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Bacterial pollution in the water comes in particular from Escherichia coli and fecal coliforms, responsible for gastroenteritis and diarrhea, intestinal streptococci or enterococci (urinary tract infections and peritonitis), salmonella which can cause serious gastroenteritis, shigella (dysen-teritis, gastroenteritis), cholera vibrio (cholera). As 23 sites on the Seine and Marne Rivers (Parisian Region) would be identified as the natation competition sites for the Paris-2024 Olympic and Paralympic Games, the water quality at these sites should be seriously monitored. Numerical modeling can be considered one powerful tool to watch the water quality parameters. However, measurements show that the water quality is not homogeneous in a river cross-section, and one-dimensional (1D) models are not enough to accurately calculate the bacteriological concentration dispersion in the aquatic environments. Therefore, a two-dimensional model has been developed by coupling the TELEMAC-2D model and its water quality module WAQTEL for simulating bathing water quality in the Seine and Marne Rivers. The model was validated against in situ measurements and was compared against a 1D model. Results show that this model can simulate not only the longitudinal evolution but also the transverse dispersion of bacteriological pollutants. Then, a 3D multi-layer model has been developed around a bathing site using the TELEMAC-3D model. The result of the 3D model is promising and allows us to get a finer representation of the bacteriological concentration in three dimensions. [ABSTRACT FROM AUTHOR]

Fecal contamination of drinking water is a major health problem which accounts for many cases of diarrhea mainly in infants and foreigners. This contamination is a complex interaction of many parameters. Antibiotic resistance among bacterial isolates complicates the problem. The study was done to identify fecal contamination of drinking water by Diarrheagenic Antibiotic-Resistant Escherichia coli in Zagazig city and to trace reasons for such contamination, three hundred potable water samples were investigated for E. coli existence. Locations of E. coli positive samples were investigated in relation to population density, water source, and type of water pipe. Sixteen E. coli strains were isolated. Antibiotic sensitivity was done and enterotoxigenic, enteropathogenic, and enterohaemorrhagic virulence genes were investigated by PCR. Probability of fecal contamination correlated with higher population density, with increased distance from Zagazig water plant, and with asbestos cement water pipes. Resistance to at least one antimicrobial drug was found in all isolates. Virulence genes were detected in a rate of 26.27%, 13.13%, 20%, 6.67%, and 33.33% for LT, ST, stx1, stx2, and eae genes, respectively. This relatively high frequency of fecal contamination points towards the high risk of developing diarrhea by antibiotic resistant DEC in low socioeconomic communities particularly with old fashion distribution systems. [ABSTRACT FROM AUTHOR]

The role of environmental forcings on the fecal contamination dynamics in small coastal streams was investigated, using the Aljezur coastal stream (Portugal) as a test case. An integrated modeling approach, combining hydrological and coupled hydrodynamic-fecal indicator bacteria models, was applied to selected scenarios of relevant forcings, on the basis of previously calibrated and validated model applications. The forcings investigated included: tides, winds, waves, atmospheric pressure, bathymetry, river flow, and upstream fecal bacteria concentrations; this study targeted the characterization of the relative importance of these factors on the fecal contamination dynamics, to enhance our understanding of small coastal streams water quality dynamics. Results highlight distinct patterns for dry and wet periods. Fecal bacteria transport is mainly forced by tidal propagation, in dry periods; and by freshwater flows, during wet periods. The analysis reveals a complex hydraulic and water quality dynamic resulting from the interactions between the environmental forcings and strongly dependent on their relative strength. This analysis can be extrapolated for systems with a similar balance between the environmental factors. [ABSTRACT FROM AUTHOR]

Water-borne illness, primarily caused by fecal contamination of drinking water, is a major health burden in the state of Andhra Pradesh, India. Currently drinking water is treated at the reservoir level and supplied on alternate days, necessitating storage in households for up to 48 hrs. We hypothesized that fecal contamination occurs principally during storage due to poor water handling. In this study we tested for coliform bacteria in water samples collected at distribution points as household storage containers were filled, and then tested containers in the same households 24-36 hours after collection. We also conducted an observational survey to make an assessment of water handling and hygiene. Ninety-two percent (47/51) of samples tested at supply points were adequately chlorinated and bacterial contamination was found in two samples with no residual chlorine. Samples collected from household storage containers showed an increase in contamination in 18/50 houses (36%). Households with contaminated stored samples did not show significant differences in demographics, water handling, hygiene practices, or sanitation. Nevertheless, the dramatic increase in contamination after collection indicates that until an uninterrupted water supply is possible, the point at which the biggest health impact can be made is at the household level. [ABSTRACT FROM AUTHOR]

: Robert Bain and colleagues conduct a systematic review and meta-analysis to assess whether water from “improved” sources is less likely to contain fecal contamination than “unimproved” sources and find that access to an “improved source” provides a measure of sanitary protection but does not ensure water is free of fecal contamination. Please see later in the article for the Editors' Summary [ABSTRACT FROM AUTHOR]

In order to identify the origin of the fecal 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 bacteriophage genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, and bird) feces. Two human-specific markers (HF183 and HF134), one ruminant-specific marker (CF193′), and one pig-specific marker (PF163) 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 feces and 96% of pig feces. 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 bacteriophages were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird feces 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 fecal 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 Escherichia coli or enterococcus concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers. [ABSTRACT FROM AUTHOR]

Research on microbiological groundwater quality was conducted in Chile in a rural watershed that has almost no other water source. Forty-two wells were randomly selected and levels of indicator bacteria - total coliforms (IC), fecal coliforms (FC), and fecal streptococci (FS) - were repeatedly measured during the four seasons of 2005. The aim of this study was to characterize microbiological groundwater quality, relate indicator levels to certain watershed features and management characteristics which are likely to affect water quality. The dynamics of seasonal temporal contamination was determined with statistical analyses of indicator organism concentrations. Nonparametric tests were used to analyze relationships between bacterial indicators in well water and other variables. TC, FC, and FS were found in all samples indicating the wells had been contaminated with human and animal fecal material. The frequency distribution of microorganisms fitted a logistic distribution. The concentrations appeared to be temporal and levels varied between seasons with higher concentrations in winter. The cause of contamination could be linked to the easy access of domestic animals to the wells and to the permeable well casing material. Local precipitation runoff directly influenced the bacterial concentrations found in the wells. [ABSTRACT FROM AUTHOR]

