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Microbial Source Tracking:In Search of Human, Gull, and
Dog
Mauricio Larenas
12th Annual Great Lakes BeachAssociation Conference
October 16-18, 2012
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Fecal Indicators
Used to determine the presence of pathogenic organisms
Typical Fecal IndicatorsE. ColiEnteroccocus
Fecal ColiformBacteroidales (EPA Method B)
Useful for the detection of general fecal pollution
Provide no insight into the potential sources of fecal
pollution
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testingsamples
Project results andoutcomes
Conclusion
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Pathways for Contamination
Industrial PlantsFarm wasteUrban runoff SewageSeptic LeaksWildlife
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testingsamples
Project results andoutcomes
Conclusion
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ProblemState of Delaware wasobserving high levels of Enterococcus at an
estuarine inlandrecreational beach
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
Project Case Study
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Project Case Study
GoalDetermine if Human , Dog or Seagull fecalsources contributed to the high Enterococcuslevels
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
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FundingFunding source for Delaware project:
EPA Region 3 BEACH Act grantDelaware Environmental Laboratory
Michigan funding:Nonpoint Source Program Grants
DESCRIPTION: Michigan's Department of Environmental Qualityadministers a Nonpoint Source (NPS) Program that assists state,federal, and local partners to restore water bodies impaired byNPS pollution and protect high quality waters from impairmentscaused by NPS pollution.
Michigan Sea GrantDESCRIPTION: Michigan Sea Grant, a cooperative program of theUniversity of Michigan and Michigan State University, is part of theNational Sea Grant College Program. It aims to promote betterunderstanding, conservation and use of Michigans coastalresources. Michigan Sea Grant funds integrated research projectsdesigned to foster science-based decisions about the use andconservation of Great Lakes resources.
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
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Validate the sensitivity of two biomarkers
Human Polyomavirus- USFRaw untreated wastewater from 3 wastewater treatmentfacilities in Sussex County, DE
ResultsSewage tested positive for HPyVs
Gull Biomarker - USF/EPA37 fecal samples from herring and/or laughing gulls testedindividually
Results84% of the samples tested positive for the gull associatedmarker
Validation of local fecal samplesMicrobial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
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PlanUse 1 Seagull, 1 Dog, and 2 Human tests todetermine which host(s) was contributing tofecal contamination49 Samples taken through the summer monthsand sent to Source Molecular for analysis
Equal number of samples analyzed by USFSamples taken in knee deep water 3 times a week at
3 locations
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
Project Case Study
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PlanDNREC would test for general fecal indicator
Enterococcus concentrations - Enterolert
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
Project Case Study
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Submitting SamplesMicrobial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
Samples where filtered at the Delaware DNREClaboratory and submitted frozen on dry ice
Samples typically submitted as 500mL water samplesovernight
Sterile bottles and packing material provided in mostcases
Serves as convenience and to maintain QA/QC
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Microbial Source Tracking
Host gut conditions and natural selection create uniquegenetics between host microbial communities.
Cow and Chicken microbial communities are not the same
Genetic differences are identified and used as the basis forfecal host discrimination
Host specific genes are detected using qPCRExponentially copies the sequence of interest.Monitors and calculates the starting quantity
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
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What hosts can MST detect?Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
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Presence/Absence MST ResultsMicrobial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
Turnaround time: 5-10 working daysDetermines +/- of host specific fecal indicators
Multiple tests targeting the same host (e.g. Human) allow confirmationof results
Valuable for screening multiple hosts prior to quantification
Less expensive than quantification
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Quantification MST Results
Turnaround time: 5-10 working days
Reports the genetic copiesfrom host specific fecalindicators
These values are used tointerpret the hosts relativecontribution to fecalcontamination
Results reported as:NegativeTrace
Minor ContributorMajor Contributor
A general fecal indicator canalso be quantified todetermine the generalcontamination
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
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ResultsDog
4% of the samples were positiveQuantification values showed few dog specific genetic
copiesHuman Bacteroidetes (HF183)
8% of the samples were positiveQuantification values showed few human specificgenetic copies
Seagull98% of the samples were positive
Quantification values showed abundant seagull specificgenetic copies
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
Project Case Study
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Project conclusions and outcomesSeagull was likely a major contributor the fecalpollution detected at the beach
Human was a possible contributor but occurringsporadically
Dog was not a likely fecal pollution contributor
The purpose of the MST study was to confirm the
visual observations that seagulls contributed to theelevated levels of Enterococcus (Delaware s waterquality indicator) at this beach. Based on MST results,there will be no change to management actions atthe site.
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
Project Case Study
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Project Considerations
Are markers available for the potential fecal sources in thewater system?
Are the markers properly validated?Cross reactivity and sensitivity for those markers
What is the scale and budget for the project?
Microbes used for MST markers may not have a directcorrelation to traditional fecal indicators
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
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Microbial Source TrackingMicrobial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
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About Source Molecular
Established in 2002 to fill the void between research andimplementation
Active in the research and development of newapproaches
Strict focus on Microbial Source Tracking
Detection of human, cattle, swine, bird, gull, goose, chicken,dog, deer, ruminant and horse possible
Pending ISO 17025 accreditation
Microbial Source TrackingIn Search ofHuman, Gull, and Dog
Fecal pollution problem
Microbial sourcetracking plan
Submitting and testing
samples
Project results andoutcomes
Conclusion
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4985 SW 74th CourtMiami, Florida 33155 USATelephone: (786) 220-0379
Mauricio [email protected]
Case Study: Microbial Source Tracking at Tower Road Bayside Beach, Rehoboth Bay, Sussex County, Delaware2011 (Dr. Humphries, DE DNREC; Dr. Harwood, USF; and Mr. Larenas, Source Molecular)