Markers Of Fecal Contamination Research Articles

A comparative study between low- and high-tech methods for the detection and mitigation of illicit connections in stormwater systems.

Illicit connections of wastewater to stormwater systems are the main drawback of separate sewer systems, as they lead to a direct discharge of untreated wastewater to the aquatic environment. Consequently, several inspection methods have been developed for detecting illicit connections. This study simultaneously applied several low- and high-tech methods for the detection of illicit connections in the same catchment (De Heuvel, the Netherlands). The methods included mesh wire screens for capturing coarse contamination, measurements of electroconductivity and temperature, sampling and quantification of Escherichia coli and extended-spectrum ß-lactamase-producing E. coli (ESBL-EC), DNA analysis via quantitative polymerase chain reaction for human-, dog-, and bird-specific fecal indicators, and distributed temperature sensing. Significant illicit connections could be identified using all methods. Nonetheless, hydraulic conditions and, predominantly, the sewage volume determine whether a misconnection can be detected by especially the low-tech methods. Using these results, the identified misconnections were repaired and biological and DNA analyses were repeated. Our results demonstrate that there were no changes in E. coli or ESBL-EC before and after mitigation, suggesting that these common markers of fecal contamination are not specific enough to evaluate the performance of mitigation efforts. However, a marked decrease in human wastewater markers (HF183) was observed.

Metagenomic Analysis of Microbial Contamination in the U.S. Portion of the Tijuana River Watershed.

The Tijuana River watershed is binational, flowing from Tijuana, Mexico into San Diego and Imperial Beach, USA. Aging sewage and stormwater infrastructure in Tijuana has not kept pace with population growth, causing overflows into this watershed during major rainfall or equipment failures. The public health consequences of this impaired watershed on the surrounding communities remain unknown. Here, we performed untargeted metagenomic sequencing to better characterize the sewage contamination in the Tijuana River, identifying potential pathogens and molecular indicators of antibiotic resistance in surface waters. In 2019-2020, water samples were collected within 48 h of major rainfall events at five transborder flow sites and at the mouth of the river in the US portion of the Tijuana River and estuary. After filtration, DNA was extracted and sequenced, and sequences were run through the Kaiju taxonomic classification program. A pathogen profile of the most abundant disease-causing microbes and viruses present in each of the samples was constructed, and specific markers of fecal contamination were identified and linked to each site. Results from diversity analysis between the sites showed clear distinction as well as similarities between sites and dates, and antibiotic-resistant genes were found at each site. This serves as a baseline characterization of microbial exposures to these local communities.

Endotoxin as a Marker for Water Quality

Water quality testing is vital to protect human health. Current testing relies mainly on culture-based detection of faecal indicator organisms such as Escherichia coli (E.coli). However, bacterial cultures are a slow process, taking 24-48 h and requiring specialised laboratories and trained personnel. Access to such laboratories is often sparse in developing countries and there are many fatalities deriving from poor water quality. Endotoxin is a molecular component of Gram-negative bacterial cell walls and can be used to detect their presence in drinking water. The current study used a novel assay (BacterisK) to rapidly detect endotoxin in various water samples and correlate the results with E. coli content measured by culture methods. The data generated by the BacterisK assay are presented as an 'endotoxin risk' (ER). The ER values correlate with E. coli and thus endotoxin can be used as a marker of faecal contamination in water. Moreover, the BacterisK assay provides data in near real-time and can be used in situ allowing water quality testing at different spatial and temporal locations. We suggest that BacterisK can be used as a convenient risk assessment tool to assess water quality where results are required quickly or access to laboratories is lacking.