The objective of this study was to combine knowledge of environmental, topographical, meteorological, and anthropologic factors in the Río Grande de Arecibo (RGA) watershed in Puerto Rico with information provided by microbial source tracking (MST) to map hot spots (i.e., likely sources) of fecal contamination. Water samples were tested for the presence of human and bovine fecal contamination in addition to fecal indicator bacteria and correlated against several land uses and the density of septic tanks, sewers, and latrines. Specifically, human sources were positively correlated with developed (r = 0.68), barren land uses (r = 0.84), density of septic tanks (r = 0.78), slope (r = 0.63), and the proximity to wastewater treatment plants (WWTPs) (r = 0.82). Agricultural land, the number of upstream National Pollution Discharge Elimination System (NPDES) facilities, and density of latrines were positively associated with the bovine marker (r = 0.71; r = 0.74; and r = 0.68, respectively). Using this information, we provided a hot spot map, which shows areas that should be closely monitored for fecal contamination in the RGA watershed. The results indicated that additional bovine assays are needed in tropical regions. We concluded that meteorological, topographical, anthropogenic, and land cover data are needed to evaluate and verify the performance of MST assays and, therefore, to identify important sources of fecal contamination in environmental waters. [ABSTRACT FROM AUTHOR]

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 (glass cuvettes, 30min exposure) or (polymethacrylate cuvettes, 5min 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. [Copyright &y& Elsevier]

This document is an editorial titled "Advancing molecular diagnostic tools for robust surveillance of microbial water quality." It emphasizes the importance of monitoring microbial water quality for human, animal, and environmental health. The presence of microbial pollutants in water can have negative impacts, including waterborne outbreaks. The editorial discusses the use of molecular diagnostic tools, such as next-generation sequencing and CRISPR technology, for detecting and assessing microbial contamination in water. It also highlights the importance of integrated assessment tools and modeling for evaluating and predicting microbial water quality and health risks. The document concludes by emphasizing the need for coordinated and validated methods to ensure effective and reliable water quality assessment. [Extracted from the article]

Microbial source tracking (MST) and chemical source tracking (CST) markers were utilized to identify fecal contamination in harvested rainwater and gutter debris samples. Throughout the sampling period, Bacteroides HF183 was detected in 57.5 % of the tank water samples and 95 % of the gutter debris samples, while adenovirus was detected in 42.5 and 52.5 % of the tank water and gutter debris samples, respectively. Human adenovirus was then detected at levels ranging from below the detection limit to 316 and 1253 genome copies/μL in the tank water and debris samples, respectively. Results for the CST markers showed that salicylic acid (average 4.62 μg/L) was the most prevalent marker (100 %) in the gutter debris samples, caffeine (average 18.0 μg/L) was the most prevalent in the tank water samples (100 %) and acetaminophen was detected sporadically throughout the study period. Bacteroides HF183 and salicylic acid (95 %) and Bacteroides HF183 and caffeine (80 %) yielded high concurrence frequencies in the gutter debris samples. In addition, the highest concurrence frequency in the tank water samples was observed for Bacteroides HF183 and caffeine (60 %). The current study thus indicates that Bacteroides HF183, salicylic acid and caffeine may potentially be applied as source tracking markers in rainwater catchment systems in order to supplement fecal indicator analyses. [ABSTRACT FROM AUTHOR]

Background: Packaged water products provide an increasingly important source of water for consumption. However, recent studies raise concerns over their safety. Objectives: To assess the microbial safety of packaged water, examine differences between regions, country incomes, packaged water types, and compare packaged water with other water sources. Methods: We performed a systematic review and meta-analysis. Articles published in English, French, Portuguese, Spanish and Turkish, with no date restrictions were identified from online databases and two previous reviews. Studies published before April 2014 that assessed packaged water for the presence of Escherichia coli, thermotolerant or total coliforms were included provided they tested at least ten samples or brands. Results: A total of 170 studies were included in the review. The majority of studies did not detect fecal indicator bacteria in packaged water (78/141). Compared to packaged water from upper-middle and high-income countries, packaged water from low and lower-middle-income countries was 4.6 (95% CI: 2.6–8.1) and 13.6 (95% CI: 6.9–26.7) times more likely to contain fecal indicator bacteria and total coliforms, respectively. Compared to all other packaged water types, water from small bottles was less likely to be contaminated with fecal indicator bacteria (OR = 0.32, 95%CI: 0.17–0.58) and total coliforms (OR = 0.10, 95%CI: 0.05, 0.22). Packaged water was less likely to contain fecal indicator bacteria (OR = 0.35, 95%CI: 0.20, 0.62) compared to other water sources used for consumption. Conclusions: Policymakers and regulators should recognize the potential benefits of packaged water in providing safer water for consumption at and away from home, especially for those who are otherwise unlikely to gain access to a reliable, safe water supply in the near future. To improve the quality of packaged water products they should be integrated into regulatory and monitoring frameworks. [ABSTRACT FROM AUTHOR]

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Almost half of the world's population relies on non-networked water supply services, which necessitates in-home water storage. It has been suggested that dirty hands play a role in microbial contamination of drinking water during collection, transport, and storage. However, little work has been done to evaluate quantitatively the association between hand contamination and stored water quality within households. This study measured levels of E. coli, fecal streptococci, and occurrence of the general Bacteroidales fecal DNA marker in source water, in stored water, and on hands in 334 households among communities in Dar es Salaam, Tanzania, where residents use non-networked water sources. Levels of fecal contamination on hands of mothers and children were positively correlated to fecal contamination in stored drinking water within households. Household characteristics associated with hand contamination included mother's educational attainment, use of an improved toilet, an infant in the household, and dissatisfaction with the quantity of water available for hygiene. In addition, fecal contamination on hands was associated with the prevalence of gastrointestinal and respiratory symptoms within a household. The results suggest that reducing fecal contamination on hands should be investigated as a strategy for improving stored drinking water quality and health among households using non-networked water supplies. [ABSTRACT FROM AUTHOR]

