UNDERGRADUATE RESEARCH SEMESTER/EXPLORATORY GRANT APPLICATION Budget Worksheet

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1 UNDERGRADUATE RESEARCH SEMESTER/EXPLORATORY GRANT APPLICATION Budget Worksheet BUDGET ITEM Department or College Funds Outside Agency Funds Persona l Funds Undergrad. Research Funds GRAND TOTAL Materials Five antibiotics (300 each of erythromycin, vancomycin, gentamycin, tetracycline, ciprofloxacine) $324.15(from receipt) (150 discs of each of the 5 antibiotics) $ (150 discs of each of the 5 antibiotics) $ gm Mueller Hinton (two bottles) $91.33 (one bottle) $91.33 (one bottle) $ gm Tryptic Soy agar (1 bottle) $47.31(one bottle) $47.31 Tryptic soy broth 500 gm (1 bottle) $102 (one bottle) $102 Petri Dishes 30 pkgs of ten per pack (150x15mm) Cotton swabs 2 pkgs (200 per pkg) 2 pkg 10 ml pipettes (50 per pkg) 4 pkg 5 ml pipettes (50 per pkg) 4 pkg 1 ml pipettes (50 per pkg) Petri dishes 300 plates (100 X 15) (25 per pkg) $43.95 (15 pkgs) $23.50 (1pkg) $33.65 (1pkg) $40.80 (2 pkg) $38.90 (2 pkg) $ (150 plates) $43.95 (15 pkgs) $23.50 (1pkg) $33.65 (1pkg) $40.80 (2 pkg) $38.90 (2 pkg) $ (150 plates) $87.90 $47.00 $67.30 $81.60 $ $45.48 Bile esculin azide agar $ (1 bottle) $ 91.60

2 Equipment Mileage to gather Data (.36 per mile) 198 miles x $0.36= $71.28 $71.28 GRAND TOTAL $ $71.28 $ $1, NOTES Equipment and left-over materials purchased with this grant will remain the property of WSU. You may not request money for gas purchases for travel. WSU reimburses travel expenses at a set mileage rate only.

3 Project Description Enterococcus, a bacterial genus that normally inhabits the gastrointestinal tract of many animals, can be pathogenic to humans causing illnesses such as septicemia or urinary tract infections. One important complication of those infected with Enterococcus is the fact that this bacterium often has high levels of antibiotic resistance making effective treatment of patients more difficult (Aarestrup). This organism is able to survive outside of animals even in adverse conditions such as hypersaline environments like those found in the Great Salt Lake (GSL) (Fisher and Kagie). In this experiment, we will collect enterococci from three different bodies of water. These water sources will be obtained from an alpine lake, an urban freshwater pond (Beus Pond in Ogden, Utah), and the GSL. Water will be cultured on appropriate media and isolates will be tested to determine if they are members of the genus Enterococcus. We will then determine if there are differences in antibiotic resistance patterns of the enterococci from these three sources. We hypothesize there will be differences in antibiotic resistance based on the fact of differing amounts of human impact on these three bodies of water and therefore possible exposure to antibiotics. Both Beus Pond and the GSL could contain enterococci from people treated with antibiotics, although this could be greatly diluted by the amount of enterococci from animal sources, especially birds. (Both of these waters contain a significant number of avian populations). The alpine lake should contain few, if any, enterococci from humans, and instead the vast majority of its enterococci should be of animal origin. Another important difference is that the GSL water receives effluent from sewage treatment plants that treat hospital sewage, obviously a concentrated source as many patients are given antibiotic therapy daily in this setting. It is also likely that hypersaline conditions of the GSL could select for or against strains

4 that are antibiotic resistant. Although previous work has been done noting that there are antibiotic resistant strains of enterococci in the GSL, no one to our knowledge has ever compared the enterococci isolated from the GSL with those isolated from other freshwater sources (Merrigan). To complete this study, we will finish collection of water from the last source (the alpine lake) and isolate and confirm any enterococci present. The goal of this study is to compare the three bacterial libraries for different physical characteristics and test for their antibiotic resistant patterns of five antibiotics. It is expected that the bacterial pools will be different in terms of their antibiotic resistance patterns. In addition, results of this study could help estimate any risk that recreational or occupational exposure to the GSL or the other sources may have in terms of this pathogen. To our knowledge, no one has researched this question as little is known about organisms, especially Enterococcus, from the Great Salt Lake, nor have comparisons been made to isolates from freshwater sources. Our role in this research project is to test this hypothesis. Since we were trained by Dr. Karen Nakaoka concerning the methods, we will operate mainly independently. Dr. Nakaoka will guide and consult with us as we proceed with this project. Our training and experiences have come from the course and laboratory work in our Microbiology major and training by Dr. Nakaoka. These courses include: Principles of Microbiology, Microbial Procedures, Microbial Genetics, Microbial Physiology, Microbial Ecology, Immunology, Medical Microbiology, Food Microbiology, Genetics, and two years of Chemistry. All of these courses have had labs. The results of this work will be presented at the annual WSU Undergraduate Research Symposium and a paper submitted to ERGO. We hope to present this research at the Utah

