Investigation of Escherichia coli Investigation of Escherichia coli in freshwater sources using in freshwater sources using

membrane filtration and Rep-membrane filtration and Rep-PCR DNA fingerprintingPCR DNA fingerprinting

Workshop AgendaWorkshop Agenda

IntroductionIntroduction Hands on: Load gelsHands on: Load gels Discussion of ProcedureDiscussion of Procedure Hands on: Filter Water Hands on: Filter Water Discussion of contexts to use labDiscussion of contexts to use lab Hands on: Results of GelHands on: Results of Gel

Diseases Associated with Diseases Associated with Recreational Waters in the US Recreational Waters in the US (1)(1)

GastroenteritisGastroenteritis– nausea, vomiting, abdominal cramps, fever, headache, nausea, vomiting, abdominal cramps, fever, headache,

diarrheadiarrhea Bacteria: Bacteria: E. coli O157:H7, SalmonellaE. coli O157:H7, Salmonella Parasite: Parasite: Cryptosporidium parvumCryptosporidium parvum Viruses: Norwalk-like virusViruses: Norwalk-like virus

DermatitisDermatitis– Skin infectionSkin infection

Bacteria: Bacteria: Pseudomonas aeruginosaPseudomonas aeruginosa MeningoencephalitisMeningoencephalitis

– hemorrhage and brain damagehemorrhage and brain damage Amoeba: Amoeba: Naegleria fowleriNaegleria fowleri

What is the source of microbial What is the source of microbial contamination?contamination?

Waste from mammals and birdsWaste from mammals and birds Storm water runoffStorm water runoff Untreated sewageUntreated sewage

How do you make sure recreational How do you make sure recreational water is safe?water is safe?

Strong correlation between the presence of Strong correlation between the presence of indicator bacteria and gastroenteritis indicator bacteria and gastroenteritis (9)(9)

Indicator BacteriaIndicator Bacteria– Present in waste water when pathogen presentPresent in waste water when pathogen present– Present in greater number than pathogenPresent in greater number than pathogen– Survives for a time in waterSurvives for a time in water– Unable to multiply in waterUnable to multiply in water– Non-pathogenicNon-pathogenic– Can be detected reliably, rapidly and cheaplyCan be detected reliably, rapidly and cheaply

E. coliE. coli is a standard indicator bacterium for fresh is a standard indicator bacterium for fresh waterwater

Test for Indicator BacteriaTest for Indicator Bacteria

– Fecal coliforms Fecal coliforms Facultative aerobesFacultative aerobes Gram negative Gram negative Nonspore-formingNonspore-forming Rod shapedRod shaped Ferment lactose and produce gas within 48hr. At 35Ferment lactose and produce gas within 48hr. At 35ooCC ExamplesExamples: Klebsiella pneumoniae, Enterobacter aerogenes, : Klebsiella pneumoniae, Enterobacter aerogenes,

Citrobacter sp., Escherichia coliCitrobacter sp., Escherichia coli

– Enterococci – fresh and marine waterEnterococci – fresh and marine water

– E. coliE. coli – fresh water – fresh water

USEPA, 1986aUSEPA, 1986a

NWC Monitoring Program NWC Monitoring Program

Use EPA methods and standards to monitor Use EPA methods and standards to monitor Lake Johanna for elevated levels of Lake Johanna for elevated levels of E. coli E. coli

EPA StandardsEPA Standards

Content shall not exceed 235 Content shall not exceed 235 E. coliE. coli per per 100ml of water100ml of water

andand

Geometric mean of not less than 5 samples, Geometric mean of not less than 5 samples, equally spaced over a 30 day period, should equally spaced over a 30 day period, should not exceed 126 not exceed 126 E. coliE. coli per 100 ml of water per 100 ml of water

Testing Lake Johanna for Testing Lake Johanna for E. coliE. coli

Collect Water SamplesCollect Water Samples– Three sitesThree sites– Tested weeklyTested weekly– Tested in duplicateTested in duplicate

SitesSites Tested 3 sites around Lake Tested 3 sites around Lake

Johanna once a week for 4 weeks:Johanna once a week for 4 weeks:– NWC swimming beachNWC swimming beach– Public beachPublic beach– Drain PipeDrain Pipe

NWC Private Beach

Drain Pipe Location

Public Access/ Beach

• Obtain a 50 mL water sample from Lake JohannaObtain a 50 mL water sample from Lake Johanna• Filter the water sample through a 0.45Filter the water sample through a 0.45μμm membranem membrane• Transfer filter to a membrane theromotolerant Transfer filter to a membrane theromotolerant E.coli E.coli

(mTEC) agar(mTEC) agar

(1)(1)

Water Testing ProcedureWater Testing Procedure

Water Testing Procedure cont.Water Testing Procedure cont. Incubate mTEC plate at 37 °C for 2 hours then Incubate mTEC plate at 37 °C for 2 hours then

transfer it to 45 °C and incubate overnighttransfer it to 45 °C and incubate overnight

(1)(1)

Water Testing Procedure cont.Water Testing Procedure cont. Transfer membrane onto another petri plate Transfer membrane onto another petri plate

containing 1-2 mL of urea substrate. Colonies containing 1-2 mL of urea substrate. Colonies that remain yellow are possibly that remain yellow are possibly E.coli.E.coli.

