Microbial Source Tracking Using Quantitative and Digital PCR To Identify Sources of Fecal Contamination in Stormwater, River Water, and Beach Water in a Great Lakes Area of Concern

ABSTRACTAreas of concern (AOCs) around the Great Lakes are characterized by historic and ongoing problems with microbial water quality, leading to beneficial use impairments (BUIs) such as beach postings and closures. In this study, we assessed river and beach sites within the Rouge River watershed, associated stormwater outfalls, and at Rouge Beach. The concentrations ofEscherichia colias well as human- and gull-specific qPCR microbial source tracking (MST) markers were assessed at all sites. A preliminary comparison of digital PCR (dPCR) methodologies for both MST markers was conducted regarding sensitivity and specificity. Within the watershed, the outfalls were found to be a prominent source of human fecal contamination, with two outfalls particularly affected by sewage cross-connections. However, the occurrence of human fecal contamination along Rouge Beach and in the lower portions of the watershed was largely dependent on rain events. Gull fecal contamination was the predominant source of contamination at the beach, particularly during dry weather. The multiplex human/gull dPCR methodology used in this study tended to be more sensitive than the individual quantitative PCR (qPCR) assays, with only a slight decrease in specificity. Both dPCR and qPCR methodologies identified the same predominance of human and gull markers in stormwater and beach locations, respectively; however, the dPCR multiplex assay was more sensitive and capable of detecting fecal contamination that was undetected by qPCR assays. These results demonstrate the dPCR assay used in this study could be a viable tool for MST studies to increase the ability to identify low levels of fecal contamination.IMPORTANCEFecal contamination of recreational water poses a persistent and ongoing problem, particularly in areas of concern around the Great Lakes. The identification of the source(s) of fecal contamination is essential for safeguarding public health as well as guiding remediation efforts; however, fecal contamination may frequently be present at low levels and remain undetectable by certain methodologies. In this study, we utilized microbial source tracking techniques using both quantitative and digital PCR assays to identify sources of contamination. Our results indicated high levels of human fecal contamination within stormwater outfalls, while lower levels were observed throughout the watershed. Additionally, high levels of gull fecal contamination were detected at Rouge Beach, particularly during drier sampling events. Furthermore, our results indicated an increased sensitivity of the digital PCR assay to detect both human and gull contamination, suggesting it could be a viable tool for future microbial source tracking studies.

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Use of Bacteroidales Microbial Source Tracking To Monitor Fecal Contamination in Fresh Produce Production

Applied and Environmental Microbiology ◽

10.1128/aem.02891-13 ◽

2013 ◽

Vol 80 (2) ◽

pp. 612-617 ◽

Cited By ~ 14

Author(s):

Kruti Ravaliya ◽

Jennifer Gentry-Shields ◽

Santos Garcia ◽

Norma Heredia ◽

Anna Fabiszewski de Aceituno ◽

...

Keyword(s):

Fresh Produce ◽

Fecal Contamination ◽

Microbial Source Tracking ◽

Source Tracking ◽

Rrna Gene ◽

Specific Marker ◽

Production Environment ◽

Content Type ◽

Food Borne

ABSTRACTIn recent decades, fresh and minimally processed produce items have been associated with an increasing proportion of food-borne illnesses. Most pathogens associated with fresh produce are enteric (fecal) in origin, and contamination can occur anywhere along the farm-to-fork chain. Microbial source tracking (MST) is a tool developed in the environmental microbiology field to identify and quantify the dominant source(s) of fecal contamination. This study investigated the utility of an MST method based onBacteroidales16S rRNA gene sequences as a means of identifying potential fecal contamination, and its source, in the fresh produce production environment. The method was applied to rinses of fresh produce, source and irrigation waters, and harvester hand rinses collected over the course of 1 year from nine farms (growing tomatoes, jalapeño peppers, and cantaloupe) in Northern Mexico. Of 174 samples, 39% were positive for a universalBacteroidalesmarker (AllBac), including 66% of samples from cantaloupe farms (3.6 log10genome equivalence copies [GEC]/100 ml), 31% of samples from tomato farms (1.7 log10GEC/100 ml), and 18% of samples from jalapeño farms (1.5 log10GEC/100 ml). Of 68 AllBac-positive samples, 46% were positive for one of three human-specific markers, and none were positive for a bovine-specific marker. There was no statistically significant correlation betweenBacteroidalesand genericEscherichia coliacross all samples. This study provides evidence thatBacteroidalesmarkers may serve as alternative indicators for fecal contamination in fresh produce production, allowing for determination of both general contamination and that derived from the human host.

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Comparative microbial source tracking methods for identification of fecal contamination sources at Sunnyside Beach in the Toronto region area of concern

Journal of Water and Health ◽

10.2166/wh.2016.296 ◽

2016 ◽

Vol 14 (5) ◽

pp. 839-850 ◽

Cited By ~ 13

Author(s):

Zachery R. Staley ◽

Thomas A. Edge

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Fecal Contamination ◽

Bird Species ◽

Microbial Source Tracking ◽

Source Tracking ◽

Pcr Marker ◽

E Coli ◽

Remediation Strategies ◽

Humber River ◽

Area Of Concern

Several beaches within the Toronto region area of concern have persistent issues with fecal contamination, causing a beach beneficial use impairment (BUI). In this study, Escherichia coli, including ampicillin-resistant strains, were enumerated via culturable and quantitative polymerase chain reaction (qPCR) methods. Microbial source tracking (MST) markers (for general Bacteroidales, human, ruminant/cow, gull, and dog) were detected and enumerated via PCR and qPCR to identify sources of fecal contamination at Sunnyside Beach and in the Humber River. Human, cow, and dog markers had good host-specificity, while gull markers sometimes amplified a few other bird species. The ruminant endpoint PCR marker amplified a variety of other animal species rendering it less useful. Both human and gull fecal contamination were prevalent in the Humber River, while Sunnyside Beach was predominantly impacted by gull fecal contamination. Human sewage impacts were more prevalent in the lower Humber River, particularly in Black Creek. However, to reduce Sunnyside beach postings, reducing bird fecal contamination in the river and at the beach would be necessary. When there are high levels of E. coli throughout a beachshed, an MST toolbox approach can add value to discriminate source(s) of E. coli contamination and guide decisions relating to public health risk and remediation strategies.