Abstract Coliphage have been proposed as indicators of fecal contamination in recreational waters because they better reflect the persistence of pathogenic viruses in the environment and through wastewater treatment than traditional fecal indicator bacteria. Herein, we conducted a systematic literature search of peer-reviewed publications to identify coliphage density data (somatic and male-specific, or MSC) in raw wastewater and ambient waters. The literature review inclusion criteria included scope, study quality, and data availability. A non-parametric two-stage bootstrap analysis was used to estimate the coliphage distributions in raw wastewater and account for geographic region and season. Additionally, two statistical methodologies were explored for developing coliphage density distributions in ambient waters, to account for the nondetects in the datasets. In raw wastewater, the analysis resulted in seasonal density distributions of somatic coliphage (SC) (mean 6.5 log 10 plaque forming units (PFU)/L; 95% confidence interval (CI): 6.2–6.8) and MSC (mean 5.9 log 10 PFU/L; 95% CI: 5.5–6.1). In ambient waters, 49% of MSC samples were nondetects, compared with less than 5% for SC. Overall distributional estimates of ambient densities of coliphage were statistically higher for SC than for MSC (mean 3.4 and 1.0 log 10 PFU/L, respectively). Distributions of coliphage in raw wastewater and ambient water will be useful for future microbial risk assessments. Highlights • Systematic review conducted of coliphage levels in raw wastewater and ambient waters. • Studies conducted in 19 countries provided 2124 individual coliphage data points. • Somatic coliphage outnumber MSC densities in raw wastewater and in ambient waters. • Overall coliphage densities were significantly higher in colder seasons. • Coliphage distributions will inform quantitative microbial risk assessment efforts. [ABSTRACT FROM AUTHOR]

Repetitive element sequence-based polymerase chain reaction (rep-PCR) is one of the commonest methods used to identify sources of fecal contamination of water systems. In this work, BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) was used to discriminate Escherichia coli strains originating from different animals and water sources, and the suitability of the technique for bacterial source tracking (BST) was evaluated. A total of 214 strains from humans, 150 strains from animals, 55 strains from sewage and 77 strains from water bodies were analyzed by the BOX-PCR technique. When maximum similarity between the fingerprints was used, a correct classification rate of 84% was achieved for strains from human and animal sources. Furthermore, 95% of the strains found in sewage were classified as being from human sources by at least one of the four classification tools used. Classification of the strains found in water bodies in the State of São Paulo was based on the fingerprints obtained for human and animal sources. Most of the sampling sites appeared to be affected by mixed sources of fecal contamination. The use of BOX-PCR for BST could be especially valuable in developing countries, where simplicity and cost are important considerations. [Copyright &y& Elsevier]

This paper reports a reconnaissance survey of the concentrations of sterol compounds (as indicators of fecal contamination) in a large water supply system in southeast Australia comprising a network of rivers, channels, and drains. Levels of coprostanol and cholestanol were determined in surface water and bottom sediment using gas chromatography–mass spectrometry analysis across 17 strategic sampling sites and over 12 months. Clear differences in the levels of fecal contamination were observed among sites. Four sites routinely contained high levels of the fecal indicator sterols indicated from surface water and sediment sample analysis. Coprostanol concentrations at each location varied from 0 ng/L at the reference site to 11,327 ng/L in a surface water sample of a drain directly downstream of a knackery. The majority of the sites contained coprostanol in the range of 500 to 800 ng/L. Since no fecal-associated sterol compounds were detected at the external reference sites, these were assumed to be free from fecal contamination. Sewage water discharge and/or substantial water runoff maybe the principal factors contributing to fecal contamination of the supply drains and channels. [ABSTRACT FROM AUTHOR]

In a continuing effort to develop inexpensive source tracking methods to detect human fecal contamination in environmental waters, targeted sampling was combined with fluorometry. Targeted sampling works by identifying hot spots of fecal contamination through multiple samplings over ever-decreasing distances. Fluorometry identifies human fecal contamination by detecting optical brighteners, primarily from laundry detergents. Because organic matter fluoresces and interferes with fluorometry, two locations were chosen for sampling: waters relatively low in organic matter at Mayaguez Bay, Puerto Rico, and waters relatively high in organic matter at St. Simons Island, Georgia. In Puerto Rico, targeted sampling and fluorometry quickly and easily identified two hot spots of human fecal contamination in the Yaguez River, which flows through the city of Mayaguez. Another source tracking method, detection of the esp gene, confirmed their human origin. On St. Simons Island, targeted sampling and fluorometry identified two hot spots of potential human fecal contamination. Detection of the rap gene confirmed the human origin of one site but not the other, most likely because background organic matter fluorescence interfered with fluorometry. A separate experiment showed that adding a 436-nm emission filter to the fluorometer reduced this background fluorescence by > 50%. With the 436-nm filter in place, another sampling was conducted on St. Simons Island, and the second hot spot was identified as fecal contamination from birds. As long as the fluorometer was equipped with a 436-mn filter and organic matter concentrations were considered, targeted sampling combined with fluorometry was a relatively inexpensive method for identifying human fecal contamination in water. [ABSTRACT FROM AUTHOR]

Escherichia coli (E. coli) is a widely used indicator of fecal contamination in water bodies. External contact and subsequent ingestion of bacteria coming from fecal contamination can lead to harmful health effects. Since E. coli data are sometimes limited, the objective of this study is to use secondary information in the form of turbidity to improve the assessment of E. coli at unmonitored locations. We obtained all E. coli and turbidity monitoring data available from existing monitoring networks for the 2000-2006 time period for the Raritan River Basin, New Jersey. Using collocated measurements, we developed a predictive model of E. coli from turbidity data. Using this model, soft data are constructed for E. coli given turbidity measurements at 739 space/time locations where only turbidity was measured. Finally, the Bayesian Maximum Entropy (BME) method of modern space/time geostatistics was used for the data integration of monitored and predicted E. coli data to produce maps showing E. coli concentration estimated daily across the river basin. The addition of soft data in conjunction with the use of river distances reduced estimation error by about 30%. Furthermore, based on these maps, up to 35% of river miles in the Raritan Basin had a probability of E. coli impairment greater than 90% on the most polluted day of the study period. [ABSTRACT FROM AUTHOR]

Fecal contamination of water bodies causes a public health problem and economic loss. To control such contamination management actions need to be guided by sound science. From 2007–2009 a study was undertaken to determine the sources of fecal bacteria contamination to the marine waters adjoining the Town of Wrightsville Beach, North Carolina, USA. The research effort included sampling for fecal coliform and Enterococcus bacteria, sampling for optical brighteners, dye studies, and use of molecular bacterial source tracking techniques including polymerase chain reaction (PCR) and terminal restriction fragment polymorphism (T-RFLP) fingerprinting of the Bacteroides–Prevotella group. Of the 96 samples collected from nine locations during the study, the water contact standard for Enterococcus was exceeded on 13 occasions. The T-RFLP fingerprint analyses demonstrated that the most widespread source of fecal contamination was human, occurring in 38% of the samples, with secondary ruminant and avian sources also detected. Optical brightener concentrations were low, reflecting a lack of sewage line leakage or spills. A lack of sewer leaks and lack of septic systems in the town pointed toward discharge from boat heads into the marine waters as the major cause of fecal contamination; this was supported by dye studies. Based on these data, the Town initiated action to have the U.S. Environmental Protection Agency declare the coastal waters (out to 3 nautical miles), the nearby Atlantic Intracoastal Waterway and its tributaries a no-discharge zone (NDZ) to alleviate the human fecal pollution. The Town garnered supporting resolutions from other local communities who jointly petitioned the North Carolina Department of Environmental and Natural Resources. This State regulatory agency supported the local government resolutions and sent an application for an NDZ to the EPA in April 2009. The EPA concurred, and in February 2010 the coastal waters of New Hanover County, NC, became the first marine area on the U.S. eastern seaboard between Delaware and the Florida Keys to be declared a no-discharge zone. [Copyright &y& Elsevier]