(Typical Results)(Typical Results)

a)a) b) b) Figure 4. Colonies after growth on mTEC Figure 4. Colonies after growth on mTEC

(a) and transfer to urease substrate (b).(a) and transfer to urease substrate (b). All of the colonies were yellow after growth All of the colonies were yellow after growth

on mTEC and two of these colonies turned on mTEC and two of these colonies turned pink/red after transfer to urease substrate. pink/red after transfer to urease substrate. These red colonies are NOT These red colonies are NOT E. coliE. coli, the , the ones that remained yellow are ones that remained yellow are E. coliE. coli and and should be counted. should be counted.

Remove membrane from mTEC and Remove membrane from mTEC and soak in Urea Substratesoak in Urea Substrate

Tests for the presence of UreaseTests for the presence of Urease– Contains urea and phenol redContains urea and phenol red – E. coliE. coli is urease negative is urease negative

Urea Ammoniaurease

E. coli levels in Lake Johanna – E. coli levels in Lake Johanna – summer 2009summer 2009

E. coli Levels Levels in Lake Johanna for the Summer of 2005

0

20

40

60

80

100

120

140

5/24

/05

6/1/

05

6/7/

05

6/14

/05

6/21

/05

6/28

/05

7/6/

05

7/13

/05

7/19

/05

7/26

/05

8/2/

05

8/10

/05

8/17

/05

Date

Nu

mb

er o

f E

. co

li C

olo

nie

s p

er

100m

l of

Wat

er

Public Beach 1Public Beah 2NWC Beah 1NWC Beah 2Storm Drain 1Storm Drain 2

E. coli levels in Lake Johanna for the Summer of 2006

020406080

100120140160

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Date

Nu

mb

er

of

E. c

oli

Co

lon

ies

/10

0m

l of

Wa

ter

Public Beach 1

Public Beach 2

NWC Beach 1

NWC Beach 2

Storm Drain 1

Storm Drain 2

0102030405060708090

E. c

oli/

10

0 m

l

5/28

/200

7

6/4/

2007

6/11

/200

7

6/18

/200

7

6/25

/200

7

7/2/

2007

7/9/

2007

7/16

/200

7

7/23

/200

7

7/30

/200

7

8/6/

2007

Date

Lake Johanna E. coli levels - 2007

Storm Drain

NWC Beach

Public Beach

ConclusionsConclusions

Contamination of Lake Johanna has not been a Contamination of Lake Johanna has not been a problem 2005-2009.problem 2005-2009.

Continue to monitor in 2010Continue to monitor in 2010– Plan of action if elevated levels detectedPlan of action if elevated levels detected

Notify appropriate individuals, post warnings, retest waterNotify appropriate individuals, post warnings, retest water

Continued preventionContinued prevention– Clean up after petsClean up after pets– Don’t feed ducks/geeseDon’t feed ducks/geese– Avoid swimming after heavy Avoid swimming after heavy

rainfallrainfall– No poopy diapers!No poopy diapers!

CaveatsCaveats

Current methods are not entirely satisfactoryCurrent methods are not entirely satisfactory– 24 hour delay in obtaining results24 hour delay in obtaining results

Newly developed hand held sensor for detecting Newly developed hand held sensor for detecting E. E. colicoli is available is available (12)(12)

– Sampling is only done once per weekSampling is only done once per week Tests for chronic problemsTests for chronic problems

ReferencesReferences1.1. Madigan, M.T. Martinko, J.M. Brock Biology of Microorganisms. Pearson Prentice Hall Publishing, 11Madigan, M.T. Martinko, J.M. Brock Biology of Microorganisms. Pearson Prentice Hall Publishing, 11 thth ed. p. 915- ed. p. 915-

921.921.2.2. Steverman B. Issue Update – Bacteria at beaches are the target of monitoring effort. Star Tribune 2005 July 6. Steverman B. Issue Update – Bacteria at beaches are the target of monitoring effort. Star Tribune 2005 July 6.

Retrieved from News Bank database.Retrieved from News Bank database.3.3. [anonymous]. Lake Superior lakewide management plan 2006. Available from: [anonymous]. Lake Superior lakewide management plan 2006. Available from:

www.epa.gov/glnpo/lakesuperior/2006/ls_chapter5_upd_2006.pdfwww.epa.gov/glnpo/lakesuperior/2006/ls_chapter5_upd_2006.pdf Accessed 2007 Apr 11. Accessed 2007 Apr 11.4.4. Duchschere K. Roseville beach closed because of Duchschere K. Roseville beach closed because of E. coliE. coli levels. Star Tribune 2006 July 28. Retrieved 2007 Apr 11 levels. Star Tribune 2006 July 28. Retrieved 2007 Apr 11

from Access World News database.from Access World News database.5.5. Brunswick M. Lake Harriet beach closed over E. coli levels in open. Star Tribune 2005 June 12. Retrieved from Brunswick M. Lake Harriet beach closed over E. coli levels in open. Star Tribune 2005 June 12. Retrieved from