5 Academy of Sciences, the Intermountain Branch of the American Society of Microbiology, or elsewhere.

6 Project Methods & Timeline The first half of this project was started in this past summer semester and the other half will be completed by the end of this fall semester in December, if funded. Water has been collected from the GSL and from Beus Pond and enterococci have been isolated, confirmed, frozen and await further testing. The water samples from the Alpine Lake will be collected in October and collected on a 0.45 µm membrane filter via the Milipore system. This water sample will be filtered within one hour of its collection, the filters will be placed onto ment media agar plates and allowed to incubate for 2 days. Colonies typical of Enterococcus colonies growing on the ment agar plates will be transferred to bile esculin azide agar plates and incubated for 2 days. Finally the colonies positive for growth on this media and that hydrolyze bile esculin with black precipitate will be transferred to tryptic soy agar (TSA) plates. The colonies growing on TSA will be tested for their catalase activity and gram-stained to confirm that they are of the genus Enterococcus. These isolates will be frozen until we are ready to perform the Kirby-Bauer disk diffusion method (Bauer), screening testing for resistance against five antibiotics (erythromycin, vancomycin, gentamycin, tetracycline, ciprofloxacine). The water from these three sources has/will be analyzed for ph, total dissolved solids, conductivity and temperature) as well. Budget Explanation The items listed on the budget worksheet under the department or college funds have/ will be provided by the Microbiology department and were/are for the first half of this experiment. The items listed under the undergraduate research funds are those items needed for the second half of this experiment. Without these item funded by the Undergraduate Research funds, this research may not be possible.

7 Bibliography Aarestrup F.M. et al. Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark. Diagnostic microbiology and infectious Disease 37: (2000). Bauer, A.W. et al. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology 45 (4): (1966). Fisher, Katie and Carol Phillips. The ecology, epidemiology and virulence of Enterococcus. Microbiology 155: (2009). Kagie, Scott, Lorowitz, William and Nakaoka, Karen. Factors affecting the isolation of Enterococcus-like organism from the Great Salt Lake. Utah Academy of Sciences, Arts & Letters 85:69-87 (2008) Merrigan, Stephen, Lorowitz, William and Nakaoka, Karen. Bacterial Source tracking of Enterococcus sp. and Escherichia coli isolated from the Great Salt Lake using antibiotic resistance analysis. Utah Academy of Sciences, Arts & Letters 86:57-71 (2009)

8 UNDERGRADUATE RESEARCH SEMESTER/EXPLORATORY GRANT APPLICATION Additional Questions 1. What funding have you received from OUR in the past, Where has your previous project been disseminated. We have not received any prior funding. 2. Is this project part of a required course? If so, please indicate the support (monetary and in-kind) provided for this project by the academic department. Half of this research was done under a class guidance,the second half is for the proposal. 3. What additional sources of funding have been solicited? Is your department willing/able to fund any equipment they will be retaining? The Microbiology department has provided funding for some of the materials needed. 4. Where do you plan to disseminate the results of this project At the upcoming annual WSU Undergraduate Research Symposium and a paper submitted to ERGO. We hope to present this research at the Utah Academy of Sciences, the Intermountain Branch of the American Society of Microbiology, or elsewhere. 5. If you are requesting a stipend, please list all significant time commitments (5+ hours per week) that you expect to maintain over the duration of your project including, for example, class and work schedules. No stipend is being requested.

9 UNDERGRADUATE RESEARCH SEMESTER/EXPLORATORY GRANT APPLICATION FACULTY MENTOR RECOMMENDATION FORM Student Name (last, first): Jorgenson, Jennifer and Badley, Ashley Project Title: _ Antibiotic resistance of enterococci from hypersaline and fresh water Mentor Directions: After carefully reviewing the proposal and assessing both the viability of this project and the qualifications of the student requesting funding, answer the questions found below. Please expand the sections as necessary (do not attach separate letter). If the project involves the use of human subjects or protected animals, be sure the student secures IRB or ACUC approval. If the project receives funding, it is your responsibility to work closely with the student, monitor the ongoing progress of the project and budget, and evaluate the project s results. Failure to do so will jeopardize funding for this project and any future projects. 1. How long and in what capacity have you known this student? Jennifer is a senior microbiology major and was a student in three of my classes, all of which she earned an A. I have known her for about two years. From what I know about her, she has all the microbiology skills required to complete this project. Ashley is also a senior and has taken two classes from me. Based on this, she also has all the skills required to research this area. 2. Briefly describe the proposed project. Is this part of a larger research project? Is this part of a course? If so, how is the project apart from the nature and scope of activities normally taken for the course (Please attach a copy of your course syllabus)? Jennifer did some of this research for a summer course, Microbiology This semester, she will complete this project proposal with the hope to present this work at some local/ regional conferences. Ashley will only participate this fall semester. 3. Give an assessment of the project s significance to the student s discipline and of the project s educational and/or professional benefit to the student. This project will investigate the differences in antibiotic resistance between enterococci isolated from hypersaline water versus freshwater. It has never been done. Thus, any knowledge of this will be an important contribution to this field. IT will be of significance to know if the hypersaline environment selects for organisms that are resistant to antibiotics. The enterococci are the only pathogen that is found consistently in the Great Salt Lake and which has potential to colonize humans and to cause disease. 4. Comment on the qualifications of the student to successfully complete this project, both in terms of the project s scope and its time frame. The students will be able to complete the lab research this semester and finish the analysis and presentations next semester. 5. Comment on the justification and appropriateness of the project budget, including the necessity of a stipend (if requesting one). The budget is appropriate. 6. Describe your role in the project. I helped design the project and expect to assist with the lab research as needed. Since I trained Jennifer as part of the summer course, I know that she is competent and capable of doing all the procedures. Thus, I will mainly be involved with problem solving and data analysis. 7. Include anything else that you think will be helpful to the committee in evaluating this application. This project DOES x DOES NOT require review by the WSU Institutional Review Board for Human Subjects or the WSU Animal Care and Use Committee.

10 Project Mentor Signature Date Campus Mail Code Phone Extension