Human Fecal Contamination Research Articles

Sustainable microbial water quality monitoring programme design using phage-lysis and multivariate techniques

Contamination of surface waters is a pervasive threat to human health, hence, the need to better understand the sources and spatio-temporal variations of contaminants within river catchments. River catchment managers are required to sustainably monitor and manage the quality of surface waters. Catchment managers therefore need cost-effective low-cost long-term sustainable water quality monitoring and management designs to proactively protect public health and aquatic ecosystems. Multivariate and phage-lysis techniques were used to investigate spatio-temporal variations of water quality, main polluting chemophysical and microbial parameters, faecal micro-organisms sources, and to establish ‘sentry’ sampling sites in the Ouse River catchment, southeast England, UK. 350 river water samples were analysed for fourteen chemophysical and microbial water quality parameters in conjunction with the novel human-specific phages of Bacteroides GB-124 (Bacteroides GB-124). Annual, autumn, spring, summer, and winter principal components (PCs) explained approximately 54%, 75%, 62%, 48%, and 60%, respectively, of the total variance present in the datasets. Significant loadings of Escherichia coli, intestinal enterococci, turbidity, and human-specific Bacteroides GB-124 were observed in all datasets. Cluster analysis successfully grouped sampling sites into five clusters. Importantly, multivariate and phage-lysis techniques were useful in determining the sources and spatial extent of water contamination in the catchment. Though human faecal contamination was significant during dry periods, the main source of contamination was non-human. Bacteroides GB-124 could potentially be used for catchment routine microbial water quality monitoring. For a cost-effective low-cost long-term sustainable water quality monitoring design, E. coli or intestinal enterococci, turbidity, and Bacteroides GB-124 should be monitored all-year round in this river catchment.

Determination of the prevalence of synthesis and/or absorption of cholesterol in a Portuguese population: Preliminary study in volunteer healthy students from Coimbra University

The effects of agricultural activities on stream water quality were assessed by nitrogen analysis, further investigated by gas chromatography mass spectrometry (GC-MS) sterol analysis (including chemometric analysis), and characterized by bacterial source tracking (BST). Surface water samples were collected from five sites, throughout the agriculturally-influenced Nathan Creek watershed, British Columbia, Canada and a nearby control site between October 2005 and March 2006. From a total of 48 samples, Canadian Water Quality Guidelines were exceeded nineteen times for nitrate (NO3−; guideline value: 2.94 mg/L N) and four times for un-ionized ammonia (NH3; guideline value 0.019 mg/L N). Gas chromatography mass spectrometry single ion monitoring (GC-MS SIM) analysis of 18 sterols showed that five fecal sterols (coprostanol, episoprostanol, cholesterol, cholestanol, desmosterol) were detected at all sites except the control site (where only cholesterol, cholestanol and desmosterol were detected). Three phytosterols (campesterol, stigmasterol and β-sitosterol) were also detected at all sites while the hormone estrone was present at one site on two occasions at concentrations of 0.01 and 0.04 μg/L. Chemometric analysis (principal component analysis and cluster analysis) grouped sites based on their similarities in sterol composition. Analysis of ten sterol ratios (seven for identifying human fecal contamination and four for differentiating sources of fecal contamination) showed multiple instances of human and animal contamination for every site but the control site. Application of a Bacteroides-BST method confirmed contamination from ruminant animals, pigs and dogs in varying combinations at all impact sites. Together, these results confirmed the impact of agricultural activities on the Nathan Creek watershed and support a need for better land management practices to protect water quality and aquatic life.

Sewage Treatment Plants Efficiencies in Removal of Sterols and Sterol Ratios as Indicators of Fecal Contamination Sources

This study assessed the efficiency of sewage treatment plants (STPs) in removing sterols based on chemical analyses of both influents and effluents. Samples from 3s and three tertiary plants were collected and analyzed by gas chromatography mass spectrometry for 23 individual sterols including mestranol, norethindrone, equol, estrone, equilin, norgestrel, 17α-ethinylestradiol, 17α-estradiol, 17β-estradiol, estriol, dihydrocholesterol (cholestanol), coprostanol, epicoprostanol, cholesterol, desmosterol, campesterol, stigmasterol, β-sitosterol, coprostanone, cholestanone, epicholestanol, stigmastanol, and 24-ethylcoprostanol. The percentage of sterols remaining in effluent samples (compared to influent samples) ranged from 0% to 80% and varied among sterol compounds and with STP location and treatment type. Differences in the efficiency of sterol removal for secondary and tertiary STPs were statistically significant. Although the concentration of sterol compounds differed between influents and effluents, sterol abundances remained the same. The most abundant sterol detected was cholesterol, followed by the fecal sterol coprostanol, and the plant sterols 24-ethylcoprostanol and β-sitosterol. For three STPs, the hormone estrone was detected in effluents at concentrations of 0.03–0.05 μg L−1. Ten sterol ratios specific for human fecal contamination and eight sterol ratios for differentiating among multiple sources of fecal contamination were calculated and showed that 12 ratios for influent and nine ratios for effluent were successful for human fecal source tracking. Based on sterol ratio values in this study, new criteria for identification of human fecal contamination were suggested.

