Microbial risk assessment for recreational use of the Chicago area waterway system.

A microbial risk assessment was conducted to estimate the human health risks from incidental contact recreational activities such as canoeing, boating and fishing in the Chicago Area Waterway System (CAWS) receiving secondary treated, but non-disinfected, effluent from three municipal water reclamation plants. Actual concentrations of the pathogens (pathogenic E. coli [estimated], Giardia, Cryptosporidium, adenovirus, norovirus, enteric virus) detected from the waterway field data collection at locations upstream and downstream of the effluent outfall during dry and wet weather conditions within the recreation season were included in the risk assessment. The results under the current treatment scheme with no disinfection indicated that the total expected gastrointestinal illness (GI) rate per 1000 incidental contact recreational exposure events during combined weather (dry and wet) conditions ranged from 0.10 to 2.78 in the CAWS, which is below the eight illnesses per 1000 swimmers considered tolerable by the United States Environmental Protection Agency. Wet weather conditions contribute to elevated pathogen load to the CAWS; therefore this study determined that disinfecting the effluents of three major WRPs that discharge to the CAWS would result in an extremely small reduction in the aggregate recreation season risk to incidental contact recreators.

Isolation and characterization of a Geobacillus thermoleovorans strain from an ultra-deep South African gold mine.

A thermophilic facultative bacterial isolate was recovered from 3.2km depth in a gold mine in South Africa. This isolate, designated GE-7, was cultivated from pH 8.0, 50 degrees C water from a dripping fracture near the top of an exploration tunnel. GE-7 grows optimally at 65 degrees C and pH 6.5 on a wide range of carbon substrates including cellobiose, hydrocarbons and lactate. In addition to O(2), GE-7 also utilizes nitrate as an electron acceptor. GE-7 is a long rod-shaped bacterium (4-6microm longx0.5microm wide) with terminal endospores and flagella. Phylogenetic analysis of GE-7 16S rDNA sequence revealed high sequence similarity with G. thermoleovorans DSM 5366(T) (99.6%), however, certain phenotypic characteristics of GE-7 were distinct from this and other previously described strains of G. thermoleovorans.

Physical versus chemical effects on bacterial and bromide transport as determined from on site sediment column pulse experiments.

Twenty-eight bacterial and Br transport experiments were performed in the field to determine the effects of physical and chemical heterogeneity of the aquifer sediment. The experiments were performed using groundwater from two field locations to examine the effects of groundwater chemistry on transport. Groundwater was extracted from multilevel samplers and pumped through 7-cm-long columns of intact sediment or repacked sieved and coated or uncoated sediment from the underlying aquifer. Two bacterial strains, Comamonas sp. DA001 and Paenibacillus polymyxa FER-02, were injected along with Br into the influent end of columns to examine the effect of cell morphology and cell surface properties on bacterial transport. The effects of column sediment grain size and mineral coatings coupled with groundwater geochemistry were also investigated. Significant irreversible attachment of DA001 was observed in the Fe oxyhydroxide-coated columns, but only in the suboxic groundwater where the concentrations of dissolved organic carbon (DOC) were ca. 1 ppm. In the oxic groundwater where DOC was ca. 8 ppm, little attachment of DA001 to the Fe oxyhydroxide-coated columns was observed. This indicates that DOC can significantly reduce bacterial attachment due electrostatic interactions. The larger and more negatively charged FER-02 displayed increasing attachment with decreasing grain size regardless of DOC concentration, and modeling of FER-02 attachment revealed that the presence of Fe and Al coatings on the sediment also promoted attachment. Finally, the presence of Al coatings and Al containing minerals appeared to significantly retard the Br tracer regardless of the concentration of DOC. These findings suggest that DOC in shallow oxic groundwater aquifers can significantly enhance the transport of bacteria by reducing attachment to Fe, Mn and Al oxyhydroxides. This effect appears to be profound for weakly and strongly charged hydrophilic bacteria and may contribute to differences in observations between laboratory experiments versus field-scale investigations particularly if the groundwater pH remains subneutral and Fe oxyhydroxide phases exist. These observation validate the novel approach taken in the experiments outlined here of performing laboratory-scale experiments on site to facilitate the use of fresh groundwater and thus be more representative of in situ groundwater conditions.

