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Developing and applying biological tools to address environmental concerns and discover new resources through collaborative interdisciplinary research and education.

Pyroprinting: City Of Pismo Beach
Fecal Source Tracking at Pismo Beach

All of the E. coli investigations are an outgrowth of a real-world research project commissioned by the City of Pismo Beach 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.

Presentations & Publications: Read Entire Pyroprinting Study | View Pyroprinting Poster (PDF) | About Pyroprinting (PDF)
The Cal Poly Pyroprints Project
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E. coli Strain Demographics and Transmission in Cattle

Our working hypothesis is that the environment plays a significant role in the microbial flora of cattle. We expect bulls from the same farm will be more likely to share E. coli strains with one another than those of different farms. Furthermore, we predict that when bulls from different farms cohabitate, the E. coli strain demographics will shift so that the pen will be a more significant predictor of the strain isolated from bulls than the farm from which they originated. E. coli were collected from bulls that arrived at Cal Poly from farms across the state. Two isolates were taken from bulls when they first arrived, then another two isolates from the same bulls after cohabitating for four months. A total of 380 bulls were sampled in this manner over the summers of 2011 and 2012. Preliminary results support our hypotheses. E. coli isolated from bulls when they first arrived showed many common strains from the same farm and very few from different farms. Conversely, E. coli isolated after bulls had cohabited for 4 months showed more strains in common across farms, common to the pens these bulls inhabited.

Presentations & Publications: Read Entire E. coli Strain Demographics and Transmission in Cattle Study (PDF) 
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Investigating Changes in Prevalent Human Escherichia coli Strains with Different Collection Methods Using Pyroprinting: A Novel MST Method

Microbial Source Tracking (MST) is the science of identifying the source of a bacterial species to its host species. This practice is used frequently in applied science to identify sources of contamination in the environment like beaches or lakes, for example, but MST techniques can also be used to study microbial population dynamics in specific organisms. In this study, a new MST method was developed to investigate the variation of Escherichia coli strains in humans. The new approach uses pyrosequencing to generate DNA fingerprints (or pyroprints) based on the sequences from two polymorphic regions within the ribosomal RNA operon of E. coli. Seven copies of the ribosomal RNA operons are present in the E. coli genome and each possesses two highly variable regions called Intergenic Transcribed Spacers (ITS). These regions of DNA are non-coding, and thus are able to accumulate nucleotide changes. The pyroprinting MST method developed by our lab capitalizes on these differences to create patterns of data by simultaneously sequencing all seven copies of each ITS: the 16-23S rRNA region (ITS 1) and 23-5S rRNA region (ITS 2). A "match" between E. coli isolates is determined when the patterns are nearly identical at both ITS 1 and 2. A cluster developed by matching isolates is considered to be a unique strain.

Presentations & Publications: Read Entire 'A Novel MST Method' Study (PDF)
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Investigating the Dominant Escherichia coli Strain in Lambs and Ewes Using Pyroprinting: A Novel Method for Strain Identification

Escherichia coli is a bacterium commonly found in the intestines and feces of animals. Due to the vast number of strains of E. coli, a specific host can often be identified by the dominant strain they carry in their intestines. This study looked at differences in dominant E. coli strains between ewes and their lambs. Although the lambs were sired by a single ram from a flock of ewes, the lambs were separated from the ewes after weaning. As a result, whether the dominant E. coli strains were stably maintained after early transfer events from the ewes to the lambs or if the lambs acquired their own dominant strain from the environment after separation from the ewes was able to be determined.

E. coli strains were isolated from fecal samples and categorized, using a new method referred to as pyroprinting. Pyroprinting involves simultaneously pyrosequencing the polymorphic, non-coding regions of all seven copies of E. coli’s ribosomal RNA operons. The accumulation of mutations over time in these regions results in strain-specific patterns in the pyrogram output, referred to as a pyroprint. E. coli isolates purified from ewe or lamb fecal samples were classified into strains when pyroprints from both regions were matched based on Pearson correlation analysis using 99% as the lower threshold.

