Abstract: Recent work has suggested that bacteriophages infecting Bacteroides are a potential tool for faecal source tracking, but that different host strains may be needed for different geographic areas. This study used a recently identified strain of Bacteroides (GB-124) to detect human sources of faecal pollution in a river catchment in southeast England (UK). A total of 306 river water, municipal wastewater and animal samples were obtained over a 16-month period. Bacteriophages capable of infecting GB-124 were present in all municipal wastewaters but were not detected in faecal samples from animals, and were detected at significantly lower levels (P< 0.001) in river waters directly downstream of a dairy farm. This last observation was despite the presence of high levels of faecal indicator bacteria at this site. The study suggests that GB-124 appears to be specific to human faeces. As such it may represent an effective and low-cost method of faecal source identification.

Abstract: We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were sequenced and were determined to define 289 different genomic DNA regions. These putative human-specific fecal bacterial DNA sequences were then analyzed by dot blot hybridization, which confirmed that 98% were present in the source human fecal microbial community and absent from the original pig fecal DNA extract. Comparative sequence analyses of these sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26 of the candidate sequences predicted to encode factors involved in interactions with host cells were then used in the PCR and did not amplify markers in DNA from any additional pig fecal specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal sources, with more than half amplifying markers only in specimens from dogs or cats. Four assays were more specific, detecting markers only in specimens from humans, including those from 18 different human populations examined. We then demonstrated the potential utility of these assays by using them to detect human fecal contamination in several impacted watersheds.

Abstract: Bacteroidales host-specific PCR offers a rapid method of diagnosing fecal pollution in water and identifying sources of input. To assess human health risks from exposure to fecal pathogens, however, Bacteroidales markers should be detectable when pathogens are present. To determine if Bacteroidales general, human-, ruminant-, and swine-specific markers correlate with certain fecal pathogens, we conducted a retrospective study on water samples for which the presence of E. coli O157:H7, Salmonella spp., and Campylobacter spp. had been determined. We found a positive relationship between detection of the Bacteroidales general fecal marker and presence of the pathogens. Detection of ruminant-specific markers predicted E. coli O157: H7 occurrence. There was a significant increase in the likelihood of detecting Salmonella when a ruminant marker was present, and Campylobacter spp. when human markers were present. For pathogens such as E. coli O157: H7 that are strongly associated with particular hosts, Bacteroidales host-specific markers can estimate the likelihood of pathogen occurrence, enabling more accurate health risk assessments.

Abstract: One of the problems associated with the use of ambient water quality standards in surface water regulation is the difficulty of identifying and regulating nonpoint source pollution, making such standards unenforceable, especially at the local level. We used the Escherichia coli indicator to locate the most contaminated reaches in rural South Carolina's Bush River watershed (297 km(2), 186 stream-km). We divided the watershed into 20 smaller reaches and sampled each reach multiple times, but restricted each sampling round to one day. We located four low order creek reaches, representing just nine stream-km, where we observed geometric mean E. coli densities of over 1250 E. coli/100 mL; in each case, the source of the contamination (riparian grazing of cattle) was easily identifiable. On the Bush River itself, we observed a step change in one reach where geometric means increased from 106 E. coli/100 mL to 565 E. coli/100 mL over the reach's 10 km length. In this case, the sources of contamination were not as obvious as in the lower order streams; in this case, more advanced Microbial Source Tracking techniques will be required to identify the sources. Nevertheless, this sampling protocol helped locate polluted reaches and provided decision-makers with reasonable justifications for concrete action in deciding where (or where not) to install conservation practices and where more sophisticated (and expensive) MST techniques were warranted.

Abstract: Two public beaches (Anderson and Hilton) in Newport News, Virginia, were frequently closed to swimming in 2004 due to high Enterococcus spp. counts that exceeded the regulatory standard. The microbial source tracking (MST) methods of antibiotic resistance analysis (ARA) and fluorometry (to detect optical brighteners) were used in the summer of 2004 to determine the origins of fecal pollution at the two beaches. Both MST methods detected substantial human-origin pollution at the two beaches, in locations producing consistently high levels of Enterococcus spp. Investigations by municipal officials led to the fluorometric detection and subsequent repair of sewage infrastructure problems at both beaches. The success of the mitigation efforts was confirmed during the summer of 2005 using ARA and fluorometry, with the results cross-validated by pulsed-field gel electrophoresis (PFGE).

Abstract: A collection of 366 Escherichia coli strains from 10 host groups and surface waters were tested for the presence of 15 virulence genes associated with strains causing intestinal and extra-intestinal infections. The virulence genes included eaeA, VT1, 2 and 2e, LT1, ST1 and 2, Einv gene, EAgg gene, CNF1 and 2, papC, O111 and O157 side chain LPS. Of the 262 strains obtained from nine different hosts, 39 (15%) carried one or more of these virulence genes. These included six strains from humans, two from horses, eight from dogs, two from ducks, five from cattle, seven from chickens, four from pigs, two from sheep and three from deer. Of the remaining 104 strains obtained from water samples, 10 (10%) also carried one or more of the tested virulence genes. Of these, six had identical biochemical phenotypes (BPTs) to strains isolated from humans (two strains), dogs (two strains), chickens (one strain) and sheep (one strain) with 4 BPTs also carrying same virulence genes. Our results indicate that the sources of clinically important E. coli strains found in surface waters due to faecal contamination can be predicted by using a combination of biochemical fingerprinting method and the detection of virulence genes. From the public health point of view this information will be of great importance for evaluating the risk associated with public use of the catchment.

Abstract: The recent implementation of the Revised Bathing Water Directive in the European Union has highlighted the need for development of effective methods to differentiate between sources of faecal contamination. It had previously been shown that amplification of 16S rRNA genes of host-specific Bacteriodales species using the HF183F and CF128F primers could be used as markers for human and bovine faecal contamination in the United States. This paper determined the sensitivity and specificity of these markers in four Atlantic Rim countries (France, Ireland, Portugal and the United Kingdom) to evaluate their usefulness in determining the origin of faecal contamination. It was shown that the HF183F marker displayed high sensitivity (80–100%) and specificity (91–100%), and is reliable as an indication of human faecal contamination. The CF128F marker displayed 100% sensitivity in all four countries. However, strong regional variations in specificity (41–96%) were observed, highlighting the need for local validation before this marker is employed in source tracking of faecal contamination.

Abstract: Among the seven assemblages identified in Giardia duodenalis species, only assemblages A and B infect humans and numerous other mammals as well. On the other hand, assemblage E is considered to be host restricted to livestock. The aim of the present study was to compare the presence of G. duodenalis assemblages A, B and E in wastewater samples from two municipal treatment plants (n=24) and one slaughterhouse (n=12). Thus, PCR assays targeting the tpi gene were developed to detect specifically these three G. duodenalis assemblages. Assemblages A and B were detected in urban wastewater with a predominance of assemblage A, especially for one treatment plant. Concerning slaughterhouse wastewater, assemblage A was found in 58% of the samples, whereas assemblage B was not detected. Assemblage E was not detected in urban wastewater, but was found in 92% of the samples from slaughterhouse. Thus, combination of assemblages A and B seemed to indicate a human contamination origin, while combination of assemblages A and E appeared to correspond to a livestock contamination origin.

Abstract: The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine-specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%), but did not amplify a product in 47 bacterial and archaeal stock cultures, 477 environmental bacterial isolates, sewage, and water samples from a bovine waste lagoon and polluted creek. Amplification was observed in only 1 sheep out of 260 human and non-swine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10-6 g of wet pig feces in 500 mL of PBS and 10-4 g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.

Abstract: Water samples from sites potentially impacted by septic tanks, cattle, sewage treatment plant (STP) and natural forests were collected at regular monthly intervals and within 48 h of rainfall events between October 2004 and June 2006. All samples (n=296) were analysed for faecal coliforms and faecal sterols including coprostanol, epicoprostanol, cholestanol, cholesterol and 24-ethylcoprostanol. Faecal sterol ratios were used to assign human and/or herbivore contamination sources and to estimate their percentage relative contributions in water samples. The catchments had significantly different profiles of designated contamination origins (p<0.05), which were consistent with land use patterns. The STP impacted site had the highest incidence of human contamination assignations and the highest mean levels of coprostanol, whilst the forested site had the highest incidence of uncontaminated samples and the lowest mean concentration of coprostanol. Coprostanol concentrations were not always correlated with faecal coliform counts.

Abstract: Aims: The aim of the study was the development of a sensitive human-specific quantitative real-time PCR assay for microbial faecal source tracking (MST) in alpine spring water. The assay detects human-specific faecal DNA markers (BacH) from 16S rRNA gene sequences from the phylum Bacteroidetes using TaqMan® minor groove binder probes. Methods and Results: The qualitative and quantitative detection limits of the PCR assay were 6 and 30 marker copies, respectively. Specificity was proved by testing 41 human faeces and waste water samples and excluding cross-amplification from 302 animal faecal samples from Eastern Austria. Marker concentrations in human faecal material were in the range from 6·6 × 109 to 9·1 × 1010 marker equivalents per gram. The method was sensitive enough to detect a few 100 pg of faeces in faecal suspensions. The assay was applied on water samples from an alpine karstic spring catchment area and the results reflected the expected levels of human faecal influence. Conclusions: The method exhibited sufficient sensitivity to allow quantitative source tracking of human faecal impact in the investigated karstic spring water. Significance and Impact of the Study: The developed method constitutes the first quantitative human-specific MST tool sensitive enough for investigations in ground and spring water.

Abstract: Epilithic periphyton communities were sampled at three sites on the Minnesota shoreline of Lake Superior from June 2004 to August 2005 to determine if fecal coliforms and Escherichia coli were present throughout the ice-free season. Fecal coliform densities increased up to 4 orders of magnitude in early summer, reached peaks of up to 1.4 x 105 CFU cm–2 by late July, and decreased during autumn. Horizontal, fluorophore-enhanced repetitive-PCR DNA fingerprint analyses indicated that the source for 2% to 44% of the E. coli bacteria isolated from these periphyton communities could be identified when compared with a library of E. coli fingerprints from animal hosts and sewage. Waterfowl were the major source (68 to 99%) of periphyton E. coli strains that could be identified. Several periphyton E. coli isolates were genotypically identical (92% similarity), repeatedly isolated over time, and unidentified when compared to the source library, suggesting that these strains were naturalized members of periphyton communities. If the unidentified E. coli strains from periphyton were added to the known source library, then 57% to 81% of E. coli strains from overlying waters could be identified, with waterfowl (15 to 67%), periphyton (6 to 28%), and sewage effluent (8 to 28%) being the major potential sources. Inoculated E. coli rapidly colonized natural periphyton in laboratory microcosms and persisted for several weeks, and some cells were released to the overlying water. Our results indicate that E. coli from periphyton released into waterways confounds the use of this bacterium as a reliable indicator of recent fecal pollution.

Abstract: As the requirements of the Water Framework Directive (WFD) and the US Clean Water Act (USCWA) for the maintenance of microbiological water quality in 'protected areas' highlight, there is a growing recognition that integrated management of point and diffuse sources of microbial pollution is essential. New information on catchment microbial dynamics and, in particular, the sources of faecal indicator bacteria found in bathing and shellfish harvesting waters is a pre-requisite for the design of any 'programme of measures' at the drainage basin scale to secure and maintain compliance with existing and new health-based microbiological standards. This paper reports on a catchment-scale microbial source tracking (MST) study in the Leven Estuary drainage basin, northwest England, an area for which quantitative faecal indicator source apportionment empirical data and land use information were also collected. Since previous MST studies have been based on laboratory trials using 'manufactured' samples or analyses of spot environmental samples without the contextual microbial flux data (under high and low flow conditions) and source information, such background data are needed to evaluate the utility of MST in USCWA total maximum daily load (TMDL) assessments or WFD 'Programmes of Measures'. Thus, the operational utility of MST remains in some doubt. The results of this investigation, using genotyping of Bacteroidetes using polymerase chain reaction (PCR) and male-specific ribonucleic acid coliphage (F + RNA coliphage) using hybridisation, suggest some discrimination is possible between livestock- and human-derived faecal indicator concentrations but, in inter-grade areas, the degree to which the tracer picture reflected the land use pattern and probable faecal indicator loading were less distinct. Interestingly, the MST data was more reliable on high flow samples when much of the faecal indicator flux from catchment systems occurs. Whilst a useful supplementary tool, the MST information did not provide quantitative source apportionment for the study catchment. Thus, it could not replace detailed empirical measurement of microbial flux at key catchment outlets to underpin faecal indicator source apportionment. Therefore, the MST techniques reported herein currently may not meet the standards required to be a useful forensic tool, although continued development of the methods and further catchment scale studies could increase confidence in such methods for future application.

Abstract: Multiplex real-time PCR amplifying fecal mitochondrial DNA (mtDNA) combined with rapid, crude DNA preparations are promising additions to surface water source tracking methods. Amplification of eukaryotic mitochondrial DNA identifies the fecal source directly and can be used in conjunction with other intestinal microbial methods to characterize effluents. Species-specific primers and dual-labeled probes for human, swine, and bovine NADH dehydrogenase subunit 5 (ND5) genes were created for multiplex real-time PCR in feces and effluent slurries. The linear range of the multiplex assay was 10(2)-10(7) mtDNA copies for human, bovine, and swine effluent in combination (equal volumes). PCR amplification efficiencies for bovine, human, and swine mtDNA when assayed in combination were 93, 107, and 92% respectively. Linear regression correlation coefficients (r2) were 0.99 for all standard curves except for human mtDNA in combination (r2 = 0.95). Multiplex amplification of bovine, human, and swine mtDNA (ND5) exhibited no cross-reactions between the effluents from three species of interest. Also, no cross-reactions were observed with effluents of other vertebrates: sheep, goat, horse, dog, cat, Canada goose, broiler, layer, turkey, and tilapia. Performed as a blind test, the PCR operator was able to correctly identify all but two effluent challenge samples (10/12 or 83% correct) with no false positives (22/22 or 100% correct). The multiplex assay had a tendency to detect the species of highest mtDNA concentration only. Better detection of all three species in a combination of human, bovine, and swine effluents was accomplished by running each real-time PCR primer/ probe set singly. Real-time PCR detection limit was calculated as 2.0 x 10(6) mitochondrial copies or 0.2 g of human feces per 100 mL effluent. Some carry-over mtDNA PCR signal from consumed beef, but not pork, was found in feces of human volunteers.

Abstract: Three independent microbial source tracking (MST) methods were applied to a small urban subwatershed in Orange County, California. Fifty-seven water samples collected over summer 2002 were analyzed for human adenovirus and enterovirus. Enterococci and E. coli were isolated for antibiotic resistance analysis (ARA) and for PCR identification of human- and animal-specific toxin genes, respectively. All water samples were PCR negative for human enteroviruses and E. coli human-specific toxin gene. E. coli toxin markers revealed the presence of toxin genes specific to bird, rabbit, and cow. Enterococci ARA results supported this conclusion and indicated that fecal bacteria from bird and wild animal feces as well as soil were the predominant source found in the watershed. An E. coli, isolated from the watershed and inoculated back into the heat-sterilized storm drain water, increased 4 log units within 6 days. Collectively, these results suggest that bird and wild animal feces, soil amendments, and/or fecal coliform growth in the storm drain are the major contributors to the fecal bacterial pollution in downstream areas. However, human adenoviruses were detected on two occasions. Fecal bacterial concentrations were not elevated on these two occasions, suggesting that the elevated levels of fecal indicator bacteria in this small watershed could be unrelated to the source of human adenovirus.

Abstract: In this study, we applied a genome fragment enrichment (GFE) method to select for genomic regions that differ among different fecal metagenomes. Competitive DNA hybridizations were performed between chicken fecal DNA and pig fecal DNA (CP) and between chicken fecal DNA and an avian DNA composite consisting of turkey, goose, and seagull fecal DNA extracts (CB) to enrich for chicken-specific DNA fragments. A total of 471 non-redundant chicken metagenomic sequences were retrieved and analyzed. All of the clone sequences were similar to prokaryotic genes, of which more than 60% could not be assigned to previously characterized functional roles. In general terms, sequences assigned characterized functional roles were associated with cellular processes (11.7%), metabolism (11.0%) and information storage and processing (13.4%). Approximately 53% of the non-redundant sequences are similar to genes present in intestinal bacteria belonging to Clostridia (20.9%), Bacteroidetes (15.0%), and Bacilli (17.3%). Twenty-five sequences from the CP and CB clone libraries were selected to develop chicken fecal-specific PCR assays. These assays were challenged against fecal DNA extracted from 21 different animal species, including mammals and birds. The results from the host-specificity studies showed that 12 of the assays had a high degree of specificity to chicken feces. In addition, three assays were specific to chicken and turkey while another four assays tested positive to more than two avian species, suggesting a broader distribution of some of the enriched gene fragments among different avian fecal microbial communities. Fecal pollution signals were detected using chicken-specific assays in contaminated water samples, although the PCR assays showed different detection limits. These results indicate the need for multiple assays to detect poultry fecal sources of pollution. The competitive DNA hybridization approach used in this study can rapidly select for numerous chicken fecal metagenomic regions that can be used as potential genetic markers for fecal source tracking.

Abstract: We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28–54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3–5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19–24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards.