Lachnospiraceae and Bacteroidales Alternative Fecal Indicators Reveal Chronic Human Sewage Contamination in an Urban Harbor

The complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within the Lachnospiraceae family, which was closely related to the genus Blautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for human Bacteroidales (based on the HF183 genetic marker), total Bacteroidales spp., and enterococci and the conventional Escherichia coli and enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and human Bacteroidales increased specificity to detect sewage compared to general indicators, and the relationship to a human pathogen group suggests that the use of these alternative indicators will improve assessments for human health risks in urban waters.

Comparison of the Host Specificities of Two Bacteroidales Quantitative PCR Assays Used for Tracking Human Fecal Contamination

The sewage-associated real-time quantitative PCR (qPCR) assays BacHum and HF183 SYBR were compared for specificity against local fecal sources. Both assays were equally sensitive to sewage, but BacHum showed substantially more false-positive results for cat, dog, gull, and raccoon feces.

Occurrence of water-borne enteric viruses in two settlements based in Eastern Chad: analysis of hepatitis E virus, hepatitis A virus and human adenovirus in water sources

Hepatitis E virus (HEV) is a common cause of water-borne acute hepatitis in areas with poor sanitation. In 2004 an outbreak of HEV infection affected around 2,000 people in Eastern Chad (Dar Sila). This paper describes the decrease in the incidence of acute jaundice syndrome (AJS) from 2004 until 2009 when a mean incidence of 0.48 cases/1,000 people/year was recorded in the region. Outbreaks of AJS were identified in some of the camps in 2007 and 2008. Moreover, water samples from drinking water sources were screened for human adenoviruses considered as viral indicators and for hepatitis A virus and HEV. Screening of faecal samples from donkeys for HEV gave negative results. Some of the samples were also analysed for faecal coliforms showing values before disinfection treatment between 3 and >50 colony forming units per 100 mL. All water samples tested were negative for HEV and HAV; however, the presence of low levels of human adenoviruses in 4 out of 16 samples analysed indicates possible human faecal contamination of groundwater. Consequently, breakdowns in the treatment of drinking water and/or increased excretion of hepatitis viruses, which could be related to the arrival of a new population, could spread future outbreaks through drinking water.

Quantification of Pathogens and Sources of Microbial Indicators for QMRA in Recreational Waters

This study has increased knowledge about relationships between pathogen indicators, source identifiers and pathogens to support Quantitative Microbial Risk Assessment (QMRA) efforts and the implementation of revised recreational water quality criteria. Data gaps pertaining to waterborne pathogens and indicators in fecally-impacted discharges to recreational waters were identified and filled by targeted monitoring campaigns in three geographic regions. Study design involved simultaneous detection of pathogens in water samples using a combination of genetic, culture- and microscopy-based methodologies for Salmonella, Campylobacter jejuni, Vibrio cholerae, Cryptosporidium, Giardia, Toxoplasma gondii, adenoviruses, enteroviruses, noroviruses, rotaviruses, Bacteroidales, Enterococcus, and Escherichia coli. The project team worked closely with experts and stakeholder groups in order to advance the understanding and applicability of QMRA for risk management purposes. QMRA analyses revealed Norovirus as the most dominant health risk followed by rotavirus. Norovirus and Enterococcus both had significant correlations with a number of pathogens in discharges. Using qPCR data on the fecal source identifier Bacteroidales a new model can predict the true amount of human fecal contamination in a water sample by relating a human-associated genetic marker to a universal assay for fecal sources. The model output can then be used to implement and evaluate management options intended to restore microbial water quality. This title belongs to WERF Research Report Series . ISBN: 9781843395430 (eBook)

Evaluation of microbiological water quality in the Pettaquamscutt River (Rhode Island, USA) using chemical, molecular and culture-dependent methods

