Development of Microbial Specific Genetic Markers to Track Sources of Fecal Pollution

Project Description: High levels of coliform bacteria and nutrients have been found in surface waters of the Laguna de Santa Rosa watershed. The City of Santa Rosa is interested in determining the sources of fecal pollution. This project develops polymerase chain reaction (PCR) techniques using microbial specific genetic markers to identify the source (i.e., human, ruminants) of the bacteria, and tests water samples from Copeland Creek near the SSU Campus to identify the source of bacterial contaminants.

Duration: Fall 2013 - Spring 2014

Type of Educational Activities: Service-learning, independent research

Project Faculty: Mike Cohen (Biology)

Partners:

  • City of Santa Rosa Environmental Compliance Inspector Caden Hare
  • Mami Kainuma (Consultant).

Participating Courses:

  • Biology 338 Environmental Microbiology and Biotechnology - 18 students
  • Biology 494 Independent Research - 1 student (Dipali Vasadia)

Extracted DNA was amplified by Polymerase Chain Reaction (PCR) using various published fecal source-associated molecular markers in prokaryotes (16S rRNA gene) and eukaryotes (mitochondrial DNA) to identify general and species-specific fecal contaminations. Among nine different molecular markers tested, three from the order Bacteroidales were chosen that showed consistent species-specific (human and ruminants) contamination results.  The amplified PCR products were purified for sequence analysis to validate the presence of Bacteroidales 16S rRNA gene.

Samples were collected from 3 sites on Copeland Creek on March 15, 2014 after a rain storm. E. coli were detected in the samples using (IDEXX Quanti-Tray/2000). Fecal sources were collected from the 3 samples using a 0.45 uM filter and cotton swabs, and stored at 20C until DNA extraction.

Filter/swab retantate was resuspended in water and DNA extracted using a newly developed inexpensive method (TENS buffer and 3M NaOAC followed by phenol-chloroform extraction) and a standard manufacturer protocol (Powersoil DNR extraction kit, Mo Bio). Bacterial 16S ribosomal RNA were amplified from each sample using universal primers. An additional amplification was made from a known bacterial source to serve as a positive control. Nested PCR was performed using universal bacteriodes 16S rRNA gene as a template and primers for ruminant-specific and human-specific fecal detection. (See Visalie et al. 2014 in "Resulting Data & Reports for more details).

Field collection and laboratory techniques were undertaken by SSU students under the guidance of Drs. Mike Cohen and Mami Kainuma.

Results Summary: The newly developed inexpensive extraction method yielded good quality DNA from human but not ruminant feces. Ruminant fecal contamination was detected from samples at all sites; human fecal contamination was detected in the upstream and West Bridge sites, but not East Bridge site. Levels were well below the US EPA regulatory limit for recreational waters.

Data (see data disclaimer)

Presentations:

  • Establishing Whodunit: Application of molecular markers for fecal source tracking. Dipali Vasadia, Mami Kainuma, and Spring 2014 Biology 338 (Advisor: Michael Cohen, Biology 338 Environmental Microbiology and Biotechnology and Student Internship). Vasadia et al. 2014 (pdf; 13 Mb); 2014 WATERS abstracts

Scientific Papers

Lane, D.J. 1991. In: Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt, E. and Goodfellow, M. Eds.) pp. 115-147.

Bernhard, A.E. and K.G. Field. 2000. Appl. Environ. Microbiol. 66:1587-1594.

Bernhard, A.E. and K.G. Field. 2000. Appl. Environ. Microbiol. 66:4571-4574.

EPA 2012. Recreational Water Quality Criteria. Office  of Water. Dec 2012,8220-F-12-061.