Abstract: The literature on microbial source tracking (MST) suggests that DNA analysis of fecal samples leads to more reliable determinations of bacterial sources of surface water contamination than antibiotic resistance analysis (ARA). Our goal is to determine whether the increased reliability, if any, in library-based MST developed with DNA data is sufficient to justify its higher cost, where the bacteria source predictions are used in TMDL surface water management programs. We describe an application of classification trees for MST applied to ARA and DNA data from samples collected in the Potomac River Watershed in Maryland. Conclusions concerning the comparison of ARA and DNA data, although preliminary at the current time, suggest that the added cost of obtaining DNA data in comparison to the cost of ARA data may not be justified, where MST is applied in TMDL surface water management programs

Abstract: Aims: The goal of the study was to improve the fidelity of library-dependent bacterial source tracking efforts in determining sources of faecal pollution. The first objective was to compare the fidelity of source assignments using Escherichia coli vs Enterococcus spp. The second objective was to determine the efficacy of using thresholds during source assignments to reduce the rate of misassignments when nonlibrary isolates (i.e. isolates from animals not used in building the identification library) are present. Methods and Results: E. coli and Enterococcus isolates from 784 human, cow, deer, dog, chicken, and gull faecal samples were fingerprinted using BOX-PCR. Jack-knife analysis of the fingerprints showed that the overall rate of correct assignment (ORCA) of 867 E. coli isolates was 67% compared with 82% for 1020 Enterococcus isolates. In a separate blind test using similarity value and quality factor thresholds, the ORCA of 130 E. coli and 131 Enterococcus isolates were 70% and 98%, respectively. The use of these thresholds reduced misassignment of 262 nonlibrary enterococcal isolates from horses, goats, pigs, bats, squirrels, ducks, geese, and migratory song birds. Misassignment was reduced from 100% when thresholds were not used, to 47% using similarity threshold alone, and to 12% when both thresholds were used. Conclusions: The use of enterococci provides higher rates of correct source assignment compared with E. coli. The use of similarity thresholds to decide whether to accept source assignments made by computer programmes reduces the rate of misassignment of nonlibrary isolates. Significance and Impact of the Study: Although both E. coli and Enterococcus spp. are still used in microbial source tracking, the use of enterococci should be preferred over the use of E. coli in DNA fingerprint-based efforts. In addition, because environmental isolates are not limited to those from animals used to build source tracking libraries, similarity thresholds should be used during source assignments to reduce the rate of misassignments

Abstract: Antibiotic resistance analysis (ARA), frequency of sampling, and seasonality were evaluated in a rural Virginia watershed dominated by cattle. The selected watershed (Mill Creek) was 3767ha in size, included two small communities (one sewered and one unsewered), and several farms that when combined contained over 3800 beef and dairy cattle. Monthly monitoring of fecal coliforms at two sampling sites in Mill Creek from January to December, 2001, revealed that the recreational standard (1000 colony forming units, CFUs/100ml) was exceeded a total of eight times for a 33% violation rate at each site. In addition, stream samples were collected weekly for 4 consecutive weeks during seasonal high flows (March) and seasonal low flows (September-October), plus daily for 7 consecutive days within the weekly schedules for a combined total of 60 stream samples (30 at each of two sites). The recreational standard was exceeded once during seasonal high flow and nine times during seasonal low flow. Microbial source tracking (MST) was performed by ARA to assess the impact of cattle on water quality within the different sampling routines. The resistance patterns of 2880 water isolates and 1158 known source (host-origin) isolates were determined with seven antibiotics at 28 different concentrations. The 1158 isolate database was reduced to 562 unique isolates when clonal ARA patterns were removed. This database of 562 unique isolates had an average rate of correct classification (ARCC) of 95.4%, and several statistical procedures confirmed the library as accurate and representative. Sixty-five percent of 50 challenge-set isolates from sources, but not samples, used in the library were correctly identified. The 562 unique pattern database was used to classify Escherichia coli isolates from water samples into six host source categories. The ARA results showed that cattle were the major source of pollution in the stream and cattle were the dominant source in over 60% of the water samples. Sampling frequency and seasonality had no effect on the MST results, as cattle dominated both seasons and samplings. Deer were a minor contributor in the summer (high water demand), and geese were a minor contributor in the winter when migratory flocks were observed moving through the watershed. An unexpected human allocation was found, especially under seasonal high flow conditions. The exact origin of this human allocation is not known. This project demonstrated that a host-origin library, based on a phenotypic method, could be developed for a well-defined watershed and was both representative of the sources in the watershed and performed reasonably well against a challenge set.

Abstract: Multiple microbial source-tracking methods were investigated to determine the source of elevated Escherichia coli levels at Bayfront Park Beach in Hamilton Harbour, Lake Ontario. E. coli concentrations were highest in wet foreshore sand (114,000 CFU/g dry sand) and ankle-depth water (177,000CFU/100mL), declining rapidly in deeper waters. Many gull and geese droppings were enumerated each week on the foreshore sand within 2m of the waterline. Both antimicrobial resistance analysis and rep-PCR DNA fingerprinting of E. coli collected at the beach and nearby fecal pollution sources indicated that E. coli in sand and water samples were predominantly from bird droppings rather than from pet droppings or municipal wastewater. Both methods indicated a trend of decreasing bird contamination, and increasing wastewater contamination, moving offshore from the beach. When foreshore sand was treated as a reservoir and secondary source of E. coli, waterborne E. coli were found to be more similar to sand isolates than bird or wastewater isolates out to 150m offshore. Multiple lines of evidence indicated the importance of bird droppings and foreshore sand as primary and secondary sources of E. coli contamination in beach water at Bayfront Park.

Abstract: Given known limitations of current microbial source-tracking (MST) tools, emphasis on small, simple study areas may enhance interpretations of fecal contamination sources in streams. In this study, three MST tools—Escherichia coli repetitive element polymerase chain reaction (rep-PCR), coliphage typing, and Bacteroidales 16S rDNA host-associated markers—were evaluated in a selected reach of Plum Creek in south-central Nebraska. Water-quality samples were collected from six sites. One reach was selected for MST evaluation based on observed patterns of E. coli contamination. Despite high E. coli concentrations, coliphages were detected only once among water samples, precluding their use as a MST tool in this setting. Rep-PCR classification of E. coli isolates from both water and sediment samples supported the hypothesis that cattle and wildlife were dominant sources of fecal contamination, with minor contributions by horses and humans. Conversely, neither ruminant nor human sources were detected by Bacteroidales markers in most water samples. In bed sediment, ruminant- and human-associated Bacteroidales markers were detected throughout the interval from 0 to 0.3 m, with detections independent of E. coli concentrations in the sediment. Although results by E. coli-based and Bacteroidales-based MST methods led to similar interpretations, detection of Bacteroidales markers in sediment more commonly than in water indicates that different tools to track fecal contamination (in this case, tools based on Bacteroidales DNA and E. coli isolates) may have varying relevance to the more specific goal of tracking the sources of E. coli in watersheds. This is the first report of simultaneous, toolbox approach application of a library-based and marker-based MST analyses to flowing surface water.

Abstract: The effect of a stormwater conveyance system on indicator bacteria levels at a Florida beach was assessed using microbial source tracking methods, and by investigating indicator bacteria population structure in water and sediments. During a rain event, regulatory standards for both fecal coliforms and Enterococcus spp. were exceeded, contrasting with significantly lower levels under dry conditions. Indicator bacteria levels were high in sediments under all conditions. The involvement of human sewage in the contamination was investigated using polymerase chain reaction (PCR) assays for the esp gene of Enterococcus faecium and for the conserved T antigen of human polyomaviruses, all of which were negative. BOX-PCR subtyping of Escherichia coli and Enterococcus showed higher population diversity during the rain event; and higher population similarity during dry conditions, suggesting that without fresh inputs, only a subset of the population survives the selective pressure of the secondary habitat. These data indicate that high indicator bacteria levels were attributable to a stormwater system that acted as a reservoir and conduit, flushing high levels of indicator bacteria to the beach during a rain event. Such environmental reservoirs of indicator bacteria further complicate the already questionable relationship between indicator organisms and human pathogens, and call for a better understanding of the ecology, fate and persistence of indicator bacteria.

Abstract: Watershed management programs often rely on monitoring for a large number of water quality parameters to define contaminant issues. While coliforms have traditionally been used to identify microbial contamination, these indicators cannot discriminate among potential contaminant sources. Microbial source tracking (MST) can provide the missing link that implicates the sources of contamination. The objective of this study was to use a weight-of-evidence approach (land use analysis using GIS, sanitary surveys, traditional water quality monitoring, and MST targets) to identify sources of pollution within a watershed that contains a raw drinking water source. For the study watersheds, statistical analyses demonstrated that one measure each of particulate matter (turbidity, particle counts), organic matter (total organic carbon, dissolved organic carbon, UV(254) absorbance), and indicator organisms (fecal coliforms, enterococci) were adequate for characterizing water quality. While these traditional parameters were useful for assessing overall water quality, they were not intended to differentiate between microbial sources at different locations. In contrast, the MST targets utilized (Rhodococcus coprophilus, sorbitol-fermenting Bifidobacteria, and male-specific coliphages) pinpointed specific sources of microbial pollution. However, these targets could not be used for routine monitoring due to a high percentage of non-detects.

Abstract: The ongoing development of microbial source tracking has made it possible to identify contamination sources with varying accuracy, depending on the method used. The purpose of this study was to test the efficiency of the antibiotic resistance analysis (ARA) method under low resistance by tracking the fecal sources at Turkey Creek, Oklahoma exhibiting this condition. The resistance patterns of 772 water-isolates, tested with nine antibiotics, were analyzed by discriminant analysis (DA) utilizing a five-source library containing 2250 isolates. The library passed various representativeness tests; however, two of the pulled-sample tests suggested insufficient sampling. The resubstitution test of the library individual sources showed significant isolate misclassification with an average rate of correct classification (ARCC) of 58%. These misclassifications were explained by low antibiotic resistance (Wilcoxon test P < 0.0001). Seasonal DA of stream E. coli isolates for the pooled sources human/livestock/deer indicated that in fall, the human source dominated (P < 0.0001) at a rate of 56%, and that human and livestock respective contributions in winter (35 and 39%), spring (43 and 40%), and summer (37 and 35%) were similar. Deer scored lower (17-28%) than human and livestock at every season. The DA was revised using results from a misclassification analysis to provide a perspective of the effect caused by low antibiotic resistance and a more realistic determination of the fecal source rates at Turkey Creek. The revision increased livestock rates by 13-14% (0.04 </= P </= 0.06), and decreased human and deer by 6-7%. Negative misclassification into livestock was significant (0.04 </= P </= 0.06). Low antibiotic resistance showed the greatest effect in this category.

Abstract: Fluorometry identifies human fecal contamination by detecting optical brighteners in environmental waters. Because optical brighteners are sensitive to sunlight, we determined if we could improve fluorometry by exposing water samples to ultraviolet (UV) light to differentiate between optical brighteners and other fluorescing organic compounds. Optical brighteners were likely present when the relative percentage difference in fluorometric value of the water before and after UV light exposure was >30% (glass cuvettes, 30 min exposure) or >15% (polymethacrylate cuvettes, 5 min exposure). In a blind study, we correctly identified the presence or absence of optical brighteners in 178 of 180 (99%) of the samples tested with a more expensive field fluorometer and in 175 of 180 (97%) of the samples tested with a less expensive handheld fluorometer. In the field, the method correctly identified two negative and three positive locations for human fecal contamination. When combined with counts of fecal bacteria, the new fluorometric method may be a simple, quick, and easy way to identify human fecal contamination in environmental waters.

Abstract: The goals of this study were to (i) identify issues that affect the ability of discriminant function analysis (DA) of antimicrobial resistance profiles to differentiate sources of fecal contamination, (ii) test the accuracy of DA from a known-source library of fecal Escherichia coli isolates with isolates from environmental samples, and (iii) apply this DA to classify E. coli from surface water. A repeated cross-sectional study was used to collect fecal and environmental samples from Michigan livestock, wild geese, and surface water for bacterial isolation, identification, and antimicrobial susceptibility testing using disk diffusion for 12 agents chosen for their importance in treating E. coli infections or for their use as animal feed additives. Nonparametric DA was used to classify E. coli by source species individually and by groups according to antimicrobial exposure. A modified backwards model-building approach was applied to create the best decision rules for isolate differentiation with the smallest number of antimicrobial agents. Decision rules were generated from fecal isolates and applied to environmental isolates to determine the effectiveness of DA for identifying sources of contamination. Principal component analysis was applied to describe differences in resistance patterns between species groups. The average rate of correct classification by DA was improved by reducing the numbers of species classifications and antimicrobial agents. DA was able to correctly classify environmental isolates when fewer than four classifications were used. Water sample isolates were classified by livestock type. An evaluation of the performance of DA must take into consideration relative contributions of random chance and the true discriminatory power of the decision rules.

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Abstract: It is well documented that microbial contamination of coastal waters poses a significant risk to human health through recreational exposure and consumption of shellfish. Identifying the source of microbial contamination (microbial source tracking) plays a dominant role in enabling effective management and remediation strategies. One method used to determine the source of the contamination is quantification of the ratio of the four subgroups of F+-specific RNA coliphages (family Leviviridae) in impacted water samples. Because of typically low concentrations in the environment, enrichment assays are performed prior to detection, even though differential replication rates have been reported. These assays are also compromised by differential loss of phage infectivity among subgroups after release into the environment, thus obscuring the initial ratio. Here, a culture-independent multiplex real-time reverse transcriptase-PCR (RT-PCR) protocol for the simultaneous quantification of all four subgroups of F+-specific RNA coliphages using novel primer sets and molecular beacons is presented. This assay is extremely sensitive, achieving detection with as few as 10 copies of isolated coliphage RNA, and is linear for a minimum of six orders of magnitude. During survival experiments, the real-time RT-PCR technique was able to quantify coliphages in seawater when culture-based double agar layer assay failed. While infectivity was lost at different rates at the subgroup level, decay constants in seawater, calculated using the real-time RT-PCR estimates, did not vary among subgroups. The accurate determination of the in situ concentration of F+-specific RNA coliphages using this method will facilitate more effective remediation strategies for impacted environments.

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Abstract: Recent molecular evidence suggests that different species and/or genotypes of Cryptosporidium display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidium species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidium parasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidium muskrat genotype II, Cryptosporidium cervine genotype, C. baileyi, C. parvum, Cryptosporidium muskrat genotype I, the Cryptosporidium fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidium parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidium parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium.

Abstract: Simple, rapid, and reliable fecal indicator tests are needed to better monitor and manage ambient waters and treated waters and wastes. Antibody-coated polymeric bead agglutination assays can fulfill these needs and are inexpensive and portable for nonlaboratory settings, and their reagents can be stored at ambient temperatures for months. The goal of this study was to develop, optimize, and validate a rapid microbial water quality monitoring assay using F+ coliphage culture, latex agglutination, and typing (CLAT) to detect F+ coliphage groups with antibody-coated particles. Rapid (180 min) F+ coliphage culture gave comparable results to those with the 16- to 24-h culture time used in EPA method 1601 and was amenable to CLAT assay detection. CLAT was performed on a cardboard card by mixing a drop of coliphage enrichment culture with a drop of antibody-coated polymeric beads as the detection reagent. Visual agglutination or clumping of positive samples occurred in <60 seconds. The CLAT assay had sensitivities of 96.4% (185/192 samples) and 98.2% (161/164 samples) and specificities of 100% (34/34 samples) and 97.7% (129/132 samples) for F+ RNA and DNA coliphages, respectively. CLAT successfully classified F+ RNA coliphages into serogroups typically obtained from human (groups II and III) and animal (groups I and IV) fecal sources, in similar proportions to those obtained with a nucleic acid hybridization assay. This novel group-specific antibody-based particle agglutination technique for rapid and simple detection and grouping of F+ coliphages provides a new and improved tool for monitoring the microbiological quality of drinking, recreational, shellfishing, and other waters.

Abstract: Escherichia coli is currently used as an indicator of fecal pollution and to assess water quality. While several genotypic techniques have been used to determine potential sources of fecal bacteria impacting waterways and beaches, they do not allow for the rapid analysis of a large number of samples in a relatively short period of time. Here we report that gene probes identified by Hamilton and colleagues (M. J. Hamilton, T. Yan, and M. J. Sadowsky, Appl. Environ. Microbiol. 72:4012-4019, 2006) were useful for the development of a high-throughput and quantitative macroarray hybridization system to determine numbers of E. coli bacteria originating from geese/ducks. The procedure we developed, using a QBot robot for picking and arraying of colonies, allowed us to simultaneously analyze up to 20,736 E. coli colonies from water samples, with minimal time and human input. Statistically significant results were obtained by analyzing 700 E. coli colonies per water sample, allowing for the analysis of approximately 30 sites per macroarray. Macroarray hybridization studies done on E. coli collected from water samples obtained from two urban Minnesota lakes and one rural South Carolina lake indicated that geese/ducks contributed up to 51% of the fecal bacteria in the urban lake water samples, and the level was below the detection limit in the rural lake water sample. This technique, coupled with the use of other host source-specific gene probes, holds great promise as a new quantitative microbial source tracking tool to rapidly determine the origins of E. coli in waterways and on beaches

Abstract: Based on the comparative 16S rRNA gene sequence analysis of fecal DNAs, we identified one human-, three cow-, and two pig-specific Bacteroides-Prevotella 16S rRNA genetic markers, designed host-specific real-time polymerase chain reaction (real-time PCR) primer sets, and successfully developed real-time PCR assay to quantify the fecal contamination derived from human, cow, and pig in natural river samples. The specificity of each newly designed host-specific primer pair was evaluated on fecal DNAs extracted from these host feces. All three cow-specific and two pig-specific primer sets amplified only target fecal DNAs (in the orders of 9-11 log(10) copies per gram of wet feces), showing high host specificity. This real-time PCR assay was then applied to the river water samples with different fecal contamination sources and levels. It was confirmed that this assay could sufficiently discriminate and quantify human, cow, and pig fecal contamination. There was a moderate level of correlation between the Bacteroides-Prevotella group-specific 16S rRNA gene markers with fecal coliforms (r (2) = 0.49), whereas no significant correlation was found between the human-specific Bacteroides 16S rRNA gene with total and fecal coliforms. Using a simple filtration method, the minimum detection limits of this assay were in the range of 50-800 copies/100 ml. With a combined sample processing and analysis time of less than 8 h, this real-time PCR assay is useful for monitoring or identifying spatial and temporal distributions of host-specific fecal contaminations in natural water environments.

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Abstract: In order to identify the origin of the faecal contamination observed in French estuaries, two library-independent microbial source tracking (MST) methods were selected: (i) Bacteroidales host-specific 16S rRNA gene markers and (ii) F-specific RNA bacteriophages genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, bird) faeces. Two human-specific (HF183 and HF134), one ruminant-specific (CF193') and one pig-specific (PF163) markers showed a high level of specificity (> 90%). However, the data suggest that the proposed ruminant-specific CF128 marker would be better described as an animal marker, as it was observed in all bovine and sheep faeces and 96% of pig faeces. F RNA bacteriophages were detected in only 21% of individual fecal samples tested, in 60% of pig slurries but in all sewage samples. Most detected F RNA bacteriophage were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird faeces, and from pig slurries. Both MST methods were applied to 28 water samples collected from three watersheds at different times. Classification of water samples as subject to human, animal or mixed faecal contamination was more frequent when using Bacteroidales markers (82.1% of water samples) than by bacteriophage genotyping (50%). The ability to classify a water sample increased with increasing E. coli or enterococci concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers.