We evaluated microbiological water quality in the Pettaquamscutt River (Rhode Island, USA), an estuarine river. Fecal coliform (FC) and enterococci (FE) bacteria, presence of Bifidobacterium adolescentis DNA (indicating human fecal contamination), and optical brightener (OB) fluorescence (associated with laundry detergents) were determined for 14 stations from May to September 2010. Six stations had high counts of FE and FC, and the presence of B. adolescentis DNA and high OB fluorescence indicated human fecal contamination – four had septic systems as likely sources of contamination; the others were in sewered areas. The ability of FC and FE to indicate human fecal contamination was assessed against a positive B. adolescentis test. FC and FE had false positive rates of 25% and 17%, respectively, and false negatives of 44% for FC and 63% for FE. Inclusion of molecular and chemical indicators should improve tracking of human fecal contamination sources in the river.

Source tracking faecal contamination in an urbanised and a rural waterway in the Nelson-Tasman region, New Zealand

Microbial contamination of New Zealand's rivers, lakes and coastal waters can pose a risk to human health through both recreational contact and the consumption of contaminated shellfish. Microbial source tracking (MST) methods provide a means to identify potential contaminant sources, which can lead to high faecal indicator bacteria concentrations and elevated human health risk because of associated pathogens. Eight MST markers, including general, ruminant and human-associated Bacteroidales markers, a duck-associated E2 marker, a gull-associated Catellicoccus marimammalium marker and three additional human markers [Enterococcus faecium esp gene, Methanobrevibacter smithii nifH gene, and human polyomaviruses (HPyVs)] were tested for host specificity and sensitivity using an array of animal faecal samples of known origin and wastewater samples. The cross-reactivity identified for some of the markers, although limited, signals a need to validate overseas markers further in New Zealand before employing them in field studies. Application of MST markers on water samples collected from an urbanised section of the Maitai River (Nelson, New Zealand) identified the presence of faecal contamination originating from humans, ruminants and birds. Human faecal contamination was present in the lower section of the Matai River, and in stormwater drains entering the river, in association with elevated faecal coliform concentrations. Application of the markers to the rural Little Sydney Stream (near Motueka, Tasman district, NZ) identified faecal contamination derived from ruminants, which is consistent with the agricultural (pasture) use of the catchment. This study indicates that some of the markers developed overseas can be used effectively to track sources of microbial contamination in New Zealand watersheds.

Effective detection of human adenovirus in hawaiian waters using enhanced pcr methods

BackgroundThe current criteria for recreational water quality evaluation are primarily based on measurements of fecal indicator bacteria growth. However, these criteria often fail to predict the presence of waterborne human pathogenic viruses. To explore the possibility of direct use of human enteric viruses as improved human fecal contamination indicators, human adenovirus (HAdV) was tested as a model in this study.FindingsIn order to establish a highly sensitive protocol for effective detection of HAdV in aquatic environments, sixteen published PCR primer sets were re-optimized and comparatively evaluated. Primer sets nehex3deg/nehex4deg, ADV-F/ADV-R, and nested PCR primer sets hex1deg/hex2deg and nehex3deg/nehex4deg were identified to be the most sensitive ones, with up to 1,000 fold higher detection sensitivity compared to other published assays. These three PCR protocols were successfully employed to detect HAdV in both treated and untreated urban wastewaters, and also in 6 of 16 recreational water samples collected around the island of Oahu, Hawaii.ConclusionsFindings from this study support the possible use of enteric viruses for aquatic environmental monitoring, specifically for the essential routine monitoring of Hawaiian beach waters using the optimized PCR protocol to detect HAdV at certain water sites to ensure a safe use of recreational waters.

Novel Bacteroides host strains for detection of human- and animal-specific bacteriophages in water

Bacteriophages active against specific Bacteroides host strains were shown to be suitable for detection of human faecal pollution. However, the practical application of this finding is limited because some specific host strains were restricted to certain geographic regions. In this study, novel Bacteroides host strains were isolated that discriminate human and animal faecal pollution in Switzerland. Two strains specific for bacteriophages present in human faecal contamination and three strains specific for bacteriophages indicating animal faecal contamination were evaluated. Bacteriophages infecting human strains were exclusively found in human wastewater, whereas animal strains detected bacteriophages only in animal waste. The newly isolated host strains could be used to determine the source of surface and spring water faecal contamination in field situations. Applying the newly isolated host Bacteroides thetaiotaomicron ARABA 84 for detection of bacteriophages allowed the detection of human faecal contamination in spring water.