Evidence for detachment of indigenous bacteria from aquifer sediment in response to arrival of injected bacteria.

Two bacterial strains isolated from the aquifer underlying Oyster, Va., were recently injected into the aquifer and monitored using ferrographic capture, a high-resolution immunomagnetic technique. Injected cells were enumerated on the basis of a vital fluorescence stain, whereas total cell numbers (stained target cells plus unstained target and antigenically similar indigenous bacteria) were identified by cell outlines emanating from fluorophore-conjugated antibodies to the two target strains. The arrival of injected bacteria at the majority of monitored sampling ports was accompanied by simultaneous temporary increases in unstained cell counts that outnumbered the injected bacteria by 2- to 100-fold. The origin and mechanism of appearance of the unstained cells are considered.

Comparison of methods for monitoring bacterial transport in the subsurface.

The purpose of this study was to compare in a laboratory experiment, a suite of methods developed to track viable bacteria during field transport experiments. The criteria for development and selection of these methods included: (1) the ability to track bacteria within the environment from which they were isolated; (2) the lack of any effect upon the viability or the transport characteristics of the strain; (3) low detection limits; (4) a quantification range that covered several orders of magnitude; and (5) an analytical cost and turnover time commensurate with the analysis of several thousands of samples in a few months. The approaches developed included: enumeration of bacteria labeled with a vital fluorescent stain (CFDA/SE) using microplate spectrofluorometry, flow cytometry, and ferrographic (immunomagnetic) capture; enumeration of highly (13)C-enriched bacteria using combustion-IRMS; and quantitative PCR. These methods were compared to direct microscopic enumeration and plate counts during a bacterial transport experiment performed in an intact sediment core and designed to simulate the field experiment. Four of the seven methods had equivalent recoveries for the breakthrough of a pulse of bacteria eluting from a 50-cm long sediment core, and all of the methods detected the arrival of cells in the effluent prior to the conservative tracer. Combustion IRMS and ferrographic enumeration had the lowest quantification limits (approximately 2 to 20 cells/ml), whereas microplate spectrofluorometry had the highest quantification limit (approximately 10(5) cells/ml). These methods have the potential for numerous applications beyond tracking bacteria injected into the subsurface.

Ferrographic tracking of bacterial transport in the field at the narrow channel focus area, Oyster, VA.

The first results from an innovative bacterial tracking technique, ferrographic capture, applied to bacterial transport in groundwater are reported in this paper. Ferrographic capture was used to analyze samples during an October 1999 bacterial injection experiment at the Narrow Channel focus area of the South Oyster site, VA. Data obtained using this method showed that the timing of bacterial breakthrough was controlled by physical (hydraulic conductivity) heterogeneity in the vertical dimension as opposed to variation in sedimentsurface or aqueous chemical properties. Ferrographic tracking yielded results that compared well with results from other tracking techniques over a concentration range of 8 orders of magnitude and provided a low detection limit relative to most other bacterial tracking techniques. The low quantitation limit of this method (approximately 20 cells/mL) allowed observation of transport of an adhesion-deficient bacterium over distances greater than 20 m in the fine sand aquifer underlying this site.

Utility of high performance liquid chromatography/electrospray/mass spectrometry of polar lipids in specifically Per-13C labeled Gram-negative bacteria DA001 as a tracer for acceleration of bioremediation in the subsurface.

