Using molecular biology tools to investigate fecal contamination

Tami L. Sivy, Ph.D.

Professor of Chemistry

Quick review of standard methods

Rapid DNA testing for beach closings

Genetic methods for microbial source tracking

Presentation TopicsWhy am I speaking during this session on beach closings BUI?

Fecal indicator bacteria

Fecal coliforms--bacterial indicator of feces contamination

--typically Escherichia coli are measured

Standard EPA culture method

Colilert culture method measures E. coli levels after overnight growth

4-Methylumbelliferyl β-D-glucuronide (MUGlcU) *Allowable level for full body human contact is < 300 CFU/100 mL

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Rapid DNA testing:Taqman qPCR relies on a fluorescent reporter

http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/real-time-pcr/taqman-probe-based-gene-expression-analysis.html

(qPCR: quantitative Polymerase Chain Reaction)

A= Reporter Q = Ouenche,

s1,and OiSJ)hu.-:en,ent O ~

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Cleavage

*'' ~. ,. ,. •• ,. -r .. Poly1n erization

* J Compl&ted ' \ ·- --, •• ,. ••

•• -r ••

Collect water sample

Transport to lab

Filter water sample Run qPCR

Extract DNA

Rapid DNA testing

Obtain results

Analyze dataFluorescence a 1/CT

CT a 1/[DNA]

Adapted from MSU figure

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Validation study

Standardized prototol, Master same standard Standardized data

prescribed • wrve5, • quality

reagents, positive & atteptanc;e

reference negative criteria

and c;ontrol control data materials

Water Research Volume 156, 1 June 2019, Pages 456-464

ELSLYlER

Standardized data quality acceptance criteria for a rapid Escherichia coli qPCR method (Draft Method C) for water quality monitoring at recreational beaches

Mano Sivaganesan •, Tiong Gim Aw h, Shannon Briggs<, Erin Dreelin d, Asli Asian °, Samuel Dorevitch f, Abhilasha

Shreslha f, Natasha Isaacs S,Julie Kinzelman ", Greg l<leinheinz ;. Rachel Noblei, Rick Rediske •, Brian Scull k, Susan

Rosenberg 1, Barbara Weberman 1, Tami Sivy m, Ben Southwell n, Shawn Siefring O ••• Richard Haugland O P.. B

l!I Show more

https:/ /d oi.org/10.1016/j.watres .2019 .03.011 Get rights and content

1.863 log10

DNA copies/reaction

Acceptance level for full body human contact

The results are entered in Michigan Beach Guard each afternoon for sampled beaches.

qPCR sampling sites now

Map produced by Chris Vandenberg, MDEQ

Microbial Source Tracking project

Health Department Officers

Makes final decisions for policy and public health.

Sanitarian and students

Conducts monitoring and surveys

Laboratory

Analyzes water samples, reports results to

Health Department

AOC Coordinator, EGLE, USEPAOversees project with regulatory and financial

recommendations, receives quarterly and final reports

Project/ QAPP Manager

Supervises sampling and laboratory

measurements, prepares necessary reports

The objective of the project has been (and will be) to identify locations and sources of fecal contamination that negatively impact Saginaw Bay,

especially at sites that the public uses for recreational purposes

Source trackingCan we determine where the contamination originates?

Bacteriodes

We can exploit subtle differences in gene sequences of bacteria that grow in varying hosts. Here, we use primers designed for the 16S rDNA from Bacteroides from host organisms.

www.biotechniques.org

Identify source(s)'t10

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qPCR standard curves for each marker

y = -3.3267x + 39.11R² = 0.9991

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6 7

Ruminant

y = -3.389x + 39.918R² = 0.9986

0

5

10

15

20

25

30

35

40

0.E+00 1.E+00 2.E+00 3.E+00 4.E+00 5.E+00 6.E+00 7.E+00

Human

y = -3.3635x + 38.622R² = 0.9988

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6 7

Bovine

RumBac2 HF183

CowM2

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Our home: the Saginaw Bay Watershed

Iosco County source tracking sampling sites 2018

Site # Site Name

19Tawas State Park - Day

Use

20Tawas State Park -

Campground

21 County Road

22 East Tawas

23 1st Street

24 Tawas City

25 Gateway

26 Douglas Drive

27 Alabaster

Example of pilot study results

0

0.2

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1.2

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1.6

1.8

2

0.0

500.0

1000.0

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2500.0

7/24 8/7 8/14

Sou

rce (log1

0 D

NA

cop

ies/reaction

)

Co

liler

t (C

FU/1

00

mL)

Iosco County: 1st Street Bridge (Tawas River)

Colilert Human Ruminant

0

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1

1.2

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0

500

1000

1500

2000

2500

6/19 6/26 7/3 7/10 7/17 7/24 7/31 8/7 8/23

Sou

rce (log1

0 D

NA

cop

ies/reaction

)

Co

liler

t (C

FU/1

00

mL)

Iosco County: Gateway Park Beach

Colilert Human Ruminant

0

0.2

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1

1.2

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2

0

500

1000

1500

2000

2500

7/31 8/7 8/14 8/23

Sou

rce (log1

0 D

NA

cop

ies/reaction

)

Co

liler

t (C

FU/1

00

mL)

Iosco County: Douglas Drive Beach

Colilert Human Ruminant

0

0.2

0.4

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1

1.2

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2

0.0

500.0

1000.0

1500.0

2000.0

2500.0

7/24 7/31 8/7 8/14 8/23

Sou

rce (log1

0 D

NA

cop

ies/reaction

)

Co

liler

t (C

FU/1

00

mL)

Iosco County: State Park Day Use Beach

Colilert Human Ruminant

L • • • •

- - - I L _ L - -• • • •

Starting to piece things together…

0

0.2

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Gateway Douglas Dr Tawas State Park- Day Use 1st Street

Sou

rce

(lo

g10

DN

A c

op

ies/

reac

tio

n

Co

liler

t (C

FU/1

00

mL)

Rain event 8/7: Iosco County

Colilert Ruminant Human

Bio-Rad digital droplet PCR system

Photo provided by Matt Flood, MSU

.-.......... ~ .... -

ddPCR

Oncotarget. 2017; 8:85234-85251.

qPCR B ddPCR L partitioning

PCR - - --""'•► - - -

- -- - --20,0CIO droplets

analysl1J '

quantllllllve .._,..,eaUll'M.,o,tll.. __ __, ______ ..,.

•

analy1Jls . •

-""'•►0 posJtivo cJroplot 0 negative drople

What now???

Filters have been stored for source tracking analysisfrom beaches and inlets for the entire season of 2019

New markers!

Continue optimization of ddPCR methods

Consider upstream sites for analysis

My students: Olivia Bishop, Trenton Vogel, Emily Greeson, April Lukowski, Marc Dean

Bruce HartEGLE: Shannon BriggsOGL: John RileyMSU: Jean Pierre Nshimyimana, Matt Flood, Erin DreelinEPA: Rich Haugland, Orin Shanks, Mano Sivaganesan, Kevin Oshima

Health Departments: Joel Strasz, Joel Kwiatkowski, Tip MacGuire, Don Rousseau

Sea Grant: Meaghan GassSVSU: Saginaw Bay Environmental Science Institute,Chemistry Department, Sponsored Programs

Acknowledgements