Distributions of Fecal Markers in Wastewater from Different Climatic Zones for Human Fecal Pollution Tracking in Australian Surface Waters

ABSTRACTRecreational and potable water supplies polluted with human wastewater can pose a direct health risk to humans. Therefore, sensitive detection of human fecal pollution in environmental waters is very important to water quality authorities around the globe. Microbial source tracking (MST) utilizes human fecal markers (HFMs) to detect human wastewater pollution in environmental waters. The concentrations of these markers in raw wastewater are considered important because it is likely that a marker whose concentration is high in wastewater will be more frequently detected in polluted waters. In this study, quantitative PCR (qPCR) assays were used to determine the concentrations of fecal indicator bacteria (FIB)Escherichia coliandEnterococcusspp., HFMsBacteroidesHF183, human adenoviruses (HAdVs), and polyomaviruses (HPyVs) in raw municipal wastewater influent from various climatic zones in Australia.E. colimean concentrations in pooled human wastewater data sets (from various climatic zones) were the highest (3.2 × 106gene copies per ml), followed by those of HF183 (8.0 × 105gene copies per ml) andEnterococcusspp. (3.6 × 105gene copies per ml). HAdV and HPyV concentrations were 2 to 3 orders of magnitude lower than those of FIB and HF183. Strong positive and negative correlations were observed between the FIB and HFM concentrations within and across wastewater treatment plants (WWTPs). To identify the most sensitive marker of human fecal pollution, environmental water samples were seeded with raw human wastewater. The results from the seeding experiments indicated thatBacteroidesHF183 was more sensitive for detecting human fecal pollution than HAdVs and HPyVs. Since the HF183 marker can occasionally be present in nontarget animal fecal samples, it is recommended that HF183 along with a viral marker (HAdVs or HPyVs) be used for tracking human fecal pollution in Australian environmental waters.

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Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

Applied and Environmental Microbiology ◽

10.1128/aem.02354-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 196-202 ◽

Cited By ~ 7

Author(s):

Jennifer Weidhaas ◽

Sirisha Mantha ◽

Elliott Hair ◽

Bina Nayak ◽

Valerie J. Harwood

Keyword(s):

Poultry Litter ◽

Water Quality Monitoring ◽

Quality Monitoring ◽

Source Tracking ◽

Environmental Waters ◽

Fecal Indicator ◽

Content Type ◽

Gene Copies ◽

Laboratory Microcosm ◽

Host Feces

ABSTRACTWater quality monitoring techniques that target microorganisms in the orderBacteroidalesare potential alternatives to conventional methods for detection of fecal indicator bacteria.Bacteroidalesand members of the genusBacteroideshave been the focus of microbial source tracking (MST) investigations for discriminating sources of fecal pollution (e.g., human or cattle feces) in environmental waters. For accurate source apportionment to occur, one needs to understand both the abundance ofBacteroidesin host feces and the survival of these host-associated microbial markers after deposition in the environment. Studies were undertaken to evaluate the abundance, persistence, and potential for growth ofBacteroidalesoriginating from poultry litter under oxic and anoxic environmental conditions.Bacteroidalesabundance, as determined by quantitative PCR (qPCR) with GenBac primers and probe, increased 2 to 5 log gene copies ml−1and 2 log gene copies g litter−1under most conditions during incubation of poultry litter in a variety of laboratory microcosm and field mesocosm studies. DNA sequencing of theBacteroidalesorganisms in the litter identified taxa with sequences corresponding exactly to the GenBac primer and probe sequences and that were closely related toBacteroides uniformis,B. ovatus, andB. vulgatus. These results suggest that MST studies using qPCR methods targetingBacteroidalesin watersheds that are affected by poultry litter should be interpreted cautiously. Growth ofBacteroidalesoriginating from poultry litter in environmental waters may occur whileBacteroidalesgrowth from other fecal sources declines, thus confounding the interpretation of MST results.

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Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

Applied and Environmental Microbiology ◽

10.1128/aem.02583-15 ◽

2016 ◽

Vol 82 (5) ◽

pp. 1625-1635 ◽

Cited By ~ 11

Author(s):

Xiang Li ◽

Valerie J. Harwood ◽

Bina Nayak ◽

Jennifer L. Weidhaas

Keyword(s):

Legionella Pneumophila ◽

Microbial Source Tracking ◽

Fecal Pollution ◽

Marine Water ◽

Treated Wastewater ◽

Human Polyomavirus ◽

Source Tracking ◽

Marker Genes ◽

Content Type ◽

Gene Copies

ABSTRACTPathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% forEscherichia coliand human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such asLegionella pneumophila,Shigella flexneri, andCampylobacter fetuswere detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment.