Abstract: Library-dependent (LD) (biochemical fingerprinting of Escherichia coli and enterococci) and library-independent (LI) (PCR detection of human-specific biomarkers) methods were used to detect human faecal pollution in three non-sewered catchments. In all, 550 E. coli isolates and 700 enterococci isolates were biochemically fingerprinted from 18 water samples and compared with metabolic fingerprint libraries of 4508 E. coli and 4833 enterococci isolates. E. coli fingerprints identified human unique biochemical phenotypes (BPTs) in nine out of 18 water samples; similarly, enterococci fingerprints identified human faecal pollution in 10 water samples. Seven samples were tested by PCR for the detection of biomarkers. Human-specific HF134 Bacteroides and enterococci surface protein (esp) biomarkers were detected in five samples. Four samples were also positive for HF183 Bacteroides biomarker. The combination of biomarkers detected human faecal pollution in six out of seven water samples. Of the seven samples analysed for both the indicators/markers, at least one indicator/marker was detected in every sample. Four of the seven PCR-positive samples were also positive for one of the human-specific E. coli or enterococci BPTs. The results indicated human faecal pollution in the studied sub-catchments after storm events. LD and LI methods used in this study complimented each other and provided additional information regarding the polluting sources when one method failed to detect human faecal pollution. Therefore, it is recommended that a combination of methods should be used to identify the source(s) of faecal pollution where possible

Abstract: We developed a suspension array to enhance the ability to use library-independent genetic markers for bacterial source tracking. Six markers from Enterococcus spp. were selected to distinguish between cattle, humans, and cervids. Multiplex PCR was used to amplify fecal markers and resulting products were biotinylated and fragmented by nick translation followed by hybridization to polystyrene beads. Six populations of beads were included simultaneously in each assay where beads were labeled with an oligonucleotide probe complementary to one of the six library-independent markers. Hybridized products were detected on the beads using a 2-laser flow cytometer in a 96-well format. Testing with previously characterized strains showed that the assay could achieve 100% diagnostic sensitivity and >95% diagnostic specificity. Results from water samples were congruent for conventional PCR. Serial dilutions of template DNA demonstrated that the bench top analytic sensitivity of the entire assay was equivalent to <1600 cells. Suspension arrays permit greater certainty of product identification and this format can be expanded to include many additional markers.

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Abstract: In urbanized areas, contaminated storm sewers can feed high bacterial levels into free-flowing streams and rivers. Although illicit connections sometimes cause contamination, urban wildlife and free-roaming domesticated or feral pets may be another source. After eliminating illicit connections as sources of high levels of Escherichia coli in two storm sewers tributary to the Huron River in southeast Michigan, the roles of urban wildlife, pets, humans, and birds were investigated using a sequence-based bacterial source tracking technology. After enumeration, E. coli were isolated from water samples collected during spring to fall, 2005. Sequences in the gene beta-glucuronidase of each isolate were compared to sequences of reference strains from humans, raccoons, pets (cats and dogs), and birds. The highest percentage source for six of ten events was pets (ANOVA, p=0.005). Among isolates attributed to pets, strains from cats occurred more frequently on seven of nine events in which pets had a non-zero probability. High raccoon percentages (up to 60%) occurred in late summer and fall, and varied significantly more than in the spring (F-test), possibly reflecting urban raccoon den-site mobility. The sequence-based bacterial source tracking method suggests that feces from pets and raccoons are important contributors to urban storm sewers

Abstract: Most library-dependent bacterial source tracking studies using Escherichia coli (E. coli) have focused on strain diversity of isolates obtained from known human and animal faecal sources for library development. In contrast, this study evaluated the genotype variation of E. coli isolated from natural surface water using pulsed field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) to better understand these naturally occurring populations. A total of 650 water samples were collected over a nine month period from eleven sampling stations from Lake Waco and Belton Lake in Central Texas. Of the 650 water samples collected, 412 were positive for E. coli, yielding a total of 631 E. coli isolates (1–12 isolates collected per sample). PFGE and ERIC-PCR patterns were successfully generated for 555 isolates and were compared using the curve-based Pearson's product–moment correlation coefficient. The 555 E. coli isolates represented 461 PFGE genotypes, with 84% (386/461) of the genotypes being represented by individual isolates. The remaining 75 genotypes were represented by 2–5 isolates each. Using ERIC-PCR, the 555 E. coli isolates represented 175 genotypes, with 63% (109/175) of the genotypes being represented by individual isolates. In contrast to the PFGE results, two ERIC-PCR genotypes represented 37% of the E. coli isolates, (83 and 124 isolates, respectively), and were found throughout the watersheds both spatially and temporally. Based on the PFGE genotype diversity of water isolates, there is little evidence that a small number of environmentally-adapted E. coli represent dominant populations in the studied waterbodies. However, with the lower discriminatory power technique ERIC-PCR, an opposing conclusion might have been drawn. These results emphasize the importance of considering the resolving power of the source tracking technique being used when assessing strain diversity and geographical stability

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Abstract: AIMS: The aim of this study was to assess geographical variation in multiple antibiotic resistance (MAR) profiles of livestock Escherichia coli as well as to evaluate the ability of MAR profiles to differentiate sources of faecal pollution. METHODS AND RESULTS: More than 2000 E. coli isolates were collected from water retention ponds and manure of swine, poultry, beef and dairy farms in south, central and north Florida, and analysed for MAR using nine antibiotics. There were significant differences in antibiotic resistance of E. coli by season and livestock type for more than one antibiotic, but regional differences were significant only for ampicillin. Over the three regions, discriminant analysis using MAR profiles correctly classified 27% of swine, 49% of poultry, 56% of beef and 51% of dairy isolates. CONCLUSIONS: Regional variations in MAR combined with moderate discrimination success suggest that MAR profiles of E. coli may only be marginally successful in identifying sources of faecal pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the existence of regional and seasonal differences in MAR profiles as well as the limited ability of MAR profiles to discriminate among livestock sources.

Abstract: The Adenoviridae and Polyomaviridae families comprise a wide diversity of viruses which may be excreted for long periods in feces or urine. In this study, a preliminary analysis of the prevalence in the environment and the potential usefulness as source-tracking tools of human and animal adenoviruses and polyomaviruses has been developed. Molecular assays based on PCR specifically targeting human adenoviruses (HAdV), porcine adenoviruses (PAdV), bovine adenoviruses (BAdV), and bovine polyomaviruses (BPyV) were applied to environmental samples including urban sewage, slaughterhouse, and river water samples. PAdV and BPyV were detected in a very high percentage of samples potentially affected by either porcine or bovine fecal contamination, respectively. However, BAdV were detected in only one sample, showing a lower prevalence than BPyV in the wastewater samples analyzed. The 22 slaughterhouse samples with fecal contamination of animal origin showed negative results for the presence of HAdV. The river water samples analyzed were positive for the presence of both human and animal adenoviruses and polyomaviruses, indicating the existence of diverse sources of contamination. The identities of the viruses detected were confirmed by analyses of the amplified sequences. All BPyV isolates showed a 97% similarity in nucleotide sequences. This is the first time that PAdV5, BAdV6, and BPyV have been reported to occur in environmental samples. Human and porcine adenoviruses and human and bovine polyomaviruses are proposed as tools for evaluating the presence of viral contamination and for tracking the origin of fecal/urine contamination in environmental samples.

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Abstract: Nonpoint source fecal contamination is a concern for drinking water supplies worldwide. In this study, 4812 E. coli isolates were classified to source. Results of this experiment show that the fecal coliform (FC) counts varied by year, month, and site, for each of the watersheds sampled. For both years, the lowest FC counts tended to be at the highest elevation sites followed by the drinking water intake sites at the lowest elevation. The highest FC counts tended to be at the mid-elevation sites on BX, Deer, and Duteau Creeks. The sources of E. coli varied significantly with stream for 2003 and 2004 (P < 0.001, df = 39), although the main sources of E. coli (avian, deer/elk, canine, rodent, bovine, and bear) tended to be similar between watersheds. The dominant sources of E. coli changed from 2003 (avian, deer/elk, and canine) to 2004 (avian, bovine, and rodent). It is important to look at the results of more than 1 year of source tracking data to get a better picture of the dominant sources within a watershed. Overall, wildlife was the largest contributor of E. coli to the watersheds in both 2003 (> 84%) and 2004 (> 73%).

Abstract: Little information has been gathered on the effect of avian species on the microbial water quality in constructed wetlands. To address this concern, fecal pollution from nonpoint and point sources was evaluated in a constructed wetland in Tolleson, Arizona. Antibiotic resistance profiling and biochemical fingerprinting were performed on 325 Escherichia coli isolates, collected from key points in the wetlands. Multivariate statistical analysis was used to interpret the data for samples collected on October 3 and December 12, 2000, and January 16, 2001. It was found that the passerine population was the major source of the Escherichia coli in the water samples collected in the wetlands on October 3 and December 12, 2000, whereas the regrowth in the treated municipal wastewater was the main source on January 16, 2001. This information is useful in providing data for operators in the monitoring of wetlands created for wastewater treatment and wildlife habitat.

Abstract: Spring water from alpine catchments are important water resources but they can be vulnerable against faecal contamination. Potential faecal contamination sources are wildlife populations, pasturing activities, or alpine tourism. Unfortunately, no faecal source tracking method is available to date which is sensitive enough for appropriate spring water monitoring and source allocation. Our purpose was to develop a Duplex Scorpion real-time PCR approach for the specific and sensitive quantification of Bacteroides sp. 16S rDNA fragments from human and cattle origin. By the developed approach, detection of plasmids, carrying the respective biomarker sequence, was possible over a range of more than seven orders of magnitudes down to six copy numbers per PCR assay. Furthermore, the Duplex Scorpion real-time PCR allowed the specific quantification down to 50 targets in plasmid spiked spring water matrices. Results indicate that microbial source tracking appears feasible in spring water habitats by probe-based real-time PCR technologies. However, preliminary testing of the established approach on faecal samples collected from a representative alpine habitat did not allow unambiguous source allocation in all cases. In the future, the available sequence database has thus to be widened to allow reliable source tracking in alpine spring watersheds and even expand this approach to other potential faecal sources.

Abstract: Microbial source tracking (MST) methods need to be rapid, inexpensive and accurate. Unfortunately, many MST methods provide a wealth of information that is difficult to interpret by the regulators who use this information to make decisions. This paper describes the use of classification tree analysis to interpret the results of a MST method based on fatty acid methyl ester (FAME) profiles of Escherichia coli isolates, and to present results in a format readily interpretable by water quality managers. Raw sewage E. coli isolates and animal E. coli isolates from cow, dog, gull, and horse were isolated and their FAME profiles collected. Correct classification rates determined with leaveone-out cross-validation resulted in an overall low correct classification rate of 61%. A higher overall correct classification rate of 85% was obtained when the animal isolates were pooled together and compared to the raw sewage isolates. Bootstrap aggregation or adaptive resampling and combining of the FAME profile data increased correct classification rates substantially. Other MST methods may be better suited to differentiate between different fecal sources but classification tree analysis has enabled us to distinguish raw sewage from animal E. coli isolates, which previously had not been possible with other multivariate methods such as principal component analysis and cluster analysis.

Abstract: The objective of this study was to investigate the host-specific differences in fatty acid methyl ester (FAME) profiles of fecal coliforms (FC). A known-source library was constructed with 314 FC isolates cultured from 6 possible sources of fecal pollution; 99 isolates from sewage; 29 from bovine; 29 from poultry; 50 from swine; 46 from waterfowl; and 61 from deer. It was found that the hydroxy FAMEs 12:0 2 OH, 12:03 OH, and 14:02 OH were exclusively associated with isolates of human origin. On the other hand, 3 saturated FAMEs, 10:0, 15:0, and 18:0 were found only in isolates from non-human sources, 15:0 being associated with livestock samples only. In addition to the presence of these signature FAMEs, the mean relative masses of 16:1 omega7c and 16:1 ISO/14:03 OH were significantly different between the isolates of human and non-human origins. A linear discriminant function differentiated FC isolates of human origin from those of livestock and wildlife origin at 99% accuracy. These results strongly suggest that the FAME profiles of FC show statistically significant host specificity and may have the potential to be used as a phenotypic microbial source tracking tool.

Abstract: A comparison of the efficacy of an existing large metabolic fingerprint database of enterococci and Escherichia coli with a locally developed database was undertaken to identify the sources of faecal contamination in a coastal lake, in southeast Qld., Australia. The local database comprised of 776 enterococci and 780 E. coli isolates from six host groups. In all, 189 enterococci and 245 E. coli biochemical phenotypes (BPTs) were found, of which 118 and 137 BPTs were unique (UQ) to host groups. The existing database comprised of 295 enterococci UQ-BPTs and 273 E. coli UQ-BPTs from 10 host groups. The representativeness and the stability of the existing database were assessed by comparing with isolates that were external to the database. In all, 197 enterococci BPTs and 179 E. coli BPTs were found in water samples. The existing database was able to identify 62.4% of enterococci BPTs and 64.8% of E. coli BPTs as human and animal sources. The results indicated that a representative database developed from a catchment can be used to predict the sources of faecal contamination in another catchment with similar landuse features within the same geographical area. However, the representativeness and the stability of the database should be evaluated prior to its application in such investigation.

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Abstract: Criteria for sub-typing of microbial organisms by DNA sequencing proposed by Olive and Bean were applied to several genes in Escherichia coli to identify targets for the development of microbial source tracking assays. Based on the aforementioned criteria, the icd (isocitrate dehydrogenase), and putP (proline permease) genes were excluded as potential targets due to their high rates of horizontal gene transfer; the rrs (16S rRNA) gene was excluded as a target due to the presence of multiple gene copies, with different sequences in a single genome. Based on the above criteria, the mdh (malate dehydrogenase) gene was selected as a target for development of a microbial source tracking assay. The mdh assay was optimized to analyze a 150 bp fragment corresponding to residues G191 to R240 (helices H10 and H11) of the Mdh catalytic domain. 295 fecal isolates (52 horse, 50 deer, 72 dog, 52 seagull and 69 human isolates) were sequenced and analyzed. Target DNA sequences for isolates from horse, dog plus deer, and seagull formed identifiable groupings. Sequences from human isolates, aside from a low level (ca. 15%) human specific sequence, did not group; nevertheless, other hosts could be distinguished from human. Positive and negative predictive values for two- and three-way host comparisons ranged from 60% to 90% depending on the focus host. False positive rates were below 10%. Multiple E. coli isolates from individual fecal samples exhibited high levels of sequence homogeneity, i.e. typically only one to two mdh sequences were observed per up to five E. coli isolates from a single fecal sample. Among all isolates sequenced from fecal samples from each host, sequence homogeneity decreased in the following order: horse>dog>deer>human and gull. For in-library isolates, blind analysis of fecal isolates (n=12) from four hosts known to contain host specific target sequences was 100% accurate and 100% reproducible for both DNA sequence and host identification. For blind analysis of non-library isolates, 18/19 isolates (94.7%) matched one or more library sequences for the corresponding host. Ten of eleven geographical outlier fecal isolates from Florida had mdh sequences that were identical to in-library sequences for the corresponding host from California. The mdh assay was successfully applied to environmental isolates from an underground telephone vault in California, with 4 of 5 isolates matching sequences in the mdh library. 146 sequences of the 645bp mdh fragment from five host sources were translated into protein sequence and aligned. Seven unique Mdh protein sequences, which contained eight polymorphic sites, were identified. Six of the polymorphic sites were in the NAD+ binding domain and two were in the catalytic domain. All of the polymorphic sites were located in surface exposed regions of the protein. None of the non-silent mutations of the Mdh protein were in the 150bp mdh target. The advantages and disadvantages of the assay compared to established source tracking methods are discussed.

Abstract: The microbiological contamination of waterways by pathogenic microbes has been, and is still, a persistent public safety concern in the United States and in most countries of the world. As most enteric pathogens are transmitted through the fecal-oral route, fecal pollution is generally regarded as the major contributor of pathogens to waterways. Fecal pollution of waterways can originate from wastewater treatment facilities, septic tanks, domestic- and wild-animal feces, and pets. Because enteric pathogens are derived from human or animal sources, techniques capable of identifying and apportioning fecal sources have been intensively investigated for use in remediation efforts and to satisfy regulatory concerns. Pollution of human origin is of the most concern, since human feces is more likely to contain human-specific enteric pathogens. Fecal indicator bacteria have been used successfully as the primary tool for microbiologically based risk assessment. However measurement of fecal indicator bacteria does not define what pathogens are present, or define the sources of these bacteria. Microbial source tracking (MST) methods that have the ability to differentiate among sources of fecal pollution are currently under development. These methods will ultimately be useful for risk assessment purposes and to aid regulatory agencies in developing strategies to remediate microbiologically impaired waterways.

Abstract: This research used a weight-of-evidence approach to evaluate sources of contaminants in a drinking water watershed that serves as part of the City of Boston's water supply. The approach incorporated land use analysis using GIS, sanitary surveys, traditional water quality monitoring and microbial source tracking (MST) tools. Case-study tributaries were selected based on elevated faecal coliform counts. Land use analysis and sanitary surveys were used to identify suspected microbial sources, including residential septic systems, agricultural animal operations, commercial/industrial operations and wildlife activity. Sampling sites were selected to hydrologically isolate potential contamination sources. Samples were collected seasonally over 1 year and analysed for traditional and MST parameters. Results demonstrated that both septic systems and a horse stable were contributing microbial loads in the first tributary. In the second tributary, septic systems from the townhouses were contributing microbial loads while a plant nursery was contributing organic matter. This evidence was used to evaluate best management practices to mitigate the contamination.

Abstract: This manuscript describes the use of a recently developed microbial source tracking (MST) technique to determine sources of fecal bacteria (enterococci) from four separate European countries. The investigation aimed to evaluate whether the origin of bacterial populations from France, Spain, and Sweden (n = 456) could be predicted using a library of antibiotic resistance patterns of enterococci of known origin assembled in the United Kingdom (n = 2739). Bacterial isolates were exposed to a battery of 31 antibiotic tests and classified into source groups using discriminant function analysis (DFA). Results showed that while on average 72% of the U.K. isolates could be correctly classified as originating from either municipal wastewater (MW), livestock, or wild birds, only 43% of non-U.K. isolates could be successfully classified into the same source categories. The results suggested that patterns of resistance amongst isolates contained in the U.K. library were not representative of those found in the other locations and that it may not be possible to share libraries over large distances, such as those in this study. Future MST studies using antibiotic resistance analysis (ARA) in Europe may therefore require the assembly of watershed specific libraries, increasing the cost of such studies.

Abstract: Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 microl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking "toolbox."