Routine water quality monitoring practices based on the enumeration of culturable Escherichia coli provides no information about the source or age of fecal pollution. An emerging strategy is to use culturable E. coli and the DNA markers of Bacteroidales complementarily for microbial source tracking. In this study, we consistently observed in seawater microcosms of 3 different conditions that culturable E. coli decayed faster ( T99 = 1.14 - 4.29 days) than Bacteroidales DNA markers did ( T99 = 1.81 - 200.23 days). Concomitantly, the relative concentration between Bacteroidales DNA markers and culturable E. coli increased over time in all treatments. Particularly, the increase during the early stage of the experiments (before T99 of E. coli was reached) was faster than during the later stage (after T99 of E. coli was attained). We propose that the tracking of the relative concentration between Bacteroidales DNA markers and culturable E. coli provides an opportunity to differentiate a pollution that is relatively fresh from one that has aged. This method, upon further investigation and validation, could be useful in episodic pollution events where the surge of E. coli concentration causes noncompliance to the single sample maximum criterion that mandates high frequency follow-up monitoring. [ABSTRACT FROM AUTHOR]

Gulls are prevalent in beach environments and can be a major source of fecal contamination. Gulls have been shown to harbor a high abundance of fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, which can be readily detected as part of routine beach monitoring. Despite the ubiquitous presence of gull fecal material in beach environments, the associated microbial community is relatively poorly characterized. We generated comprehensive microbial community profiles of gull fecal samples using Roche 454 and Illumina MiSeq platforms to investigate the composition and variability of the gull fecal microbial community and to measure the proportion of FIB. Enterococcaceae and Enterobacteriaceae were the two most abundant families in our gull samples. Sequence comparisons between short-read data and nearly full-length 16S rRNA gene clones generated from the same samples revealed Catellicoccus marimammalium as the most numerous taxon among all samples. The identification of bacteria from gull fecal pellets cultured on membrane-Enterococcus indoxyl-β-D-glucoside (mEI) plates showed that the dominant sequences recovered in our sequence libraries did not represent organisms culturable on mEI. Based on 16S rRNA gene sequencing of gull fecal isolates cultured on mEI plates, 98.8% were identified as Enterococcus spp., 1.2% were identified as Streptococcus spp., and none were identified as C. marimammalium. Illumina deep sequencing indicated that gull fecal samples harbor significantly higher proportions of C. marimammalium 16S rRNA gene sequences (>50-fold) relative to typical mEI culturable Enterococcus spp. C. marimammalium therefore can be confidently utilized as a genetic marker to identify gull fecal pollution in the beach environment. [ABSTRACT FROM AUTHOR]

While many current microbial source tracking (MST) methods rely on the use of specific molecular marker genes to identify sources of fecal contamination, these methods often fail to determine all point and nonpoint contributors of fecal inputs into waterways. In this study, we developed a new library-dependent MST method that uses pyrosequencing-derived shared operational taxonomy units (OTUs) to define sources of fecal contamination in waterways. A total 56,841 pyrosequencing reads of 16S rDNA obtained from the feces of humans and animals were evaluated and used to compare fecal microbial diversity in three freshwater samples obtained from the Yeongsan river basin in Jeonnam Province, South Korea. Sites included an urbanized agricultural area (Y1) (Escherichia coli counts ≥ 1600 CFU/100 mL), an open area (Y2) with no major industrial activities (940 CFU/100 mL), and a typical agricultural area (Y3) (= 1600 CFU/100 mL). Data analyses indicated that the majority of bacteria in the feces of humans and domesticated animals were comprised of members of the phyla Bacteroidetes or Firmicutes, whereas the majority of bacteria in wild goose feces and freshwater samples were classified to the phylum Proteobacteria. Analysis of OTUs shared between the fecal and environmental samples suggested that the potential sources of the fecal contamination at the sites were of human and swine origin. Quantification of fecal contamination was also examined by comparing the density of pyrosequencing reads in each fecal sample within shared OTUs. Taken together, our results indicated that analysis of shared OTUs derived from barcoded pyrosequencing reads provide the necessary resolution and discrimination to be useful as a next generation platform for microbial source tracking studies. [ABSTRACT FROM AUTHOR]

As a representative urban stream in Korea, the Gwangju (GJ) stream suffers from chronic fecal contamination. In this study, to characterize levels of fecal pollution in the GJ stream, the monthly monitoring data for seven years (from 2001 to 2007) and the hourly monitoring data from two field experiments were examined with respect to seasonal/daily variations and spatial distribution under wet and dry weather conditions. This research revealed that concentrations of fecal indicator bacteria strongly varied depending on the prevalent meteorological conditions. That is, during the dry daytime, fecal indicator bacteria concentrations decreased due to inactivation from solar irradiation, but rapidly increased in the absence of sunlight, suggesting external source inputs. In addition, bacterial concentrations substantially increased during rainfall events, due probably to a major contribution from combined sewer overflow. The observations in this study can be useful for implementing fecal pollution management strategies and for predicting fecal contamination as a function of meteorological conditions. [ABSTRACT FROM AUTHOR]

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 "Bacteroidales" 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution. [ABSTRACT FROM AUTHOR]

