Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Escherichia coli (E. coli) is a normal component of human, domesticated animal, and wildlife feces so when found in drinking and natural water supplies, E. coli has been used historically to indicate that human pathogens may also be present and serving as a primary indicator of fecal contamination of a water source. Although most E. coli are considered to be harmless, continued food- and beverage-related disease outbreaks involving pathogenic, or disease-causing, E. coli continue to be reported. This project focused on linkage of E. coli pathogens to environmental isolates held in our in-house databases derived from humans as well as domesticated animals (chickens, cows, dogs, horses, goats, pigs, and sheep) and wildlife (deer, raccoons, and geese). Eighteen (18) E. coli NotI pulsed-field gel electrophoresis (PFGE) databases including single-region collections from West Virginia, Ohio, and Iowa as well as combinations of single-region databases were challenged with 51 Shiga-toxin producing E. coli (STEC) isolates from human cases of disease to determine matching efficiency to these databases. Each isolate was previously serologically typed where 26 of 76 were determined to be O antigen 157 and 23 determined to be O157: H7. All 51 isolates were tested to confirm their identity as E. coli against a battery of biochemical substrates. Strong lactose fermentation, or the lack thereof, and reaction to a modified Colilert solution were noted. Finally, each pathogen was tested using an in-house method to demonstrate if signs of virulence, moderate virulence and a high degree of virulence could be determined when tested with the worm, Caenorhabditis elegans (C. elegans). NotI PFGE was performed on all STEC isolates and challenged to find the closest match in a database held in our laboratory consisting of 14,900 E.coli from chickens (n=1569), cow (n=2398), deer (n=2230), dog (n=382), goat (n=161), goose (n=1888), horse (n=368), human (n=1819), pig (n=2110), raccoon (n=1578), and sheep (n=397). These E. coli that make up this database are from known sources that were derived from defined regions of West Virginia, Ohio, and Iowa. Several graduate students contributed to these studies and will enter their respective professional careers with a greater appreciation of research and our goal of developing techniques which will serve to protect the environment as well as human and animal health and well-being. PARTICIPANTS: PI's on this project included Dr. Pamela Staton, Dr. Hongwei Yu, and Dr. Terry Fenger. Research technicians included Jennifer Ross-Wilkinson, Conan Goolsby, Tara Fry, and Megan Bartley. Graduate students included Amanda Hoffman, Dishari Mukherjee, and Stephanie Johnson. All received IRB training in the protection of human subjects as well as training in specialized techniques pertinent to the project and safety. TARGET AUDIENCES: Our target audiences include the environmental sciences, agriculture community, microbiology, community awareness groups, high school students, and other audiences with an interest in environment, human and animal protection. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Thirty-five of 51 or 69% were not strong lactose fermenters and therefore did not demonstrate a green metallic sheen on eosin methylene blue (EMB) agar when incubated at 35 degrees C for 18 to 24 hours. This demonstrates that use of colony appearance on EMB agar as a screening method for E. coli will result in under-representation of some strains of E. coli. Thirteen of 51 or 25% produced a negative modified Colilert result suggesting that some E. coli in our environmental testing were missed and therefore not included in the databases. It would be beneficial to go back to the original samples to determine the degree to which Shiga toxin-producing E. coli could be isolated. While this would improve our technical knowledge, it would also serve to demonstrate the degree to which this pathogen is present in the environment. While it may be beneficial to pursue this, these future studies were not the focus of this particular study. Nevertheless, taken together, use of a specialized medium for isolating pathogenic E. coli should be included in our screening procedures. In addition, application of the C. elegans pathogen screening method revealed eight of 51 pathogens tested that showed highly virulent results, with 19 pathogens demonstrating moderately virulent results and 24 showing no affect on the worm. We believe these results are promising for the C. elegans pathogen screening method. Our goal is that all STEC would show some degree of virulence with respect to worm behavior. Currently, 53% of all isolates tested demonstrated some degree of virulence using this assay. We are currently in the developmental validation phase of this procedure wherein further optimization may be possible. Plans for optimizing this procedure include modification of pathogen placement on agar plates and optimization of the number of worms applied and evaluated. Evaluation of increased incubation temperature at various stages of the procedure as well as agar modifications for better visualization of worm behavior is also planned. As this method is inexpensive to perform, we believe it shows promise for screening environmental water samples for pathogens without the labor intense and costly testing for multiple pathogens which is often beyond the scope of environmental water testing facilities. Additionally, NotI PFGE was performed on each pathogen and challenged to find its best match in a database containing 14,900 E. coli isolates of known source. All pathogens found a best-match in the database with all but 6 finding a similarity match of 80% similarity or greater. Pathogens matched to humans (n=8), domesticated animals (cow n=10; chicken n=4; pig n=5; sheep n=6; horse n=1; goat n=2), and wildlife (raccoon n=5; deer n=3; goose n=7). Such a method of matching pathogens isolated from human disease outbreaks may provide investigative clues when outbreaks of unknown origin occur and should be further evaluated. Once the C. elegans pathogen screening procedure is validated, we will publish our findings through scientific meeting poster presentations and/or publication in a peer-reviewed journal.
Publications
- No publications reported this period
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