GENETIC DIVERSITY OF CATTLE INTESTINAL BACTERIA DETECTED BY HIGH-OUTPUT SEQUENCING

The gut microbiota and its development for the health of pets are currently being actively developed. The composition of the gut microbiome is found in the reduction of food intake, the state of the immune system of animals, productivity and growth in livestock content. Also found in the colon are unusual pathogen phyla that can serve as markers of fecal contamination in the environment. We conducted a study of the intestinal microbiome of 12 animals divided into two groups - calves and adult cows. Bacteria of taxa of taxa Actinobacteriota, Bacteroidota, Campilobacterota, Chloroflexi, Cyanobacteria, Desulfobacterota, Fibrobacterota, Firmicutes, Fusobacterota, Halobacterota, Elusimicrobiota, Euryarchaeota, Proteobacteria, Patescibacteria, Spirochaetota, Thermoplasmatota, Verrucomicrobia and a large number of unclassified bacteria were identified.It has been shown that the intestinal microbiome of calves differs from that of adult cows, and diarrhea affects the composition of the intestines of young animals, reducing the biodiversity of the inhabitants. In calves, the Shannon index ranged from 3.18 to 4.3, in adult animals from 4.41 to 5.24. Comparison of the gut microbiomes of healthy calves and calves with diarrhea was carried out using the Hutcheson t-test, the difference was significant (P<<<0.0001). > The main phyla of calf intestinal bacteria are Bacteroidota and Firmicutes, moreover, the diversity and number of microbial lines of Bacteroidota increases with age. Firmicutes of the families Lactobacillae and Lactobacillales_fa, as well as the family Selenomonadaceae, are markers of the juvenile age of animals. Calf-specific Bacteroidota are representatives of Tannerellaceae and Marinifilaceae. The microbiome of adult animals at the phylum level is distinguished by the presence of bacteria Verrucomicrobiota, Desulfobacterota, archaea Methanobacteria and Methanomicrobia. At the level of families and genera, the formed microbiome of cows has unique representatives of Bacteroidota and Firmicutes.Thus, we have presented data on the main representatives of healthy intestinal bacteria of cows and calves, which can later be used to diagnose the physiological state of animals, as well as in environmental studies to detect fecal environmental pollution.

Passive Samplers, a Powerful Tool to Detect Viruses and Bacteria in Marine Coastal Areas

The detection of viruses and bacteria which can pose a threat either to shellfish health or shellfish consumers remains difficult. The current detection methods rely on point sampling of water, a method that gives a snapshot of the microorganisms present at the time of sampling. In order to obtain better representativeness of the presence of these microorganisms over time, we have developed passive sampling using the adsorption capacities of polymer membranes. Our objectives here were to assess the feasibility of this methodology for field detection. Different types of membrane were deployed in coastal waters over 2 years and the microorganisms tested using qPCR were: human norovirus (NoV) genogroups (G)I and II, sapovirus, Vibrio spp. and the species Vibrio alginolyticus, V. cholerae, V. vulnificus, and V. parahaemolyticus, OsHV-1 virus, and bacterial markers of fecal contamination. NoV GII, Vibrio spp., and the AllBac general Bacteroidales marker were quantified on the three types of membrane. NoV GII and OsHV-1 viruses followed a seasonal distribution. All membranes were favorable for NoV GII detection, while Zetapor was more adapted for OsHV-1 detection. Nylon was more adapted for detection of Vibrio spp. and the AllBac marker. The quantities of NoV GII, AllBac, and Vibrio spp. recovered on membranes increased with the duration of exposure. This first application of passive sampling in seawater is particularly promising in terms of an early warning system for the prevention of contamination in oyster farming areas and to improve our knowledge on the timing and frequency of disease occurence.

Evaluation of CrAssphage Marker for Tracking Fecal Contamination in River Water in Nepal

Enteric viruses in the aquatic environment are a concern due to the potential for waterborne disease transmission to humans. In Nepal, the Bagmati River serves as a source of drinking and irrigation water; therefore, the detection of waterborne enteric pathogens is integral to maintaining human health. The objective of this study was to quantify the crAssphage marker in surface water samples from the Bagmati River between November 2015 and September 2016. Concentrations of crAssphage were then compared to those of other enteric viruses and indicator organisms found in the samples in order to examine the potential of crAssphage as a marker for fecal contamination. CrAssphage was detected in 17% (1/6) of samples from Sundarijal, 100% (6/6) of samples from Thapathali, and 100% (6/6) samples from Chovar, with the highest average concentrations recorded in May 2016 and the lowest average concentrations recorded in September 2016. Overall, crAssphage was present in 72% (13/18) of samples and was strongly correlated with the presence of fecal indicator bacteria Escherichia coli (r = 0.89) and Enterococcus (r = 0.92) and several enteric viruses. The strongest viral correlations were to salivirus (r = 0.84), pepper mild mottle virus (r = 0.77), Aichi virus 1 (r = 0.75), enteroviruses (r = 0.76), and tobacco mosaic virus (r = 0.71). These results provide evidence for the potential use of crAssphage as a marker for human fecal contamination in river water.

