Interaction of Escherichia coli and Soil Particles in Runoff

A laboratory-scale model system was developed to investigate the transport mechanisms involved in the horizontal movement of bacteria in overland flow across saturated soils. A suspension of Escherichia coli and bromide tracer was added to the model system, and the bromide concentration and number of attached and unattached E. coli cells in the overland flow were measured over time. Analysis of the breakthrough curves indicated that the E. coli and bromide were transported together, presumably by the same mechanism. This implied that the E. coli was transported by advection with the flowing water. Overland-flow transport of E. coli could be significantly reduced if the cells were preattached to large soil particles (>45 μm). However, when unattached cells were inoculated into the system, the E. coli appeared to attach predominantly to small particles (<2 μm) and hence remained unattenuated during transport. These results imply that in runoff generated by saturation-excess conditions, bacteria are rapidly transported across the surface and have little opportunity to interact with the soil matrix.     

Erosion and subsequent transport state of Escherichia coli from cowpats

Processes by which fecal bacteria enter overland flow and their transportation state to surface waters are poorly understood, making the effectiveness of measures designed to intercept this pathway, such as vegetated buffer strips, difficult to predict. Freshly made and aged (up to 30 days) cowpats were exposed to simulated rainfall, and samples of the cowpat material and runoff were collected. Escherichia coli in the runoff samples were separated into attached (to particles) and unattached fractions, and the unattached fraction was analyzed to determine if the cells were clumped. Within cowpats, E. coli grew for 6 to 14 days, rather than following a typical logarithmic die-off curve. E. coli numbers in the runoff correlated with numbers inside the cowpat. Most of the E. coli organisms eroded from the cowpats were transported as single cells, and only a small percentage (about 8%) attached to particles. The erosion of E. coli from cowpats and the state in which the cells were transported did not vary with time within a single rainfall event or over time as the cowpats aged and dried out. These findings indicate that cowpats can remain a significant source of E. coli in overland flow for more than 30 days. As well, most of the E. coli organisms eroded from cowpats will occur as readily transportable single cells.     

Erosion and Subsequent Transport State of Escherichia coli from Cowpats

Processes by which fecal bacteria enter overland flow and their transportation state to surface waters are poorly understood, making the effectiveness of measures designed to intercept this pathway, such as vegetated buffer strips, difficult to predict. Freshly made and aged (up to 30 days) cowpats were exposed to simulated rainfall, and samples of the cowpat material and runoff were collected. Escherichia coli in the runoff samples were separated into attached (to particles) and unattached fractions, and the unattached fraction was analyzed to determine if the cells were clumped. Within cowpats, E. coli grew for 6 to 14 days, rather than following a typical logarithmic die-off curve. E. coli numbers in the runoff correlated with numbers inside the cowpat. Most of the E. coli organisms eroded from the cowpats were transported as single cells, and only a small percentage (about 8%) attached to particles. The erosion of E. coli from cowpats and the state in which the cells were transported did not vary with time within a single rainfall event or over time as the cowpats aged and dried out. These findings indicate that cowpats can remain a significant source of E. coli in overland flow for more than 30 days. As well, most of the E. coli organisms eroded from cowpats will occur as readily transportable single cells.     

Numbers and transported state of Escherichia coli in runoff direct from fresh cowpats under simulated rainfall

AIMS: To investigate the number of Escherichia coli in runoff derived directly from fresh cowpats and to determine if the E. coli are attached to dense particles, in flocs or as individual cells. METHODS AND RESULTS: Three cowpats were collected monthly from the same farm for 13 months and the number of E. coli in them estimated. A rainfall simulator was used to generate runoff from the individual cowpats, which was fractioned to determine the transported state of any E. coli present. The number of E. coli in the cowpat runoff was highly variable and was strongly correlated with the number of E. coli in the cowpat. Only a small percentage (approx. 8%) of the E. coli in runoff were attached to dense (>1.3 g ml(-1)) particles and there was no evidence of flocculation of the cells. CONCLUSIONS: Escherichia coli in runoff from cowpats are transported predominantly as individual cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Mitigation strategies to reduce the number of faecal bacteria in overland flow from agricultural land need to be designed to trap single bacterial cells.     