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Abstract: A rapidly growing method to identify origins of nonpoint source (NPS) pollution is microbial source tracking (MST). Current MST research utilizes either an organism's genetic or physiological traits to establish source identification. To determine if an MST method based on fecal bacterial species composition can be used to determine sources of NPS pollution, samples from known NPS contributors (human, cattle, poultry, and swine) were collected and analyzed for fecal coliform (FC) and fecal streptococci (FS). Five colonies from each bacterial type were randomly selected, isolated and identified using phenotypic profiles. The species composition was calculated from these data and analyzed statistically via discriminant analysis. The rates of correct classification (RCC) for FC species composition patterns were 64, 71, 47 and 70% for cattle, human, poultry and swine, respectively. The RCC for FS species composition patterns were 87, 86, 74, and 83% for cattle, human, poultry, and swine, respectively. The average rate of correct classification for samples from all known sources was significantly higher (P=0.05) for FS species composition data (82%) than for FC (63%). The average rate of correct classification was increased when the FC and FS species composition data was combined (93%). The results from this study indicate that a phenotypic MST methodology based on species composition of dominant fecal bacteria may be useful in determining major contributors to NPS pollution. Based on the average rates of correct classification, the use of FS species composition patterns appears to be more useful in identifying source than the use of FC patterns.

Abstract: The contamination of waterways with fecal material is a persistent threat to public health. Identification of the sources of fecal contamination is a vital component for abatement strategies and for determination of total maximum daily loads. While phenotypic and genotypic techniques have been used to determine potential sources of fecal bacteria in surface waters, most methods require construction of large known-source libraries, and they often fail to adequately differentiate among environmental isolates originating from different animal sources. In this study, we used pooled genomic tester and driver DNAs in suppression subtractive hybridizations to enrich for host source-specific DNA markers for Escherichia coli originating from locally isolated geese. Seven markers were identified. When used as probes in colony hybridization studies, the combined marker DNAs identified 76% of the goose isolates tested and cross-hybridized, on average, with 5% of the human E. coli strains and with less than 10% of the strains obtained from other animal hosts. In addition, the combined probes identified 73% of the duck isolates examined, suggesting that they may be useful for determining the contribution of waterfowl to fecal contamination. However, the hybridization probes reacted mainly with E. coli isolates obtained from geese in the upper midwestern United States, indicating that there is regional specificity of the markers identified. Coupled with high-throughput, automated macro- and microarray screening, these markers may provide a quantitative, cost-effective, and accurate library-independent method for determining the sources of genetically diverse E. coli strains for use in source-tracking studies. However, future efforts to generate DNA markers specific for E. coli must include isolates obtained from geographically diverse animal hosts.

Abstract: Received for publication August 26, 2005. Most bacterial source tracking (BST) methods are too expensive for most communities to afford. We developed targeted sampling as a prelude to BST to reduce these costs. We combined targeted sampling with three inexpensive BST methods, Enterococcus speciation, detection of the esp gene, and fluorometry, to confirm the sources of fecal contamination to beaches on Georgia's Jekyll and Sea Islands during calm and stormy weather conditions. For Jekyll Island, the most likely source of contamination was bird feces because the percentage of Ent. faecalis was high (30%) and the esp gene was not detected. For the Sea Island beach during calm conditions, the most likely sources of fecal contamination were leaking sewer lines and wildlife feces. The leaking sewer lines were confirmed with fluorometry and detection of the esp gene. For the Sea Island beach during stormflow conditions, the most likely sources of fecal contamination were wildlife feces and runoff discharging from two county-maintained pipes. For the pipes, the most likely source of contamination was bird feces because the percentage of Ent. faecalis was high (30%) and the esp gene was not detected. Sediments were also a reservoir of fecal enterococci for both Jekyll and Sea Islands. Combining targeted sampling with two or more BST methods identified sources of fecal contamination quickly, easily, and inexpensively. This combination was the first time targeted sampling was conducted during stormy conditions, and the first time targeted sampling was combined with enterococcal speciation, detection of the esp gene, and fluorometry.

Abstract: Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 µl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking "toolbox."

Abstract: The Adenoviridae and Polyomaviridae families comprise a wide diversity of viruses which may be excreted for long periods in feces or urine. In this study, a preliminary analysis of the prevalence in the environment and the potential usefulness as source-tracking tools of human and animal adenoviruses and polyomaviruses has been developed. Molecular assays based on PCR specifically targeting human adenoviruses (HAdV), porcine adenoviruses (PAdV), bovine adenoviruses (BAdV), and bovine polyomaviruses (BPyV) were applied to environmental samples including urban sewage, slaughterhouse, and river water samples. PAdV and BPyV were detected in a very high percentage of samples potentially affected by either porcine or bovine fecal contamination, respectively. However, BAdV were detected in only one sample, showing a lower prevalence than BPyV in the wastewater samples analyzed. The 22 slaughterhouse samples with fecal contamination of animal origin showed negative results for the presence of HAdV. The river water samples analyzed were positive for the presence of both human and animal adenoviruses and polyomaviruses, indicating the existence of diverse sources of contamination. The identities of the viruses detected were confirmed by analyses of the amplified sequences. All BPyV isolates showed a 97% similarity in nucleotide sequences. This is the first time that PAdV5, BAdV6, and BPyV have been reported to occur in environmental samples. Human and porcine adenoviruses and human and bovine polyomaviruses are proposed as tools for evaluating the presence of viral contamination and for tracking the origin of fecal/urine contamination in environmental samples.

Abstract: The objectives of this study were to elucidate spatial and temporal dynamics in source-specific Bacteroidales 16S rRNA genetic marker data across a watershed; to compare these dynamics to fecal indicator counts, general measurements of water quality, and climatic forces; and to identify geographic areas of intense exposure to specific sources of contamination. Samples were collected during a 2-year period in the Tillamook basin in Oregon at 30 sites along five river tributaries and in Tillamook Bay. We performed Bacteroidales PCR assays with general, ruminant-source-specific, and human-source-specific primers to identify fecal sources. We determined the Escherichia coli most probable number, temperature, turbidity, and 5-day precipitation. Climate and water quality data collectively supported a rainfall runoff pattern for microbial source input that mirrored the annual precipitation cycle. Fecal sources were statistically linked more closely to ruminants than to humans; there was a 40% greater probability of detecting a ruminant source marker than a human source marker across the basin. On a sample site basis, the addition of fecal source tracking data provided new information linking elevated fecal indicator bacterial loads to specific point and nonpoint sources of fecal pollution in the basin. Inconsistencies in E. coli and host-specific marker trends suggested that the factors that control the quantity of fecal indicators in the water column are different than the factors that influence the presence of Bacteroidales markers at specific times of the year. This may be important if fecal indicator counts are used as a criterion for source loading potential in receiving waters.

Abstract: Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.

Abstract: Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host-specific markers. Here we describe the application of a genome fragment enrichment (GFE) method to identify host-specific genetic markers from fecal microbial community DNA. As a proof of concept, bovine fecal DNA was challenged against a porcine fecal DNA background to select for bovine-specific DNA sequences. Bioinformatic analyses of 380 bovine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode membrane-associated and secreted proteins. Oligonucleotide primers capable of annealing to select Bacteroidales-like bovine GFE sequences exhibited extremely high specificity (>99%) in PCR assays with total fecal DNAs from 279 different animal sources. These primers also demonstrated a broad distribution of corresponding genetic markers (81% positive) among 148 different bovine sources. These data demonstrate that direct metagenomic DNA analysis by the competitive solution hybridization approach described is an efficient method for identifying potentially useful fecal genetic markers and for characterizing differences between environmental microbial communities.

Abstract: A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum Bacteroidetes. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 x 109 marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples from a karstic catchment area at levels matching a gradient from negligible to considerable ruminant fecal influence (from not detectable to 105 marker equivalents per liter).

Abstract: The ubiquity of fecal indicator bacteria such as Escherichia coli and Enterococcus spp. in urban environments makes tracking of fecal contamination extremely challenging. A multitiered approach was used to assess sources of fecal pollution in Ballona Creek, an urban watershed that drains to the Santa Monica Bay (SMB) near Los Angeles, Calif. A mass-based design at six main-stem sites and four major tributaries over a 6-h period was used (i) to assess the flux of Enterococcus spp. and E. coli by using culture-based methods (tier 1); (ii) to assess levels of Enterococcus spp. by using quantitative PCR and to detect and/or quantify additional markers of human fecal contamination, including a human-specific Bacteroides sp. marker and enterovirus, using quantitative reverse transcriptase PCR (tier 2); and (iii) to assess the specific types of enterovirus genomes found via sequence analysis (tier 3). Sources of fecal indicator bacteria were ubiquitous, and concentrations were high, throughout Ballona Creek, with no single tributary dominating fecal inputs. The flux of Enterococcus spp. and E. coli averaged 109 to 1010 cells h–1 and was as high at the head of the watershed as at the mouth prior to discharge into the SMB. In addition, a signal for the human-specific Bacteroides marker was consistently detected: 86% of the samples taken over the extent during the study period tested positive. Enteroviruses were quantifiable in 14 of 36 samples (39%), with the highest concentrations at the site furthest upstream (Cochran). These results indicated the power of using multiple approaches to assess and quantify fecal contamination in freshwater conduits to high-use, high-priority recreational swimming areas.

Abstract: Escherichia coli is the most completely characterized prokaryotic model organism and one of the dominant indicator organisms for food and water quality testing, yet comparatively little is known about the structure of E. coli populations in their various hosts. The diversities of E. coli populations isolated from the feces of three host species (human, cow, and horse) were compared by two subtyping methods: ribotyping (using HindIII) and antibiotic resistance analysis (ARA). The sampling effort required to obtain a representative sample differed by host species, as E. coli diversity was consistently greatest in horses, followed by cattle, and was lowest in humans. The diversity of antibiotic resistance patterns isolated from individuals was consistently greater than the diversity of ribotypes. E. coli populations in individuals sampled monthly, over a 7- to 8-month period, were highly variable in terms of both ribotypes and ARA phenotypes. In contrast, E. coli populations in cattle and humans were stable over an 8-h period. Following the cessation of antibiotic therapy, the E. coli population in the feces of one human experienced a rapid and substantial shift, from a multiply antibiotic-resistant phenotype associated with a particular ribotype to a relatively antibiotic-susceptible phenotype associated with a different ribotype. The high genetic diversity of E. coli populations, differences in diversity among hosts, and temporal variability all indicate complex population dynamics that influence the usefulness of E. coli as a water quality indicator and its use in microbial source tracking studies.

Abstract: Typing of F-specific RNA (FRNA) coliphages has been proposed as a useful method for distinguishing human from animal fecal contamination in environmental samples. Group II and III FRNA coliphages are generally associated with human wastes, but several exceptions have been noted. In the present study, we have genotyped and partially sequenced group III FRNA coliphage field isolates from swine lagoons in North Carolina (NC) and South Carolina (SC), along with isolates from surface waters and municipal wastewaters. Phylogenetic analysis of a region of the 5' end of the maturation protein gene revealed two genetically different group III FRNA subclusters with 36.6% sequence variation. The SC swine lagoon isolates were more closely related to group III prototype virus M11, whereas the isolates from a swine lagoon in NC, surface waters, and wastewaters grouped with prototype virus Q-beta. These results suggest that refining phage genotyping systems to discriminate M11-like phages from Q-beta-like phages would not necessarily provide greater discriminatory power in distinguishing human from animal sources of pollution. Within the group III subclusters, nucleotide sequence diversity ranged from 0% to 6.9% for M11-like strains and from 0% to 8.7% for Q-beta-like strains. It is demonstrated here that nucleotide sequencing of closely related FRNA strains can be used to help track sources of contamination in surface waters. A similar use of phage genomic sequence information to track fecal pollution promises more reliable results than phage typing by nucleic acid hybridization and may hold more potential for field applications.

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.

Abstract: Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50 Enterococcus isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four Enterococcus species (Enterococcus casseliflavus, E. faecalis, E. hirae, or E. mundtii). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of library-independent markers.

Abstract: Various statistical classification methods, including discriminant analysis, logistic regression, and cluster analysis, have been used with antibiotic resistance analysis (ARA) data to construct models for bacterial source tracking (BST). We applied the statistical method known as classification trees to build a model for BST for the Anacostia Watershed in Maryland. Classification trees have more flexibility than other statistical classification approaches based on standard statistical methods to accommodate complex interactions among ARA variables. This article describes the use of classification trees for BST and includes discussion of its principal parameters and features. Anacostia Watershed ARA data are used to illustrate the application of classification trees, and we report the BST results for the watershed.

Abstract: Aims: The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards. Methods and Results: Coliphages were isolated from 117 surface water samples by single agar layer (SAL) and enrichment presence/absence (EP/A) methods. Confirmed F+RNA coliphages were typed for microbial source tracking using a library-independent approach. Concentrations of somatic coliphages using 37 and 44·5°C incubation temperatures were found to be significantly different and the higher temperature may be more specific for faecal contamination. The EP/A technique detected coliphages infecting Escherichia coli Famp in 38 (66%) of the 58 surface water samples negative for F+ coliphages by the SAL method. However, coliphages isolated by EP/A were found to be less representative of coliphage diversity within a sample. Among the 2939 coliphage isolates tested from surface water and known source samples, 813 (28%) were found to be F+RNA. The majority (94%) of surface water F+RNA coliphage isolates typed as group I. Group II and/or III viruses were identified from 14 surface water stations, the majority of which were downstream of wastewater discharges. These sites were likely contaminated by human-source faecal pollution. Conclusions: The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses. Significance and Impact of the Study: This study supports the premise that coliphage typing can provide useful, but not absolute, information to distinguish human from animal sources of faecal pollution. Furthermore, the comparison of coliphage isolation methods detailed in this study should provide valuable information to those wishing to incorporate coliphage detection into water quality assessments.

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Abstract: To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify any unique populations in chickens, cows, deer, dogs, geese, horses, humans, pigs, and seagulls. The group appears to be a good potential fecal indicator in all groups tested except for avians. Cluster analysis of Bacteroides-Prevotella community T-RFLP profiles indicates that Bacteroides-Prevotella populations from samples of the same host species are much more similar to each other than to samples from different source species. We were unable to identify unique peaks that were exclusive to any source species; however, for most host species, at least one T-RFLP peak was identified to be more commonly found in that species, and a combination of peaks could be used to identify the source. T-RFLP profiles obtained from water spiked with known-source feces contained the expected diagnostic peaks from the source. These results indicate that the approach of identifying Bacteroides-Prevotella molecular markers associated with host species might be useful in identifying sources of fecal contamination in the environment.

Abstract: A metabolic fingerprint database of enterococci and Escherichia coli from 10 host groups of animals was developed to trace the sources of fecal contamination in surface waters. In all, 526 biochemical phenotypes (BPTs) of enterococci and 530 E. coli BPTs were obtained from 4,057 enterococci and 3,728 E. coli isolates tested. Of these, 231 Enterococcus BPTs and 257 E. coli BPTs were found in multiple host groups. The remaining 295 Enterococcus BPTs and 273 E. coli BPTs were unique to individual host groups. The database was used to trace the sources of fecal contamination in a local creek. The mean diversities (Di) of enterococci (Di = 0.76 ± 0.05) and E. coli (Di = 0.88 ± 0.04) were high (maximum 1) in water samples, indicating diverse sources of fecal contamination. Overall, 71% of BPTs of enterococci and 67% of E. coli BPTs from water samples were identified as human and animal sources. Altogether, 248 Enterococcus BPTs and 282 E. coli BPTs were found in water samples. Among enterococci, 26 (10%) BPTs were identical to those of humans and 152 BPTs (61%) were identical to those of animals (animal BPTs). Among E. coli isolates, 36 (13%) BPTs were identical to those of humans and 151 (54%) BPTs were identical to those of animals. Of the animal BPTs, 101 (66%) Enterococcus BPTs and 93 (62%) E. coli BPTs were also unique to individual animal groups. On the basis of these unique Enterococcus BPTs, chickens contributed 14% of contamination, followed by humans (10%), dogs (7%), and horses (6%). For E. coli, humans contributed 13% of contamination, followed by ducks (9%), cattle (7%), and chickens (6%). The developed metabolic fingerprint database was able to distinguish between human and animal sources as well as among animal species in the studied catchment.

Abstract: Lake Michigan surface waters impacted by fecal pollution were assessed to determine the occurrence of genetic markers for Bacteroides and Escherichia coli. Initial experiments with sewage treatment plant influent demonstrated that total Bacteroides spp. could be detected by PCR in a 25- to 125-fold-higher dilution series than E. coli and human-specific Bacteroides spp., which were both found in similar dilution ranges. The limit of detection for the human-specific genetic marker ranged from 0.2 CFU/100 ml to 82 CFU/100 ml culturable E. coli for four wastewater treatment plants in urban and rural areas. The spatial and temporal distributions of these markers were assessed following major rain events that introduced urban storm water, agricultural runoff, and sewage overflows into Lake Michigan. Bacteroides spp. were detected in all of these samples by PCR, including those with <1 CFU/100 ml E. coli. Human-specific Bacteroides spp. were detected as far as 2 km into Lake Michigan during sewage overflow events, with variable detection 1 to 9 days postoverflow, whereas the cow-specific Bacteroides spp. were detected in only highly contaminated samples near the river outflow. Lake Michigan beaches were also assessed throughout the summer season for the same markers. Bacteroides spp. were detected in all beach samples, including 28 of the 74 samples that did not exceed 235 CFU/100 ml of E. coli. Human-specific Bacteroides spp. were detected at three of the seven beaches; one of the sites demonstrating positive results was sampled during a reported sewage overflow, but E. coli levels were below 235 CFU/100 ml. This study demonstrates the usefulness of non-culture-based microbial-source tracking approaches and the prevalence of these genetic markers in the Great Lakes, including freshwater coastal beaches.

Abstract: Fecal coliforms and enterococci are indicator organisms used worldwide to monitor water quality. These bacteria are used in microbial source tracking (MST) studies, which attempt to assess the contribution of various host species to fecal pollution in water. Ideally, all strains of a given indicator organism (IO) would experience equal persistence (maintenance of culturable populations) in water; however, some strains may have comparatively extended persistence outside the host, while others may persist very poorly in environmental waters. Assessment of the relative contribution of host species to fecal pollution would be confounded by differential persistence of strains. Here, freshwater and saltwater mesocosms, including sediments, were inoculated with dog feces, sewage, or contaminated soil and were incubated under conditions that included natural stressors such as microbial predators, radiation, and temperature fluctuations. Persistence of IOs was measured by decay rates (change in culturable counts over time). Decay rates were influenced by IO, inoculum, water type, sediment versus water column location, and Escherichia coli strain. Fecal coliform decay rates were significantly lower than those of enterococci in freshwater but were not significantly different in saltwater. IO persistence according to mesocosm treatment followed the trend: contaminated soil > wastewater > dog feces. E. coli ribotyping demonstrated that certain strains were more persistent than others in freshwater mesocosms, and the distribution of ribotypes sampled from mesocosm waters was dissimilar from the distribution in fecal material. These results have implications for the accuracy of MST methods, modeling of microbial populations in water, and efficacy of regulatory standards for protection of water quality.