Exposure to Human-Associated Chemical Markers of Fecal Contamination and Self-Reported Illness among Swimmers at Recreational Beaches

Anthropogenic chemicals have been proposed as potential markers of human fecal contamination in recreational water. However, to date, there are no published studies describing their relationships with illness risks. Using a cohort of swimmers at seven U.S. beaches, we examined potential associations between the presence of chemical markers of human fecal pollution and self-reported gastrointestinal (GI) illness, diarrhea, and respiratory illness. Swimmers were surveyed about their beach activities, water exposure, and baseline symptoms on the day of their beach visit, and about any illness experienced 10-12 days later. Risk differences were estimated using model-based standardization and adjusted for the swimmer's age, beach site, sand contact, rainfall, and water temperature. Sixty-two chemical markers were analyzed from daily water samples at freshwater and marine beaches. Of those, 20 were found consistently. With the possible exception of bisphenol A and cholesterol, no chemicals were consistently associated with increased risks of illness. These two chemicals were suggestively associated with 2% and 1% increased risks of GI illness and diarrhea in both freshwater and marine beaches. Additional research using the more sensitive analytic methods currently available for a wider suite of analytes is needed to support the use of chemical biomarkers to quantify illness risk and identify fecal pollution sources.

Quantification of pathogens and markers of fecal contamination during storm events along popular surfing beaches in San Diego, California

Along southern California beaches, the concentrations of fecal indicator bacteria (FIB) used to quantify the potential presence of fecal contamination in coastal recreational waters have been previously documented to be higher during wet weather conditions (typically winter or spring) than those observed during summer dry weather conditions. FIB are used for management of recreational waters because measurement of the bacterial and viral pathogens that are the potential causes of illness in beachgoers exposed to stormwater can be expensive, time-consuming, and technically difficult. Here, we use droplet digital Polymerase Chain Reaction (digital PCR) and digital reverse transcriptase PCR (digital RT-PCR) assays for direct quantification of pathogenic viruses, pathogenic bacteria, and source-specific markers of fecal contamination in the stormwater discharges. We applied these assays across multiple storm events from two different watersheds that discharge to popular surfing beaches in San Diego, CA. Stormwater discharges had higher FIB concentrations as compared to proximal beaches, often by ten-fold or more during wet weather. Multiple lines of evidence indicated that the stormwater discharges contained human fecal contamination, despite the presence of separate storm sewer and sanitary sewer systems in both watersheds. Human fecal source markers (up to 100% of samples, 20-12440 HF183 copies per 100 ml) and human norovirus (up to 96% of samples, 25-495 NoV copies per 100 ml) were routinely detected in stormwater discharge samples. Potential bacterial pathogens were also detected and quantified: Campylobacter spp. (up to 100% of samples, 16-504 gene copies per 100 ml) and Salmonella (up to 25% of samples, 6-86 gene copies per 100 ml). Other viral human pathogens were also measured, but occurred at generally lower concentrations: adenovirus (detected in up to 22% of samples, 14-41 AdV copies per 100 ml); no enterovirus was detected in any stormwater discharge sample. Higher concentrations of avian source markers were noted in the stormwater discharge located immediately downstream of a large bird sanctuary along with increased Campylobacter concentrations and notably different Campylobacter species composition than the watershed that had no bird sanctuary. This study is one of the few to directly measure an array of important bacterial and viral pathogens in stormwater discharges to recreational beaches, and provides context for stormwater-based management of beaches during high risk wet-weather periods. Furthermore, the combination of culture-based and digital PCR-derived data is demonstrated to be valuable for assessing hydrographic relationships, considering delivery mechanisms, and providing foundational exposure information for risk assessment.

Quantification of pathogens and markers of fecal contamination during storm events along popular surfing beaches in San Diego, California

Quantification of pathogens and markers of fecal contamination during storm events along popular surfing beaches in San Diego, California

A prolonged, community-wide cholera outbreak associated with drinking water contaminated by sewage in Kasese District, western Uganda

