Larry D. McKay

Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water

ABSTRACT Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2 = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.

Evaluation of chloride and pesticide transport in a fractured clayey till using large undisturbed columns and numerical modeling

Saturated groundwater flow and tracer experiments using fluorescent dye, chloride, and the herbicides mecoprop and simazine were carried out in the laboratory using three large-diameter (0.5 m) undisturbed columns of fractured clayey till. Hydraulic conductivity of the columns ranged from 10−5 m/s in the shallowest column (1 m dept)) to 10−7 m/s in the deepest column (4 m depth) and were similar to field-measured values for these deposits. Results of the tracer experiments are consistent with a conceptual model of advective transport along the fractures combined with diffusion into the fine-grained matrix between the fractures. Arrival of the chloride tracer in the effluent was highly retarded relative to fracture flow velocities calculated on the basis of the cubic law and measured values of fracture spacing and hydraulic conductivity. The herbicides were more strongly retarded than the chloride at low flow rates, but at higher flow rates the herbicides arrived with the chloride, indicating the influence of nonequilibrium sorption of the herbicides to fracture walls and the matrix solids. The columns were dismantled following the tracer experiments and mapping under UV light showed that nearly all of the visible, weathered fractures (and the few root holes in the case of the shallowest sample) were active transport pathways, with the dye appearing mainly on the fracture surfaces and as a “rim” in the adjacent matrix. Concentration profiles measured perpendicular to the fracture surfaces showed that the herbicides had also moved into the matrix, apparently by diffusion. Simulations of solute transport with a discrete fracture flow/matrix diffusion model showed that the simulations could be “fit” to the data if all of the visible fractures were hydraulically active, but could not be fit if all or most of the flow was channelled through just the primary fractures (defined by prominent oxidation stains). Simulations with an equivalent porous media (EPM) model could not fit the data using the measured total porosity as the effective porosity. The simulations could likely be fit with a smaller value of effective porosity, but this would limit applicability to field situations because fitted effective porosity is expected to change with physical scale and residence time of the solute in the soil.

Comparison of fecal indicators with pathogenic bacteria and rotavirus in groundwater

Groundwater is routinely analyzed for fecal indicators but direct comparisons of fecal indicators to the presence of bacterial and viral pathogens are rare. This study was conducted in rural Bangladesh where the human population density is high, sanitation is poor, and groundwater pumped from shallow tubewells is often contaminated with fecal bacteria. Five indicator microorganisms (E. coli, total coliform, F+RNA coliphage, Bacteroides and human-associated Bacteroides) and various environmental parameters were compared to the direct detection of waterborne pathogens by quantitative PCR in groundwater pumped from 50 tubewells. Rotavirus was detected in groundwater filtrate from the largest proportion of tubewells (40%), followed by Shigella (10%), Vibrio (10%), and pathogenic E. coli (8%). Spearman rank correlations and sensitivity-specificity calculations indicate that some, but not all, combinations of indicators and environmental parameters can predict the presence of pathogens. Culture-dependent fecal indicator bacteria measured on a single date did not predict total bacterial pathogens, but annually averaged monthly measurements of culturable E. coli did improve prediction for total bacterial pathogens. A qPCR-based E. coli assay was the best indicator for the bacterial pathogens. F+RNA coliphage were neither correlated nor sufficiently sensitive towards rotavirus, but were predictive of bacterial pathogens. Since groundwater cannot be excluded as a significant source of diarrheal disease in Bangladesh and neighboring countries with similar characteristics, the need to develop more effective methods for screening tubewells with respect to microbial contamination is necessary.

Factors Influencing the Persistence of Fecal Bacteroides in Stream Water

Laboratory microcosm experiments were used to assess the effects of environmental parameters on the persistence of the Bacteroides 16S rRNA genes derived from equine fecal samples in stream water to investigate the utility of Bacteroides spp. as fecal indicator organisms. Quantitative real-time polymerase chain reaction (qPCR) was used to measure gene concentrations over time with treatments designed to compare filtered vs. unfiltered stream water, fecal aggregate size, initial fecal concentrations, and water temperatures. Comparison of Bacteroides16S rRNA genes/mL in microcosms constructed with unfiltered stream water and filtered stream water indicated that stream water filtration to remove indigenous microorganisms followed by temperature had the largest effects on gene persistence. First-order exponential decay functions were fitted to the data from each microcosm constructed using unfiltered stream water, and the decay constants (k) ranged from 0.0071 h(-1) in the microcosms incubated at 5 degrees C to 0.0336 h(-1) in a set of microcosms incubated at 25 degrees C. Analysis of k calculated from the 10 experimental treatments indicated that k is more highly correlated to temperature than initial Bacteroides 16S rRNA gene starting concentrations. The equation resulting from graphing k (as the dependent variable) vs. temperature (as the independent variable) best fit a peak, Gaussian, 3 parameter function with a maximum decay at 30 degrees C, a r(2) of 0.83 and all parameters were significant (P < 0.0015). Thus this data suggest that factors that reduce biological activity, such as physical removal of stream microorganisms by filtration and low temperature, result in slower Bacteroides 16S rRNA gene decay.