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Cross-Comparison of Human Wastewater-Associated Molecular Markers in Relation to Fecal Indicator Bacteria and Enteric Viruses in Recreational Beach Waters

Applied and Environmental Microbiology ◽

10.1128/aem.00028-17 ◽

2017 ◽

Vol 83 (8) ◽

Cited By ~ 29

Author(s):

B. Hughes ◽

D. J. Beale ◽

P. G. Dennis ◽

S. Cook ◽

W. Ahmed

Keyword(s):

Water Samples ◽

Enteric Viruses ◽

Human Adenovirus ◽

Fecal Pollution ◽

Treated Wastewater ◽

Recreational Water ◽

Environmental Waters ◽

Fecal Indicator ◽

Content Type ◽

Beach Water

ABSTRACT Detection of human wastewater contamination in recreational waters is of critical importance to regulators due to the risks posed to public health. To identify such risks, human wastewater-associated microbial source tracking (MST) markers have been developed. At present, however, a greater understanding of the suitability of these markers for the detection of diluted human wastewater in environmental waters is necessary to predict risk. Here, we compared the process limit of detection (PLOD) and process limit of quantification (PLOQ) of six human wastewater-associated MST markers (Bacteroides HF183 [HF183], Escherichia coli H8 [EC H8], Methanobrevibacter smithii nifH, human adenovirus [HAdV], human polyomavirus [HPyV], and pepper mild mottle virus [PMMoV]) in relation to a fecal indicator bacterium (FIB), Enterococcus sp. 23S rRNA (ENT 23S), and three enteric viruses (human adenovirus serotypes 40/41 [HAdV 40/41], human norovirus [HNoV], and human enterovirus [EV]) in beach water samples seeded with raw and secondary-treated wastewater. Among the six MST markers tested, HF183 was the most sensitive measure of human fecal pollution and was quantifiable up to dilutions of 10−6 and 10−4 for beach water samples seeded with raw and secondary-treated wastewater, respectively. Other markers and enteric viruses were detected at various dilutions (10−1 to 10−5). These MST markers, FIB, and enteric viruses were then quantified in beach water (n = 12) and sand samples (n = 12) from South East Queensland (SEQ), Australia, to estimate the levels of human fecal pollution. Of the 12 sites examined, beach water and sand samples from several sites had quantifiable concentrations of HF183 and PMMoV markers. Overall, our results indicate that while HF183 is the most sensitive measure of human fecal pollution, it should be used in conjunction with a conferring viral marker to avoid overestimating the risk of gastrointestinal illness. IMPORTANCE MST is an effective tool to help utilities and regulators improve recreational water quality around the globe. Human fecal pollution poses significant public health risks compared to animal fecal pollution. Several human wastewater-associated markers have been developed and used for MST field studies. However, a head-to-head comparison in terms of their performance to detect diluted human fecal pollution in recreational water is lacking. In this study, we cross-compared the performance of six human wastewater-associated markers in relation to FIB and enteric viruses in beach water samples seeded with raw and secondary-treated wastewater. The results of this study will provide guidance to regulators and utilities on the appropriate application of MST markers for tracking the sources of human fecal pollution in environmental waters and confer human health risks.

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

Applied and Environmental Microbiology ◽

10.1128/aem.05480-11 ◽

2011 ◽

Vol 77 (19) ◽

pp. 6972-6981 ◽

Cited By ~ 79

Author(s):

Ryan J. Newton ◽

Jessica L. VandeWalle ◽

Mark A. Borchardt ◽

Marc H. Gorelick ◽

Sandra L. McLellan

Keyword(s):

Sequence Analysis ◽

Genetic Marker ◽

Microbial Community Composition ◽

Fold Increase ◽

Fecal Pollution ◽

Plate Count ◽

Rrna Gene ◽

Fecal Indicators ◽

Fecal Indicator ◽

Content Type

ABSTRACTThe 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 theLachnospiraceaefamily, which was closely related to the genusBlautia. 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 humanBacteroidales(based on the HF183 genetic marker), totalBacteroidalesspp., and enterococci and the conventionalEscherichia coliand 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 humanBacteroidalesincreased 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.

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Decay of Fecal Indicator Bacterial Populations and Bovine-Associated Source-Tracking Markers in Freshly Deposited Cow Pats

Applied and Environmental Microbiology ◽

10.1128/aem.02203-13 ◽

2013 ◽

Vol 80 (1) ◽

pp. 110-118 ◽

Cited By ~ 29

Author(s):

Adelumola Oladeinde ◽

Thomas Bohrmann ◽

Kelvin Wong ◽

S. T. Purucker ◽

Ken Bradshaw ◽

...