BackgroundIn May 2015, a cholera outbreak that had lasted 3 months and infected over 100 people was reported in Kasese District, Uganda, where multiple cholera outbreaks had occurred previously. We conducted an investigation to identify the mode of transmission to guide control measures.MethodsWe defined a suspected case as onset of acute watery diarrhoea from 1 February 2015 onwards in a Kasese resident. A confirmed case was a suspected case with Vibrio cholerae O1 El Tor, serotype Inaba cultured from a stool sample. We reviewed medical records to find cases. We conducted a case-control study to compare exposures among confirmed case-persons and asymptomatic controls, matched by village and age-group. We conducted environmental assessments. We tested water samples from the most affected area for total coliforms using the Most Probable Number (MPN) method.ResultsWe identified 183 suspected cases including 61 confirmed cases of Vibrio cholerae 01; serotype Inaba, with onset between February and July 2015. 2 case-persons died of cholera. The outbreak occurred in 80 villages and affected all age groups; the highest attack rate occurred in the 5–14 year age group (4.1/10,000). The outbreak started in Bwera Sub-County bordering the Democratic Republic of Congo and spread eastward through sustained community transmission. The first case-persons were involved in cross-border trading. The case-control study, which involved 49 confirmed cases and 201 controls, showed that 94% (46/49) of case-persons compared with 79% (160/201) of control-persons drank water without boiling or treatment (ORM-H=4.8, 95% CI: 1.3–18). Water collected from the two main sources, i.e., public pipes (consumed by 39% of case-persons and 38% of control-persons) or streams (consumed by 29% of case-persons and 24% control-persons) had high coliform counts, a marker of faecal contamination. Environmental assessment revealed evidence of open defecation along the streams. No food items were significantly associated with illness.ConclusionsThis prolonged, community-wide cholera outbreak was associated with drinking water contaminated by faecal matter and cross-border trading. We recommended rigorous disposal of patients’ faeces, chlorination of piped water, and boiling or treatment of drinking water. The outbreak stopped 6 weeks after these recommendations were implemented.

Evidence of massive river pollution in the tropical megacity Jakarta as indicated by faecal steroid occurrence and the seasonal flushing out into the coastal ecosystem

In the rapidly growing coastal megacities of developing and emerging countries, the implementation of new sewage treatment facilities is often not keeping pace with the enormous population growth, leading to a deterioration of the urban water resources. Only very few studies discuss faecal water contamination in these urban areas. Our study area Jakarta, the capital of Indonesia, is such a megacity. We investigated chemical markers of faecal contamination in water and sediments from the rivers and canals flowing through Jakarta. Moreover, also the spatial distribution of faecal markers in Jakarta Bay, the coastal ecosystem that receives all urban river discharges, was assessed. The concentrations of the faecal steroid coprostanol in river water ranged from 0.45 to 24.2 µg L−1, and in sediments from 0.3 to 400 µg g−1, reflecting the problem of inadequate sewage treatment capacities in the city. The steroid distribution in Jakarta Bay in May 2013 as compared to dry season data indicates a flushing out of particle-associated pollutants from the urban rivers far offshore during the precedent rainy season, where the city experienced a severe flood. This flushing out of particle-associated pollutants during times of heavy rainfall as observed in our study is a pollutant transport mechanism that is important for all tropical coastal systems. The associated impacts on sensitive coastal habitats might become more severe in the future, as a consequence of the expected climate change effects on monsoon variability.

Optimization of the skimmed-milk flocculation method for recovery of adenovirus from sludge

Return of treated sludge to the environment poses concerns and has stimulated the development of studies on viral monitoring in this matrix, in order to assess its potential risks for public health. Human adenovirus (HAdV) has been identified as a putative viral marker of faecal contamination due to its stability and resistance to the sewage treatment process. The aim of this study was to optimize the organic flocculation procedure in order to establish an appropriate methodology for HAdV recovery from sewage sludge samples. Four protocols (A-D) have been proposed, with changes in the initial sample dilution, in the stirring time and in the final concentration of skimmed-milk. A single sludge sample was obtained in Wastewater Treatment Plant (WWTP) and divided into aliquots. In each protocol, three aliquots were inoculated with HAdV and bacteriophage PP7 and a non-inoculated one was used as negative control. Viral load and recovery rate were determined by quantitative PCR. HAdV recovery rate varied between the protocols tested (p=0.016) and the best result was obtained through the protocol C. In order to confirm this result a field study with activated, thickened and digested sludge samples was carried out. Different types of sludge were obtained in two WWTPs and processed using protocol C. HAdV was detected in all samples, with a similar or higher viral load than those obtained with other concentration techniques already applied to sludge. Protocol C proved to be really efficient, with the advantage of showing low cost and practicability in routine laboratories.