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Abstract: Bacteriophages infecting Bacteroides are potentially a good tool for fecal source tracking, but different Bacteroides host strains are needed for different geographic areas. A feasible method for isolating Bacteroides host strains for phages present in human fecal material is described. Useful strains were identified for application in Spain and the United Kingdom. One strain, GA-17, identified as Bacteroides thetaiotaomicron, was tested in several locations in Europe with excellent performance in Southern Europe.

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Abstract: Repetitive extragenic palindromic PCR fingerprinting of Escherichia coli is one microbial source tracking approach for identifying the host source origin of fecal pollution in aquatic systems. The construction of robust known-source libraries is expensive and requires an informed sampling strategy. In many types of farming systems, waste is stored for several months before being released into the environment. In this study we analyzed, by means of repetitive extragenic palindromic PCR using the enterobacterial repetitive intergenic consensus primers and comparative analysis using the Bionumerics software, collections of E. coli obtained from a dairy farm and from a swine farm, both of which stored their waste as a slurry in holding tanks. In all fecal samples, obtained from either barns or holding tanks, the diversity of the E. coli populations was underrepresented by collections of 500 isolates. In both the dairy and the swine farms, the diversity of the E. coli community was greater in the manure holding tank than in the barn, when they were sampled on the same date. In both farms, a comparison of stored manure samples collected several months apart suggested that the community composition changed substantially in terms of the detected number, absolute identity, and relative abundance of genotypes. Comparison of E. coli populations obtained from 10 different locations in either holding tank suggested that spatial variability in the E. coli community should be accounted for when sampling. Overall, the diversity in E. coli populations in manure slurry storage facilities is significant and likely is problematic with respect to library construction for microbial source tracking applications.

Abstract: A molecular method based on the detection of mitochondrial DNA from various animal species was developed to track the origin of surface water pollutions, and to differentiate human and animal sources. Mitochondrial DNA sequences were used to design PCR primers specific for human, bovine, ovine and porcine DNA using single, multiplex and nested PCR protocols. The primers were tested with DNA extracted from untreated domestic sewage, agricultural soils run-off, swine farm effluents and water from two rivers with known pollution sources. At least one of the four species was detected in most of these samples. The limit of detection in wastewater was 10(3)-10(4) cells L(-1) with a multiplex PCR protocol. This is the first report of a method using eukaryotic genetic DNA to detect and differentiate animal DNA from fecal sources in water. This innovative method is simple and could be used to quickly differentiate sources of pollution in a watershed

Abstract: Several genotypic and phenotypic microbial source tracking (MST) methods have been proposed and utilized to differentiate groups of microorganisms, usually indicator organisms, for the purpose of tracking sources of fecal pollution. Targeting of host-specific microorganisms is one of the approaches currently being tested. These methods are useful as they circumvent the need to isolate individual microorganisms and do not require the establishment of reference databases. Several studies have demonstrated that the presence and distribution of Enterococcus spp. in feces seems to be influenced by the host species. Here, we present a method for detection of genetic sequences in culturable enterococci capable of identifying human sources of fecal pollution in the environment. The human fecal pollution marker designed in this study targets a putative virulence factor, the enterococcal surface protein (esp), in Enterococcus faecium. This gene was detected in 97% of sewage and septic samples but was not detected in any livestock waste lagoons or in bird or animal fecal samples. Epidemiological studies in recreational and groundwaters have shown enterococci to be useful indicators of public health risk for gastroenteritis. By identifying the presence of human fecal pollution, and therefore the possible presence of human enteric pathogens, this marker allows for further resolution of the source of this risk.

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Abstract: Rapid population growth and urban development along waterways and coastal areas have led to decreasing water quality. To examine the effects of upstream anthropogenic activities on microbiological water quality, methods for source-specific testing are required. In this study, molecular assays targeting human enteroviruses (HEV), bovine enteroviruses (BEV), and human adenoviruses (HAdV) were developed and used to identify major sources of fecal contamination in the lower Altamaha River, Georgia. Two-liter grab samples were collected monthly from five tidally influenced stations between July and December 2002. Samples were analyzed by reverse transcription- and nested-PCR. PCR results were confirmed by dot blot hybridization. Eleven and 17 of the 30 surface water samples tested positive for HAdV and HEV, respectively. Two-thirds of the samples tested positive for either HEV or HAdV, and the viruses occurred simultaneously in 26% of samples. BEV were detected in 11 of 30 surface water samples. Binary logistic regression analysis showed that the presence of both human and bovine enteric viruses was not significantly related to either fecal coliform or total coliform levels. The presence of these viruses was directly related to dissolved oxygen and streamflow but inversely related to water temperature, rainfall in the 30 days preceding sampling, and chlorophyll-a concentrations. The stringent host specificity of enteric viruses makes them good library-independent indicators for identification of water pollution sources. Viral pathogen detection by PCR is a highly sensitive and easy-to-use tool for rapid assessment of water quality and fecal contamination when public health risk characterization is not necessary.

Abstract: Analyses for the presence of indicator organisms provide information on the microbiological quality of water. Indicator organisms recommended by the United States Environmental Protection Agency for monitoring the microbiological quality of water include Escherichia coli, a thermotolerant coliform found in the feces of warm-blooded animals. These bacteria can also be isolated from environmental sources such as the recreational and pristine waters of tropical rain forests in the absence of fecal contamination. In the present study, E. coli isolates were compared to E. coli K12 (ATCC 29425) by restriction fragment length polymorphism using pulsed-field gel electrophoresis. Theoretically, genomic DNA patterns generated by PFGE are highly specific for the different isolates of an organism and can be used to identify variability between environmental and fecal isolates. Our results indicate a different band pattern for almost every one of the E. coli isolates analyzed. Cluster analysis did not show any relations between isolates and their source of origin. Only the discriminant function analysis grouped the samples with the source of origin. The discrepancy observed between the cluster analysis and discriminant function analysis relies on their mathematical basis. Our validation analyses indicate the presence of an artifact (i.e., grouping of environmental versus fecal samples as a product of the statistical analyses used and not as a result of separation in terms of source of origin) in the classification results; therefore, the large genetic heterogeneity observed in these E. coli populations makes the grouping of isolates by source rather difficult, if not impossible.

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Abstract: The ability to identify sources of fecal pollution plays a key role in the analysis of human health risk and the implementation of water resource management strategies. One approach to this problem involves the identification of bacterial lineages or gene sequences that are found exclusively in a particular host species or group. We used subtractive hybridization to enrich for target host-specific fecal Bacteroidales rRNA gene fragments that were different from those of very closely related reference (subtracter) host sources. Target host rRNA gene fragments were hybridized to subtracter rRNA gene fragments immobilized in a microplate well, and target sequences that did not hybridize were cloned and sequenced for PCR primer design. The use of microplates for DNA immobilization resulted in a one-step subtractive hybridization in which the products could be directly amplified with PCR. The new host-specific primers designed from subtracted target fragments differentiated among very closely related Bacteroidales rRNA gene sequences and distinguished between similar fecal sources, such as elk and cow or human and domestic pet (dog).

Abstract: A bacterial primer set, known to produce a 542-bp amplicon specific for Bacteroides thetaiotaomicron, generated this product in PCR with 1 ng of extracted DNA from 92% of 25 human fecal samples, 100% of 20 sewage samples, and 16% of 31 dog fecal samples. The marker was not detected in 1 ng of fecal DNA from 61 cows, 35 horses, 44 pigs, 24 chickens, 29 turkeys, and 17 geese.

Abstract: In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST) of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study Enterococcus sp. isolates originating from deer, bovine, gull, and human sources were examined using microarrays. Isolates were subjected to Box PCR amplification and the resulting amplification products labeled with Cy5. Fluorescent-labeled templates were hybridized to in-house constructed nonamer oligonucleotide microarrays consisting of 198 probes. Microarray hybridization profiles were obtained using the ArrayPro image analysis software. Principal Components Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were compared for their ability to visually cluster microarray hybridization profiles based on the environmental source from which the Enterococcus sp. isolates originated. The PCA was visually superior at separating origin-specific clusters, even for as few as 3 factors. A Soft Independent Modeling (SIM) classification confirmed the PCA, resulting in zero misclassifications using 5 factors for each class. The implication of these results for the application of random oligonucleotide microarrays for BST is that, given the reproducibility issues, factor-based variable selection such as in PCA and SIM greatly outperforms dendrogram-based similarity measures such as in HCA and K-Nearest Neighbor KNN.

Abstract: AIMS: To identify the sources of faecal contamination in investigated surface waters and to determine the significance of onsite wastewater treatment systems (OWTS) as a major contributor to faecal contamination. METHODS AND RESULTS: Antibiotic resistance patterns (ARP) were established for a library of 717 known Escherichia coli source isolates obtained from human, domesticated animals, livestock and wild sources. Eight commonly used antibiotics, including amoxicillin, cephalothin, erythromycin, gentamicin, ofloxacin, chlortetracycline, tetracycline and moxalactam, at four different concentrations were used to obtain ARPs for E. coli isolates. Discriminant analysis (DA) was used to differentiate between the ARP of sources isolates. The developed ARP library was found to be adequate for discriminating human from nonhuman isolates, and was used to classify 256 enumerated E. coli isolates collected from monitored surface water locations. CONCLUSIONS: The resulting ARP DA indicated that a majority of the faecal contamination in more rural areas was nonhuman; however, the percentage of human isolates increased significantly in urbanized areas using OWTS for wastewater treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study signifies the feasibility of using ARP for source tracking faecal contamination in surface waters, and linking faecal contamination to OWTS. The information will enable regulatory authorities to implement appropriate management practices to reduce the contamination of water resources caused by high densities and failing OWTS.

Abstract: The purpose of this study was to examine host distribution patterns among fecal bacteria in the order Bacteroidales, with the goal of using endemic sequences as markers for fecal source identification in aquatic environments. We analyzed Bacteroidales 16S rRNA gene sequences from the feces of eight hosts: human, bovine, pig, horse, dog, cat, gull, and elk. Recovered sequences did not match database sequences, indicating high levels of uncultivated diversity. The analysis revealed both endemic and cosmopolitan distributions among the eight hosts. Ruminant, pig, and horse sequences tended to form host- or host group-specific clusters in a phylogenetic tree, while human, dog, cat, and gull sequences clustered together almost exclusively. Many of the human, dog, cat, and gull sequences fell within a large branch containing cultivated species from the genus Bacteroides. Most of the cultivated Bacteroides species had very close matches with multiple hosts and thus may not be useful targets for fecal source identification. A large branch containing cultivated members of the genus Prevotella included cloned sequences that were not closely related to cultivated Prevotella species. Most ruminant sequences formed clusters separate from the branches containing Bacteroides and Prevotella species. Host-specific sequences were identified for pigs and horses and were used to design PCR primers to identify pig and horse sources of fecal pollution in water. The primers successfully amplified fecal DNAs from their target hosts and did not amplify fecal DNAs from other species. Fecal bacteria endemic to the host species may result from evolution in different types of digestive systems.

Abstract: Sources of Enterococcus faecalis isolates from Pensacola Beach, FL. were identified using a library-based approach by applying the statistical method of average similarity to single and composite data sets generated from separate analyses. Data sets included antibiotic resistance analysis (ARA), rep-fingerprints, and fatty acid methyl ester (FAME) profiles. Use of a composite data set composed of ARA and rep-fingerprints, added to the confidence of the identifications. The addition of FAME data to composite data sets did not add to the confidence of identifications. Source identification was performed to better understand risk associated with higher densities of enterococci found in swash zone interstitial water (SZIW) as compared to adjacent bathing water on Pensacola Beach, FL. The "swash zone" is that area of the beach continually washed over by waves. As the potential sources of enterococci were limited in this environment, only two library units, sea gull and human, were constructed. Identification of the beach isolates using a composite data set indicated a sea gull origin. The clonality of the beach isolates suggested that the beach environment selects certain subspecies of E. faecalis.

Abstract: The goal of the study was to determine which similarity coefficient and statistical method to use to produce the highest rate of correct assignment (RCA) in repetitive extragenic palindromic PCR-based bacterial source tracking. In addition, the use of standards for deciding whether to accept or reject source assignments was investigated. The use of curve-based coefficients Cosine Coefficient and Pearson's Product Moment Correlation yielded higher RCAs than the use of band-based coefficients Jaccard, Dice, Jeffrey's x, and Ochiai. When enterococcal and Escherichia coli isolates from known sources were used in a blind test, the use of maximum similarity produced consistently higher RCAs than the use of average similarity. We also found that the use of a similarity value threshold and/or a quality factor threshold (the ratio of the average fingerprint similarity within a source to the average similarity of this source's isolates to an unknown) to decide whether to accept source assignments of unknowns increases the reliability of source assignments. Applying a similarity value threshold improved the overall RCA (ORCA) by 15 to 27% when enterococcal fingerprints were used and 8 to 29% when E. coli fingerprints were used. Applying the quality factor threshold resulted in a 22 to 32% improvement in the ORCA, depending on the fingerprinting technique used. This increase in reliability was, however, achieved at the expense of decreased numbers of isolates that were assigned a source.

Abstract: Antibiotic resistance was examined in 462 Escherichia coli isolates from surface waters and fecal pollution sources around Hamilton, Ontario. Escherichia coli were resistant to the highest concentrations of each of the 14 antibiotics studied, although the prevalence of high resistance was mostly low. Two of 12 E. coli isolates from sewage in a CSO tank had multiple resistance to ampicillin, ciprofloxacin, gentamicin, and tetracycline above their clinical breakpoints. Antibiotic resistance was less prevalent in E. coli from bird feces than from municipal wastewater sources. A discriminant function calculated from antibiotic resistance data provided an average rate of correct classification of 68% for discriminating E. coli from bird and wastewater fecal pollution sources. The preliminary microbial source tracking results suggest that, at times, bird feces might be a more prominent contributor of E. coli to Bayfront Park beach waters than municipal wastewater sources.

Abstract: Aims: The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples. Methods and Results: Antibiotic resistance patterns and HindIII ribotypes of Escherichia coli (n = 997), and antibiotic resistance patterns of Enterococcus spp. (n = 3657) were used to construct libraries from sewage samples and from faeces of seagulls, dogs, cats, horses and humans within the watershed. The three libraries were analysed to determine the accuracy of host source prediction. The internal accuracy of the libraries (average rate of correct classification, ARCC) with six source categories was 44% for E. coli ARA, 69% for E. coli ribotyping and 48% for Enterococcus ARA. Each library's predictive ability towards isolates that were not part of the library was determined using a blinded proficiency panel of 97 E. coli and 99 Enterococcus isolates. Twenty-eight per cent (by ARA) and 27% (by ribotyping) of the E. coli proficiency isolates were assigned to the correct source category. Sixteen per cent were assigned to the same source category by both methods, and 6% were assigned to the correct category. Addition of 2480 E. coli isolates to the ARA library did not improve the ARCC or proficiency accuracy. In contrast, 45% of Enterococcus proficiency isolates were correctly identified by ARA. Conclusions: None of the methods performed well enough on the proficiency panel to be judged ready for application to environmental samples. Significance and Impact of the Study: Most microbial source tracking (MST) studies published have demonstrated library accuracy solely by the internal ARCC measurement. Low rates of correct classification for E. coli proficiency isolates compared with the ARCCs of the libraries indicate that testing of bacteria from samples that are not represented in the library, such as blinded proficiency samples, is necessary to accurately measure predictive ability. The library-based MST methods used in this study may not be suited for determination of the source(s) of faecal pollution in large, urban watersheds

Abstract: Aims: An assessment of microbial densities in an urbanized Florida watershed was performed during a period of changing rainfall patterns to investigate the role of climate coupled with urbanization in declining water quality. Methods and Results: Concentrations of traditional and alternative faecal indicators were assessed by standard methods over 24 months. Sources of faecal contamination were determined by antibiotic resistance analysis (ARA) of faecal coliform (FC) bacteria. Composite indices of indicator organisms based on a suite of microbial measurements were used to quantify pollution impacts in the river. ARA results found that FC from wild animal sources dominated during the drought, and the relative frequency of FC from human sources increased after cumulative rainfall increased to near-normal levels. Conclusions: Changes observed in faecal indicator densities and in FC sources during changing rainfall patterns strongly suggest a role of precipitation on the sources and extent of microbial pollution in urbanized coastal watersheds. Significance and Impact of the Study: Bacterial source tracking coupled with a composite index of microbial contamination resulted in a more complete picture of microbial pollution within the river, as opposed to the general practice of reliance on one indicator organism. Improved land use decisions in urban areas are necessary to insure maintenance of coastal environmental health under changing climate patterns and population density.

Abstract: Public health departments bear the responsibility for investigating recreational water-associated disease outbreaks. Tracking the source of the disease is often problematic, however, because routine monitoring of recreational waters (for bacterial counts) is not source specific. The intent of the project reported here was to monitor Escherichia coli levels in a small recreational lake in Iowa and to determine their source. The authors monitored water samples for E. coli and used phenotypic methods to analyze multiple samples of lake water, well water, and known fecal sources. Moderate to high levels of E. coli were found in lake water samples from the swimming area throughout the summer. The highest levels of E. coli were found after rainfall events in both lake water samples and samples taken from monitoring wells. Phenotypic analyses indicated that likely sources of E. coli in the lake included both human and wildlife (goose) fecal material. The authors also found that the phenotype used to characterize E. coli isolated from geese frequenting this lake could not be used to characterize E. coli isolated from geese in a neighboring watershed. Identifying the source of fecal material will help authorities implement the proper preventive measures to avoid fecal contamination of the lake in the future.

Abstract: Aims: To compare antibiotic resistance and ribotyping patterns ability to identify triplicate isolates sent from a group of 40 Escherichia coli taken from seven host sources. Methods and Results: Of the 120 isolates, 22 isolates were resistant to ampicillin, streptomycin, tetracycline and trimethoprim and 98 isolates were susceptible. Antibiotic patterns identified 33 of the triplicates and three of the six groups had isolates from multiple hosts. Ribotyping divided the isolates into 27 ribotype groups with all triplicates grouped into the same ribotype group with one host per group. Conclusions: Antibiotic susceptibility pattern placed 98 of the isolates in a single group with 50% of the antibiotic susceptibility pattern groups containing multiple host species. Ribotyping groups were host specific with each host having one to seven ribotype groups. Significance and Impact of the Study: Antibiotic susceptibility pattern groups have been used for environmental source identification and faecal pollution tracking, however these groups do not always distinguish between host species. Stability of the markers is a potential concern and this system can only be used if antibiotic resistance levels are high in the isolates studied. All isolates have a ribotype group which was stable and like other molecular methods has advantages over antibiotic susceptibility pattern groups which uses a phenotypic method

Abstract: In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Qß, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.