Fecal Contamination of Shallow Tubewells in Bangladesh Inversely Related to Arsenic

The health risks of As exposure due to the installation of millions of shallow tubewells in the Bengal Basin are known, but fecal contamination of shallow aquifers has not systematically been examined. This could be a source of concern in densely populated areas with poor sanitation because the hydraulic travel time from surface water bodies to shallow wells that are low in As was previously shown to be considerably shorter than for shallow wells that are high in As. In this study, 125 tubewells 6−36 m deep were sampled in duplicate for 18 months to quantify the presence of the fecal indicator Escherichia coli. On any given month, E. coli was detected at levels exceeding 1 most probable number per 100 mL in 19−64% of all shallow tubewells, with a higher proportion typically following periods of heavy rainfall. The frequency of E. coli detection averaged over a year was found to increase with population surrounding a well and decrease with the As content of a well, most likely because of downward transport of E. coli associated with local recharge. The health implications of higher fecal contamination of shallow tubewells, to which millions of households in Bangladesh have switched in order to reduce their exposure to As, need to be evaluated.

Effects of Contaminant Concentration, Aging, and Soil Properties on the Bioaccessibility of Cr(III) and Cr(VI) in Soil

Contaminated soils at numerous U.S. Department of Defense, Department of Energy, and other industrial facilities often contain huge inventories of toxic metals such as chromium. Ingestion of soil by children is often the primary risk factor that drives the need for remediation. Site assessments are typically based solely on total soil-metal concentrations and do not consider the potential for decreased bioaccessibility due to metal sequestration by soil. The objectives of this research are to investigate the effect of soil properties on the bioaccessibility of Cr(III) and Cr(VI) as a function of contaminant concentration and aging. The A and upper B horizons of two well-characterized soils, representative of Cr-contaminated soils in the southeastern United States, were treated with varying concentration of Cr(III) and Cr(VI) and allowed to age. The bioaccessibility of the contaminated soils was measured over a 200-d time period using a physiologically based extraction test (PBET) that was designed to simulate the digestive process of the stomach. The sorption of Cr(III) and Cr(VI) varied significantly as a function of soil type and horizon, and the oxidation state of the contaminant. Solid phase concentrations with Cr(III) were significantly greater than Cr(VI) for any given initial Cr concentration. This is consistent with the mechanisms of Cr(III) vs. Cr(VI) sequestration by the soils, where the formation of Cr(III)-hydroxides can result in the accumulation of large mass fractions of contaminant on mineral surfaces. Overall, Cr bioaccessibility decreased with duration of exposure for all soils and at all solid phase concentrations, with aging effects being more pronounced for Cr(III). The decrease in Cr bioaccessibility was rapid for the first 50 d and then slowed dramatically between 50 and 200 d. In general, the effects of Cr solid phase concentration on bioaccessibility was small, with Cr(III) showing the most pronounced effect; higher solid phase concentrations resulted in a decrease in bioaccessibility. Chemical extraction methods and X-ray Adsorption Spectroscopy analyses suggested that the bioaccessibility of Cr(VI) was significantly influenced by reduction processes catalyzed by soil organic carbon. Soils with sufficient organic carbon had lower Cr bioaccessibility values (∼10 to 20%) due to an enhanced reduction of Cr(VI) to Cr(III). In soils where organic carbon was limited and reduction processes were minimal, the bioaccessibility of Cr(VI) dramatically increased (∼60 to 70%).

Transport and retention of a bacteriophage and microspheres in saturated, angular porous media: Effects of ionic strength and grain size

Eight saturated column experiments were conducted to examine the effects of solution chemistry and grain size on the transport of colloids through crushed silica sand. Two sizes of colloids, 0.025-microm bacteriophage (MS-2) and 1.5-microm carboxylated microspheres, were used as surrogates for the transport of pathogenic viruses and bacteria, respectively. Increasing the Ca(2+) concentration from 1 to 4.8 mM (along with background monovalent ions) resulted in complete attenuation (>6-log decrease in C/C(0)) of MS-2, but caused only a 1-log reduction (C/C(0)=0.1) in the concentration of the microspheres. Decreasing grain size from medium sand (d(50)=0.70 mm) to fine sand (d(50)=0.34 mm) resulted in substantial decreases in effluent concentrations of both the MS-2 (5-log decrease) and microspheres (>2.5-log decrease). Comparison of observed colloid retention to that predicted by a recently published correlation equation for colloid filtration revealed that the model can considerably underpredict (by 4 orders of magnitude or more) colloid retention by angular sand over distances as short as 20 cm. This indicates that state-of-the-art colloid filtration models are still limited in applicability to natural systems.

