Investigation of Escherichia coli in freshwater sources using membrane filtration and Rep- PCR DNA fingerprinting

Workshop Agenda  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  Facultative aerobes  Gram negative  Nonspore-forming  Rod shaped  Ferment lactose and produce gas within 48hr. At 35 o C  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 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 0.45μm membraneFilter the water sample through a 0.45μm membrane Transfer filter to a membrane theromotolerant E.coli (mTEC) agarTransfer filter to a membrane theromotolerant E.coli (mTEC) agar(1) Water Testing Procedure

Water Testing Procedure cont.  Incubate mTEC plate at 37 °C for 2 hours then transfer it to 45 °C and incubate overnight (1)

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. (Typical Results) (Typical Results)

 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 Ammonia urease

E. coli levels in Lake Johanna – summer 2009

Conclusions  Contamination of Lake Johanna has not been a problem  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 1.Madigan, M.T. Martinko, J.M. Brock Biology of Microorganisms. Pearson Prentice Hall Publishing, 11 th ed. p Steverman B. Issue Update – Bacteria at beaches are the target of monitoring effort. Star Tribune 2005 July 6. Retrieved from News Bank database. 3.[anonymous]. Lake Superior lakewide management plan Available from: Accessed 2007 Apr Duchschere K. Roseville beach closed because of E. coli levels. Star Tribune 2006 July 28. Retrieved 2007 Apr 11 from Access World News database. 5.Brunswick M. Lake Harriet beach closed over E. coli levels in open. Star Tribune 2005 June 12. Retrieved from News Bank database. 6.Coleman N. One thing is clear: Increasingly, state’s waters aren’t. Star Tribune 2005 Aug 7. Retrieved from Access World News database. 7.Collins T. Carver County – Lake Minnewashta beach closed for tests. Star Tribune 2005 July 15. Retrieved from News Bank database. 8.Lerner M. E. coli closes Coon Lake Beach in Anoka County. Star Tribune 2005 July 9. Retrieved from News Bank database. 9.USEPA (1986a) Ambient water quality criteria for bacteria Office of Water Regulations and Standards, Criteria and Standards Division, Washington, DC. EPA-440/5-84/ Madigan, M.T. Martinko, J.M. Brock Biology of Microorganisms. Pearson Prentice Hall Publishing, 11 th ed. p Talaro, KP, Talaro, A. Foundations in Microbiology. McGraw Hill Publishing. 4 th ed. p Detecting bacteria with mechanical cantelievers. Science Daily. Nov. 1, Available from Accessed The rep-PCR genomic fingerprinting. Available from Accessed Vierstraete, Andy. “Principle of the PCR.” 11 Aug University of Ghent. 1 May Snustad, Simmons, Principles of Genetics. Wiley Publishing, 3 rd ed. p 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

(12)

Polymerase Chain Reaction (PCR) (13)

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

Agarose gel electrophoresis (14)

Agarose gel electrophoresis (15)

rep-PCR DNA fingerprint 1kb lab DH5  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?