The genetic characteristics among Escherichia coli strains can be grouped by origin of isolation. Then, it is possible to use the genotypes as a tool to determine the source of water contamination. The aim of this study was to define water aptitude for human consumption in a rural basin and to assess the diversity of E. coli water populations. Thus, it was possible to identify the main sources of fecal contamination and to explore linkages with the hydrogeological environment and land uses. The bacteriological analysis showed that more than 50% of samples were unfit for human consumption. DNA fingerprinting analysis by BOX-PCR indicated low genotypic diversity of E. coli isolates taken from surface water and groundwater. The results suggested the presence of a dominant source of fecal contamination. The relationship between low genotypic diversity and land use would prove that water contamination comes from livestock. The genetic diversity of E. coli isolated from surface water was less than that identified in groundwater because of the different hydraulic features of both environments. Furthermore, each one of the two big strain groups identified in this basin is located in different sub-basins, showing that hydrological dynamics exerts selective pressure on bacteria DNA. [ABSTRACT FROM AUTHOR]

The presence of the hepatitis A virus was studied in spring and river water samples, and heterotrophic plate count, total and fecal coliforms, fecal streptococci, and sulfite-reducing Clostridium were analyzed, in addition to physical-chemical indicators of fecal contamination (conductivity, chemical oxygen demand, nitrate, nitrite, ammonia, phosphate and soluble proteins). The virus was concentrated by means of the aluminium hydroxide adsorption-precipitation method. Following the extraction of RNA, reverse transcriptase PCR, semi-nested PCR and hybridization were performed. No hepatitis A virus was detected in any of the spring water samples, yet 69.6% of the river water samples were positive. There were no significant differences in the mean values of the microbiological indicators when the positive and the negative river samples were compared, which confirms that there is no quantitative relationship between microbiological indicators of fecal contamination and the presence of hepatitis A virus. This was confirmed through a spatial analysis of the river water samples: the point of wastewater discharge was reflected in a high percentage of hepatitis A virus positive samples, regardless of the microbiological indicators. Among the physical-chemical indicators, only conductivity and chemical oxygen demand were significantly correlated with all microbiological indicators. Nitrate and nitrite show limited value as indicators of fecal contamination, and ammonia, phosphate and soluble proteins correlated best with heterotrophic plate count, total coliforms, fecal coliforms and fecal streptococci, which are microbiological indicators of short-term fecal pollution. [ABSTRACT FROM AUTHOR]

Health risks associated with sewage-contaminated recreational waters are of important public health concern. Reliable water monitoring systems are therefore crucial. Current recreational water quality criteria rely predominantly on the enumeration of bacterial indicators, while potentially dangerous viral pathogens often remain undetected. Human enteric viruses have been proposed as alternative indicators; however, their detection is often hindered by low viral concentrations present in the environment. Reported here are novel and effective laboratory protocols for viral concentration and highly sensitive and optimized RT-PCR for the efficient detection of enteroviruses, an important enteric virus subset, in Hawaiian environmental waters. Eighteen published enterovirus primer pairs were comparatively evaluated for detection sensitivity. The primer set exhibiting the lowest detection limit under optimized conditions, EQ-1/EQ-2, was validated in a field survey of 22 recreational bodies of water located around the island of Oahu, Hawaii. Eleven sites tested positive for enterovirus, indicating fecal contamination at these locations. As an additional means of viral concentration, shellfish were collected from 9 sample sites and subjected to dissection, RNA extraction, and subsequent RT-PCR. Shellfish tissue from 6 of 9 sites tested positive for enterovirus. The techniques implemented here are valuable resources to aid accurate reflection of microbial contamination in Hawaii's environmental waters. [ABSTRACT FROM AUTHOR]

Human faecal contamination poses a widespread hazard for human health. In urban areas, sewer leakage may be an important cause of faecal pollution to surface water. Faecal indicator bacteria (FIB) are the most widely used indicators to monitor surface water quality. However, assessing whether a water body is meeting water quality criteria is made difficult by the high variability of FIB concentrations over time. In this study, the variation of FIB concentration in surface water from tropical urban catchments is investigated. Eleven urban sub-catchments were sampled hourly over 24-hr and samples analysed for FIB. It was found that FIB show a diurnal pattern that is characterised by daytime FIB concentrations that are significantly higher than nighttime FIB concentrations. This observed diurnal variation of FIB closely follows that of sewer flows and contrasts with observations in rural streams where FIB concentrations are known to be low in the daytime and high during the night. Field tracer tests provide qualitative evidence of sewage exfiltration and transport to drains via preferential flow paths. The diurnal FIB variation and field tracer tests indicate the likelihood of surface water contamination due to leaking sewers. The results further suggest that contamination of surface-water drains is likely a widespread problem in tropical urban areas due to extensive drainage networks and the persistence of FIB under tropical conditions. Because of FIB variation over time, the time at which samples are collected is important in being able to capture the daily maximum and minimum FIB concentrations. The Kruskal–Wallis test shows that hourly sampling from 04:00 to 07:00 and from 12:00 to 15:00 results in significantly different FIB concentration (minimum and maximum, respectively). Furthermore, the Wilcoxon-Mann-Whitney test shows that sampling at 12:00 and 14:00 results in significantly higher FIB concentrations, while sampling at 05:00 and 04:00 or 05:00 and 06:00 results in significantly lower FIB concentrations, than sampling at other hours of the day. [ABSTRACT FROM AUTHOR]

Hepatitis A virus (HAV) strains found in selected South African (SA) surface waters were characterised to establish what HAV types are circulating in the environment, thus reflecting circulation in the surrounding communities. Surface water samples used for irrigation or domestic purposes, and water samples from the outflow of wastewater plants were collected from six provinces. Viruses were recovered from the samples using a glass wool adsorption-elution method and then further concentrated using polyethylene glycol/sodium chloride precipitation. After automated nucleic acid extraction, samples were analysed for HAV by real-time reverse-transcriptase polymerase chain reaction. HAV strains were genotyped by nucleotide sequence analysis of the capsid gene VP1 and the VP1/P2B junction. HAVs were detected in 76% (16/21) of the surface water samples and in 37% (19/51) of the samples from the wastewater plants. Strains were characterised from 32 of the 35 samples and classified within genotype IB. The presence of genotype IB in the water sources confirms human faecal contamination. Hence, these faecally-contaminated water sources may be a potential transmission route of HAV infection and a potential source of contamination of irrigated fresh produce in SA. [ABSTRACT FROM AUTHOR]

