectives of this work were to compare the attachment of fecal coliforms (FC) to different soils and soil fractions and to assess the effect of bovine manure on FC attachment to soil and soil fractions. Three sand fractions of different sizes, the silt fraction, and the clay fraction of loam and sandy clay loam soils were separated and used along with soil samples in batch attachment experiments with water-FC suspensions and water-manure-FC suspensions. In the absence of manure colloids, bacterial attachment to soil, silt, and clay particles was much higher than the attachment to sand particles having no organic coating. The attachment to the coated sand particles was similar to the attachment to silt and clay. Manure colloids in suspensions decreased bacterial attachment to soils, clay and silt fractions, and coated sand fractions, but did not decrease the attachment to sand fractions without the coating. The low attachment of bacteria to silt and clay particles in the presence of manure colloids may cause predominantly free-cell transport of manure-borne FC in runoff.

Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10(5) cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10-10(4) cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.

Quantitative Detection of Escherichia coli O157 in Surface Waters by Using Immunomagnetic Electrochemiluminescence

ABSTRACT A protocol for the quantitative detection of Escherichia coli O157 in raw and concentrated surface waters using immunomagnetic electrochemiluminescence (IM-ECL) was developed and optimized. Three antibody sandwich formats were tested: commercial anti-O157:H7 IM beads, IM beads made in-house with a polyclonal anti-O157:H7 immunoglobulin G (IgG), or IM beads made in-house with a monoclonal anti-O157:H7 IgG coupled with a polyclonal anti-O157:H7 IgG to which an electrochemiluminescent label (TAG) was attached. The monoclonal IM bead-polyclonal TAG format was chosen for optimization because it gave lower background levels and linear regression slopes of ca. 1.0, indicative of a constant ECL signal per cell. The dynamic range was ca. 101 to 105 cells ml−1 in phosphate-buffered saline and in raw water samples. The monoclonal IM beads selectively captured E. coli O157 cells in the presence of ca. 108 cells of a non-O157 strain of E. coli ml−1. Background ECL signals from concentrated (100-fold) water samples were substantially higher and more variable than raw water samples. The background signal was partially eliminated by the addition of polyvinylpolypyrrolidone. Successive cell capture incubations, termed sequential bead capture (SBC), were optimized for establishing baseline ECL values for individual water samples. The linear dynamic range with SBC was ca. 102 to 105E. coli O157 cells ml of concentrated water−1. To validate the protocol, 10-liter surface water samples were spiked with ca. 5,000E. coli O157 (Odwalla) cells and concentrated by vortex filtration, and 1- or 3-ml aliquots were analyzed by IM-ECL. Differential ECL signals (SBC) from 1- and 3-ml samples were statistically significant and were generally consistent with standard curves for these cell concentrations. Enrichments were conducted with aliquots of spiked raw water and concentrated water using EC broth and minimal lactose broth (MLB). All tubes with concentrated water became turbid and gave a positive ECL response for E. coli O157 (>10,000 ECL units); MLB gave a somewhat higher detection rate with spiked raw water. The potential sensitivity of the IM-ECL assay is ca. 25 E. coli O157 cells ml of raw water−1, 25 cells 100 ml of 100-fold concentrated water−1, or 1 to 2 viable cells liter−1 with concentration and enrichment. The IM-ECL assay appears suitable for routine analysis and screening of water samples.

Survival of Escherichia coli in cowpats in pasture and in laboratory conditions

Aims:  To compare survival of Escherichia coli and faecal coliforms (FC) in bovine faeces deposited in a pasture or incubated in a controlled laboratory environment at temperatures within the same range.