Impact of population and latrines on fecal contamination of ponds in rural Bangladesh

A majority of households in Bangladesh rely on pond water for hygiene. Exposure to pond water fecal contamination could therefore still contribute to diarrheal disease despite the installation of numerous tubewells for drinking. The objectives of this study are to determine the predominant sources (human or livestock) of fecal pollution in ponds and examine the association between local population, latrine density, latrine quality and concentrations of fecal bacteria and pathogens in pond water. Forty-three ponds were analyzed for E. coli using culture-based methods and E. coli, Bacteroidales and adenovirus using quantitative PCR. Population and sanitation spatial data were collected and measured against pond fecal contamination. Humans were the dominant source of fecal contamination in 79% of the ponds according to Bacteroidales measurements. Ponds directly receiving latrine effluent had the highest concentrations of fecal indicator bacteria (up to 10⁶ Most Probable Number (MPN) of culturable E. coli per 100 mL). Concentrations of fecal indicator bacteria correlated with population surveyed within a distance of 30-70 m (p<0.05) and total latrines surveyed within 50-70 m (p<0.05). Unsanitary latrines (visible effluent or open pits) within the pond drainage basin were also significantly correlated to fecal indicator concentrations (p<0.05). Water in the vast majority of the surveyed ponds contained unsafe levels of fecal contamination attributable primarily to unsanitary latrines, and to lesser extent, to sanitary latrines and cattle. Since the majority of fecal pollution is derived from human waste, continued use of pond water could help explain the persistence of diarrheal disease in rural South Asia.

Natural attenuation of trichloroethylene in fractured shale bedrock.

This paper describes one of the first well-documented field examples of natural attenuation of trichloroethylene (TCE) in groundwater in a fractured shale bedrock. The study was carried out adjacent to a former waste burial site in Waste Area Grouping 5 (WAG5) on the Oak Ridge Reservation, Oak Ridge, TN. A contaminant plume containing TCE and its daughter products were detected downgradient from the buried waste pits, with most of the contamination occurring in the upper 6 m of the bedrock. The monitoring well array consists of a 35-m-long transect of multilevel sampling wells, situated along a line between the waste pits and a seep which discharges into a small stream. Concentrations of volatile organic carbons (VOCs) were highest in the waste trenches and decreased with distance downgradient towards the seep. Sampling wells indicated the presence of overlapping plumes of TCE, cis-dichloroethylene (cDCE), vinyl chloride (VC), ethylene, ethane, and methane, with the daughter products extending further downgradient than the parent (TCE). This type of distribution suggests anaerobic biodegradation. Measurements of redox potential at the site indicated that iron-reduction, sulfate reduction, and potentially methanogensis were occurring and are conducive to dechlorination of TCE. Bacteria enrichment of groundwater samples revealed the presence of methanotrophs, methanogens, iron-reducing bacteria and sulfate-reducing bacteria, all of which have previously been implicated in anaerobic biodegradation of TCE. 16S rDNA sequence from DNA extracted from two wells were similar to sequences of organisms previously implicated in the anaerobic biodegradation of chlorinated solvents. The combined data strongly suggest that anaerobic biodegradation of the highly chlorinated compounds is occurring. Aerobic biodegradation may also be occurring in oxygenated zones, including near a seep where groundwater exits the site, or in the upper bedrock during seasonal fluctuations in water table elevation and oxygen levels.

Fractal characterization of fracture networks: An improved box‐counting technique

given by N / rD , where N is the number of boxes containing one or more fractures and r is the box size, then the network is considered to be fractal. However, researchers are divided in their opinion about which is the best box-counting algorithm to use, or whether fracture networks are indeed fractals. A synthetic fractal fracture network with a known D value was used to develop a new algorithm for the box-counting method that returns improved estimates of D. The method is based on identifying the lower limit of fractal behavior (rcutoff) using the condition ds/dr ! 0, where s is the standard deviation from a linear regression equation fitted to log(N) versus log(r) with data for r < rcutoff sequentially excluded. A set of 7 nested fracture maps from the Hornelen Basin, Norway was used to test the improved method and demonstrate its accuracy for natural patterns. We also reanalyzed a suite of 17 fracture trace maps that had previously been evaluated for their fractal nature. The improved estimates of D for these maps ranged from 1.56 ± 0.02 to 1.79 ± 0.02, and were much greater than the original estimates. These higher D values imply a greater degree of fracture connectivity and thus increased propensity for fracture flow and the transport of miscible or immiscible chemicals.