Presentations & Publications: Read Entire 'A Novel Method for Strain Identification' Study (PDF)
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Chronology-Sensitive Hierarchical Clustering of Pyrosequenced DNA Samples of E. coli: A Case Study

Our research group is developing an efficient, cost-effective, librarydependent Microbial Source Tracking method to create DNA fingerprints for different strains of E. coli using pyrosequencing; which we refer to as pyroprinting. In a pilot study, pyroprinting was used to investigate the variation in E. coli. Characterizing E. coli populations and their variation in humans is important not only to build an MST library but also to further understand the human interaction with this commensal organism.

Presentations & Publications: Read Entire 'Chronology-Sensitive Hierarchical Clustering of Pyrosequenced DNA Samples of E. coli' Study(PDF) | View Poster (PDF) | View Presentation (PDF)
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Investigating Temporal Strain Diversity in Human E. coli Populations Using Pyroprinting: A Novel Strain Identification Method

Escherichia coli (E. coli) is a well-studied commensal bacterium which inhabits the intestinal tract of some birds and most mammals, including humans. E. coli is also an indicator of fecal contamination when found in environments such as watersheds, lakes, beaches, and recreational water. It is necessary for health and environmental protection agencies to be able to track a source of fecal contamination at the species level because dangerous interspecific pathogens can be transferred through contaminated water. Thus, E. coli is often used for Microbial Source Tracking (MST), a field in which methods of strain differentiation are used to identify and associate strains with a host species. 
Current strain differentiation methods include phenotypic and genotypic microbial analysis. Genotypic methods are advantageous compared to phenotypic methods since they use differences in nucleic acids to distinguish between strains of E. coli with greater sensitivity. However, current methods can be labor intensive or financially expensive, and many have issues in reproducibility when identifying known E. coli strains.

Presentations & Publications: Read Entire 'Investigating Temporal Strain Diversity in Human E. coli Populations Using Pyroprinting' Study (PDF)
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Gene Expression Profiling of the Human Intestinal Epithelial Innate Immune Response to Two Major Vibrio Pathogens

Diarrheal diseases caused by bacteria, viruses, and parasites rank fourth in fatality rate among all other diseases around the globe, causing at least 3 million deaths a year. Vibrio cholerae and V. parahaemolyticus are two major pathogens that elicit diarrheal related diseases through very different mechanisms. The goal of this investigation is to compare and expand our knowledge of the innate immune response to these Vibrio pathogens. As such, the human intestinal epithelial cell line, Caco-2, was challenged for 2 hours with the two Vibrio species, and mRNA expression of 84 different genes involved in inflammation or autoimmune disease was quantified using a pathway focused real time reverse transcription PCR array. Appropriate controls for the quantitative PCR for genomic DNA contamination and analyses of housekeeping genes are included in these arrays. We found both V. cholerae and V. parahaemolyticus most notably induce over expression (10-fold or higher) of the following cytokines and chemokines; TNF-α, IL-7, CXCL-1 (GRO-α), CXCL-2 (GRO-β), CXCL-10 (IP-10), CCL-2 (MCP-1) and CXCL-11 (I-TAC). Of the expected proinflammatory cytokines TNF-α was significantly increased (over 50 fold) whereas IL-1 and IL-6 were only increased by about 4 fold. In addition, many neutrophil chemoattractants (CXCL family) were upregulated, but not IL-8. We were surprised and intrigued by the high level (more than 50 fold) of IL-7 gene expression induced by both species as IL-7 is generally thought of as a hematopoietic cytokine. Because V. parahaemolyticus causes inflammatory diarrheal disease and V. cholerae causes noninflammatory diarrhea, we were particularly interested in differences in gene expression between the two. The most notable of these was the induction of LIGHT (TNFSF14) observed in V. parahaemolyticus but not V. cholerae. In summary, we observed a many proinflammatory cytokines and chemokines upregulated by both Vibrio species in Caco-2 cells. We will use these array data to guide further investigations confirming our initial observations and probing the functional significance of these inflammatory pathways in both species.