Overland flow delivery of faecal bacteria to a headwater pastoral stream

AIMS: To quantify and derive statistical relationships with which to predict the delivery of faecal bacteria (Escherichia coli) to a pastoral stream, by overland flow. METHODS AND RESULTS: A large-scale (1050 m2) rainfall simulator, located upon a steep (18 degrees) grazed hillside in New Zealand, was used to simulate 11 heavy rainfall events. Overland flow was generated and sampled throughout each event, before discharging to a headwater stream. The samples were subsequently analysed to determine the concentration of E. coli. Statistical analysis showed that the time elapsed since the last period of grazing was a statistically significant predictor of both the total number (load) and concentrations of E. coli in overland flow. Between 10(5) and 10(8)E. coli per m2 of hillside were delivered to the stream within overland flow during each event, and peak concentrations ranged between 10(3) and 10(7) most probable number per 100 ml. CONCLUSIONS: Under heavy rainfall on steep pastoral land, overland flow can transport substantial levels of faecal bacteria to streams. Under such conditions, it is unlikely that vegetated buffer strips will be particularly effective at attenuating bacteria within overland flow. SIGNIFICANCE AND IMPACT OF THE STUDY: This work has improved understanding of the importance of overland flow as a process contributing to the contamination of pastoral streams by faecal bacteria. In addition, the predictive relationships derived can be incorporated within catchment models.     

Importance of Preferential Flow and Soil Management in Vertical Transport of a Biocontrol Strain of Pseudomonas fluorescens in Structured Field Soil

The large-scale release of wild-type or genetically modified bacteria into the environment for control of plant diseases or for bioremediation entails the potential risk of groundwater contamination by these microorganisms. For a model study on patterns of vertical transport of bacteria under field conditions, the biocontrol strain Pseudomonas fluorescens CHA0, marked with a spontaneous resistance to rifampin (CHA0-Rif), was applied to a grass-clover ley plot (rotation grassland) and a wheat plot. Immediately after bacterial application, heavy precipitation was simulated by sprinkling, over a period of 8 h, 40 mm of water containing the mobile tracer potassium bromide and the dye Brilliant Blue FCF to identify channels of preferential flow. One day later, a 150-cm-deep soil trench was dug and soil profiles were prepared. Soil samples were extracted at different depths of the profiles and analyzed for the number of CHA0-Rif cells and the concentration of bromide and Brilliant Blue FCF. Dye coverage in the soil profiles was estimated by image analysis. CHA0 was present at 10(sup8) CFU/g in the surface soil, and 10(sup6) to 10(sup7) CFU/g of CHA0 was detected along macropores between 10 and 150 cm deep. Similarly, the concentration of the tracer bromide along the macropores remained at the same level below 20 cm deep. Dye coverage in lower soil layers was higher in the ley than in the wheat plot. In nonstained parts of the profiles, the number of CHA0-Rif cells was substantially smaller and the bromide concentration was below the detection limit in most samples. We conclude that after heavy rainfall, released bacteria are rapidly transported in large numbers through the channels of preferential flow to deeper soil layers. Under these conditions, the transport of CHA0-Rif is similar to that of the conservative tracer bromide and is affected by cultural practice.     

Leaching of Cryptosporidium parvum oocysts, Escherichia coli, and a Salmonella enterica serovar Typhimurium bacteriophage through intact soil cores following surface application and injection of slurry

Increasing amounts of livestock manure are being applied to agricultural soil, but it is unknown to what extent this may be associated with contamination of aquatic recipients and groundwater if microorganisms are transported through the soil under natural weather conditions. The objective of this study was therefore to evaluate how injection and surface application of pig slurry on intact sandy clay loam soil cores influenced the leaching of Salmonella enterica serovar Typhimurium bacteriophage 28B, Escherichia coli, and Cryptosporidium parvum oocysts. All three microbial tracers were detected in the leachate on day 1, and the highest relative concentration was detected on the fourth day (0.1 pore volume). Although the concentration of the phage 28B declined over time, the phage was still found in leachate at day 148. C. parvum oocysts and chloride had an additional rise in the relative concentration at a 0.5 pore volume, corresponding to the exchange of the total pore volume. The leaching of E. coli was delayed compared with that of the added microbial tracers, indicating a stronger attachment to slurry particles, but E. coli could be detected up to 3 months. Significantly enhanced leaching of phage 28B and oocysts by the injection method was seen, whereas leaching of the indigenous E. coli was not affected by the application method. Preferential flow was the primary transport vehicle, and the diameter of the fractures in the intact soil cores facilitated transport of all sizes of microbial tracers under natural weather conditions.     