Keyword(s):

Decay Rate ◽

Fecal Indicator Bacteria ◽

Decay Rates ◽

Indicator Bacteria ◽

Source Tracking ◽

Fecal Indicator ◽

Bacterial Populations ◽

Content Type ◽

E Coli ◽

Decay Dynamic

ABSTRACTUnderstanding the survival of fecal indicator bacteria (FIB) and microbial source-tracking (MST) markers is critical to developing pathogen fate and transport models. Although pathogen survival in water microcosms and manure-amended soils is well documented, little is known about their survival in intact cow pats deposited on pastures. We conducted a study to determine decay rates of fecal indicator bacteria (Escherichia coliand enterococci) and bovine-associated MST markers (CowM3, Rum-2-bac, and GenBac) in 18 freshly deposited cattle feces from three farms in northern Georgia. Samples were randomly assigned to shaded or unshaded treatment in order to determine the effects of sunlight, moisture, and temperature on decay rates. A general linear model (GLM) framework was used to determine decay rates. Shading significantly decreased the decay rate of theE. colipopulation (P< 0.0001), with a rate of −0.176 day−1for the shaded treatment and −0.297 day−1for the unshaded treatment. Shading had no significant effect on decay rates of enterococci, CowM3, Rum-2-bac, and GenBac (P> 0.05). In addition,E. colipopulations showed a significant growth rate (0.881 day−1) in the unshaded samples during the first 5 days after deposition. UV-B was the most important parameter explaining the decay rate ofE. colipopulations. A comparison of the decay behaviors among all markers indicated that enterococcus concentrations exhibit a better correlation with the MST markers thanE. coliconcentrations. Our results indicate that bovine-associated MST markers can survive in cow pats for at least 1 month after excretion, and although their decay dynamic differs from the decay dynamic ofE. colipopulations, they seem to be reliable markers to use in combination with enterococci to monitor fecal pollution from pasture lands.

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Evaluation of Bovine Feces-Associated Microbial Source Tracking Markers and Their Correlations with Fecal Indicators and Zoonotic Pathogens in a Brisbane, Australia, Reservoir

Applied and Environmental Microbiology ◽

10.1128/aem.03234-12 ◽

2013 ◽

Vol 79 (8) ◽

pp. 2682-2691 ◽

Cited By ~ 35

Author(s):

W. Ahmed ◽

T. Sritharan ◽

A. Palmer ◽

J. P. S. Sidhu ◽

S. Toze

Keyword(s):

Host Specificity ◽

Water Samples ◽

Microbial Source Tracking ◽

Fecal Pollution ◽

Decay Rates ◽

Source Tracking ◽

Content Type ◽

Zoonotic Pathogens ◽

Maximum Value ◽

Bovine Feces

ABSTRACTThis study was aimed at evaluating the host specificity and host sensitivity of two bovine feces-associated bacterial (BacCow-UCD and cowM3) and one viral [bovine adenovirus (B-AVs)] microbial source tracking (MST) markers by screening 130 fecal and wastewater samples from 10 target and nontarget host groups in southeast Queensland, Australia. In addition, 36 water samples were collected from a reservoir and tested for the occurrence of all three bovine feces-associated markers along with fecal indicator bacteria (FIB),Campylobacterspp.,Escherichia coliO157, andSalmonellaspp. The overall host specificity values of the BacCow-UCD, cowM3, and B-AVs markers to differentiate between bovine and other nontarget host groups were 0.66, 0.88, and 1.00, respectively (maximum value of 1.00). The overall host sensitivity values of these markers, however, in composite bovine wastewater and individual bovine fecal DNA samples were 0.93, 0.90, and 0.60, respectively (maximum value of 1.00). Among the 36 water samples tested, 56%, 22%, and 6% samples were PCR positive for the BacCow-UCD, cowM3, and B-AVs markers, respectively. Among the 36 samples tested, 50% and 14% samples were PCR positive for theCampylobacter16S rRNA andE. coliO157rfbEgenes, respectively. Based on the results, we recommend that multiple bovine feces-associated markers be used if possible for bovine fecal pollution tracking. Nonetheless, the presence of the multiple bovine feces-associated markers along with the presence of potential zoonotic pathogens indicates bovine fecal pollution in the reservoir water samples. Further research is required to understand the decay rates of these markers in relation to FIB and zoonotic pathogens.