Presentations & Publications: Read Entire 'Gene Expression Profiling of the Human Intestinal Epithelial Innate Immune Response to Two Major Vibrio Pathogens' Study (PDF)
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Investigating Temporal Strain Diversity in Human E. Coli Populations Using Pyroprinting: A Novel Strain Identifiation Method

Professors and students in Cal Poly's Biological Sciences, Chemistry and Biochemistry, and Computer Science departments are working together with the university's Environmental Biotechnology Institute (CAB) 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. 

Presentations & Publications: View 'Investigating Temporal Strain Diversity in Human E. Coli Populations Using Pyroprinting' Presentation (PDF)
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Pyroprinting Sensitivity Analysis on the GPU

Microbial Source Tracking (MST) is a field in which microbial strains are identified and associated with a specific host source (e.g., human, canine, avian, etc). Identifying the hosts of microbial strains lies at the heart of many studies of bacterial contamination in the environment. Being able to determine which host species is responsible, e.g., for fecal contamination of a creek, allows the parties involved to develop specific measures for addressing the contamination.

Presentations & Publications: Read Entire 'Pyroprinting Sensitivity Analysis on the GPU' Study (PDF) | View 'Pyroprinting Sensitivity Analysis on the GPU' Poster (PDF)
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Utility of Terminal Restriction Fragment Analysis on Raw Milk to Evaluate Milk Quality

The quality of pasteurized milk and other finished dairy products has been previously shown to correlate with raw milk quality. Several conventional culture-dependent methods are available to evaluate raw milk quality yet there is an unacceptable level of variability in the methodology and interpretation of these tests. There is a critical need to arrive at a consensus on one or more tests that can produce results with the most relevance in assessing and/or predicting the final quality of dairy products. We reasoned that a culture-independent method should be included to understand the entire bacterial community in raw milk. To this end, the objective was to evaluate the utility of Terminal Restriction Fragment (TRF) analysis in assessing raw milk quality.

Presentations & Publications: Read Entire 'Utility of Terminal Restriction Fragment Analysis on Raw Milk to Evaluate Milk Quality' Study (PDF)
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Virulence Characterization of Viable but Nonculturable Vibrio parahaemolyticus

Under unfavorable conditions, some bacteria have demonstrated the ability to enter into a viable but nonculturable (VBNC) state, possibly as a survival mechanism. Vibrio parahaemolyticus, a foodborne pathogen commonly associated with seafood, could exist in VBNC state under nutrient starvation or low temperature conditions. Standard enumeration method of viable V. parahaemolyticus from food samples is dependent on the ability of the isolates to grow on laboratory media. However, VBNC cells of V. parahaemolyticus do not form colonies on standard media and therefore their presence is overlooked. VBNC cells may resuscitate and thus regain virulence under proper conditions, such as in the human GI track. Therefore, the goal of this study is to 1) optimize methods to enumerate VBNC cells of V. parahaemolyticus, and 2) to characterize and compare virulence between VBNC and vegetative cells. V. parahaemolyticus strain, RIMD2210633, a clinical O3:K6 isolate, was subjected to both nutrient (modified Morita mineral salt solution) and temperature (5°C) stresses to reach the VBNC state. Cells were considered nonculturable when no CFU was formed after plating 0.1 ml on Trypticase Soy Agar (TSAS ) followed by incubation at 25°C. Live/Dead® BacLightTM kit was used to determine cell viability. The microscopic method was optimized to detect viable V. parahaemolyticus by adjusting the ratio of STYO 9 and propidium iodide from 1:1 to 1:3. The expression of virulence marker genes, tdh2 and escU, were measured by using quantitative PCR. The levels of expression for each gene were normalized according to the geometric mean of results obtained for the control gene pvsA. Our study shows that VBNC cells expressed comparable levels of these virulence genes.

Presentations & Publications: Read Entire 'Virulence Characterization of Viable but Nonculturable Vibrio parahaemolyticus' Study (PDF)
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