The risk to human health of the annual sandhill crane (Grus canadensis) migration through Nebraska, which is thought to be a major source of fecal pollution of the central Platte River, is unknown. To better understand potential risks, the presence of Campylobacter species and three fecal indicator bacterial groups (Enterococcus spp., Escherichia coli, and Bacteroidetes) was assayed by PCR from crane excreta and water samples collected during their stopover at the Platte River, Nebraska, in 2010. Genus-specific PCR assays and sequence analyses identified Campylobacter jejuni as the predominant Campylobacter species in sandhill crane excreta. Campylobacter spp. were detected in 48% of crane excreta, 24% of water samples, and 11% of sediment samples. The estimated densities ofEnterococcus spp. were highest in excreta samples (mean, 4.6 × 108 cell equivalents [CE]/g), while water samples contained higher levels of Bacteroidetes(mean, 5.1 × 105 CE/100 ml). Enterococcus spp., E. coli, and Campylobacter spp. were significantly increased in river water and sediments during the crane migration period, with Enterococcus sp. densities (∼3.3 × 105 CE/g) 2 to 4 orders of magnitude higher than those of Bacteroidetes (4.9 × 10³ CE/g), E. coli (2.2 × 10³ CE/g), and Campylobacter spp. (37 CE/g). Sequencing data for the 16S rRNA gene and Campylobacter species-specific PCR assays indicated that C. jejuni was the major Campylobacter species present in water, sediments, and crane excreta. Overall, migration appeared to result in a significant, but temporary, change in water quality in spring, when there may be a C. jejuni health hazard associated with water and crops visited by the migrating birds. [ABSTRACT FROM AUTHOR]

Fecal contaminations of inland and coastal waters induce risks to human health and economic losses. To improve water management, specific markers have been developed to differentiate between sources of contamination. This study investigates the relative decay of fecal indicator bacteria (FIB, Escherichia coli and enterococci) and six human-associated markers (two bacterial markers: Bacteroidales HF183 (HF183) and Bifidobacterium adolescentis (BifAd); one viral marker: genogroup II F-specific RNA bacteriophages (FRNAPH II); three chemical markers: caffeine and two fecal stanol ratios) in freshwater and seawater microcosms seeded with human wastewater. These experiments were performed in darkness, at 20 °C and under aerobic conditions. The modeling of the decay curves allows us (i) to compare FIB and markers and (ii) to classify markers according to their persistence in seawater (FRNAPH II < HF183, stanol ratios < BifAd, caffeine) and in freshwater (HF183, stanol ratios < FRNAPH II < BifAd < caffeine). Although those results depend on the experimental conditions, this study represents a necessary step to develop and validate an interdisciplinary toolbox for the investigation of the sources of fecal contaminations. [ABSTRACT FROM AUTHOR]

The current water quality monitoring and regulation approaches use fecal indicator bacteria (FIB) to indirectly assess health risks from fecal pathogens. Direct detection of waterborne pathogens is expected to provide more accurate and comprehensive risk assessment, which however has been hindered by the lack of methods for simultaneous detection of the numerous waterborne pathogens. This study aimed to develop a mPCR-NGS approach that uses the high sequencing depth of NGS and sequence-based detection to significantly increase the multiplex level of mPCR for direct pathogen detection in water. Individual PCR primers were designed for 16 target marker genes of nine different bacterial pathogens, and an optimal combination of primers with least primer complementarities was identified for the multiplex setting. Using an artificial tester sample, the mPCR system was optimized for annealing temperature and primer concentration, and bioinformatic procedures were developed to directly detect the target marker gene amplicons in NGS sequence reads, which showed simultaneous detection of 14 different target genes in one reaction. The effectiveness of the developed mPCR-NGS approach was subsequently demonstrated on DNA extracts from stream water samples and their counterparts that were spiked with various target pathogen DNA, and all target genes spiked into the environmental water samples were successfully detected. Several key issues for further improving the mPCR-NGS approach were also identified and discussed. [ABSTRACT FROM AUTHOR]

The article offers information on the study that focuses on the source tracking faecal contamination in an urbanized and a rural waterway in the Nelson-Tasman region, New Zealand. It offers an outline of the methods used in the research including marker validation, and field microbial source tracking (MST) study.

The decay of fecal bacteria in surface water often follows a biphasic pattern with the apparent first-order rate constant relatively high during a first phase and lower in a second one. This could be the result of population heterogeneity in resistance due to various mechanisms (different strains, genetically or nongenetically differentiated cells, different growth or cell cycle stage, clumping, hardening), or the specific decay rate could be directly or indirectly affected by the cell density (e.g., quorum sensing). All these mechanisms can theoretically produce a biphasic decay pattern and are consistent with the literature. However, they are fundamentally different and lead to different behavior of mechanistic total maximum daily load models, so identifying the correct mechanism is important. This technical note presents the results of a study aimed at determining if a density effect is involved. Laboratory decay experiments with pure strain Escherichia coli cells in phosphate buffer were conducted over a range of initial densities. The results show that the rate constant changes after a certain time, rather than at a certain density, which is inconsistent with a density effect. As the experiments were performed with a pure strain, the resistant fraction cannot be attributed to a different strain. Further research is needed to identify the mechanism responsible for the population heterogeneity. [ABSTRACT FROM AUTHOR]

The four subgroups of F-specific RNA bacteriophages (I–IV) have been proposed as potential tracers for faecal source tracking. Groups II and III predominate in human sources while groups I and IV are most abundant in animal sources. The four subgroups of naturally occurring F-specific RNA bacteriophages were identified in different samples by plaque hybridization with genotype-specific probes and the persistence of each subgroup was evaluated. The proportions of the F-specific RNA bacteriophage subgroups were measured in wastewaters, after inactivation in surface waters or after wastewater treatment and in mixtures of wastewater of human and animal origin. Our results indicate that phage groups differ in their persistence in the environment and to different disinfecting treatments. The greater survival of subgroups I and II in treated samples hinders the interpretation of results obtained with F-specific RNA bacteriophages. The phages of subgroups III and IV were the least resistant to all treatments. These results should be considered when using genotypes of F-specific RNA as sole tracers for faecal source tracking. [Copyright &y& Elsevier]

As the introduction and promotion of dehydrating toilets progresses, the safety of handling and reuse of their biosolids remains a question. A detailed study to understand the storage conditions and the fate of selected faecal indicators was conducted on four urine diverting dehydrating toilet units, using ash as a major additive, in Kathmandu Valley, Nepal. Presumptive Escherichia coli, total coliforms, enterococci and different fractions of Clostridium perfringens were investigated under field storage conditions. In addition, chemo-physical and chemical (carbon, nitrogen, phosphorous content) parameters were investigated. Observed temperature was low in all the four toilets with a median of 24.0°C, which was in the same range as the ambient temperature. pH was below the desired range of .9 and moisture level was very high (>60%). No single factor of the studied chemo-physical and chemical parameters could be found by statistical analysis to have accounted for the reduction of the indicators in any of the toilets. By time series analysis of the investigated strata in the faecal heaps (n = 96), the determined reduction rate showed increasing persistence characteristics for E. coli, coliforms and enterococci with respective average log10 reduction of -0.4, -0.3 and -0.2 per month ( p < 0.001). No significant reduction was observed for the different fractions of C. perfringens determined for the non-pasteurised and pasteurised fraction at 60°C and 85°C. 72% of randomly selected and analysed samples (n = 36) were found to contain helminthes eggs. The used 6 months storage time did not prove sufficient to reach appropriate safety levels for handling and reuse of the biosolids. [ABSTRACT FROM AUTHOR]