Abstract: Repetitive element anchored PCR was used to evaluate the genetic profiles of Escherichia coli isolated from surface water contaminated with urban stormwater, sanitary sewage, and gull feces to determine if strains found in environmental samples reflect the strain composition of E. coli obtained from host sources. Overall, there was less diversity in isolates collected from river and beach sites than with isolates obtained from human and nonhuman sources. Unique strain types comprised 28.8, 29.2, and 15.0% of the isolate data sets recovered from stormwater, river water, and beach water, respectively. In contrast, 50.4% of gull isolates and 41.2% of sewage isolates were unique strain types. River water, which is expected to contain E. coli strains from many diffuse sources of nonpoint source pollution, contained strains most closely associated with other river water isolates that were collected at different sites or on different days. However, river sites impacted by sewage discharge had approximately 20% more strains similar to sewage isolates than did sites impacted by stormwater alone. Beach sites with known gull fecal contamination contained E. coli most similar to other beach isolates rather than gull isolates collected at these same sites, indicating underrepresentation of possible gull strains. These results suggest large numbers of strains are needed to represent contributing host sources within a geographical location. Additionally, environmental survival may influence the composition of strains that can be recovered from contaminated waters. Understanding the ecology of indicator bacteria is important when interpreting fecal pollution assessments and developing source detection methodology.

Abstract: High levels of fecal bacteria are a concern for recreational waters; however, the source of contamination is often unknown. This study investigated whether direct sequencing of a bacterial gene could be utilized for detecting genetic differences between bacterial strains for microbial source tracking. A 525-nucleotide segment of the gene for ß-glucuronidase (uidA) was sequenced in 941 Escherichia coli isolates from the Clinton River–Lake St. Clair watershed, 182 E. coli isolates from human and animal feces, and 34 E. coli isolates from a combined sewer. Environmental isolates exhibited 114 alleles in 11 groups on a genetic tree. Frequency of strains from different genetic groups differed significantly (p < 0.03) between upstream reaches (Bear Creek–Red Run), downstream reaches, and Lake St. Clair beaches. Fecal E. coli uidA sequences exhibited 81 alleles that overlapped with the environmental set. An algorithm to assign alleles to different host sources averaged approximately 75% correct classification with the fecal data set. Using the same algorithm, the percent of environmental isolates assignable to humans decreased significantly between Bear Creek–Red Run (30 ± 3%) and the beaches (17 ± 2%) (p < 0.05). Birds accounted for approximately 50% of assignable environmental isolates. For combined sewer isolates, the same algorithm assigned 51% to humans. These experiments demonstrate differences in the frequency of different E. coli strains at different locations in a watershed, and provide a "proof in principle" that sequence-based data can be used for microbial source tracking.

Abstract: A renewed emphasis on source water protection and watershed management has resulted from recent amendments and initiatives under the Safe Drinking Water Act and the Clean Water Act. Knowledge of the impact of land use choices on source water quality is critical for efforts to properly manage activities within a watershed. This study evaluated qualitative relationships between land use and source water quality and the quantitative impact of season and rainfall events on water quality parameters. High levels of specific conductance tended to be associated with dense residential development, while organic carbon was elevated at several forested sites. Turbidity was generally higher in more urbanized areas. Source tracking indicators were detected in samples where land use types would predict their presence. Coliform levels were statistically different at the 95 percent confidence levels for winter versus summer conditions and dry versus wet weather conditions. Other water quality parameters that varied with season were organic carbon, turbidity, dissolved oxygen, and specific conductance. These results indicate that land use management can be effective for mitigating impacts to a water body; however, year- round, comprehensive data are necessary to thoroughly evaluate the water quality at a particular site.

Abstract: Surface water impairment by fecal coliform bacteria is a water quality issue of national scope and importance. In Virginia, more than 400 stream and river segments are on the Commonwealth's 2002 303(d) list because of fecal coliform impairment. Total maximum daily loads (TMDLs) will be developed for most of these listed streams and rivers. Information regarding the major fecal coliform sources that impair surface water quality would enhance the development of effective watershed models and improve TMDLs. Bacterial source tracking (BST) is a recently developed technology for identifying the sources of fecal coliform bacteria and it may be helpful in generating improved TMDLs. Bacterial source tracking was performed, watershed models were developed, and TMDLs were prepared for three streams (Accotink Creek, Christians Creek, and Blacks Run) on Virginia's 303(d) list of impaired waters. Quality assurance of the BST work suggests that these data adequately describe the bacteria sources that are impairing these streams. Initial comparison of simulated bacterial sources with the observed BST data indicated that the fecal coliform sources were represented inaccurately in the initial model simulation. Revised model simulations (based on BST data) appeared to provide a better representation of the sources of fecal coliform bacteria in these three streams. The coupled approach of incorporating BST data into the fecal coliform transport model appears to reduce model uncertainty and should result in an improved TMDL.

Abstract: Amplified fragment length polymorphism (AFLP) and enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) genomic fingerprinting assays were compared for their ability to differentiate Escherichia coli isolates obtained from various host sources, and with respect to their pathogenicity. One hundred and ten verotoxigenic, enterotoxigenic and non-pathogenic E. coli isolates obtained from cattle, humans and pigs were used in this study. The AFLP assay was shown to be highly effective in predicting both the host source and pathogenicity of the E. coli isolates. A stepwise discriminant function analysis showed that 91.4, 90.6 and 97.7% of the human, bovine and pig isolates were classified into the correct host types, respectively. The analysis also distinguished the non-pathogenic E. coli from the verocytotoxigenic and enterotoxigenic virulence phenotypes at 100, 100 and 90.9% accuracy, respectively. Sixty-two E. coli strains from the collection were subjected to the ERIC-PCR fingerprinting analysis. Using this method, only 28.6, 0 and 75.0% of the human, bovine and pig isolates were classified into the correct host types, respectively. Overall, the AFLP method was able to ascribe host source with a high level of confidence and readily discriminate pathogenic from non-clinical isolates of E. coli

Abstract: In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.

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Abstract: 16S rDNA clone libraries were evaluated for detection of fecal source-identifying bacteria from a collapsed equine manure pile. Libraries were constructed using universal eubacterial primers and Bacteroides-Prevotella group-specific primers. Eubacterial sequences indicated that upstream and downstream water samples were predominantly β- and γ-Proteobacteria (35 and 19%, respectively), while the manure library consisted predominantly of Firmicutes (31%) and previously unidentified sequences (60%). Manure-specific eubacterial sequences were not detectable beyond 5 m downstream of the pile, suggesting either poor survival or high dilution rates. In contrast, Bacteroides and Prevotella sp. sequences were detected both in manure and downstream using group-specific primers. Novel sequences from Bacteroides and Prevotella analysis produced an equine-specific phylogenetic cluster as compared to previous data sets obtained for human and bovine samples. While these results suggest that some anaerobic fecal bacteria might be potential identifiers for use in source-tracking applications, a comprehensive examination of environmental sequences within these species should be performed before methods targeting these bacterial groups are applied to watersheds for development of microbial source-tracking protocols.

Abstract: The Santa Ana River (SAR), CA and adjacent wetlands have been identified as potential sources of fecal indicator bacteria (FIB) to the surf zone at Huntington Beach, CA. A suite of fecal steroids, including coprostanol (COP), epicoprostanol (eCOP), cholesterol (CHOE), cholestanol (CHOA), alpha-cholestanone (aONE), beta-cholestanone (bONE), beta-sitosterol (bSIT), stigmasterol (STIG), stigmastanol (STAN), and campesterol (CAM), were used as chemical markers to examine whether sewage was a significant source of FIB within the lower Santa Ana River watershed. A total of 54 water samples were collected from three locations in the intertidal zone near the mouth of the Santa Ana River at different tidal stages. Steroid ratios in SAR samples were different from those found in raw and treated sewage from a local wastewater treatment plant or in nearby effluent plume and did not appear to be influenced by the sampling location, daily tides, and spring/neap tidal cycle. The characteristics of steroid ratios suggested a diagenetic ratherthan a biogenic source forthe COP content of the samples. The log-based concentrations of COP and FIB in the SAR samples were not significantly correlated, inconsistent with sewage being the source of FIB in the study area. In addition, multivariate statistical analysis showed that the concentrations of FIB were better correlated with bird fecal steroids than with the typical sewage sterols. The results implied that sewage was not a significant source of fecal steroids, and therefore perhaps FIB to the study area. Instead, birds may be one possible source of the intermittently high levels of FIB observed in the lower Santa Ana River watershed and the nearby surf zone.

Abstract: A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E. coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E. coli was very great and is most likely accounting for our inability to correctly classify many environmental E. coli isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.

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Abstract: Antibiotic resistance profiling (ARP) is a potentially useful method for distinguishing faecal bacteria according to host source. This phenotypic approach has cost benefits over genotypic methods, but existing protocols are time-consuming and manual data handling is open to human error. A simplified, low-cost approach to the ARP technique was developed that used automated data recording techniques combined with simple statistical analyses to compare isolates of the genus Enterococcus from various faecal sources. An initial battery of 21 antibiotics (at up to four concentrations) was chosen for source discrimination. Images of growth or non-growth in microplate wells were stored as bitmaps and converted to binary data to form a database of known antibiotic resistance profiles. Discriminant function analysis (DFA) showed that the average rate of isolates correctly classified by the database (wastewater vs non-wastewater) was 86%. Once the more discriminating antibiotics and their concentrations had been identified, it was possible to reduce the number of tests from 80 to 18 whilst increasing the number of correctly classified human isolates. ARP could offer a low-cost and rapid means of identifying sources of faecal pollution. As such, the technique may be of particular benefit to developing countries, where water quality may have a significant impact on health and where cost is a major factor when choosing environmental management technology.

Abstract: Microbial source tracking (MST) uses various approaches to classify fecal-indicator microorganisms to source hosts. Reproducibility, accuracy, and robustness of seven phenotypic and genotypic MST protocols were evaluated by use of Escherichia coli from an eight-host library of known-source isolates and a separate, blinded challenge library. In reproducibility tests, measuring each protocol's ability to reclassify blinded replicates, only one (pulsed-field gel electrophoresis; PFGE) correctly classified all test replicates to host species; three protocols classified 48-62% correctly, and the remaining three classified fewer than 25% correctly. In accuracy tests, measuring each protocol's ability to correctly classify new isolates, ribotyping with EcoRI and PvuII approached 100% correctclassification but only 6% of isolates were classified; four of the other six protocols (antibiotic resistance analysis, PFGE, and two repetitive-element PCR protocols) achieved better than random accuracy rates when 30-100% of challenge isolates were classified. In robustness tests, measuring each protocol's ability to recognize isolates from nonlibrary

Abstract: In many parts of the world, coliform counts in recreational waters are unacceptably high. In an attempt to rectify this problem, programmes are under way to develop methods that will allow the sources of the faecal contamination thought to be responsible for these elevated counts to be identified. The success of these efforts depends on the validity of several assumptions that underlie many of the proposed methods. One of the critical assumptions is that the clonal composition of the coliform species being monitored in a water body reflects the clonal composition of the species in the host populations responsible for the faecal inputs into that water body. To determine the extent to which among-strain variation in a coliform species might invalidate this assumption, a series of simple mathematical models was proposed and analysed. The first series of models assumed that all cells of species were identical. The question posed was - is the density of a coliform species in a body of water linearly related to the rate at which cells of the species enter the water body via faecal production? The results of these models suggest that, over a wide range of conditions, cell densities in the water body are linearly related to the rate at which cells enter the water body as a result of faecal contamination. This outcome occurs whether or not cells are capable of division in the external environment. When the rate of cell division depends on the concentration of available nutrients then, when nutrient input rates are 'high' and rates of faecal contamination are 'low', this linear relationship does not hold. The second series of models assumed that the coliform species consists of different strains and that these strains differ in their performance in the external environment. The results of these multistrain models show that the relative abundance of strains in the external environment is unlikely to reflect their relative abundance in the faecal inputs to the environment. Consequently, statements such as - domestic animals are responsible for 30% and wildlife for 70% of the faecal inputs to a water body - may well be meaningless.

Abstract: Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

Abstract: Escherichia coli isolates were obtained from common host sources of fecal pollution and characterized by using repetitive extragenic palindromic (REP) PCR fingerprinting. The genetic relationship of strains within each host group was assessed as was the relationship of strains among different host groups. Multiple isolates from a single host animal (gull, human, or dog) were found to be identical; however, in some of the animals, additional strains occurred at a lower frequency. REP PCR fingerprint patterns of isolates from sewage (n = 180), gulls (n = 133), and dairy cattle (n = 121) were diverse; within a host group, pairwise comparison similarity indices ranged from 98% to as low as 15%. A composite dendrogram of E. coli fingerprint patterns did not cluster the isolates into distinct host groups but rather produced numerous subclusters (approximately >80% similarity scores calculated with the cosine coefficient) that were nearly exclusive for a host group. Approximately 65% of the isolates analyzed were arranged into host-specific groups. Comparable results were obtained by using enterobacterial repetitive intergenic consensus PCR and pulsed-field gel electrophoresis (PFGE), where PFGE gave a higher differentiation of closely related strains than both PCR techniques. These results demonstrate that environmental studies with genetic comparisons to detect sources of E. coli contamination will require extensive isolation of strains to encompass E. coli strain diversity found in host sources of contamination. These findings will assist in the development of approaches to determine sources of fecal pollution, an effort important for protecting water resources and public health.

Abstract: Microbiological source tracking (MST) methods are increasingly being used to identify fecal contamination sources in surface waters, but these methods have been subjected to limited comparative testing. In this study, 22 researchers employing 12 different methods were provided sets of identically prepared blind water samples. Each sample contained one to three of five possible fecal sources (human, dog, cattle, seagull or sewage). Researchers were also provided with portions of the fecal material used to inoculate the blind water samples for use as library material. No MST method that was tested predicted the source material in the blind samples perfectly. Host-specific PCR performed best at differentiating between human and non-human sources, but primers are not yet available for differentiating between all of the non-human sources. Virus and F+ coliphage methods reliably identified sewage, but were unable to identify fecal contamination from individual humans. Library-based isolate methods correctly identified the dominant source in most samples, but also had frequent false positives in which fecal sources not in the samples were incorrectly identified as being present. Among the library-based methods, genotypic methods generally performed better than phenotypic methods.

Abstract: Received for publication March 11, 2002. Escherichia coli is a ubiquitous component of the intestinal microflora of warm-blooded animals, and is an indicator of fecal contamination of surface waters. Ribotype profiling of E. coli is one of several genotypic methods that has been developed to determine the host origin of fecal bacteria. Like most genotypic methods of source tracking, ribotyping requires a host origin database to identify environmental isolates. To determine the extent of temporal variability of ribotypes and its effect on a host origin database, E. coli isolates were obtained from fecal samples of two herds of Black Angus steers at a long-term experimental site at four sampling times from October 1999 to July 2000. Fecal samples were taken from six randomly chosen steers at each time. At a similarity index of 90% as calculated by unweighted pair-group method using arithmetic averages (UPGMA), 240 ribotypes were identified from 451 E. coli isolates. Only 20 ribotypes (8.3%), comprising 33% of the total isolates, were shared among sampling times and were considered resident ribotypes. Two of the twenty resident ribotypes appeared at three sampling times, and the remaining eighteen appeared at two. The majority of the ribotypes, therefore, were transient and unique to each sampling time and steer. Both the apparent turnover of E. coli ribotypes and a clonal diversity index of 0.97 (indicative of extensive ribotype variability) suggest the necessity of ribotyping a large number E. coli isolates per host to establish a host origin database that is independent of temporal variability, or complete enough to be effective.

Abstract: A new, simple, and specific protocol to discriminate between human and animal fecal pollution is described. The procedure is based on the detection of certain Bifidobacterium species in the samples. Two 16S rRNA gene-targeted probes are described. One of these probes (BDE) has as its target a region of the 16S rRNA gene of Bifidobacterium dentium, a Bifidobacterium species of exclusively human origin. The other probe (BAN) is based on the sequence of a region of 16S rRNA gene for several Bifidobacterium species related with animal origins. The specificity of both probes was evaluated by using 24 Bifidobacterium species, and their threshold detection limit was established by DNA-DNA hybridization. DNA-DNA hybridization with the BDE probe showed it to be specific for B. dentium, whereas that with the BAN probe showed it to be specific for B. animalis, B. asteroides, B. coryneforme, B. cuniculi, B. globosum, B. magnum, B. minimum, and B. subtile. A simple and specific protocol was also developed for the detection of their target species in environmental samples (sewage and feces). DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats. Thus, this probe is not suitable for the identification of any animal fecal pollution. Whereas all samples with human fecal pollution showed a positive DNA-DNA hybridization result with the BDE probe, none of those with animal fecal pollution did. Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin.

Abstract: Despite efforts to minimize fecal input into waterways, this kind of pollution continues to be a problem due to an inability to reliably identify nonpoint sources. Our objective was to find candidate source-specific Escherichia coli fingerprints as potential genotypic markers for raw sewage, horses, dogs, gulls, and cows. We evaluated 16S-23S rRNA intergenic spacer region (ISR)-PCR and repetitive extragenic palindromic (rep)-PCR analyses of E. coli isolates as tools to identify nonpoint fecal sources. The BOXA1R primer was used for rep-PCR analysis. A total of 267 E. coli isolates from different fecal sources were typed with both techniques. E. coli was found to be highly diverse. Only two candidate source-specific E. coli fingerprints, one for cow and one for raw sewage, were identified out of 87 ISR fingerprints. Similarly, there was only one candidate source-specific E. coli fingerprint for horse out of 59 BOX fingerprints. Jackknife analysis resulted in an average rate of correct classification (ARCC) of 83% for BOX-PCR analysis and 67% for ISR-PCR analysis for the five source categories of this study. When nonhuman sources were pooled so that each isolate was classified as animal or human derived (raw sewage), ARCCs of 82% for BOX-PCR analysis and 72% for ISR-PCR analysis were obtained. Critical factors affecting the utility of these methods, namely sample size and fingerprint stability, were also assessed. Chao1 estimation showed that generally 32 isolates per fecal source individual were sufficient to characterize the richness of the E. coli population of that source. The results of a fingerprint stability experiment indicated that BOX and ISR fingerprints were stable in natural waters at 4, 12, and 28°C for 150 days. In conclusion, 16S-23S rRNA ISR-PCR and rep-PCR analyses of E. coli isolates have the potential to identify nonpoint fecal sources. A fairly small number of isolates was needed to find candidate source-specific E. coli fingerprints that were stable under the simulated environmental conditions.

