Investigation of Escherichia coli diversity from freshwater sources using Rep-PCR DNA fingerprinting

Northwestern College

jrklein@nwc.edu

Escherichia coli is a common indicator bacterium for fecal contamination and testing for E. coli levels is a measure of recreational water quality.  Students in my microbiology course have used a membrane filtration protocol adapted from the EPA for detection and enumeration of E. coli from freshwater sources (1).  Students then use standard biochemical methods to confirm isolates as E. coli.  To enhance this laboratory experience, I sought funding from ABLE to incorporate molecular techniques for bacterial identification and analysis of strain diversity into this lab experience.  I chose to adapt Rep-PCR DNA fingerprinting to the classroom setting.  Rep-PCR is a rapid and reliable test used for determining the relatedness of E. coli isolates (2, 3).  With the ABLE grant, I have modified published protocols for Rep-PCR and used them in a unique teaching opportunity I had last summer as the co-director of a pilot program of the Summer Research Institute (SRI), run jointly by Northwestern College and Concordia College – St. Paul.  SRI serves undergraduate biology majors, high school science teachers, and underrepresented urban high school students. The goal of SRI is to foster skills in basic and applied scientific research in order to better prepare students for further education and/or careers in the sciences.  The program began with a 4 week summer research component led by myself and a faculty member from Concordia.  During the first week of the program, I taught foundational microbiology laboratory skills through guided inquiry labs.  During the second week, the students used these skills as they embarked on a small-scale independent research project investigating the water quality of a local lake of their choice.  They began by collecting water samples and identifying the numbers of E. coli in the sample.  They then performed Rep-PCR to compare the E. coli isolates they obtained from their lake and to samples from other lakes.  Finally, they wrote a report about the general quality of their chosen lake.  During the academic year, students have presented their findings in high school biology classes and local biotech companies who funded the program. 

Combined with activities I had already been using in my courses, the addition of the Rep-PCR technique resulted in a multi-component laboratory experience starting with sample collection and proceeding to molecular analysis of isolates.  In addition, activities were incorporated that promoted critical thinking, communication and interpersonal skills.  The final product was a relevant laboratory experience that provided students with necessary skills and exposed students to the most current methodology used by government, industry and academic researchers for the analysis of microbial contamination of water, food and pharmaceuticals.  I plan to implement this series of labs in my college level microbiology course. 

References

1.  Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC). 2002. EPA 821-R-02-020.  Office of Water, Washington D.C. USEPA. 

2.  Dombek, P.E., L.K. Johnson, S.T. Zimmerley, and M.J. Sadowsky. 2000.  Use of repetitive DNA sequences and PCR to differentiate Escherichia coli isolates from human and animal sources.  Appl. Environ. Microbiol. 66:2572-2577.

3.  Yang, H., R.T. Vinopal, D. Grasso, B.F. Smets.  2004.  High Diversity among Environmental Escherichia coli Isolates from a Bovine Feedlot.  Appl.Environ. Microbiol. 70:1528-1536.