- Staton, Pamela
- Marshall University Forensic Science Center
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- Can E. coli RNA provide clues of source-determination Pili will be the target of these studies as they may play a role in strain adaptation of E. coli in the colon of various species. Gene expression, through RNA analysis, from known-source E. coli will be studied to determine if previously characterized regions can provide clues to source-determination.
- What high throughput technology can be used to sequence significant numbers of known-source E. coli isolates' target regions from Objective 1 when preliminary studies show promise for source-determination Pyrosequencing, a rapid sequencing techniques/technology, will be used to characterize 30-50 bp of pili regions from collections of source-known E. coli.
- Can C. elegans serve as a Waterborne Pathogen Indicator This represents a continued focus on use of the worm, C. elegans, as a waterborne pathogen indicator. Shiga-toxin strains of E. coli are serving as the pathogen of choice in standardizing and validating this method. While the method has shown in-house potential for detecting pathogenic species, more work is needed to overcome method variability.
- Can a quantitative PCR method replace traditional filtration and culture techniques in estimating waterborne fecal pollution load Work continues to develop an optimized real-time PCR procedure that can be used to estimate fecal pollution load. A high throughput real-time PCR instrument will be used for these studies.
- Is concentration of raw water samples a reliable, or even necessary, step prior to RT-PCR analysis Tangential flow will be used to concentrate water samples prior to RT-PCR analysis. This procedure will be compared to data collected from samples collected from filters using traditional water filtration or from nate water samples.
- Is there an advantage to using a 2 enzyme PFGE restriction of known-source E. coli database isolates for source determination Current bacterial source tracking databases in our laboratory have been restricted or "cut" with NotI restriction endonuclease to produce their distinctive DNA fingerprint. XbaI is used by the CDC PulseNet system for epidemiologic purposes. We are seeking to determine if XbaI restriction holds any advantages over the current NotI PFGE method with respect to improving matching efficiency and source determination.
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- NON-TECHNICAL SUMMARY: Each year, a significant number of U.S. rivers, lakes, and estuaries continue to be classified as having exceeded fecal pollution water quality standards. Non-point-source fecal pollution is particularly difficult to characterize by major source contributor. Therefore, anyone tasked with reducing fecal pollution in a complex water body system is well aware of the importance of developing , evaluating, and standardizing microbial source tracking methods. Our primary goal is to evaluate, standardize, and validate the NotI PFGE Bacterial Source Tracking method for ultimate use by the water testing community. As a library (database)-dependent method, the PFGE BST method requires creation of a database intended to serve a given region or water body. Bringing this method out of the laboratory and into the community requires strict attention to validation procedures, cost, turn-around time, and transferability across laboratories. The ability to deliver results in a timely manner is an essential attribute of a good BST/MST method. We will investigate the use of a rapid PFGE method that models a technique approved by the Center for Disease Control National Molecular Subtyping Network for Foodborne Disease Surveillance program. Our goal is to evaluate the utility of the CDC method for MST/BST testing for use in combination with our current method. The debate continues with respect to which "indicator" microorganism best serves as a predictor of waterborne pathogen presence. While we currently use E. coli as our "indicator" of choice in PFGE BST studies, we are also interested in pursuing methods that could predict the actual virulence, i.e., disease causing potential, of a water sample. For these studies, we use the worm, C. elegans, as a model organism capable of reacting to microbial toxin as well as other pathogenic mechanisms. The search continues for unique DNA sequences that provide linkage to the animal/human source of fecal pollution. In these studies, pili, fimbriae or other cell-surface structures will be investigated from a collection of known-animal/human source E. coli. Selected for this study will be the E. coli PFGE profile(s) that predominate in each of our source-based libraries, e.g. human, cow, pig, chicken, horse, deer, raccoon, goose, and dog. Pili are microbial cell-surface structures that are known to play a role as mediators of attachment in the human intestine although most information has been based on uropathogenic strains of E. coli. As fecal pollution testing methods is our primary focus, we also search for ways that the culture-based approach to enumerating or "counting" water indicators can be improved. A high throughput, low cost, qPCR method will be evaluated in terms of replicating standard water testing data. The purpose of these studies is to ultimately determine if the post-collection, quantification step for fecal pollution estimation can be automated. Each of these activities seeks to provide essential information in an effort to expand the MST/BST and environmental testing toolbox toward the control of fecal pollution in our waterways.
Objective 1: Pili present on the surface of E. coli strains are known to play a role as mediators of attachment to human epithelial surfaces. E. coli pili are classified according to the host cell types and specific cell surface receptors to which they bind. The genetics of pili are complex as pili of different types may coexist on the same bacterium, and their expression may vary under different environmental conditions. Due to this "expression" variability, we will isolate RNA from known-source bacterial cultures for conversion to cDNA for analysis. Gel electrophoresis, real-time PCR, and pyrosequencing technique will be employed to determine if host-source correlates with pili-associated sequences.
Objective 2: Should preliminary studies of select pili sequences show promise for source determination, high throughput pyrosequencing will be used to evaluate larger collections of source-known E. coli currently present in our repository to determine the efficacy of this approach. Real-time PCR will be used for the initial studies.
Objective 3: Work will continue to optimize the C. elegans pathogen screening method which will include a specificity, sensitivity and reproducibility study using a variety of enteric pathogens such as Shigella, Salmonella, Yersinia, and Aeromonas will be tested in addition to E. coli O157:H7 currently under study.
Objective 4: qPCR E. coli methods will be tested on 2-3 real-time PCR instruments to evaluate cost, high throughput capacity, turn-around-time, and potential for automation.
Objective 5: Sensitivity, specificity, and reproducibility are important aspects of any validation study. Raw water, or simulated raw water samples, will be concentrated using tangential flow filtration and compared to non-concentrated water samples and traditionally filtered samples, for real-time PCR assays. Real-time PCR of complex samples, such as soil, often requires a unique DNA extraction step or step for removal of PCR inhibitors. If needed, a product known to efficiently extract DNA from complex samples, e.g. soil, and to remove PCR inhibitors, such as humic acid, will be evaluated. A comparison study will be performed to determine if a) water samples greater than 100 mL, which represents the traditional amount of water collected for routine water analysis, improves the sensitivity of these RT-PCR assays, and b) if products such as PowerSoil DNA Isolation Kit or PCR Inhibitor Removal Technolog (Mo Bio Laboratories, Carlsbad, CA) improve the sensitivity and reproducibility of these assays.
Objective 6: a) A published rapid PFGE procedure will be evaluated to determine if the rapid method can replace our traditional, more time consuming, method. b) An alternative restriction procedure will also be evaluated to determine if a 2-enzyme method improves matching efficiency and classification accuracy of our current BST method.
- Funding Source
- Nat'l. Inst. of Food and Agriculture
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- Sanitation and Quality Standards
- Natural Toxins
- Bacterial Pathogens
- Escherichia coli
- Meat, Poultry, Game