Abstract: This research developed a PCR method to identify swine fecal pollution in water, using a portion of the STII toxin gene from enterotoxigenic Escherichia coli as the target sequence. This method showed the gene to have a wide-spread geographical distribution and temporal stability; and the primers demonstrated high specificity, sensitivity, and reliability. A total of 110 DNA extracts from different animal fecal and human sewage samples were screened using the primers and no positives resulted. Centrifugation and filtration methods for concentrating E. coli seeded into stream, ocean, secondary effluent, and dairy lagoon waters resulted in detection limits at the femtogram and attogram levels. E. coli with the biomarker seeded into stream, ocean, and secondary effluent waters remained stable for approximately 2 weeks for all water types. Of the farm lagoon and waste samples tested, 94% were positive for the STII trait, regardless of the number of E. coli screened and 100% were positive when > or =35 E. coli isolates were screened. As the PCR product of the target sequence yielded a single band, the method is applicable to dot blot detection methodology, yielding great accuracy in determining the presence of swine fecal sources.

Abstract: Pattern recognition has been applied to environmental systems for identification of numerous pollution sources including aerosolized lead and petroleum hydrocarbons. In recent years, DNA fingerprinting has gained widespread application as a means to characterize genetic variations for such purposes as microbial source tracking. This approach, however, is strongly dependent on the statistical and image analyses applied. Several statistical analyses of rep-PCR DNA fingerprints were assessed as a means to differentiate between potential sources of fecal contamination. GelCompar II and methods based on penalized discriminant analysis (PDA) and k-nearest neighbors (KNN) classification procedures were used to differentiate between 10 source groups within a library containing DNA fingerprints of 548 Escherichia coli isolates from known human and nonhuman sources. KNN performed significantly better than PDA in a jackknife analysis, though the library was not large enough to detect significant differences between GelCompar II and the other two methods. GelCompar II and KNN both attained > or = 90% correct classification in a holdout procedure. In addition, interpoint distance analyses indicate coherency within source groups, while library randomization demonstrated that KNN does not create artificial groupings. This investigation stresses the need to understand limitations of statistical analyses used in pattern recognition of DNA fingerprints.

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Abstract: Assays for the detection and typing of adenoviruses, enteroviruses and F+ specific coliphages were performed on samples created as part of a national microbial source tracking methods comparison study. The samples were created blind to the researchers, and were inoculated with a variety of types of fecal contamination source (human, sewage, dog, seagull and cow) and mixtures of sources. Viral tracer and pathogen assays demonstrated a general ability to discriminate human from non-human fecal contamination. For example, samples inoculated with sewage were correctly identified as containing human fecal contamination because they contained human adenovirus or human enterovirus. In samples containing fecal material from individual humans, human pathogen analysis yielded negative results probably because the stool samples were taken from healthy individuals. False positive rates for the virus-based methods (0-8%) were among the lowest observed during the methods comparison study. It is suggested that virus-based source tracking methods are useful for identification of sewage contamination, and that these methods may also be useful as an indication of the public health risk associated with viral pathogens. Overall, virus-based source tracking methods are an important approach to include in the microbial source tracking 'toolbox'.

Abstract: Several commonly used statistical methods for fingerprint identification in microbial source tracking (MST) were examined to assess the effectiveness of pattern-matching algorithms to correctly identify sources. Although numerous statistical methods have been employed for source identification, no widespread consensus exists as to which is most appropriate. A large-scale comparison of several MST methods, using identical fecal sources, presented a unique opportunity to assess the utility of several popular statistical methods. These included discriminant analysis, nearest neighbour analysis, maximum similarity and average similarity, along with several measures of distance or similarity. Threshold criteria for excluding uncertain or poorly matched isolates from final analysis were also examined for their ability to reduce false positives and increase prediction success. Six independent libraries used in the study were constructed from indicator bacteria isolated from fecal materials of humans, seagulls, cows and dogs. Three of these libraries were constructed using the rep-PCR technique and three relied on antibiotic resistance analysis (ARA). Five of the libraries were constructed using Escherichia coli and one using Enterococcus spp. (ARA). Overall, the outcome of this study suggests a high degree of variability across statistical methods. Despite large differences in correct classification rates among the statistical methods, no single statistical approach emerged as superior. Thresholds failed to consistently increase rates of correct classification and improvement was often associated with substantial effective sample size reduction. Recommendations are provided to aid in selecting appropriate analyses for these types of data.

Abstract: The methods comparison study described in accompanying manuscripts demonstrated the potential value of microbial source tracking (MST) techniques, but also identified a need for method refinement. This paper provides three classes of recommendations to improve MST technology: optimization, development and evaluation. Optimization recommendations focus on library-dependent methods and include improved selection of restriction enzymes or antibiotics, better definition of appropriate library size, selection of target species and choice of statistical pattern-matching algorithms. Methods development recommendations focus on identifying new genomic targets and quantification procedures for library-independent methods. Longer-term methods development recommendations include integration of microarrays and other direct pathogen detection technology with MST. Studies defining host specificity and population dynamics should aid selection of target species during methods development. Evaluation recommendations include enhancements that should be incorporated into future methods comparison studies, along with studies to assess the value of MST results for risk characterization.

Abstract: Aims: Carbon source utilization profiles as a phenotypic fingerprinting methodology for determining sources of faecal pollution in water were evaluated. Methods and Results: Three hundred and sixty-five Enterococcus isolates were collected from known faecal sources in four different geographical regions and were identified to species with the commercial Biolog system. Discriminant analysis (DA) was used to identify the substrate-containing wells that best classified the 365 isolates by source. By using 30 of the 95 wells for the analysis, the average rate of correct classification (ARCC) by source was 92·7% for a human vs non-human two-way classification when isolates from all regions were combined into one library. Corresponding ARCCs for other classification schemes were 81·9% for a four-way classification of human vs livestock vs wildlife vs domestic pets, and 85·7% for a three-way classification without human isolates. When three individual libraries were made based on classification of sources within Enterococcus species, the ARCC was 95·3% for the Ent. faecalis library, 95·8% for the Ent. gallinarum library and 94·7% for the Ent. mundtii library. Thirty Enterococcus isolates (unknown sources) were obtained from each of three stream sites where a specific source of pollution was apparent; 90·0% of the isolates from a human-suspected source were classified as human, 86·6% were classified as livestock from a livestock-suspected site, and 93·3% were classified as wildlife from a wildlife-suspected site. Conclusions: Phenotypic fingerprinting with carbon source utilization profiles provided levels of correct classification by sources from an Enterococcus library that were in the upper range of those reported in the literature. ARCCs for three Enterococcus species-specific libraries were very high and may be the best approach for further developing this concept and methodology. Significance and Impact of the Study: The results, based on a modest Enterococcus library and a preliminary field validation test, demonstrated the potential for carbon source utilization profiles to be employed as a phenotypic method for determining sources of faecal pollution in water

Abstract: Microbial source tracking (MST) results, obtained using identical sample sets and pulsed field gel electrophoresis (PFGE), repetitive element PCR (rep-PCR) and ribotyping techniques were compared. These methods were performed by six investigators in analysis of duplicate, blind sets of water samples spiked with feces from five possible sources (sewage, human, dog, cow and seagull). Investigators were provided with samples of the fecal material used to inoculate the water samples for host origin database construction. All methods correctly identified the dominant source in the majority of the samples. Modifications of some of these methods correctly identified the dominant sources in over 90% of the samples; however, false positive rates were as high as 57%. The high false positive rates appeared to be indirectly proportional to the levels of stringency applied in pattern analysis. All the methods produced useful data but the results highlighted the need to modify and optimize these methods in order to minimize sources of error.

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Abstract: As part of a larger microbial source tracking (MST) study, several laboratories used library-based, phenotypic subtyping techniques to analyse fecal samples from known sources (human, sewage, cattle, dogs and gulls) and blinded water samples that were contaminated with the fecal sources. The methods used included antibiotic resistance analysis (ARA) of fecal streptococci, enterococci, fecal coliforms and E. coli; multiple antibiotic resistance (MAR) and Kirby-Bauer antibiotic susceptibility testing of E. coli; and carbon source utilization for fecal streptococci and E. coli. Libraries comprising phenotypic patterns of indicator bacteria isolated from known fecal sources were used to predict the sources of isolates from water samples that had been seeded with fecal material from the same sources as those used to create the libraries. The accuracy of fecal source identification in the water samples was assessed both with and without a cut-off termed the minimum detectable percentage (MDP). The libraries (approximately 300 isolates) were not large enough to avoid the artefact of source-independent grouping, but some important conclusions could still be drawn. Use of a MDP decreased the percentage of false-positive source identifications, and had little effect on the high percentage of true-positives in the most accurate libraries. In general, the methods were more prone to false-positive than to false-negative errors. The most accurate method, with a true-positive rate of 100% and a false-positive rate of 39% when analysed with a MDP, was ARA of fecal streptococci. The internal accuracy of the libraries did not correlate with the accuracy of source prediction in water samples, showing that one should not rely solely on parameters such as the average rate of correct classification of a library to indicate its predictive capabilities.

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Abstract: Recent studies suggest that host origin databases for bacterial source tracking (BST) must contain a large number of isolates because bacterial subspecies change with geography and time. A new targeted sampling protocol was developed as a prelude to BST to minimize these changes. The research was conducted on the Sapelo River, a tidal river on the Georgia coast. A general sampling of the river showed fecal enterococcal numbers ranging from <10 (below the limit of detection) to 990 colony-forming units (CFU) per 100 mL. Locations with high enterococcal numbers were combined with local knowledge to determine targeted sampling sites. Fecal enterococcal numbers around one site ranged from <10 to 24000 CFU per 100 mL. Bacterial source tracking was conducted to determine if a wastewater treatment facility at the site was responsible for this contamination. The fecal indicator bacterium was Enterococcus faecalis. Ribotyping, automated with a RiboPrinter (DuPont Qualicon, Wilmington, DE), was the BST method. Thirty-seven ribotypes were observed among 83 Ent. faecalis isolates obtained from the Sapelo River and the wastewater lagoon. Sixteen ribotypes were associated with either the river or the lagoon, and only five ribotypes (14%) were shared. Nevertheless, these five ribotypes represented 39 of the 83 Ent. faecalis isolates, almost a majority (47%). These results suggest that the fecal contamination in the river came from the wastewater treatment facility. As a prelude to BST, targeted sampling minimized subspecies changes with geography and time, and eliminated the need for a permanent host origin database by restricting BST to a small geographic area and requiring sampling to be completed in one day.

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Abstract: As part of a larger microbial source tracking (MST) study, several laboratories used library-based, phenotypic subtyping techniques to analyse fecal samples from known sources (human, sewage, cattle, dogs and gulls) and blinded water samples that were contaminated with the fecal sources. The methods used included antibiotic resistance analysis (ARA) of fecal streptococci, enterococci, fecal coliforms and E. coli; multiple antibiotic resistance (MAR) and Kirby-Bauer antibiotic susceptibility testing of E. coli; and carbon source utilization for fecal streptococci and E. coli. Libraries comprising phenotypic patterns of indicator bacteria isolated from known fecal sources were used to predict the sources of isolates from water samples that had been seeded with fecal material from the same sources as those used to create the libraries. The accuracy of fecal source identification in the water samples was assessed both with and without a cut-off termed the minimum detectable percentage (MDP). The libraries (approximately 300 isolates) were not large enough to avoid the artefact of source-independent grouping, but some important conclusions could still be drawn. Use of a MDP decreased the percentage of false-positive source identifications, and had little effect on the high percentage of true-positives in the most accurate libraries. In general, the methods were more prone to false-positive than to false-negative errors. The most accurate method, with a true-positive rate of 100% and a false-positive rate of 39% when analysed with a MDP, was ARA of fecal streptococci. The internal accuracy of the libraries did not correlate with the accuracy of source prediction in water samples, showing that one should not rely solely on parameters such as the average rate of correct classification of a library to indicate its predictive capabilities.

Abstract: The use of antibiotic resistance analysis (ARA) for microbial source tracking requires the generation of a library of isolates collected from known sources in the watershed. The size and composition of the library are critical in determining if it represents the diversity of patterns found in the watershed. This study was performed to determine the size that an ARA library needs to be to be representative of the watersheds for which it will be used and to determine if libraries from different watersheds can be merged to create multiwatershed libraries. Fecal samples from known human, domesticated, and wild animal sources were collected from six Virginia watersheds. From these samples, enterococci were isolated and tested by ARA. Based on cross-validation discriminant analysis, only the largest of the libraries (2,931 isolates) were found to be able to classify nonlibrary isolates as well as library isolates (i.e., were representative). Small libraries tended to have higher average rates of correct classification, but were much less able to correctly classify nonlibrary isolates. A merged multiwatershed library (6,587 isolates) was created and was found to be large enough to be representative of the isolates from the contributing watersheds. When isolates that were collected from the contributing watersheds approximately 1 year later were analyzed with the multiwatershed library, they were classified as well as the isolates in the library, suggesting that the resistance patterns are temporally stable for at least 1 year. The ability to obtain a representative, temporally stable library demonstrates that ARA can be used to identify sources of fecal pollution in natural waters.

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Abstract: Huntington Beach, California, one of the most popular surfing spots in the world, is plagued by sporadic, elevated levels of fecal bacteria. To assist with pollution source identification, we analyzed antibiotic resistance patterns (ARPs) of enterococci from four known sources (bird feces, urban runoff, coastal marsh sediment and sewage effluent from local sanitation district) and one unknown source (seawater) using seven antibiotics at four concentrations each. Of 2491 enterococci tested, all were resistant to at least one antibiotic at some level. Discriminant analysis indicated that the average correct classification rates for bird feces and urban runoff sources were above 80%. Sewage effluent contained mixed fecal sources. Sixty-four percent of the sewage isolates classified with the sewage category, while the other 35% of isolates were assigned evenly across the other three categories. When enterococci isolated from the seawater were classified using the known ARP database, it was evident that bird feces were the source of surf zone contamination on some days while the coastal salt marsh and sewage plume may have impacted the surf zone water quality to various degrees during other times.

Abstract: Culture-independent fecal source tracking methods have many potential advantages over library-dependent, isolate-culture methods, but they have been subjected to limited testing. The purpose of this study was to compare culture-independent, library-independent methods of fecal source tracking. Five laboratories analysed identical sets of aqueous samples that contained one or more of the following sources: sewage, human feces, dog feces, cattle feces and gull feces. Two investigators used methods based on PCR amplification of Bacteroidetes marker genes and both successfully discriminated between samples that did or did not contain human fecal material. One of these investigators was also able to identify the remaining sources, except for gull, with a low rate of false positives. A method based on E. coli toxin genes successfully identified samples containing sewage and cattle feces, but missed some samples with human feces because of low marker prevalence in individual human fecal samples. Researchers who used community terminal restriction fragment length polymorphism (T-RFLP) were limited by the amount of DNA recovered from samples, but they correctly identified human and cattle fecal contamination when sufficient DNA was obtained. Culture independent methods show considerable promise; further research is needed to develop markers for additional fecal sources and to understand the correlation of these source-tracking indicators to measures of human and environmental health.

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Abstract: Waters impacted by fecal pollution can exact high risks to human health and can result in financial losses due to closures of water systems used for recreation and for harvesting seafood. Identifying the sources of fecal pollution in water is paramount in assessing the potential human health risks involved as well as in assessing necessary remedial action. Recently, various researchers have used the ribotyping method to identify sources of bacterial indicators (Escherichia coli and enterococci) in environmental waters. While these studies have identified genotypic differences between human- and animal-derived indicators that are capable of differentiating organisms isolated from humans and various animal hosts, most have focused on organisms collected from a confined geographic area and have not addressed the question of whether these ribotype profiles are watershed specific or if they can be applied universally to organisms from other geographic locations. In this study, E. coli isolates were obtained from humans, beef cattle, dairy cattle, swine, and poultry from locations in northern, central, and southern Florida and were subjected to ribotyping analysis. The intent was to determine (i) if ribotype profiles are capable of discriminating the source of E. coli at the host species level and (ii) if the resulting fingerprints are uniform over an extended geographic area or if they can be applied only to a specific watershed. Our research indicated that, using a single restriction enzyme (HindIII), the ribotyping procedure is not capable of differentiating E. coli isolates from the different animal species sampled in this study. Results indicate, however, that this procedure can still be used effectively to differentiate E. coli as being either human or animal derived when applied to organisms isolated from a large geographic region.

Abstract: We demonstrate the application of a new PCR assay to detect and differentiate human and ruminant sources of fecal pollution in natural water samples. We tested samples collected from Tillamook Bay, Oregon, which has a long history of fecal pollution levels that exceed acceptable standards. The most likely sources are from dairy operations and ineffective sewage treatment. Using a suite of three PCR primer pairs specific for human or ruminant bacterial 16S ribosomal DNA markers, we detected at least one marker in 17 of 22 samples. In general, host-specific fecal markers were detected in areas that are heavily impacted by anthropogenic activities. Nine out of 11 sites classified as either urban or near a sewage point source were positive for the human marker while only five of these same sites were positive for ruminant markers. Conversely, 12 out of 21 sites classified as rural or agricultural use were positive for ruminant markers, while only six of these sites were positive for human pollution. This suite of host-specific genetic markers holds promise for identifying non-point source fecal pollution in coastal waters.

Abstract: Male-specific (F+) coliphages have been investigated as viral indicators of fecal contamination that may provide source-specific information for impacted environmental waters. This study examined the presence and proportions of the different subgroups of F+ coliphages in a variety of fecal wastes and surface waters with well-defined potential waste impacts. Municipal wastewater samples had high proportions of F+ DNA and group II and III F+ RNA coliphages. Bovine wastewaters also contained a high proportion of F+ DNA coliphages, but group I and IV F+ RNA coliphages predominated. Swine wastewaters contained approximately equal proportions of F+ DNA and RNA coliphages, and group I and III F+ RNA coliphages were most common. Waterfowl (gull and goose) feces contained almost exclusively F+ RNA coliphages of groups I and IV. No F+ coliphages were isolated from the feces of the other species examined. F+ coliphage recovery from surface waters was influenced by precipitation events and animal or human land use. There were no significant differences in coliphage density among land use categories. Significant seasonal variation was observed in the proportions of F+ DNA and RNA coliphages. Group I F+ RNA coliphages were the vast majority (90%) of those recovered from surface waters. The percentage of group I F+ RNA coliphages detected was greatest at background sites, and the percentage of group II F+ RNA coliphages was highest at human-impacted sites. Monitoring of F+ coliphage groups can indicate the presence and major sources of microbial inputs to surface waters, but environmental effects on the relative occurrence of different groups need to be considered.

