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Investigation of Escherichia coli
in freshwater sources using
membrane filtration and RepPCR DNA fingerprinting
Workshop Agenda
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Introduction
Hands on: Load gels
Discussion of Procedure
Hands on: Filter Water
Discussion of contexts to use lab
Hands on: Results of Gel
Diseases Associated with
Recreational Waters in the US (1)
 Gastroenteritis
– nausea, vomiting, abdominal cramps, fever, headache,
diarrhea
 Bacteria: E. coli O157:H7, Salmonella
 Parasite: Cryptosporidium parvum
 Viruses: Norwalk-like virus
 Dermatitis
– Skin infection
 Bacteria: Pseudomonas aeruginosa
 Meningoencephalitis
– hemorrhage and brain damage
 Amoeba: Naegleria fowleri
What is the source of microbial
contamination?
 Waste from mammals and birds
 Storm water runoff
 Untreated sewage
How do you make sure recreational
water is safe?
 Strong correlation between the presence of
indicator bacteria and gastroenteritis (9)
 Indicator Bacteria
–
–
–
–
–
–
Present in waste water when pathogen present
Present in greater number than pathogen
Survives for a time in water
Unable to multiply in water
Non-pathogenic
Can be detected reliably, rapidly and cheaply
 E. coli is a standard indicator bacterium for fresh
water
Test for Indicator Bacteria
– Fecal coliforms
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Facultative aerobes
Gram negative
Nonspore-forming
Rod shaped
Ferment lactose and produce gas within 48hr. At 35oC
Examples: Klebsiella pneumoniae, Enterobacter aerogenes,
Citrobacter sp., Escherichia coli
– Enterococci – fresh and marine water
– E. coli – fresh water
 USEPA, 1986a
NWC Monitoring Program
 Use EPA methods and standards to monitor
Lake Johanna for elevated levels of E. coli
EPA Standards
 Content shall not exceed 235 E. coli per
100ml of water
and
 Geometric mean of not less than 5 samples,
equally spaced over a 30 day period, should
not exceed 126 E. coli per 100 ml of water
Testing Lake Johanna for E. coli
 Collect Water Samples
– Three sites
– Tested weekly
– Tested in duplicate
Sites
 Tested 3 sites around Lake
Johanna once a week for 4
weeks:
– NWC swimming beach
– Public beach
– Drain Pipe
Public
Access/
Beach
Drain Pipe
Location
NWC
Private
Beach
Water Testing Procedure
• Obtain a 50 mL water sample from Lake
Johanna
• Filter the water sample through a 0.45μm
membrane
• Transfer
filter
to
a
membrane
theromotolerant E.coli (mTEC) agar
Water Testing Procedure cont.
 Incubate mTEC plate at 37 °C for 2 hours
then transfer it to 45 °C and incubate
overnight
Water Testing Procedure cont.
 Transfer membrane onto another petri
plate containing 1-2 mL of urea substrate.
Colonies that remain yellow are possibly
E.coli.
 a)
b)
 Figure 4. Colonies after growth on mTEC
(a) and transfer to urease substrate (b).
 All of the colonies were yellow after growth
on mTEC and two of these colonies turned
pink/red after transfer to urease substrate.
These red colonies are NOT E. coli, the
ones that remained yellow are E. coli and
should be counted.
Remove membrane from mTEC and
soak in Urea Substrate
 Tests for the presence of Urease
– Contains urea and phenol red
– E. coli is urease negative
Urea
urease
Ammonia
E. coli levels in Lake Johanna –
summer 2009
140
120
Public Beach 1
Public Beah 2
NWC Beah 1
NWC Beah 2
Storm Drain 1
Storm Drain 2
100
80
60
40
20
0
5/
24
/0
6/ 5
1/
0
6/ 5
7/
6/ 05
14
/
6/ 05
21
/
6/ 05
28
/0
7/ 5
6/
7/ 05
13
/
7/ 05
19
/
7/ 05
26
/0
8/ 5
2/
8/ 05
10
/
8/ 05
17
/0
5
Number of E. coli Colonies per
100ml of Water
E. coli Levels Levels in Lake Johanna for the Summer of 2005
Date
Number of E. coli
Colonies/100ml of Water
E. coli levels in Lake Johanna for the Summer of
2006
160
140
120
100
80
60
40
20
0
Public Beach 1
Public Beach 2
NWC Beach 1
NWC Beach 2
Storm Drain 1
Storm Drain 2
1
2
3 4
5
6 7
8 9 10 11 12 13 14
Date
E. coli/100 ml
90
80
70
60
50
40
30
20
10
0
Date
8/6/2007
7/30/2007
7/23/2007
7/16/2007
7/9/2007
7/2/2007
6/25/2007
6/18/2007
6/11/2007
6/4/2007
5/28/2007
Lake Johanna E. coli levels - 2007
Storm Drain
NWC Beach
Public Beach
Conclusions
 Contamination of Lake Johanna has not been a
problem 2005-2009.