Specific fatty acids from phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) recovered from a per 13C-labeled bacteria can be detected in environmental samples and used as measures of bacterial transport in the subsurface. Detection of palmitic acid (16:0) and oleic acid (18:1) at m/z 271 (255+16) and 299 (281+18) as negative ions in PG and PE separated by high performance liquid chromatography (HPLC) and detected after up-front collisionally induced dissociation (CID) utilizing electrospray (ES) mass spectrometry (MS) provided sufficient sensitivity and specificity for detection in the presence of the indigenous microbiota. Application of tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) was use to monitor selected transitions. MRM can increase the sensitivity so that polar lipids recovered from cell densities currently at about 10(4) cells/sample can be detected. This technology provides a non-intrusive mechanism for monitoring the distribution of bacteria added to accelerate in situ bioremediation of subsurface sediments.

Development of a vital fluorescent staining method for monitoring bacterial transport in subsurface environments.

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10(5) CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.

In situ imaging of microorganisms in geologic material.

In order to fully delineate the interactions of microorganisms with geological substrates, unequivocal identification of intact microbial cells within geologic samples is required without the disruption of either the rock texture or the relationship of the microorganisms to the mineral fabric. To achieve this objective we developed a protocol that enables the visualization of intact microbial cells in petrographic thin sections, avoids detaching the cells from their host mineral surfaces and avoids microbial contamination during the lapidary process. Propidium iodide and POPO-3, nucleic acid stains that specifically target double-stranded DNA and RNA were utilized for in situ visualization of cells in surface and subsurface basalts from northeastern Idaho. Additionally, examination of samples incubated with acetic acid-UL-14C via phosphor imagining facilitated the in situ visualization of 14C labeled biomass. Biomass observed was low (<10(7) cells/g). These observations indicate that the microbial distribution in these rocks exhibits a high degree of spatial heterogeneity at the sub-centimeter scale.

Development of radiographic and microscopic techniques for the characterization of bacterial transport in intact sediment cores from Oyster, Virginia.

The objective of this study was to ascertain the physical and mineralogical properties responsible for the retention of bacteria in subsurface sediments. The sediment core chosen for this study was a fine-grained, quartz-rich sand with minor amounts of Fe and Al hydroxides. A bacterial transport experiment was performed using an intact core collected from a recent excavation of the Butler's Bluff member of the Nassawadox formation in the borrow pit at Oyster, VA. and a 14C-labeled bacterial strain OYS2-A was selected for its relatively low adhesion. After the bacterial breakthrough was observed in the effluent, the intact core was dissected to determine the internal distribution of the injected bacteria retained in the sediment. The sediment was dried, epoxy fixed, and thin sectioned. The distribution of 14C activity in the thin sections was mapped using a phosphor screen and X-ray film. The remainder of the core was subsampled and the 14C activity of the subsamples was determined by liquid scintillation counting. The phosphor imaging technique was capable of directly imaging the distribution of radiolabeled bacteria in thin sections, because of its high sensitivity and linear response over a large activity range. The phosphor imaging signal intensity was utilized as a measure of bacterial concentration. The distribution of bacteria at the millimeter scale in the thin sections was compared to the grain size, porosity, and mineralogy as measured by scanning electron microscopy (SEM) and energy dispersive spectrum (EDS) analyses. No apparent correlation was observed between the retention or collision efficiency of bacteria in the sediment and the amount of Fe and Al hydroxides. This apparent lack of correlation can be qualitatively explained by combination of several factors including a nearly neutral surface charge of the bacterial strain, and texture of the Fe and Al hydroxides in the sediment. The combination of phosphor imaging with SEM-EDS proved to be a robust method for relating the physical and mineralogical microscopic properties of poorly indurated sediment to the distribution of adsorbed bacteria, allowing bacterial retention mechanisms to be unambiguously unraveled.

Alterations in adhesion, transport, and membrane characteristics in an adhesion-deficient pseudomonad.