Effect of bovine manure on fecal coliform attachment to soil and soil particles of different sizes

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.     

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

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.     

Development of a Distributed Watershed Contaminant Transport,Transformation, and Fate (CTT&F) Sub-model

CTT&F is a physically based, spatially distributed watershed contaminant transport, transformation, and fate sub-model designed for use within existing hydrological modeling systems. To describe the fate of contaminants through landscape media as well as spatial variations of contaminant distributions, physical transport and transformation processes in CTT&F are simulated for each cell in the model and routed to the watershed outlet. CTT&F simulates contaminant erosion from soil and transport across the land surface by overland flow. The model also simulates contaminant erosion from stream bed sediment and transport through channels in addition to transport of contaminants inputs by overland flow. CTT&F can simulate solid (granular) contaminant transport and transformation, including partitioning between freely dissolved, dissolved organic carbon (DOC) bound, and particle-sorbed phases. To demonstrate model capabilities, CTT&F was coupled with an existing distributed hydrologic model and was tested and validated to simulate RDX and TNT transport using two experimental plots. These experiments examined dissolution of solid contaminants into the dissolved phase and their subsequent transport to the plot outlet. Model results were in close agreement with measured data. Such a model provides information for decision makers to make rational decisions relevant to the fate of toxic compounds.     

Measuring mass transfer processes of octane with the help of an alkSalkB::gfp-tagged Escherichia coli

Diffusion of octane from oily droplets in different microscale settings was measured using Escherichia coli expressing the stable green fluorescent protein (GFP) from the alkB promoter of Pseudomonas oleovorans . GFP fluorescence intensities were determined quantitatively at the single-cell level after 1.0 or 2.5 h incubation and compared with different calibration series using known concentrations of octane. By immobilizing the E. coli sensor cells on the bottom glass plate of a microscope flow chamber, it was possible to monitor the diffusion process for octane in aqueous solution as a function of time and distance from non-aqueous phase droplets of octane alone or oily octane mixtures. When a gas phase was included in the flow chambers, octane transport could be demonstrated from the oily mixtures to the cells through both gas and liquid phase. Assays of non-immobilized sensor cells in microdroplets in the presence or absence of soil particles incubated with octane through the vapour phase revealed a slight reduction in the total amount of induced E. coli cells in the presence of soil. Our results indicate the power of using GFP-marked single-cell biosensors in determining microscale bioavailability of organic pollutants.     

Detection of Escherichia coli in blood using flow cytometry

A rapid method for the detection of Escherichia coli in blood has been developed. The method employs blood cell lysis, staining of bacteria with ethidium bromide, and detection of stained bacteria using flow cytometry. The detection protocol requires less than 2 h sample handling time and is not dependent on bacterial growth. This method has been applied to human donor blood specimens seeded with various E. coli concentrations and to two rabbit model systems. Bacterial detection is evident from the in vitro human blood studies at levels of 10 E. coli/ml and from in vivo rabbit model studies at less than 100 E. coli/ml.     

Uridine and cytidine transport in Escherichia coli B and transport-deficient mutants.