News Bank database.News Bank database.6.6. Coleman N. One thing is clear: Increasingly, state’s waters aren’t. Star Tribune 2005 Aug 7. Retrieved from Coleman N. One thing is clear: Increasingly, state’s waters aren’t. Star Tribune 2005 Aug 7. Retrieved from

Access World News database. Access World News database. 7.7. Collins T. Carver County – Lake Minnewashta beach closed for tests. Star Tribune 2005 July 15. Retrieved from Collins T. Carver County – Lake Minnewashta beach closed for tests. Star Tribune 2005 July 15. Retrieved from

News Bank database.News Bank database.8.8. Lerner M. E. coli closes Coon Lake Beach in Anoka County. Star Tribune 2005 July 9. Retrieved from News Bank Lerner M. E. coli closes Coon Lake Beach in Anoka County. Star Tribune 2005 July 9. Retrieved from News Bank

database.database.9.9. USEPA (1986a) Ambient water quality criteria for bacteria- 1986. Office of Water Regulations and Standards, USEPA (1986a) Ambient water quality criteria for bacteria- 1986. Office of Water Regulations and Standards,

Criteria and Standards Division, Washington, DC. EPA-440/5-84/002. Criteria and Standards Division, Washington, DC. EPA-440/5-84/002. 10.10. Madigan, M.T. Martinko, J.M. Brock Biology of Microorganisms. Pearson Prentice Hall Publishing, 11Madigan, M.T. Martinko, J.M. Brock Biology of Microorganisms. Pearson Prentice Hall Publishing, 11 thth ed. p. 677. ed. p. 677.11.11. Talaro, KP, Talaro, A. Foundations in Microbiology. McGraw Hill Publishing. 4Talaro, KP, Talaro, A. Foundations in Microbiology. McGraw Hill Publishing. 4 thth ed. p. 333. ed. p. 333.12.12. Detecting bacteria with mechanical cantelievers. Science Daily. Nov. 1, 2006. Available from Detecting bacteria with mechanical cantelievers. Science Daily. Nov. 1, 2006. Available from

http://www.sciencedaily.com/videos/2006-11-09/ Accessed 5-8-07. http://www.sciencedaily.com/videos/2006-11-09/ Accessed 5-8-07. 13.13. The rep-PCR genomic fingerprinting. Available from The rep-PCR genomic fingerprinting. Available from http://www.msu.edu/~debruijn/http://www.msu.edu/~debruijn/. Accessed 5-9-07.. Accessed 5-9-07.14.14. Vierstraete, Andy. “Principle of the PCR.” 11 Aug. 1999. University of Ghent. 1 May 2007. Vierstraete, Andy. “Principle of the PCR.” 11 Aug. 1999. University of Ghent. 1 May 2007.

<http://users.ugent.be/~avierstr/principles/pcr.html>.<http://users.ugent.be/~avierstr/principles/pcr.html>.15.15. Snustad, Simmons, Principles of Genetics. Wiley Publishing, 3Snustad, Simmons, Principles of Genetics. Wiley Publishing, 3 rdrd ed. p. 498 ed. p. 49816.16. Belk, Borden, Biology Science for Life, Pearson Prentice Hall Publishing. 2Belk, Borden, Biology Science for Life, Pearson Prentice Hall Publishing. 2 ndnd ed. p. 184 ed. p. 184

What is the source of microbial What is the source of microbial contamination?contamination?

Waste from mammals and birdsWaste from mammals and birds Untreated sewageUntreated sewage

Microbial Source TrackingMicrobial Source Tracking

Microbial Source Tracking MethodsMicrobial Source Tracking Methods

Antibiotic resistance patternsAntibiotic resistance patterns ribotyping ribotyping Rep-PCR DNA fingerprintingRep-PCR DNA fingerprinting

(12)

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

(13)

rep-PCR DNA fingerprinting of rep-PCR DNA fingerprinting of E. coliE. coli isolates to investigate strain diversityisolates to investigate strain diversity

Agarose gel electrophoresisAgarose gel electrophoresis

(14)

http://www.dnalc.org/ddnalc/resources/electrophoresis.html

Agarose gel electrophoresisAgarose gel electrophoresis

(15)

rep-PCR DNA fingerprintrep-PCR DNA fingerprint

1kb 1 2 3 4 5 6 7 8 9 10 11 12 13 lab DH5 0 1kb

ConclusionsConclusions– At least 7 distinct types of At least 7 distinct types of E. coliE. coli were identified from were identified from

Lake JohannaLake Johanna– They are different than laboratory strains of They are different than laboratory strains of E. coliE. coli

Future research questionsFuture research questions– What is the strain diversity in Lake Johanna?What is the strain diversity in Lake Johanna?– Is there a shifting spectrum of strains?Is there a shifting spectrum of strains?– How does strain diversity compare in other How does strain diversity compare in other

environments?environments?

Questions?Questions?