 Continue to monitor in 2010
– Plan of action if elevated levels detected
 Notify appropriate individuals, post warnings, retest water
 Continued prevention
– Clean up after pets
– Don’t feed ducks/geese
– Avoid swimming after heavy
rainfall
– No poopy diapers!
Caveats
 Current methods are not entirely satisfactory
– 24 hour delay in obtaining results
 Newly developed hand held sensor for detecting E.
coli is available (12)
– Sampling is only done once per week
 Tests for chronic problems
References
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11.
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16.
Madigan, M.T. Martinko, J.M. Brock Biology of Microorganisms. Pearson Prentice Hall Publishing, 11 th ed. p. 915921.
Steverman B. Issue Update – Bacteria at beaches are the target of monitoring effort. Star Tribune 2005 July 6.
Retrieved from News Bank database.
[anonymous]. Lake Superior lakewide management plan 2006. Available from:
www.epa.gov/glnpo/lakesuperior/2006/ls_chapter5_upd_2006.pdf Accessed 2007 Apr 11.
Duchschere K. Roseville beach closed because of E. coli levels. Star Tribune 2006 July 28. Retrieved 2007 Apr 11
from Access World News database.
Brunswick M. Lake Harriet beach closed over E. coli levels in open. Star Tribune 2005 June 12. Retrieved from
News Bank database.
Coleman N. One thing is clear: Increasingly, state’s waters aren’t. Star Tribune 2005 Aug 7. Retrieved from Access
World News database.
Collins T. Carver County – Lake Minnewashta beach closed for tests. Star Tribune 2005 July 15. Retrieved from
News Bank database.
Lerner M. E. coli closes Coon Lake Beach in Anoka County. Star Tribune 2005 July 9. Retrieved from News Bank
database.
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.
Madigan, M.T. Martinko, J.M. Brock Biology of Microorganisms. Pearson Prentice Hall Publishing, 11 th ed. p. 677.
Talaro, KP, Talaro, A. Foundations in Microbiology. McGraw Hill Publishing. 4 th ed. p. 333.
Detecting bacteria with mechanical cantelievers. Science Daily. Nov. 1, 2006. Available from
http://www.sciencedaily.com/videos/2006-11-09/ Accessed 5-8-07.
The rep-PCR genomic fingerprinting. Available from http://www.msu.edu/~debruijn/. Accessed 5-9-07.
Vierstraete, Andy. “Principle of the PCR.” 11 Aug. 1999. University of Ghent. 1 May 2007.
<http://users.ugent.be/~avierstr/principles/pcr.html>.
Snustad, Simmons, Principles of Genetics. Wiley Publishing, 3 rd ed. p. 498
Belk, Borden, Biology Science for Life, Pearson Prentice Hall Publishing. 2 nd ed. p. 184
What is the source of microbial
contamination?
 Waste from mammals and birds
 Untreated sewage
 Microbial Source Tracking
Microbial Source Tracking Methods
 Antibiotic resistance patterns
 ribotyping
 Rep-PCR DNA fingerprinting
Polymerase Chain Reaction
(PCR)
(13)
rep-PCR DNA fingerprinting of E. coli
isolates to investigate strain diversity
Agarose gel electrophoresis
Agarose gel electrophoresis
(15)
http://www.dnalc.org/ddnalc/resources/electrophoresis.html
rep-PCR DNA fingerprint
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10 11 12 13 lab DH5a 0 1kb
 Conclusions
– At least 7 distinct types of E. coli were identified from
Lake Johanna
– They are different than laboratory strains of E. coli
 Future research questions
– What is the strain diversity in Lake Johanna?
– Is there a shifting spectrum of strains?
– How does strain diversity compare in other
environments?
Questions?