Three mutants of Escherichia coli B which are defective in components of the transport system for uridine and uracil were isolated and utilized to study the mechanism of uridine transport. Mutant U- was isolated from a culture resistant to 77 micronM 5-fluorouracil. Mutant U-UR-, isolated from a culture of mutant U-, is resistant to 770 micronM 5-fluorouracil and 750 micronM adenosine. Mutant NUC- is resistant to 80 micronM showdomycin and has been reported previously. The characteristics of uridine transport by E. coli B and the mutants provide data supporting the following conclusions. The transport of adenosine, deoxyadenosine, guanosine, deoxyguanosine, adenine, or guanine by mutant U- and mutant U-UR- is identical with that in the parental strain. Uridine is transported by E. coli B as intact uridine. In addition, extracellular uridine is also rapidly cleaved to uracil and the ribose moiety. The latter is transported into the cells, whereas uracil appears in the medium and is transported by a separate uracil transport system. The entry of the ribose moiety of uridine is fast relative to the uracil and uridine transport processes. The Km values and the inhibitory effects of heterologous nucleosides for the transport of uridine and the ribose moiety of uridine are similar. Studies of cytidine uptake in the parental and mutant strains provide evidence that cytidine is transported by two independent systems, one of which is the same as that involved in the transport of intact uridine. Uridine inhibits but is not transported by the other system for cytidine transport. Evidence for the above conclusions was based on comparisons of the characteristics of [2-14C]uridine, [U-14C]uridine, and [2-14C]cytidine transport using E. coli B and the three transport mutants under conditions which measure initial rates. The nature of the inhibitory effects of heterologous nucleosides on the uridine transport processes and identification of extracellular components from radioactive uridine provides supportive data for the conclusions.     

Effect of chemical fertilizers on the transport ofEscherichia coli,Pseudomonas aeruginosa andSalmonella infantis through sand columns

Five commercial fertilizers, Amfos, ammonium sulfate, Kamex, Kieserit and NPK affected the transport ofEscherichia coli, Pseudomonas aeruginosa andSalmonella infantis in sand columns. The percentage of cells transported through sand without fertilizers during a 2-h period was species-dependent (0.56 forS. infantis, 3.1 forE. coli and 12.4 forP. aeruginosa). The cell transport was enhanced by Kamex for all strains tested, whereas Amfos was found to decrease the transport ofE. coli andS. infantis cells. A mathematical model revealed a relationship between the transport of cells and the pH of the sand columns with fertilizers. Columns in which the pH was decreased by the fertilizers exhibited a higher retention of cells. This points to the existence of physico-chemical surface interactions between cells and sand particles.     

Phaseolotoxin transport in Escherichia coli and Salmonella typhimurium via the oligopeptide permease.

Phaseolotoxin [(N delta-phosphosulfamyl)ornithylalanylhomoarginine], a phytotoxic tripeptide produced by Pseudomonas syringae pv. phaseolicola that inhibits ornithine carbamoyltransferase, is transported into Escherichia coli and Salmonella typhimurium via the oligopeptide transport system (Opp). Mutants defective in oligopeptide permease (Opp-) were resistant to phaseolotoxin. Spontaneous phaseolotoxin-resistant mutants (Toxr) lacked the Opp function as evidenced by their cross-resistance to triornithine and failure to utilize glycylhistidylglycine as a source of histidine. Growth inhibition by phaseolotoxin was prevented by peptides known to be transported via the Opp system and by treatment of the toxin with L-aminopeptidase. In both E. coli and S. typhimurium, Toxr mutations were cotransducible with trp, suggesting that the opp locus occupies similar positions in genetic maps of these bacteria.     

Microquantitative determination of glucose in solutions using microorganisms as the test system

A technique for determining small quantities of glucose in solutions and biological liquids is proposed. The assay can be performed in the presence of some other carbohydrates, except those which are transported into E. coli cells by means of the phosphoenol-transferase system. The method is based on pH-metric regictration of H+-ions which are released from bacteria as a result of glucose consumption. The suspension of cells of E coli st. M-17 was used as a test system. We can also recommend a commercial preparation of colibacterin, which contains lyophilized bacteria E. coli st. M-17 in the sucrose-gelatin medium. The sensitivity of the method is about 1 microgram of glucose in 5-10 microliter.     

Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2)

