Source: MARSHALL UNIVERSITY FORENSIC SCIENCE CENTER submitted to
WV WATER QUALITY: BACTERIAL SOURCE TRACING AND PATHOGEN PROFILING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0219164
Grant No.
2009-38885-20000
Project No.
WVAW-2009-04582
Proposal No.
2009-04582
Multistate No.
(N/A)
Program Code
UL
Project Start Date
Sep 1, 2009
Project End Date
Aug 31, 2011
Grant Year
2009
Project Director
Staton, P. J.
Recipient Organization
MARSHALL UNIVERSITY FORENSIC SCIENCE CENTER
1401 FORENSIC SCIENCE DRIVE
HUNTINGTON,WV 25701
Performing Department
Forensic Science
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.
Animal Health Component
(N/A)
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1120320104030%
1120320106030%
1120320110040%
Knowledge Area
112 - Watershed Protection and Management;

Subject Of Investigation
0320 - Watersheds;

Field Of Science
1060 - Biology (whole systems); 1100 - Bacteriology; 1040 - Molecular biology;
Goals / Objectives
Objective 1: 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.Objective 2: 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.Objective 3: 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.Question 4: 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. Question 5: 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.Question 6: 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.
Project Methods
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.

Progress 09/01/09 to 08/31/11

Outputs
OUTPUTS: Goals of this award focused on the development of new molecular and other microbial methods to improve detection and characterization of fecal pollution in surface waters. Escherichia coli (E. coli) is a commonly used indicator of fecal pollution. One objective of our study was to identify rapid methods of E. coli analysis and, in particular, identifying universal E. coli primers for real-time polymerase chain reaction (RT-PCR) assays. The lacZ gene coding for beta-galactosidase has been sequenced and is searchable using a number of bioinformatic programs. We tested lacZ primers (lacZITf and lacZITr) using tradition PCR against DNA extracted from E. coli 25922, E. coli K-12, E. coli O157:H7, Salmonella typhimurium, Klebsiella pneumonia, Enterobacter cloacae, Serratia marcescens, Shigella flexneri, Proteus vulgaris, and Citrobacter freundii and two additional lacZ primers (lacZF1,R1; lacZF2,R2) tested against the organisms above and Enterococcus faecalis. Each of these primer pairs targeted a region of the lacZ gene of E. coli. Additional studies targeting the uidA gene were performed. A search was also performed for a commercially available kit targeting all strains of E. coli which would be required for this type of water analysis. While several commercially available kits target E. coli O157:H7, we were only successful in identifying one commercial kit that targeted all E. coli, which was the Primer Design Genesig Kit for the Quantification of Escherichia coli (all strains).The kit was reported to have the broadest detection profile possible whilst remaining specific to the E.coli-sp genome with primers and probe sequences having 100% homology with a broad range of "clinically relevant" reference sequences. As the manufacturer stated, this kit is designed to work well with all processes that yield high quality DNA with minimal PCR inhibitors, although we can expect a myriad of PCR inhibitors from surface water samples. Our challenge was to produce high quality DNA from surface waters without PCR inhibitors. That led us to the PowerWater DNA Isolation Kit (MO BIO) which according to the manufacturer's specifications would deliver high quality genomic DNA from a variety of "filtered" water samples, including turbid waters, using their Inhibitor Removal Technology. This Inhibitor Removal Technology was reportedly capable of purifying DNA from any sample, even those with a high content of PCR inhibitors. We began our comparison of homebrew and commercial methods for adaptation to surface water testing. Our second objective involved the use of the worm, Caenorhabditis elegans (C. elegans) which is a free-living, transparent nematode (roundworm) widely used in genetic studies. These worms are easy to grow in bulk, cheap, and endure long term storage. As a multicellular eukaryote, we asked if C. elegans could serve as an indicator of waterborne pathogen presence where we used the pathogen E. coli O157:H7 and other Shiga-toxin E. coli as test organisms. An additional study performed included incorporation of the enzyme XbaI as compared to our historic use of NotI restriction enzymes into our bacterial source tracking method. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Homebrew PCR/RT-PCR analysis while targeting different regions through use of different primers, all suffered from background noise, negative control contamination, and false positives to one degree or another. It was, however, interesting that two organisms in our panel of test organisms consistently caused false positive results, e.g., Streptococcus pneumonia and Enterococcus faecalis. Promising aspects of these studies were that the assays provided reproducibly positive results for E. coli (our primary focus) when control organisms were employed but not so consistently when water samples were tested. We believe that recovery of DNA from filters used in the DNA extraction process may be one cause of lower DNA yield from water samples. While tangential flow analysis was used to concentrate water samples, the membrane-based technology also gave inconsistent results. The issue of negative control contamination may be attributed to the residual DNA of microbial species found in commercially available Taq polymerase products. Both DNase solutions and UV light treatment of Taq polymerase reagents provided promising results. This represents a significant obstacle in our laboratory as the most common microbial species for making Taq polymerase is E. coli. With residual E. coli in the Taq polymerase universally needed for PCR analysis, we are pursuing several ways to overcome this obstacle. We obtained high quality DNA from the MO BIO PowerWater kit but the yield was less than expected and needed to reach our goal for a quantitative RT-PCR assay. Addition of UNG (uracil-N-glycosylase) and Sau2A1 in our homebrew assays yielded interference that could not be overcome with instrument manipulation. We were able to identify promising primer sets as well as those which lacked sufficient specificity.In our studies, MO BIO PowerWater reagents out-performed other DNA extraction methods. PFGE studies of one and two restriction enzymes, e.g., NotI and XbaI, XbaI alone demonstrated a higher accuracy when blind animal/human-derived E. coli was challenged to find an accurate match in the database. This provides an alternative to NotI PFGE profiling of E. coli isolates when test isolates are NotI resistant, which a significant number of our isolates have been shown to be. The C. elegans procedure as it is currently performed was able to detect virulence in 108 of 310 (35%) of the Shiga-toxin E. coli (STEC) tested. Of those that were positive in this assay, 70% were E. coli O157:H7 while the remainder of these were E. coli O26:NM and O26:H11. A Colilert (IDEXX) procedure was performed on 862 STEC isolates where 32% or approximately 1/3 were Colilert negative. Colilert Reagent is a commercial product used for the simultaneous detection and confirmation of total coliforms and E.coli in water and used by drinking water facilities to detect fecal contamination in drinking water supplies. A number of animals, in particular bovine species, are known to carry such human pathogens in their feces that could potentially contaminate surface and ground water supplies.

Publications

  • No publications reported this period