The Cal Poly Pyroprints Project

Professors and students in Cal Poly's Biological Sciences, Chemistry and Biochemistry, and Computer Science departments are working with the university’s Center for Applications in Biotechnology (CAB) (formerly the Environmental Biotechnology Institute or EBI)  to develop a searchable, online library of E. coli "pyroprints" – genetic sequences that are as reliable as fingerprints when it comes to identifying different strains of E. coli.

This common bacterium with hundreds of variations is found in the intestines of mammals and birds. Several of different E. coli strains can severely sicken humans. These pathogenic strains are usually spread by fecal contamination: bird, deer, and cattle droppings as well as human feces.

Cal Poly's developing E. coli database is named CPLOP. It's a punny name that stands for the Cal Poly Library of Pyroprints. Right now it contains 2,400 pyroprints from the DNA of unique E. coli strains collected, cultured and analyzed at Cal Poly; another 2,871 E. coli strains are isolated in the Biological Sciences department freezers, waiting to be cultured and categorized.

The project is led by Biological Sciences Department Chair Chris Kitts, Biology Professor Michael Black, Chemistry Professor Anya Goodman and Computer Science Professor Alex Dehktyar.

Kitts, the CAB’s director, hopes to build the Cal Poly Library of Pyroprints into a lower-cost resource for the county, the region, the state and beyond. “There are plenty of places where people need to know ‘Who’s pooping in this water?’ ” Kitts said.

Currently, one private lab in Seattle is the only facility in the Western United States that offers E. coli “fingerprint” matching to confirm the sources of contamination in recreational waters -- at $100 per strain, Kitts said.

“If you need to fingerprint 1000 strains, which is not uncommon when you’re investigating the source of water pollution, the bill is $100,000,” Kitts said. “Our method will cost about a third of that.”

Cal Poly Professors and students head to the Bull Test corral for some E-Coli sampling
Photo copyright Cal Poly Biological Sciences 2011

To create the forensic data to fill the CPLOP database, the three departments are building a cross-disciplinary curriculum around microbial forensics involving students from freshmen to the master’s level.

It's a dirty job: since E. coli is found in the intestines of most mammals and birds, finding genetic pyroprints for the myriad of different strains involves collecting fecal samples on swabs from different host animal species. The next step is culturing those swabbed samples and growing the E. coli they contain. Students in introductory microbiology courses are currently doing this by using themselves as test subjects. In the class's lab, they culture, identify and document the multiple strains of E. coli they carry around with them.

Then, freshmen in an introductory cell and molecular biology class manipulate the DNA from the microbiology courses’ lab-grown E. coli and run it through a genetic sequencer to produce the bacteria’s “fingerprint” data, known as a “pyroprint.” The resulting pyroprints are added to the CPLOP database as references for E. coli from humans.

Students are required to repeat the sequencing of a specimen until they can get the same results three times – in order to teach them about quality control and professional research standards, as well as ensure the correct data goes into the CPLOP database.

This portion of the job is made possible by a PyroMark small scale pyrosequencer machine, a second-generation sequencer that works fast enough to produce results within 30-45 minutes, complete with a digital output. This allows students to identify and pyroprint an E. coli bacterium while in class.

The CAB was able to purchase four machines thanks to a grant from the W.M. Keck Foundation. Their use by students in undergraduate classes “is a rarity and a wonderful opportunity to expose freshmen to state-of-the-art technology in the classroom,” said Kitts.

Over in the Computer Science Department, graduate students and undergraduate students are working to create the computer-driven aspects of the CPLOP database. Right now it holds digital pyroprints of 2,400 identified E. coli bacteria and information about which host animal species they come from.

The computer science students are working to create a portion of the database that would allow users to upload a digital pyroprint of an unknown E. coli bacterium and have the database return a match, which will help identify the critter responsible for depositing it.

Undergraduate student Winne Tang gets ready to swab a sample from a bull's backside
Photo copyright Cal Poly Biological Sciences 2011

“We’re not quite to the point yet where you could enter a pyroprint and the database would tell you, ‘That’s from a red-winged blackbird,’ ” Kitts said. “But that’s where we want to be.”

More grant funding or private donations would help speed up the process of analyzing the E. coli samples currently in the department’s freezer and getting them into the CPLOP database. Grants and private funding would also enable the CAB to hire students to work during non-class time identifying and pyroprinting E. coli strains to enter into the library. Grants or donations could also be used to pay computer science students for non-class time needed to complete the digital matching function of the CPLOP database, Kitts said.

UC Davis (which is also looking for toxic E. coli strains throughout California) is collaborating with the CAB by sending wildlife fecal samples to Cal Poly to be documented, cultured, pyroprinted and added to the CPLOP library. The CAB is also working with Pacific Wildlife Care to get additional samples into the database. The Morro Bay non-profit group rescues and rehabilitates injured wild animals.

Several students are also using the pyrosequencer for their senior projects, collecting E. coli from specific animals to add to the database. One is studying the yearling bulls brought to Cal Poly every year from different parts of the state for the annual Bull Test enterprise, a project of the Animal Science Department. E. coli strains are cultured from each of the nearly 200 bulls on their arrival and regularly throughout the year to see if the bulls pass E. coli strains among the herd and which strains become predominant.

Another student is studying ground squirrels in the Bull Test area to see if the squirrels and the bulls swap E. coli. (The biggest problem so far in the science project: catching squirrels to swab.)

Tang takes her sample swab and swipes it in a Petri dish.
The sealed dish will be taken back to the Biological Sciences labs and incubated
Photo copyright Cal Poly Biological Sciences 2011

All of the E. coli investigations are an outgrowth of a real-world research project commissioned by the City of Pismo Beach with the EBI in 2007. The community needed to find out what was causing high E. coli counts in the waters around the Pismo Beach Pier. Routine sampling by state authorities was resulting in routine pollution postings under state health laws regarding coliform bacteria levels. “For a beach town, that’s not a very nice thing to have,” Kitts said.

The suspects were the city's own sewer plant, dogs, pigeons, seagulls, and humans. Working through a contract with the EBI, Kitts and his microbiology students collected water sample data along Pismo’s beaches from 2007 to 2009. They then isolated E. coli bacteria in the samples and sent them to the private lab in Seattle, which matched the E. coli to the species depositing the droppings they came from.

Sleuthing results in hand, Kitts and the students issued a report to the city in 2010. Its findings: birds (specifically, pigeons) were responsible for most of the fecal contamination. They found some human and dog contamination also, “but not in quantities anywhere near enough to result in a beach pollution posting,” Kitts said.

Ever since, requests to help with E. coli investigations keep coming in to Cal Poly's CAB. Right now, the CAB is looking into “fecal tracking” in the tunnels that carry the San Luis Obispo Creek through the city’s downtown to find the source of pollution there. The study would be funded by an EPA “urban waters” grant.

A BioSci student has proposed a project that would monitor Pennington Creek to see whether fences installed to keep grazing cattle out of the creek bed are succeeding in keeping the cattle, their manure and its E. coli out of the creek water.

“All of these projects," said Kitts, "are giving our students advanced, real-world experience in the field and in the lab."

To find out more about the Cal Poly Laboratory of Pyroprints, contact Kitts at ckitts@calpoly.edu.