Multidrug resistance protein 2 (MRP2) belongs to the ATP binding cassette family of transporters. Its substrates include organic anions and anticancer drugs. We have used transport assays with vesicles derived from Sf9 insect cells overproducing MRP2 to study the interactions of drugs, organic anions, and bile acids with three MRP2 substrates: estradiol-17-beta-d-glucuronide (E217betaG), methotrexate, and glutathione-S-dinitrophenol. Complex inhibition and stimulation patterns were obtained, different from those observed with the related transporters MRP1 and MRP3. In contrast to a previous report, we found that the rate of E217betaG transport by MRP2 increases sigmoidally with substrate concentration indicative of homotropic cooperativity. Half-maximal transport was obtained at 120 microm E217betaG, in contrast to values < 20 microm for MRP1 and 3. MRP2 stimulators, such as indomethacin and sulfanitran, strongly increased the affinity of MRP2 for E217betaG (half-maximal transport rates at 65 and 16 microm E217betaG, respectively) and shifted the sigmoidal dependence of transport rate on substrate concentration to a more hyperbolic one, without substantially affecting the maximal transport rate. Sulfanitran also stimulated MRP2 activity in cells, i.e. the transport of saquinavir through monolayers of Madin-Darby canine kidney II cells. Some compounds that stimulate E217betaG transport, such as penicillin G or pantoprazole, are not detectably transported by MRP2, suggesting that they allosterically stimulate transport without being cotransported with E217betaG. We propose that MRP2 contains two similar but nonidentical ligand binding sites: one site from which substrate is transported and a second site that regulates the affinity of the transport site for the substrate.     

Distribution of a genetically-engineered Escherichia coli population introduced into soil

The spatial localization of the cells and the DNA of a genetically-engineered Escherichia coli population introduced into soil was investigated. Inoculated soils were size fractioned and bacterial numbers and E. coli EL 1003 specific chromosomal DNA target sequences were enumerated in each fraction using plate-counting and MPN-PCR, respectively. Different numbers of either indigenous or introduced bacteria were found in each fraction indicating that their distribution in the soil was non-uniform. The distributions of the indigenous bacteria and the E. coli cells within the size fractions were significantly different: the E. coli population was mainly associated with the dispersible clay fraction (79·0%) from which only 10·7% of the indigenous bacteria were recovered. The distribution of the E. coli target DNA sequences was in agreement with the location of the cells. The different distribution of the two populations is likely to restrict genetic interactions. These results are relevant to potential interactions between native soil microflora and populations introduced into soil for competitive purposes.     

The fate of Escherichia coli and E. coli O157 in cattle slurry after application to land

The fate of both faecal Escherichia coli and E. coli O157 in slurry following application to arable and grass plots on a clay loam soil was studied. Slurry (5% dry matter) containing 5.3 x 10(4) ml(-1) E. coli and 30 E. coli O157 100 ml(-1) was spread in early March. Initially, almost all E. coli were retained in the upper layers of the soil. Escherichia coli numbers steadily declined to less than 1% of those applied by day 29, and E. coli O157 were only detected in the soil and on the grass for the first week after application. There was some transport of bacteria to deeper layers of the soil, but this was approximately 2% of the total; transport to drains over the same period was mainly associated with rainfall events and amounted to approximately 7% of applied E. coli. However, there were indications that periods of heavy rainfall could cause significant losses of E. coli by both leaching and run-off. Experimental studies showed that E. coli O157 on grass, which was subsequently ensiled in conditions allowing aerobic spoilage, could multiply to numbers exceeding 10(6) g(-1) in the silage.     

Interactions between cationic liposomes and bacteria: the physical-chemistry of the bactericidal action.

The bactericidal effect of dioctadecyldimethylammonium bromide (DODAB), a liposome forming synthetic amphiphile, is further evaluated for Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus in order to establish susceptibilities of different bacteria species towards DODAB at a fixed viable bacteria concentration (2.5 x 10(7) viable bacteria/mL). For the four species, susceptibility towards DODAB increases from E. coli to S. aureus in the order above. Typically, cell viability decreases to 5% over 1 h of interaction time at DODAB concentrations equal to 50 and 5 microm for E. coli and S. aureus, respectively. At charge neutralization of the bacterial cell, bacteria flocculation by DODAB vesicles is shown to be a diffusion-controlled process. Bacteria flocculation does not yield underestimated counts of colony forming units possibly because dilution procedures done before plating cause deflocculation. The effect of vesicle size on cell viability demonstrates that large vesicles, due to their higher affinity constant for the bacteria (45.20 m(-)) relative to the small vesicles (0.14 m(-)), kill E. coli at smaller DODAB concentrations. For E. coli and S. aureus, simultaneous determination of cell viability and electrophoretic mobility as a function of DODAB concentration yields a very good correlation between cell surface charge and cell viability. Negatively charged cells are 100% viable whereas positively charged cells do not survive. The results show a clear correlation between simple adsorption of entire vesicles generating a positive charge on the cell surfaces and cell death.