A stable adhesion-deficient mutant of Burkholderia cepacia G4, a soil pseudomonad, was selected in a sand column assay. This mutant (ENV435) was compared to the wild-type strain by examining the adhesion of the organisms to silica sand and their transport through two aquifer sediments that differed in their sand, silt, and clay contents. We compared the longitudinal transport of the wild type and the adhesion mutant to the transport of a conservative chloride tracer in 25-cm-long glass columns. The transport of the wild-type strain was severely retarded compared to the transport of the conservative tracer in a variety of aquifer sediments, while the adhesion mutant and the conservative tracer traveled at similar rates. An intact sediment core study produced similar results; ENV435 was transported at a faster rate and in much greater numbers than G4. The results of hydrophobic interaction chromatography revealed that G4 was significantly more hydrophobic than ENV435, and polyacrylamide gel electrophoresis revealed significant differences in the lipopolysaccharide O-antigens of the adhesion mutant and the wild type. Differences in this cell surface polymer may explain the decreased adhesion of strain ENV435.

Tn5 Insertion Mutants of Pseudomonas fluorescens Defective in Adhesion to Soil and Seeds.

Tn5 insertion mutants of a soil isolate, Pseudomonas fluorescens Pf0-1, were selected for decreased ability to adhere to quartz sand in a column assay. Three adhesion-deficient mutants that differed in the location of the Tn5 insertion in the chromosome were isolated and compared with the wild-type strain. One mutant, Pf0-5, was described previously as an adhesion-defective, nonmobile, flagellumless mutant (M. F. DeFlaun, A. S. Tanzer, A. L. McAteer, B. Marshall, and S. B. Levy, Appl. Environ. Microbiol. 56:112-119, 1990). Another insertion mutant, Pf0-10, was also missing flagella and the 34-kDa outer membrane protein that was absent in Pf0-5 but present in the wild-type strain. The third mutant (Pf0-15) had increased amounts of this 34-kDa outer membrane protein and more flagella than the wild-type strain. These mutants also displayed decreased ability to adhere to sterile and natural (live) soil and to a variety of plant seeds. In kinetics studies, the wild-type strain showed an initial rapid binding to seeds followed by a later slow phase of binding. The mutant strains were defective in the initial stages of attachment but did show the later slow binding. The findings indicate that the same mutations that affect binding to sand and soil also affect adhesion to plant seeds.

Development of field application vectors for bioremediation of soils contaminated with polychlorinated biphenyls.

Field application vectors (FAVs), which are a combination of a selective substrate, a host, and a cloning vector, have been developed for the purpose of expressing foreign genes in nonsterile, competitive environments in which the gene products provide no advantage to the host. Such gene products are exemplified by the enzymes for the cometabolism of polychlorinated biphenyls (PCBs) through the biphenyl degradation pathway. Attempts to use highly competent PCB-cometabolizing strains in the environment in the absence of biphenyl have not been successful, while the addition of biphenyl is limited by its human toxicity and low water solubility. Broad-substrate-specificity PCB-degradative genes (bphABC) were cloned from a naturally occurring isolate. Pseudomonas sp. strain ENV307, into broad-host-range plasmid pRK293. The resulting PCB-degrading plasmids were transferred to the FAV host Pseudomonas paucimobilis 1IGP4, which utilizes the nontoxic, water-soluble, nonionic surfactant Igepal CO-720 as a selective growth substrate. Plasmid stability in the recombinant strains was determined in the absence of antibiotic selection. PCB-degrading activity was determined by resting cell assays. Treatment of contaminated soil (10, 100, or 1,000 ppm of Aroclor 1242) by surfactant amendment (1.0% [wt/wt]Igepal CO-720 in wet soil) and inoculation with recombinant isolates of strain 1IGP4 (approximately 4 x 10(6) cells per g of soil) resulted in degradation of many of the individual PCB congeners in the absence of biphenyl.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of an Adhesion Assay and Characterization of an Adhesion-Deficient Mutant of Pseudomonas fluorescens.