Detection of human adenoviruses in organic fresh produce using molecular and cell culture-based methods

The consumption of organic fresh produce has increased in recent years due to consumer demand for healthy foods without chemical additives. However, the number of foodborne outbreaks associated with fresh produce has also increased. Contamination of food with enteric viruses is a major concern because the viruses have a low infectious dose and high persistence in the environment. Human adenovirus (HAdV) has been proposed as a good marker of faecal contamination. Therefore, the aim of this study was to evaluate the efficiency of the plaque assay (PA), real time PCR (qPCR) and integrated cell culture-RT-qPCR (ICC-RT-qPCR) for the recovery of HAdV from artificially and naturally contaminated fresh produce. Organic lettuce, strawberries and green onions were selected because these fresh products are frequently associated with foodborne outbreaks. The virus extraction efficiencies from artificially contaminated samples varied from 2.8% to 32.8% depending on the food matrix and the quantification method used. Although the HAdV recoveries determined by qPCR were higher than those determined by PA and ICC-RT-qPCR, PA was defined as the most reproducible method. The qPCR assays were more sensitive than the PA and ICC-RT-qPCR assays; however, this technique alone did not provide information about the viability of the pathogen. ICC-RT-qPCR was more sensitive than PA for detecting infectious particles in fresh produce samples. HAdV genome copies were detected in 93.3% of the analysed naturally contaminated samples, attesting to the common faecal contamination of the fresh produce tested. However, only 33.3% of the total samples were positive for infectious HAdV particles based on ICC-RT-qPCR. In conclusion, this study reported that HAdV can be an efficient viral marker for fresh produce contamination. Good detection of infectious HAdV was obtained with the ICC-RT-qPCR and PA assays. Thus, we suggest that the ICC-RT-qPCR and PA assays should be considered when quantitative microbial risk assessment (QMRA) studies are required and to establish reliable food safety guidelines.

Bioaccumulation of animal adenoviruses in the pink shrimp.

Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems.

Storm loads of culturable and molecular fecal indicators in an inland urban stream

Elevated concentrations of fecal indicator bacteria in receiving waters during wet-weather flows are a considerable public health concern that is likely to be exacerbated by future climate change and urbanization. Knowledge of factors driving the fate and transport of fecal indicator bacteria in stormwater is limited, and even less is known about molecular fecal indicators, which may eventually supplant traditional culturable indicators. In this study, concentrations and loading rates of both culturable and molecular fecal indicators were quantified throughout six storm events in an instrumented inland urban stream. While both concentrations and loading rates of each fecal indicator increased rapidly during the rising limb of the storm hydrographs, it is the loading rates rather than instantaneous concentrations that provide a better estimate of transport through the stream during the entire storm. Concentrations of general fecal indicators (both culturable and molecular) correlated most highly with each other during storm events but not with the human-associated HF183 Bacteroides marker. Event loads of general fecal indicators most strongly correlated with total runoff volume, maximum discharge, and maximum turbidity, while event loads of HF183 most strongly correlated with the time to peak flow in a hydrograph. These observations suggest that collection of multiple samples during a storm event is critical for accurate predictions of fecal indicator loading rates and total loads during wet-weather flows, which are required for effective watershed management. In addition, existing predictive models based on general fecal indicators may not be sufficient to predict source-specific genetic markers of fecal contamination.

A PCR procedure for the detection of Giardia intestinalis cysts and Escherichia coli in lettuce.

Giardia intestinalis is a pathogen associated with foodborne outbreaks and Escherichia coli is commonly used as a marker of faecal contamination. Implementation of routine identification methods of G. intestinalis is difficult for the analysis of vegetables and the microbiological detection of E. coli requires several days. This study proposes a PCR-based assay for the detection of E. coli and G. intestinalis cysts using crude DNA isolated from artificially contaminated lettuce. The G. intestinalis and E. coli PCR assays targeted the β-giardin and uidA genes, respectively, and were 100% specific. Forty lettuces from local markets were analysed by both PCR and light microscopy and no cysts were detected, the calculated detection limit was 20 cysts per gram of lettuce; however, by PCR, E. coli was detected in eight of ten randomly selected samples of lettuce. These data highlight the need to validate procedures for routine quality assurance. These PCR-based assays can be employed as alternative methods for the detection of G. intestinalis and E. coli and have the potential to allow for the automation and simultaneous detection of protozoa and bacterial pathogens in multiple samples. Significance and impact of the study: There are few studies for Giardia intestinalis detection in food because methods for its identification are difficult for routine implementation. Here, we developed a PCR-based method as an alternative to the direct observation of cysts in lettuce by light microscopy. Additionally, Escherichia coli was detected by PCR and the sanitary quality of lettuce was evaluated using molecular and standard microbiological methods. Using PCR, the detection probability of Giardia cysts inoculated onto samples of lettuce was improved compared to light microscopy, with the advantage of easy automation. These methods may be employed to perform timely and affordable detection of foodborne pathogens.