Quantitative PCR (qPCR) assays targeting the host-specific Bacteroides- Prevotella 16S rRNA genetic markers have been proposed as one of the promising approaches to identify the source of fecal contamination in environmental waters. One of the concerns of qPCR assays to environmental samples is the reliability of quantified values, since DNA extraction followed by qPCR assays are usually performed without appropriate sample process control (SPC) and internal amplification controls (IACs). To check the errors in sample processing and improve the reliability of qPCR results, it is essential to evaluate the DNA recovery efficiency and PCR amplification efficiency of the target genetic markers and correct the measurement results. In this study, we constructed a genetically-engineered Escherichia coli K12 strain (designated as strain MG1655 Δlac::kan) as sample process control and evaluated the applicability to environmental water samples. The recovery efficiency of the SPC strain MG1655 Δlac::kan was similar to that of Bacteroides fragilis JCM 11019, when DNA were extracted from water samples spiked with the two bacteria. Furthermore, the SPC was included in the qPCR assays with propidium monoazide (PMA) treatment, which can exclude the genetic markers from dead cells. No significant DNA loss was observed in the PMA treatment. The inclusion of both the SPC (strain MG1655 Δlac::kan) and IAC in qPCR assays with PMA treatment gave the assurance of reliable results of host-specific Bacteroides- Prevotella 16S rRNA genetic markers in environmental water samples. [ABSTRACT FROM AUTHOR]

Using a combination of chemical and microbiological (culture-dependent and -independent) approaches, sources of human faecal contamination were identified in two water reservoirs in Puerto Rico – Guajataca and La Plata. Fluorescence from optical brighteners (OB) – commonly found in laundry detergents – was used as an indicator of contamination from septic systems and other household discharges. Traditional indicators of faecal contamination (e.g. Escherichia coli; faecal enterococci) were enumerated, and human faecal contamination was confirmed through detection of Bifidobacterium adolescentis utilizing polymerase chain reaction (PCR)-based analyses. For Guajataca Reservoir, four of 19 sampling sites (21%) were positive for the presence of B. adolescentis under baseflow conditions. The OB fluorescence data suggested that the most likely source for three of these sites was residential, whereas the source of contamination at the remaining site, although of human origin, was probably non-residential. B. adolescentis was present in 83% (19 of 23) of the sampling sites in La Plata. The La Plata sources were more difficult to identify because samples were taken under stormflow conditions, although the presence of OB fluorescence suggested a residential origin in a number of instances. OB fluorescence and traditional bacterial indicators of faecal contamination produced a number of false positive and negative findings for both reservoirs, pointing to the importance of understanding the limitations of these tools for tropical freshwater systems. The results of this study should be useful in developing a weight-of-evidence approach for the identification of potential sources and extent of human faecal contamination in similar tropical reservoirs, a necessary step in the development of management plans to reduce or eliminate these sources. [ABSTRACT FROM AUTHOR]

We investigated the presence of Arcobacter spp. in 205 water samples of freshwater, seawater and sewage in Spain. These bacteria were present in 55.1% of the samples (113/205) and were significantly associated for the first time with bacterial indicators of fecal pollution. The dominant species in the positive samples was Arcobacter butzleri (94%) followed by Arcobacter cryaerophilus (30%) and Arcobacter skirrowii (1.8%). [ABSTRACT FROM AUTHOR]

Aims: The study aim was to determine the presence of total and faecal coliforms on kitchen surfaces, in tap water and on the hands of caregivers in households on both sides of the US–Mexico border. Methods and Results: Samples were collected in 135 randomly selected households in Ciudad Juarez, Mexico, and El Paso, Texas. Different surfaces throughout the kitchen and head of households’ hands were sampled using sterile cotton swabs moistened in D/E neutralizing solution. Sponge/dishcloth and drinking water samples were also obtained. Total and faecal coliforms were enumerated on m-Endo LES and mFC respectively. Total coliforms and Escherichia coli in drinking water samples were enumerated in accordance with the Quanti-TrayTM method. Sponge/dishcloth samples were the most commonly contaminated kitchen sites, followed by countertops and cutting boards. We recovered faecal coliforms from 14% of the hands of child caregivers, and this indicator was moderately associated with self-reported failure to wash hands after using the toilet (OR = 3·2; 95% CI: 0·9, 11·1). Conclusions: Hand washing should continue to be emphasized, and additional interventions should be directed to specific kitchen areas, such as sponges/dishcloths, tables/countertops and cutting boards. Significance and Impact of the Study: There is a need for additional interventions regarding kitchen sanitation. [ABSTRACT FROM AUTHOR]

Library-based microbial source tracking (MST) can assist in reducing or eliminating fecal pollution in waters by predicting sources of fecal-associated bacteria. Library-based MST relies on an assembly of genetic or phenotypic "fingerprints" from pollution-indicative bacteria cultivated from known sources to compare with and identify fingerprints of unknown origin. The success of the library-based approach depends on how well each source candidate is represented in the library and which statistical algorithm or matching criterion is used to match unknowns. Because known source libraries are often built based on convenience or cost, some library sources may contain more representation than others. Depending on the statistical algorithm or matching criteria, predictions may become severely biased toward classifying unknowns into the library's dominant source category. We examined prediction bias for four of the most commonly used statistical matching algorithms in library-based MST when applied to disproportionately-represented known source libraries; maximum similarity (MS), average similarity (AS), discriminant analyses (DA), and k-means nearest neighbor (k-NN). MS was particularly sensitive to disproportionate source representation. AS and DA were more robust. k-NN provided a compromise between correct prediction and sensitivity to disproportional libraries including increased matching success and stability that should be considered when matching to disproportionally-represented libraries. [ABSTRACT FROM AUTHOR]