Abstract: Molecular methods are useful both to monitor natural communities of bacteria, and to track specific bacterial markers in complex environments. Length-heterogeneity polymerase chain reaction (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) of 16S rDNAs discriminate among 16S rRNA genes based on length polymorphisms of their PCR products. With these methods, we developed an alternative indicator that distinguishes the source of fecal pollution in water. We amplify 16S rRNA gene fragments from the fecal anaerobic genus Bacteroides with specific primers. Because Bacteroides normally resides in gut habitats, its presence in water indicates fecal pollution. Molecular detection circumvents the complexities of growing anaerobic bacteria. We identified Bacteroides LH-PCR and T-RFLP ribosomal DNA markers unique to either ruminant or human feces. The same unique fecal markers were recovered from polluted natural waters. We cloned and sequenced the unique markers; marker sequences were used to design specific PCR primers that reliably distinguish human from ruminant sources of fecal contamination. Primers for more species are under development. This approach is more sensitive than fecal coliform assays, is comparable in complexity to standard food safety and public health diagnostic tests, and lends itself to automation and high-throughput. Thus molecular genetic markers for fecal anaerobic bacteria hold promise for monitoring bacterial pollution and water quality.

Abstract: Counts of Escherichia coli cells in water indicate the potential presence of pathogenic microbes of intestinal origin but give no indication of the sources of the microbial pollution. The objective of this research was to evaluate methods for differentiating E. coli isolates of livestock, wildlife, or human origin that might be used to predict the sources of fecal pollution of water. A collection of 319 E. coli isolates from the feces of cattle, poultry, swine, deer, goose, and moose, as well as from human sewage, and clinical samples was used to evaluate three methods. One method was the multiple-antibiotic-resistance (MAR) profile using 14 antibiotics. Discriminant analysis revealed that 46% of the livestock isolates, 95% of the wildlife isolates, and 55% of the human isolates were assigned to the correct source groups by the MAR method. Amplified fragment length polymorphism (AFLP) analysis, the second test, was applied to 105 of the E. coli isolates. The AFLP results showed that 94% of the livestock isolates, 97% of the wildlife isolates, and 97% of the human isolates were correctly classified. The third method was analysis of the sequences of the16S rRNA genes of the E. coli isolates. Discriminant analysis of 105 E. coli isolates indicated that 78% of the livestock isolates, 74% of the wildlife isolates, and 80% of the human isolates could be correctly classified into their host groups by this method. The results indicate that AFLP analysis was the most effective of the three methods that were evaluated.

Abstract: This research describes a method based on PCR to identify cattle fecal pollution in water using a portion of the heat labile toxin IIA (LTIIa) gene from enterotoxigenic Escherichia coli (ETEC). We describe the development of the primers and target. DNA extracts (221) from different animal fecal and human sewage samples were screened and showed no cross-reactivity. Minimum detection limits using centrifugation and filtration methods to concentrate E. coli seeded into stream, ocean, and secondary effluent waters were found to be at femtogram and attogram levels, respectively. Stability of the biomarker in stream, ocean, and secondary effluent waters was 2-4 weeks for all water types. Finally, 33 farm lagoon and waste samples were collected and 31 tested to validate the method; 93% were positive for the LTIIa trait when >1,000 E. coli were screened and 100% positive when >10(5) E. coli were screened. Prevalence of the toxin gene in the E. coli population affected the outcome of the analyses. The cow biomarker can be used in watershed studies to identify cattle waste with great accuracy if the appropriate numbers of E. coli are screened.

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Abstract: AIMS: To develop an improved method for the detection of Bifidobacterium adolescentis as an indicator of human faecal pollution. METHODS AND RESULTS: Bifidobacterium medium (BFM) was identified as the optimal medium for the recovery of bifidobacteria from human effluent. Dilutions of faeces and effluent from both humans and animals were filtered, grown on BFM and human specific B. adolescentis identified via colony hybridization with a digoxigenin (DIG)-labelled oligonucleotide probe. CONCLUSIONS: The combination of BFM with colony probing allows the detection of B. adolescentis, a specific indicator of human faecal pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: It is now technically feasible to use B. adolescentis as indicators of human faecal pollution, and studies to examine the survival and appropriateness of bifidobacteria in this role can be initiated.

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Abstract: Bacteria such as fecal coliforms are used as indicators of fecal pollution in natural waters. These bacteria are found in the feces of most wild and domestic animals and thus provide no information as to the source of fecal contamination, yet identification of indicator bacteria sources allows improved risk assessment, remediation, and total daily maximum load (TDML) assessment of environmental waters. This bacterial source tracking study was initiated in order to identify the dominant source(s) of fecal contamination in the urban watershed of Stevenson Creek in Clearwater, Florida. Five sites that represent areas where routine monitoring has previously shown high levels of fecal coliforms were sampled over 7 months. Fecal coliforms were enumerated by membrane filtration, and antibiotic resistance analysis was used to "fingerprint" a subset of randomly selected isolates and statistically match them to fingerprints of fecal coliforms from known sources (the library). A field test of the classification accuracy of the library was carried out by isolating fecal coliforms from the soil and waters surrounding a failing onsite wastewater treatment and disposal system (OSTDS). The vast majority of the isolates were classified into the human category. The major sources of fecal pollution in Stevenson Creek over the course of the study were wild animal, human, and, to a lesser extent, dog. Overall, wild animal feces were identified as the dominant source when fecal coliform levels were high, but when fecal coliform levels were low, the dominant source was identified as human. The results of this study demonstrate that the sources of fecal indicator bacteria within one urban watershed can vary substantially over temporal and spatial distances.

Abstract: Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.

Abstract: Several genotypic methods have been developed for determining the host origin of fecal bacteria in contaminated waters. Some of these methods rely on a host origin database to identify environmental isolates. It is not well understood to what degree these host origin isolates are geographically variable (i.e., cosmopolitan or endemic). This is important because a geographically limited host origin database may or may not be universally applicable. The objective of our study was to use one genotypic method, ribotyping, to determine the geographic variability of the fecal bacterium, Escherichia coli, from one location in Idaho and three locations in Georgia for cattle (Bos taurus), horse (Equus caballus), swine (Sus scrofa), and chicken (Gallus gallus domesticus). A total of 568 fecal E. coli isolates from Kimberly, ID (125 isolates), Athens, GA (210 isolates), Brunswick, GA (102 isolates), and Tifton, GA (131 isolates), yielded 213 ribotypes. The percentage of ribotype sharing within an animal species increased with decreased distance between geographic locations for cattle and horses, but not for swine and chicken. When the E. coli ribotypes among the four host species were compared at one location, the percent of unshared ribotypes was 86, 89, 81, and 79% for Kimberly, Athens, Brunswick, and Tifton, respectively. These data suggest that there is good ribotype separation among host animal species at each location. The ability to match environmental isolates to a host origin database may depend on a large number of environmental and host origin isolates that ideally are not geographically separated.

Abstract: Although water quality of the Nation's lakes, rivers and streams has been monitored for many decades and especially since the passage of the Clean Water Act in 1972, many still do not meet the Act's goal of "fishable and swimmable". While waterways can be impaired in numerous ways, the protection from pathogenic microbe contamination is most important for waters used for human recreation, drinking water and aquaculture. Typically, monitoring methods used for detecting potential pathogenic microorganisms in environmental waters are based upon cultivation and enumeration of fecal indicator bacteria (i.e. fecal coliforms, E. coli, and fecal enterococci). Currently, there is increasing interest in the potential for molecular fingerprinting methods to be used not only for detection but also for identification of fecal contamination sources. Molecular methods have been applied to study the microbial ecology of environmental systems for years and are now being applied to help improve our waters by identifying problem sources and determining the effect of implemented remedial solutions. Management and remediation of water pollution would be more cost-effective if the correct sources could be identified. This review provides an outline of the main methods that either have been used or have been suggested for use in microbial source tracking and some of the limitations associated with those methods.

Abstract: Regulatory agencies are interested in a fecal indicator bacterium with a host range limited to humans because human fecal contamination represents the greatest hazard to humans, yet is a relatively easy nonpoint source to remedy. Watersheds with human fecal contamination could be given first priority for cleanup. A fecal indicator bacterium with a host range limited to humans and a few other warm-blooded animal species would also simplify microbial source tracking because only a few animal species would be required for any host origin database. The literature suggests that the fecal indicator bacterium Enterococcus faecalis has a limited host range. On this basis, we selected this bacterium for study. Of 583 fecal streptococcal isolates obtained on Enterococcosel agar from Canada goose, cattle, deer, dog, human, chicken, and swine, 392 were considered presumptive enterococci and were subsequently speciated with the API 20 Strep system. Of these isolates, 22 were Ent. durans (5.6%), 61 were Ent. faecalis (15.6%), 98 were Ent. faecium (25.0%), 86 were Ent. gallinarum (21.9%), and 125 were unidentified (31.9%). The host range of the Ent. faecalis isolates was limited to dogs, humans, and chickens. Media were developed to isolate and identify Ent. faecalis quickly from fecal samples and this scheme eliminated Ent. faecalis isolates from dogs. When the remaining Ent. faecalis isolates were ribotyped, it was possible to differentiate clearly among the isolates from human and chicken. It may be that combining the potentially limited host range of Ent. faecalis with ribotyping is useful for prioritizing watersheds with fecal contamination.

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Abstract: Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is one of several fecal coliform bacteria that inhabit the intestines of many warm-blooded animals that sometimes contaminate water. Its presence does not specifically implicate human fecal input, therefore it is necessary to differentiate contamination sources to accurately assess health risks. E. coli were isolated from human sources (HS) and nonhuman sources (NHS) in the Apalachicola National Estuarine Research Reserve and analyzed for fatty acid methyl ester (FAME), O-serogroup, and pulsed-field gel electrophoresis (PFGE) profiles. For FAME and PFGE analyses, there was no relationship between profile and isolate source. Human source PFGE profiles were less diverse than NHS isolates, and conversely for FAME. In contrast, O-serogrouping showed less diversity for HS vs. NHS isolates, and the predominant HS O-serogroups differed significantly (P < 0.01) from those of NHS isolates.

Abstract: Fecal pollution of water resources is an environmental problem of increasing importance. Identification of individual host sources of fecal Escherichia coli, such as humans, pets, production animals, and wild animals, is prerequisite to formulation of remediation plans. Ribotyping has been used to distinguish fecal E. coli of human origin from pooled fecal E. coli isolates of nonhuman origin. We have extended application of this technique to distinguishing fecal E. coli ribotype patterns from human and seven individual nonhuman hosts. Classification accuracy was best when the analysis was limited to three host sources. Application of this technique to identification of host sources of fecal coliforms in water could assist in formulation of pollution reduction plans.

Abstract: Antibiotic resistance analysis (ARA) was used to determine if enterococci of human origin were present in a stream (Spout Run) that passes through a rural nonsewered community (Millwood, VA). Millwood consists of 82 homes, all served by individual septic systems, and Spout Run drains a 5800-ha karst topography watershed that contains large populations of livestock and wildlife. Periodic monitoring by state regulatory officials had resulted in Spout Run being placed on the Virginia impaired stream list and Millwood being categorized as an at-risk community. Stream samples were collected monthly and analyzed for fecal coliforms and enterococci (May 1999–May 2000); ARA was performed on enterococci stream isolates on a quarterly basis. All 117 stream samples were positive for fecal coliforms, and 32% exceeded the Virginia recreational water standard (1000 fecal coliforms/100 mL). A library of 1174 known source Enterococcus isolate antibiotic resistance profiles was constructed, and yielded correct classification rates of 94.6% for 203 human isolates, 93.7% for 734 livestock isolates, and 87.8% for 237 wildlife isolates. Antibiotic resistance analysis of 2012 enterococcal isolates recovered from stream samples indicated isolates of human origin appeared throughout the stream as it passed through Millwood, with a yearly average of approximately 10% human, 40% wildlife, and 50% livestock. There were no human origin isolates in samples collected upstream from Millwood, and the percent human origin isolates declined downstream from Millwood. While a human signature was found in Spout Run, it was small compared with the proportion of isolates from livestock and wildlife.

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Abstract: Our purpose was to develop a rapid, inexpensive method of diagnosing the source of fecal pollution in water. In previous research, we identified Bacteroides-Prevotella ribosomal DNA (rDNA) PCR markers based on analysis. These markers length heterogeneity PCR and terminal restriction fragment length polymorphism distinguish cow from human feces. Here, we recovered 16S rDNA clones from natural waters that were close phylogenetic relatives of the markers. From the sequence data, we designed specific PCR primers that discriminate human and ruminant sources of fecal contamination.

Abstract: The antibiotic resistance patterns of fecal streptococci and fecal coliforms isolated from domestic wastewater and animal feces were determined using a battery of antibiotics (amoxicillin, ampicillin, cephalothin, chlortetracycline, oxytetracycline, tetracycline, erythromycin, streptomycin, and vancomycin) at four concentrations each. The sources of animal feces included wild birds, cattle, chickens, dogs, pigs, and raccoons. Antibiotic resistance patterns of fecal streptococci and fecal coliforms from known sources were grouped into two separate databases, and discriminant analysis of these patterns was used to establish the relationship between the antibiotic resistance patterns and the bacterial source. The fecal streptococcus and fecal coliform databases classified isolates from known sources with similar accuracies. The average rate of correct classification for the fecal streptococcus database was 62.3%, and that for the fecal coliform database was 63.9%. The sources of fecal streptococci and fecal coliforms isolated from surface waters were identified by discriminant analysis of their antibiotic resistance patterns. Both databases identified the source of indicator bacteria isolated from surface waters directly impacted by septic tank discharges as human. At sample sites selected for relatively low anthropogenic impact, the dominant sources of indicator bacteria were identified as various animals. The antibiotic resistance analysis technique promises to be a useful tool in assessing sources of fecal contamination in subtropical waters, such as those in Florida.

Abstract: We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides-Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium and Bacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made Bacteroides-Prevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides or Prevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 × 105 to 2.8 × 107 g (dry weight) of feces/liter and 6.8 × 107 g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments.

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Abstract: Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is a ubiquitous bacterium in the intestines of warm-blooded animals and is used as an indicator of fecal pollution. However, its presence does not specifically differentiate sources of pollution. A total of 238 E. coli isolates from human sources (HS) and nonhuman sources (NHS) were collected from the Apalachicola National Estuarine Research Reserve, from associated sewage treatment plants, and directly from animals and tested for ribotype (RT) profile. HS and NHS isolates showed 41 and 61 RT profiles, respectively. At a similarity index of ca. 50%, HS and NHS isolates demonstrated four clusters, with the majority of HS and NHS isolates located in clusters C and D; isolates obtained directly from human and animal feces also could be grouped within these clusters. Discriminant analysis (DA) of RT profiles showed that 97% of the NHS isolates and 100% of the animal fecal isolates were correctly classified. The average rate of correct classification for HS and NHS isolates was 82%. We conclude that DA of RT profiles may be a useful method for identifying HS and NHS fecal pollution and may potentially facilitate management practices.

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Abstract: A total of 765 Escherichia coli isolates from point and nonpoint sources were collected from the Apalachicola National Estuarine Research Reserve, and their multiple-antibiotic-resistance (MAR) profiles were determined with 10 antibiotics. E. coli isolates from point sources showed significantly greater resistance (P < 0.05) to antibiotics and higher MAR indices than isolates from nonpoint sources. Specifically, 65 different resistance patterns were observed among point source isolates, compared to 32 among nonpoint source isolates. Examples of this contrast in MAR profiles included percentages of isolates with resistance to chlortetracycline-sulfathiazole of 33.7% and to chlortetracycline-penicillin G-sulfathiazole of 14.5% for point source isolates versus 15.4 and 1.7%, respectively, for nonpoint source isolates. MAR profile homology, based on coefficient similarity, showed that isolates from point sources were markedly more diverse than isolates from nonpoint sources. Seven clusters were observed among point source isolates, with a coefficient value of approximately 1.8. In contrast, only four clusters were observed among nonpoint source isolates. Covariance matrices of data displayed six very distinct foci representing nonpoint source E. coli isolates. Importantly, E. coli isolates obtained directly from human and animal feces also clustered among point and nonpoint sources, respectively. We conclude that E. coli MAR profiles were associated with point and nonpoint sources of pollution within Apalachicola Bay and that this method may be useful in facilitating management of other estuaries.

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Abstract: Because Bacteroides spp. are obligate anaerobes that dominate the human fecal flora, and because some species may live only in the human intestine, these bacteria might be useful to distinguish human from nonhuman sources of fecal pollution. To test this hypothesis, PCR primers specific for 16S rRNA gene sequences of Bacteroides distasonis, B. thetaiotaomicron, and B. vulgatus were designed. Hybridization with species-specific internal probes was used to detect the intended PCR products. Extracts from 66 known Bacteroides strains, representing 10 related species, were used to confirm the specificity of these PCR-hybridization assays. To test for specificity in feces, procedures were developed to prepare DNA of sufficient purity for PCR. Extracts of feces from 9 humans and 70 nonhumans (cats, dogs, cattle, hogs, horses, sheep, goats, and chickens) were each analyzed with and without an internal positive control to verify that PCR amplification was not inhibited by substances in the extract. In addition, serial dilutions from each extract that tested positive were assayed to estimate the relative abundance of target Bacteroides spp. in the sample. Depending on the primer-probe set used, either 78 or 67% of the human fecal extracts tested had high levels of target DNA. On the other hand, only 7 to 11% of the nonhuman extracts tested had similarly high levels of target DNA. An additional 12 to 20% of the nonhuman extracts had levels of target DNA that were 100- to 1,000-fold lower than those found in humans

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Abstract: When Enterococcus faecalis is isolated from fresh feces, its host range appears to be limited to humans and birds. Although E. faecalis is found in human sewage, the extent to which the bacterium is found in broiler litter and in the feces of wild birds is unclear. These results have implications for bacterial source tracking. We determined if media designed for the isolation of fecal enterococci affected this host range, and if E. faecalis was routinely found in broiler litter and in the feces of wild birds. Of five different isolation media, none affected the isolation of E. faecalis. Enterococcus faecalis was routinely found in fresh broiler feces (522 of 1092 isolates; 48%), but rarely in broiler litter (12 of 1452 isolates; <2%). Therefore, broiler litter selects against this bacterium, and broiler litter is an unlikely environmental source of this bacterium. The presence of E. faecalis in eight wild bird species was highly variable. Unless the fecal loading rate from migratory or resident wild birds is high, water samples collected during baseflow conditions with high numbers of E. faecalis may indicate human fecal contamination.