Sequencing human mitochondrial hypervariable region II as a molecular fingerprint for environmental waters.

To protect environmental water from human fecal contamination, authorities must be able to unambiguously identify the source of the contamination. Current identification methods focus on tracking fecal bacteria associated with the human gut, but many of these bacterial indicators also thrive in the environment and in other mammalian hosts. Mitochondrial DNA could solve this problem by serving as a human-specific marker for fecal contamination. Here we show that the human mitochondrial hypervariable region II can function as a molecular fingerprint for human contamination in an urban watershed impacted by combined sewer overflows. We present high-throughput sequencing analysis of hypervariable region II for spatial resolution of the contaminated sites and assessment of the population diversity of the impacting regions. We propose that human mitochondrial DNA from public waste streams may serve as a tool for identifying waste sources definitively, analyzing population diversity, and conducting other anthropological investigations.

The Beat

The Beat

Presence of Torque Teno Virus (TTV) in Tap Water in Public Schools from Southern Brazil

Torque teno virus (TTV) was surveyed in tap water collected in schools from three municipalities located in the south of Brazil. TTV genomes were found in 11.7 % (4/34) of the samples. TTV DNA was detected in 10.5 % (2/19) of the samples collected at the city of Caxias do Sul and in 25 % (2/8) of the samples from Pelotas. Those cities have a low rate of sewage treatment. All samples from Santa Cruz do Sul, which has nearly 92 % of its sewage treated, were negative. These results suggest that the amount of sewage treated may have an effect on the detection rates of TTV DNA in drinking water in a given urban area, showing a mild negative correlation (r = -0.76), when comparing the percentage of sewage treatment to the detection of TTV genomes. The detection rate of TTV was also compared with Escherichia coli, showing a strong correlation (r = 0.97), indicating that TTV may be a suitable marker of fecal contamination.

Evaluation of Chemical, Molecular, and Traditional Markers of Fecal Contamination in an Effluent Dominated Urban Stream

In this paper we present a quantitative sanitary survey of the Middle Santa Ana River, in southern California, utilizing a variety of source tracking tools, including traditional culture-dependent fecal markers (Enterococcus and Escherichia coli by IDEXX), speciation of enterococci isolates, culture-independent fecal markers (human-specific HF183 Bacteroides and Enterococcus by quantitative polymerase chain reaction, qPCR), and chemical markers of sewage and wastewater (nutrients, enantiomeric fraction (EF) of propranolol and ethylenediaminetetraacetic acid). To facilitate comparison of these different methods, data are interpreted in a loading (i.e., mass per time) framework that enables a quantitative apportionment of fecal markers and nutrients to specific source waters in the Middle Santa Ana River. Multiple lines of evidence support the hypothesis that Enterococcus in the Middle Santa Ana River originates primarily from in situ growth in streambed sediments, not from significant and persistent sources of untreated human waste. The EF of propranolol of tertiary treated wastewater effluent is in the range (0.42 to 0.71) previously reported for raw sewage, making EF of propranolol an unsuitable marker for fecal pollution, at least at this site. The human fecal marker HF183 Bacteroides was detected at a few sites, although not in a source of disinfected and tertiary treated wastewater effluent. Based on the results presented here and prior experience at other sites in southern California, HF183 Bacteroides would appear to be a candidate marker of fecal contamination for inland waters, although more qPCR measurements in disinfected wastewater effluent are needed to account for variations due to treatment plant performance and other factors. More generally, our results support the notion that regrowth of fecal indicator bacteria (FIB) in river sediments may lead to a decoupling between FIB and pathogen concentrations in the water column and thus limit the utility of FIB as an indicator of recreational waterborne illness in inland waters.