The two-dimensional finite-volume model ‘Hemat’ was refined to model the pathways, fate and decay factors of faecal indicator organisms for an integrated river and coastal basin water system. The numerical model was developed, calibrated and tested by modelling faecal indicator organism inputs from river sources. A comprehensive field data set was available including intensive offshore survey data collected during a two-week period; these were used as a time series for the verification of the hydrodynamic model. River discharges and water quality data were used as boundary and initial conditions for the model. The impact of alternative land-use management strategies on the water quality of the receiving estuarine and coastal waters was investigated by modelling a series of 14 scenarios. Generally, the model results show that the faecal indicator organism input from point sources of sewage effluent dominated during dry weather and base flow conditions. On the other hand, riverine sources (such as diffuse catchment sources) significantly dominated the concentrations in the bay both during and after rainfall events. Finally, it was found that a reduction in land used for animal husbandry can considerably reduce the critical peak concentration occurring after heavy rainfall events. [ABSTRACT FROM AUTHOR]

Though coliform bacteria are used worldwide to indicate fecal pollution of groundwater, the parameters determining the transport of Escherichia coli in aquifers are relatively unknown. We evaluated the occurrence of both straining and attachment of E. coli ATCC25922 in columns of ultra-pure, angular, saturated quartz sand. The column experiments were conducted over a wide range of porous medium sizes, column heights, input concentrations, and pore water flow velocities. Straining and attachment were examined by modelling the breakthrough curves (with HYDRUS 1D). In addition, model output was compared with measured strained and attached bacteria via column extrusion experiments (in which sand was extruded from the column and placed in excess water) and flow reversal experiments (in which the pore water flow direction was reversed, thereby dislodging strained bacteria). Our model consisted of an attachment rate coefficient and a straining rate coefficient; both of these decreased with transport distance. The straining rate coefficient also decreased in a Langmuirian way, in response to the filling of available pore space, which in turn depended on influent bacteria concentration, quartz grain diameter, and transport distance. The maximum strained fraction was 25–30% of total bacteria mass applied to the column; the maximum attached fraction was 30–35%. The fit between modelled and measured (strained and attached) bacteria masses was acceptable, as was the sensitivity of the model output to fitted parameter values. Our results lead to a new description for the time-dependent mass balance of strained bacteria, which entails using three fitting parameters. The results also imply that column experiments in combination with retention profiles (or various column lengths) are not enough to explain the retention processes in a column. Column extrusion and flow reversal experiments provide vital additional information on the occurrence and magnitude of straining. [Copyright &y& Elsevier]

We report the design and validation of new TaqMan® assays for microbial source tracking based on the amplification of fecal 16S rRNA marker sequences from uncultured cells of the order Bacteroidales. The assays were developed for the detection and enumeration of non-point source input of fecal pollution to watersheds. The quantitative “universal”Bacteroidales assay BacUni-UCD detected all tested stool samples from human volunteers (18 out of 18), cat (7 out of 7), dog (8 out of 8), seagull (10/10), cow (8/8), horse (8/8), and wastewater effluent (14/14). The human assay BacHum-UCD discriminated fully between human and cow stool samples but did not detect all stool samples from human volunteers (12/18). In addition, there was 12.5% detection of dog stool (1/8), but no cross-reactivity with cat, horse, or seagull fecal samples. In contrast, all wastewater samples were positive for the BacHum-UCD marker, supporting its designation as 100% sensitive for mixed-human source identification. The cow-specific assay BacCow-UCD fully discriminated between cow and human stool samples. There was 38% detection of horse stool (3/8), but no cross-specificity with any of the other animal stool samples tested. The dog assay BacCan-UCD discriminated fully between dog and cow stool or seagull guano samples and detected 62.5% stool samples from dogs (5/8). There was some cross-reactivity with 22.2% detection of human stool (4/18), 14.3% detection of cat stool (1/7), and 28.6% detection of wastewater samples (4/14). After validation using stool samples, single-blind tests were used to further demonstrate the efficacy of the developed markers; all assays were sensitive, reproducible, and accurate in the quantification of mixed fecal sources present in aqueous samples. Finally, the new assays were compared with previously published sequences, which showed the new methodologies to be more specific and sensitive. Using Bayes’ Theorem, we calculated the conditional probability that the four assays would correctly identify general and host-specific fecal pollution in a specific watershed in California for which 73 water samples had been analyzed. Such an approach allows for a direct comparison of the efficacy of different MST methods, including those based on library-dependent methodologies. For the universal marker BacUni-UCD, the probability that fecal pollution is present when the marker is detected was 1.00; the probability that host-specific pollution is present was 0.98, 0.84, and 0.89 for the human assay HF160F, the cow assay BacCow-UCD, and the dog assay BacCan-UCD, respectively. The application of these markers should provide meaningful information to assist with efforts to identify and control sources of fecal pollution to impaired watersheds. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Detection of the faecal pollution contribution from wildfowl is an important adjunct in determining the sources of faecal pollution in waterways. This is particularly true, where human waste and other animal faecal sources have been eliminated as the pollution source. A polymerase chain reaction (PCR) marker was developed as a duck-specific marker of faecal pollution. The semi-nested primer system targeted an unknown bacterium (E2) isolated from mallard ducks. E2 had the closest 16S rRNA sequence similarity to members of the Desulfovibrio genus, which was further confirmed by phenotypic characterisation of the bacterium. Testing of the prevalence of E2 identified the marker in 76% of duck faecal samples (n=42), 20% of swan faecal samples (n=10) and 15% of Canada geese faecal samples (n=20). It was also identified in the faeces of two out of 15 domestic goats (13%). The marker was not detected in any samples derived from human faeces or effluent, dairy cows or sheep. It is proposed that this PCR marker would be useful in conjunction with faecal taxation indicators in the determination of pollution derived from duck faecal inputs in waterways. [Copyright &y& Elsevier]

Advances in microbial source tracking (MST) have largely been driven by the need to comply with water quality standards based on traditional indicator bacteria. Recently, a number of culture-independent, and library-independent methods based on polymerase chain reaction (PCR) have been gaining popularity among source trackers. However, only a limited number of these methods have been successfully used in field applications, primarily due to the fact that many of them are still being developed. In this critical outlook, we examine different viewpoints associated with the practical use of MST to identify critical research gaps, propose a priority-based timeline to address them, and outline emerging technologies that will likely impact the future of source tracking. We propose that it is necessary to consider each of these aspects in order to advance towards a unifying framework in source identification, so that fecal pollution monitoring can be reliably used for comprehensive environmental microbial monitoring, to develop risk assessment models, and to implement and validate adequate management practices. [Copyright &y& Elsevier]