Characterization of Enterococcus faecalis-infecting phages (enterophages) as markers of human fecal pollution in recreational waters

Enterophages are a novel group of phages that specifically infect Enterococcus faecalis and have been recently isolated from environmental water samples. Although enterophages have not been conclusively linked to human fecal pollution, we are currently characterizing enterophages to propose them as viral indicators and possible surrogates of enteric viruses in recreational waters. Little is known about the morphological or genetic diversity which will have an impact on their potential as markers of human fecal contamination. In the present study we are determining if enterophages can be grouped by their ability to replicate at different temperatures, and if different groups are present in the feces of different animals. As one of the main objectives is to determine if these phages can be used as indicators of the presence of enteric viruses, the survival rate under different conditions was also determined as was their prevalence in sewage and a large watershed. Coliphages were used as a means of comparison in the prevalence and survival studies. Results indicated that the isolates are mainly DNA viruses. Their morphology as well as their ability to form viral plaques at different temperatures indicates that several groups of enterophages are present in the environment. Coliphage and enterophage concentrations throughout the watershed were lower than those of thermotolerant coliforms and enterococci. Enterophage concentrations were lower than coliphages at all sampling points. Enterophages showed diverse inactivation rates and T 90 values across different incubation temperatures in both fresh and marine waters and sand. Further molecular characterization of enterophages may allow us to develop probes for the real-time detection of these alternative indicators of human fecal pollution.

Characterizing fecal contamination in stormwater runoff in coastal North Carolina, USA

Microbial contaminants in stormwater runoff have the potential to negatively impact public health. Stormwater runoff to coastal waters is increasing in amount and rate of discharge due to loss of vegetated landscape and increasing coastal development. However, the extent and nature of microbial contamination of stormwater runoff in North Carolina (NC) has not been previously characterized. The aim of this study was to measure a range of fecal indicator bacteria (FIB) and molecular markers at three coastal sites. E. coli and Enterococcus sp. were measured in addition to molecular markers including Bacteroides Human-Specific Marker (HS) and fecal Bacteroides spp. Levels of FIB in stormwater far exceeded recreational water quality guidelines, frequently by several orders of magnitude. High concentrations of fecal Bacteroides spp. and the presence of HS indicated the presence of human fecal contamination in the stormwater runoff, but only during specific storms. Examinations of levels of fecal contamination in stormwater over multiple seasons and a range of storm conditions will allow managers to consider appropriate design of effective mitigation strategies necessary to maintain and restore coastal water quality.

Enterophages, a group of phages infecting Enterococcus faecalis, and their potential as alternate indicators of human faecal contamination

We have developed a method for the detection of viruses in environmental samples that we have called enterophages, that specifically infect Enterococcus faecalis. This method has allowed us to determine the prevalence and to study the ecology of this group of phages. The enterophages replicate at 37 degrees C, and at 41 degrees C. The presence of NaN(3) in the media inhibits the growth of background microbiota and allows an accurate, specific and rapid detection of these viruses. Enterophages were present in raw domestic sewage at lower concentrations (average 1.8 x 10(2) PFU/100 mL) than those of coliphages (average 1.7 x 10(5) PFU/100 mL). Phages were characterised by transmission electron microscopy showing icosahedral capsids, some with non-contractile tails as well as icosahedral non-tailed capsids. Different isolates had capsid sizes ranging from 20 nm to about 75 nm in diameter. These data describe a new group of phages that may serve as alternate indicators of human faecal pollution, especially in recreational waters. The ecology of these enterophages indicates that these may be strictly of human origin.

Human-specific fecal bacteria in wastewater treatment plant effluents

The objective of this study was to identify fecal bacteria able to persist after wastewater treatment and that could be used as indicators of human fecal contamination. In a first step, the diversity of Bacteroidales, Clostridiaceae, Bifidobacterium, and Bacillus– Streptococcus– Lactobacillus cluster (BSL) was analysed using a fingerprint technique (CE-SSCP) and 16S rDNA libraries in waters collected at the end of the treatment process in different urban wastewater treatment plants. For each group, dominant bacteria present in most effluents were identified. Their origin (human feces, animal feces, non-fecal) was then analysed based on data of their closest relatives in public 16S rDNA databases. Among fecal bacteria recovered in the treated effluents analysed, phylotypes close to Bifidobacterium adolescentis and Bacteroides caccae seem to be specific to human beings. Phylotypes gathering only sequences of human fecal origin were also identified among the BSL and Clostridiaceae, two bacterial groups which have been poorly investigated for bacterial source-tracking purpose. Since these bacteria were detected post-treatment in most wastewater treatment plants, they may constitute potential new indicators of fecal contamination specific to humans that could be used to track fecal contamination of surface water by sewage.