A sand column adhesion assay was developed which distinguishes the adhesion abilities of a number of pseudomonads isolated from fine sandy loam. Pseudomonas fluorescens Pf0-1 which adhered at >90% of the total cells added was subjected to transposon Tn5 insertion mutagenesis. From 2,500 Pf0-1::Tn5 mutants examined in the sand column assay, two adhesion-deficient Pf0-1 mutants showing <50% attachment were isolated. Marker exchange analysis of one of these mutants, Pf0-5, confirmed that the decreased adhesion was linked to the Tn5 insertion in the chromosome. The growth rate of Pf0-5 in enriched media and sterile soil was similar to that of the wild type; in minimal medium, however, Pf0-5 grew faster. In a soil column assay, less Pf0-5 than wild-type bacteria were recovered, suggesting a decreased ability to persist in soil. A 34-kilodalton major outer membrane protein present in the wild type was missing in Pf0-5. Transmission electron microscopy of the cell surface revealed that the wild-type possessed polar flagella which were absent in the mutant.

Correlation of nonspecific macromolecular labeling with environmental parameters during [(3)H]Thymidine incorporation in the waters of southwest florida.

During routine [(3)H]thymidine incorporation measurements of environmental samples, significant amounts of radioactivity are often incorporated into macromolecules other than DNA. Although the percentage of nonspecific labeling varies both temporally and spatially, the cause(s) of these variations remain unknown. Correlations between the percent incorporated radioactivity in DNA and a variety of experimental and environmental parameters measured in the Alfia River, Crystal River, Medard Reservoir, and Bayboro Harbor were examined. The amount of radioactivity incorporated into DNA ranged from 6 to 95% ([Formula: see text]; n=121). Nonspecific labeling began immediately upon the addition of [(3)H]thymidine and was linear over time. Labeling patterns were independent of both the amount of thymidine added and cell-size fraction. A two year study of Bayboro Harbor indicated no conclusive relationship between nonspecific labeling and seasonality. The amount of radioactivity incorporated into DNA was inversely correlated with total rates of thymidine incorporation and a strong diurnal pattern was observed in the Crystal River. No consistent relationship was observed between labeling patterns and primary productivity, chlorophylla, particulate DNA, dissolved DNA, bacterial cell numbers, temperature, salinity, and dissolved organic carbon. The only relationship with dissolved inorganic nutrients (N and P) occurred in the Crystal River. In this phosphate limited river, the percent of radioactivity incorporated into DNA was positively correlated with phosphate concentrations. These results indicate that nonspecific labeling is not dependent on any one parameter but may be a function of many interacting environmental factors or a function of the specific ambient bacterial population.

Turnover of extracellular DNA in eutrophic and oligotrophic freshwater environments of southwest Florida.

The turnover of extracellular DNA was investigated in oligotrophic springs of the Crystal River and the eutrophic Medard Reservoir of southwest Florida. The Medard Reservoir possessed large populations of bacterioplankton and phytoplankton (6.8 x 10 cells per liter and 28.6 mug of chlorophyll a per liter, respectively), while the Crystal River springs only contained a fraction of the microbial biomass found in the Medard Reservoir. Although dissolved DNA values were greater in the Medard Reservoir, higher rates of DNA removal resulted in similar extracellular DNA turnover times in both environments (9.62 +/- 3.6 h in the Crystal River and 10.5 +/- 2.1 h in the Medard Reservoir). These results indicate that regardless of trophic status or microbial standing stock, extracellular DNA turns over rapidly in subtropical planktonic freshwater environments. Therefore, recombinant DNA sequences from released genetically engineered microorganisms might not be expected to survive for long periods of time in freshwater planktonic environments.

Detection of exogenous gene sequences in dissolved DNA from aquatic environments.