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0638 - Selecting microbial source tracking markers to identify fecal contamination from agricultural sources in Laguna Lake, Philippines

10.26226/morressier.5b5199bfb1b87b000ecef785 ◽

2018 ◽

Author(s):

Windell L. Rivera, PhD

Keyword(s):

Fecal Contamination ◽

Microbial Source Tracking ◽

Source Tracking ◽

Laguna Lake

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Investigating Sources of Fecal Contamination in Storm Drain Outfalls: Application of Genotypic Microbial Source Tracking

Proceedings of the Water Environment Federation ◽

10.2175/193864717822156497 ◽

2017 ◽

Vol 2017 (6) ◽

pp. 4813-4825

Author(s):

Darshan Baral ◽

Xu Li ◽

David Admiraal ◽

Bruce Dvorak

Keyword(s):

Fecal Contamination ◽

Microbial Source Tracking ◽

Source Tracking ◽

Storm Drain

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Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes

FEMS Microbiology Reviews ◽

10.1111/1574-6976.12031 ◽

2014 ◽

Vol 38 (1) ◽

pp. 1-40 ◽

Cited By ~ 303

Author(s):

Valerie J. Harwood ◽

Christopher Staley ◽

Brian D. Badgley ◽

Kim Borges ◽

Asja Korajkic

Keyword(s):

Health Outcomes ◽

Human Health ◽

Fecal Contamination ◽

Microbial Source Tracking ◽

Source Tracking ◽

Environmental Waters

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Multi-Year Microbial Source Tracking Study Characterizing Fecal Contamination in an Urban Watershed

Water Environment Research ◽

10.2175/106143016x14798353399412 ◽

2017 ◽

Vol 89 (2) ◽

pp. 127-143 ◽

Cited By ~ 5

Author(s):

Rebecca N. Bushon ◽

Amie M.G. Brady ◽

Eric D. Christensen ◽

Erin A. Stelzer

Keyword(s):

Fecal Contamination ◽

Microbial Source Tracking ◽

Source Tracking ◽

Urban Watershed

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Evaluation of Two Library-Independent Microbial Source Tracking Methods To Identify Sources of Fecal Contamination in French Estuaries

Applied and Environmental Microbiology ◽

10.1128/aem.03003-06 ◽

2007 ◽

Vol 73 (15) ◽

pp. 4857-4866 ◽

Cited By ~ 99

Author(s):

Michèle Gourmelon ◽

Marie Paule Caprais ◽

Raphaël Ségura ◽

Cécile Le Mennec ◽

Solen Lozach ◽

...

Keyword(s):

Water Samples ◽

Fecal Contamination ◽

Microbial Source Tracking ◽

Source Tracking ◽

Rrna Gene ◽

Specific Marker ◽

Gene Markers ◽

High Level ◽

Bird Feces

ABSTRACT 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.

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Microbial Source-Tracking Reveals Origins of Fecal Contamination in a Recovering Watershed

Water ◽

10.3390/w11102162 ◽

2019 ◽

Vol 11 (10) ◽

pp. 2162 ◽

Cited By ~ 3

Author(s):

Hyatt Green ◽

Daniel Weller ◽

Stephanie Johnson ◽

Edward Michalenko

Keyword(s):

Urban Areas ◽

Bacterial Contamination ◽

Fecal Contamination ◽

Microbial Source Tracking ◽

Mitigation Strategies ◽

Source Tracking ◽

Chemical Parameters ◽

Fecal Indicator ◽

Combined Sewer ◽

Rain Events

Fecal contamination of waterbodies due to poorly managed human and animal waste is a pervasive problem that can be particularly costly to address, especially if mitigation strategies are ineffective at sufficiently reducing the level of contamination. Identifying the most worrisome sources of contamination is particularly difficult in periurban streams with multiple land uses and requires the distinction of municipal, agricultural, domestic pet, and natural (i.e., wildlife) wastes. Microbial source-tracking (MST) methods that target host-specific members of the bacterial order Bacteroidales and others have been used worldwide to identify the origins of fecal contamination. We conducted a dry-weather study of Onondaga Creek, NY, where reducing fecal contamination has been approached mainly by mitigating combined sewer overflow events (CSOs). Over three sampling dates, we measured in-stream concentrations of fecal indicator bacteria; MST markers targeting human, ruminant, and canine sources; and various physical–chemical parameters to identify contaminants not attributable to CSOs or stormwater runoff. We observed that despite significant ruminant inputs upstream, these contaminants eventually decayed and/or were diluted out and that high levels of urban bacterial contamination are most likely due to failing infrastructure and/or illicit discharges independent of rain events. Similar dynamics may control other streams that transition from agricultural to urban areas with failing infrastructure.

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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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