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Occurrence of antibiotic resistance inEscherichia colifrom surface waters and fecal pollution sources near Hamilton, Ontario

Canadian Journal of Microbiology ◽

10.1139/w05-028 ◽

2005 ◽

Vol 51 (6) ◽

pp. 501-505 ◽

Cited By ~ 64

Author(s):

Thomas A Edge ◽

Stephen Hill

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Surface Waters ◽

Municipal Wastewater ◽

Average Rate ◽

Fecal Pollution ◽

Pollution Sources ◽

Source Tracking ◽

E Coli ◽

Bird Feces

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

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Distribution of Human-Specific Bacteroidales and Fecal Indicator Bacteria in an Urban Watershed Impacted by Sewage Pollution, Determined Using RNA- and DNA-Based Quantitative PCR Assays

Applied and Environmental Microbiology ◽

10.1128/aem.02446-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 91-99 ◽

Cited By ~ 27

Author(s):

Vikram Kapoor ◽

Tarja Pitkänen ◽

Hodon Ryu ◽

Michael Elk ◽

David Wendell ◽

...

Keyword(s):

Quantitative Pcr ◽

Fecal Pollution ◽

Detection Sensitivity ◽

Rrna Gene ◽

Fecal Bacteria ◽

Combined Sewer Overflows ◽

Fecal Indicator ◽

Content Type ◽

Detection Frequency ◽

Human Specific

ABSTRACTThe identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci,Enterococcus faecalis, andEnterococcus faeciummarkers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specificBacteroidalesmarkers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specificBacteroidalesmarkers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources.

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Differential Decay of Enterococci and Escherichia coli Originating from Two Fecal Pollution Sources

Applied and Environmental Microbiology ◽

10.1128/aem.03781-12 ◽

2013 ◽

Vol 79 (7) ◽

pp. 2488-2492 ◽

Cited By ~ 30

Author(s):

Asja Korajkic ◽

Brian R. McMinn ◽

Valerie J. Harwood ◽

Orin C. Shanks ◽

G. Shay Fout ◽

...

Keyword(s):

Escherichia Coli ◽

Fecal Indicator Bacteria ◽

Fecal Pollution ◽

Pollution Sources ◽

Indicator Bacteria ◽

Fecal Indicator ◽

Content Type ◽

Marine Habitats ◽

Aquatic Mesocosms

ABSTRACTUsingin situsubtropical aquatic mesocosms, fecal source (cattle manure versus sewage) was shown to be the most important contributor to differential loss in viability of fecal indicator bacteria (FIB), specifically enterococci in freshwater andEscherichia coliin marine habitats. In this study, sunlight exposure and indigenous aquatic microbiota were also important contributors, whose effects on FIB also differed between water types.

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Multiple-tool approach of combining microbial markers with artificial and natural tracers to specify the origin of fecal contamination in a karst groundwater catchment

10.5194/egusphere-egu2020-19440 ◽

2020 ◽

Author(s):

Michael Sinreich

Keyword(s):

Fecal Contamination ◽

Spring Water ◽

Source Tracking ◽

Catchment Management ◽

Karst Groundwater ◽

Fecal Indicator ◽

Natural Tracers ◽

Groundwater Environment ◽

Fecal Markers ◽

The Impact

Diverse tools do exist to study the pathway from the source of a contamination to groundwater and related springs. The backward approach, i.e. sampling spring water to determine the origin of contamination, is more complex and requires multiple information. Microbial source tracking using host-specific markers is one of the tools, which however has shown to be insufficient as a stand-alone method, particularly in karst groundwater catchments. &#160;&#160;&#160;A karst spring in the Swiss Jura Mountains was studied with respect to the occurrence and correlation of a set of fecal indicators, including classical parameters as well as a number of bacteroidale markers. Sporadic monitoring proved the impact on spring water quality, mainly during high water stages. Additional event-focused sampling over varying recharge intensities evidenced a more detailed and divergent pattern of individual indicators. In particular, the results arose the question how to interpret peaks of human fecal markers in the rural-dominated catchment.A multiple-tool approach, complementing fecal indicator monitoring with artificial tracer experiments and natural tracers measurements, assessed the input, storage and transfer of potential contaminants in order to specify the origin of both ruminant and human fecal contaminations. Natural tracers allowed for distinguishing between water components from the saturated zone, from the soil/epikarst storage, or from freshly infiltrated rainwater. Furthermore, the breakthrough of injected dye tracers, and their remobilization during subsequent recharge events, respectively, were correlated to the occurrence of fecal markers. System&#8217;s residence time distribution over discharge, deduced from numerous former dye tracing tests, also allowed for attributing maximum travel distances to their arrival.The findings of the approach hypothesize the origin of human fecal contamination at the spring being in relationship with septic tanks undergoing concentrated overflow already at moderate rainfall intensities. Those intensities are, however, not sufficient to transport diffuse ruminant contamination through the vadose zone. Linkage with vulnerability assessment and land-use information can finally better locate the potential source points. Such toolbox provides not only useful basics for groundwater protection and catchment management, but also insight into general processes governing fate and transport of fecal contaminants in a karst groundwater environment.

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