A method for the concentration and detection of gene sequences in the dissolved DNA from freshwater and marine environments has been developed. The limit of detection in the dot blot format was 167 fg/ml (100 ml sample) for exogenous herpes simplex thymidine kinase (TK) gene that was added to artificial seawater or river water. This procedure has been used to determine the longevity and monitor progressive changes in molecular weight of a plasmid containing the TK gene added to eutrophic estuarine water. The onset of plasmid degradation as determined by change in molecular weight was rapid (within 5 min). Intact plasmid was detected for at least 4 hours and sequences hybridizable to the TK gene probe were present for up to 24 hours.

Mechanisms of DNA utilization by estuarine microbial populations.

The mechanisms of utilization of DNA by estuarine microbial populations were investigated by competition experiments and DNA uptake studies. Deoxyribonucleoside monophosphates, thymidine, thymine, and RNA all competed with the uptake of radioactivity from [H]DNA in 4-h incubations. In 15-min incubations, deoxyribonucleoside monophosphates had no effect or stimulated [H]DNA binding, depending on the concentration. The uptake of radioactivity from [H]DNA resulted in little accumulation of trichloroacetic acid-soluble intracellular radioactivity and was inhibited by the DNA synthesis inhibitor novobiocin. Molecular fractionation studies indicated that some radioactivity from [H]DNA appeared in the RNA (10 and 30% at 4 and 24 h, respectively) and protein (approximately 3%) fractions. The ability of estuarine microbial assemblages to transport gene sequences was investigated by plasmid uptake studies, followed by molecular probing. Although plasmid DNA was detected on filters after filtration of plasmid-amended incubations, DNase treatment of filters removed this DNA, indicating that there was little transport of intact gene sequences. These observations led to the following model for DNA utilization by estuarine microbial populations. (i) DNA is rapidly bound to the cell surface and (ii) hydrolyzed by cell-associated and extracellular nonspecific nucleases. (iii) DNA hydrolysis products are transported, and (iv) the products are rapidly salvaged into nucleic acids, with little accumulation into intracellular nucleotide pools.

Dynamics of extracellular DNA in the marine environment.

The production and turnover of dissolved DNA in subtropical estuarine and oligotrophic oceanic environments were investigated. Actively growing heterotrophic bacterioplankton (i.e., those capable of [3H]thymidine incorporation) were found to produce dissolved DNA, presumably through the processes of death and lysis, grazing by bacteriovores, and excretion. Production of dissolved DNA as determined by [3H]thymidine incorporation was less than or equal to 4% of the ambient dissolved DNA concentration per day. In turnover studies, the addition of [3H]DNA (Escherichia coli chromosomal) to seawater resulted in rapid hydrolysis and uptake or radioactivity by microbial populations. DNA was hydrolyzed by both cell-associated and extracellular nucleases, in both estuarine and offshore environments. Kinetic analysis performed for a eutrophic estuary indicated a turnover time for dissolved DNA as short as 6.5 h. Microautoradiographic studies of bacterial populations in Tampa Bay indicated that filamentous and attached bacteria took up most of the radioactivity from [3H]DNA. Dissolved DNA is therefore a dynamic component of the dissolved organic matter in the marine environment, and bacterioplankton play a key role in the cycling of this material.

Simplified method for dissolved DNA determination in aquatic environments.

A method was developed for the determination of dissolved DNA in aquatic environments. The method is based upon the concentration of dissolved DNA by ethanol precipitation of 0.2-mum-pore-size filtered water. The DNA in concentrated extracts was quantified by the fluorescence of Hoechst 33258-DNA complexes. Fluorescence not attributable to DNA was corrected for by DNase I digestion of the extracts and averaged 25% of the total fluorescence for all samples. The effectiveness of the procedure for concentrating dissolved DNA was demonstrated by the efficient (>90%) recovery of internal standards. Concentrations of dissolved DNA from a variety of marine and freshwater environments ranged from 0.2 to 44 mug/liter, with the highest values being obtained for estuarine and river environments. The method is simple, specific for DNA, and more sensitive than previously described methods for the determination of extracellular DNA.