Development of a quantitative PCR assay for the quantitation of bovine polyomavirus as a microbial source-tracking tool

Adenoviruses and polyomaviruses are two distinct DNA viral families that are excreted in high concentrations and distributed in human and animal populations. Targeting specific virus included in these families has proved to be a promising and useful tool for tracing specifically sources of environmental contamination. In this study, a quantitative PCR assay that is specific for bovine polyomaviruses was developed and used to determine the excretion level and concentration of bovine polyomaviruses in urine and environmental samples, including urban sewage, slaughterhouse sewage, and river water. A set of primers and a TaqMan probe were designed to target a 77-bp region of the bovine polyomavirus VP1 gene, and the conditions of the reaction were optimized. A detection limit was established at 1–10 genome copies per test tube. The assay was specific and produced negative results when samples containing human or porcine fecal contamination were analyzed. This is, to our knowledge, the first description of bovine polyomaviruses excreted in bovine urine samples (mean values of 10 4 GC/l). Bovine polyomaviruses were also detected and quantified in slaughterhouse wastewater and river waters, which shows the spread of these viruses in many environmental samples containing contamination of bovine origin. The procedure described in this paper provides a quantitative source-tracking tool for the analysis of bovine polyomaviruses as indicators of the presence of bovine contamination in environmental samples.

Lack of specificity for PCR assays targeting humanBacteroides16S rRNA gene: cross-amplification with fish feces

Methods focused on members of the genus Bacteroides have been increasingly utilized in microbial source-tracking studies for identifying and quantifying sources of nonpoint fecal contamination. We present results using standard and real-time PCR to show cross-amplification of Bacteroides 16S rRNA gene molecular assays targeting human fecal pollution with fecal DNA from freshwater fish species. All except one of the presumptively human-specific assays amplified fecal DNA from at least one fish species, and one real-time PCR assay amplified DNA from all fish species tested. Sequencing of PCR amplicons generated from fish fecal DNA using primers from the real-time assay revealed no mismatches to the human-specific probe sequences, but the nucleotide sequences of clones from fish fecal samples differed markedly from those of human feces, suggesting that the fish-related bacteria may be different strains. Our results strongly demonstrate the potential for cross-amplification of human-specific PCR assays with fish feces, and may call into question the results of studies in which these Bacteroides-specific molecular markers are used to quantify human fecal contamination in waters where fish contribute to fecal inputs.

Comment on “Environmental Occurrence of the Enterococcal Surface Protein (esp) Gene is an Unreliable Indicator of Human Fecal Contamination”

Comment on “Environmental Occurrence of the Enterococcal Surface Protein (<i>esp</i>) Gene is an Unreliable Indicator of Human Fecal Contamination”

Response to Comment on “Environmental Occurrence of the Enterococcal Surface Protein (esp) Gene is an Unreliable Indicator of Human Fecal Contamination”

Response to Comment on “Environmental Occurrence of the Enterococcal Surface Protein (<i>esp</i>) Gene is an Unreliable Indicator of Human Fecal Contamination”

Application of real-time PCR assays to genotyping of F-specific phages in river water and sediments in Japan

Genotyping of F-specific RNA phages is currently one of the most promising approaches to differentiate between human and animal fecal contamination in aquatic environments. In this study, a total of 18 river water and sediment samples were collected from the Tonegawa River basin, Japan, in order to describe the genogroup distribution of F-specific RNA and DNA phages using genogroup-specific real-time PCR assays. F-specific phages were detected in nine (100%) river water and six (67%) sediment samples. Eighty-five phage plaques were isolated from these samples and subjected to real-time PCR assays specific for the phages. F-specific RNA phages of human genogroups (II and III) were detected in 32 (38%) plaques, whereas those of animal genogroups (I and IV) were detected in 17 (20%) plaques. No correlation was observed between the genogroup distribution of F-specific RNA phages and the occurrence of human adenovirus genomes, suggesting that genotyping of the phages alone is inadequate for the evaluation of the occurrence of viruses in aquatic environments. SYBR Green-based real-time PCR assay revealed the presence of F-specific DNA phages in four (5%) plaques, which were further classified into two genogroups (fd- and f1-like phages) by sequence analysis. Thirty-two (38%) plaques were not classified as the F-specific phage genogroups, indicating the limited applicability of these real-time PCR assays to a wide range of aquatic environmental samples worldwide.