Refining Molecular Approaches For Bacterial Source Tracking (BST) In Texas

Bacterial source tracking (BST) is a new science that aids in identifying sources of human and animal fecal contamination of environmental water. There are currently two approaches used in BST, library-dependent and library-independent methods. The fecal bacterium E. coli is often used in library dependent methods because it indicates fecal contamination, has been used in human health risk assessments, and has water quality regulatory significance. A disadvantage of E. coli library dependent BST methods is that identification libraries of E. coli isolated from known fecal sources are needed to identify the sources of E. coli isolated from water samples. Further, development of an identification library for each watershed is impractical and expensive. Due to the fact that E. coli is used by regulatory agencies to determine impairment of a watershed, a quick and easy to perform detection method that differentiates human and animal specific E. coli is desirable. The library independent methods are fast and relatively easy to perform. One library independent method utilizes the polymerase chain reaction (PCR) to detect Bacteroidales bacterial species for identifying sources of fecal pollution. Disadvantages include uncertain relationships to regulated fecal indicator bacteria, human health risks, and a lack of standardized protocols. This research will focus on the refinement of two BST tools: Bacteroidales PCR assays and evaluation of enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) gene targets for the development of a library dependent E. coli BST method. Several published source tracking studies have used Bacteroidales PCR assays with favorable results. Although these studies showed tremendous promise for Bacteroidales PCR, this same conclusion was not always obtained in other laboratories. In particular, amplification of non-target animal groups has been observed, especially with the Bacteroidales PCR for ruminants and less frequently for the human marker assay. For example, cross-reactivity in reactions with the ruminant marker and fecal specimens from hogs has been observed. Studies using the human marker have also reported false positives with feces from wild animals, such as whitetail deer, coyote, raccoons, badgers, porcupines, and rabbits. To investigate some of these issues, the present research included the characterization of PCR amplicons from animal fecal DNA which cross-reacted with the Bacteroidales PCR HF183 human marker. We also evaluated a modified swine Bacteroidales PCR method (PF163 marker) to determine if a primer or probe could be developed to specifically identify fecal pollution from feral hogs. Finally, to explore the possibility of developing an E. coli library independent method, characteristics of PCR products from the library dependent enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) assay were characterized. Results from the human HF183 cross-reactivity study and the PF163 feral hog study revealed that real-time PCR (RT-PCR) and high resolution melt analysis (HRM) of PCR products can be utilized to identify DNA sequence variation. DNA sequence analysis of HF183 human marker amplicons from cross-reacting animal feces revealed that an alternative target may be needed in some cases. Sequence analysis of the Bacteroidales PF163 swine marker amplicons revealed that this marker is a reliable tool for identifying feral hog fecal pollution. Analysis of E. coli ERIC-PCR amplicons revealed the presence of multiple priming sites; however, further analysis of cloning and PCR screening protocols is needed. It was also observed that ERIC-PCR yields highly reproducible results, making this a reliable method for fingerprinting bacterial genomes. The results from this project provide a better understanding of the molecular diversity of Bacteroidales in wildlife populations, helped address cross-reactivity issues, and explored a unique approach for development of a library independent E. coli method. Overall this research will help make BST a more efficient and reliable tool for identifying sources of fecal pollution