Program Director/Principal Investigator (Last, First, Middle):
Harmsen, Allen (Investigator Dilts)
A. Specific Aims This proposal seeks to increase the enrollment of Tribal and Disadvantaged students in, as well as enhance, the biomedical science curriculum at Salish Kootenai College (SKC). It is anticipated that these students will increase the number of Tribal students matriculating from the INBRE network into careers in biomedical science and/or professional programs thereby addressing the overwhelming need for culturally aware health care providers in Reservation communities. In addition, this proposal addresses health disparities in the reservation community while aiding in the preservation of a cultural resource. Specific Aim 1: Enhance the educational opportunities available at SKC through curriculum development Specific Aim 2: Enhance the educational opportunities available at SKC by providing research experiences within SKC’s Cellular and Molecular Biology Laboratory B. Background and Significance Salish Kootenai College (SKC) is a center for learning, cultural awareness, language preservation and economic development within the Flathead Reservation, home to the Confederated Tribes of the Salish and Kootenai peoples. Innovation, adaptability and flexibility have enabled SKC to thrive as an institution, growing from a single classroom with a handful of students, to a 30-acre campus with more than 1,200 students with over 59 different tribes represented in the SKC community. Over a quarter of the students enrolled are from other tribes in Montana and the Northwest United States. Students do not have to be American Indian or descendents to attend the college and there are students of many ethnicities enrolled. Nearly 100% of the students are low income and more than 90% are first generation to college. American Indian people want to be cared for by Native health professionals who intimately know cultural norms, ways of relating to individuals, ways of maneuvering through the community resources and politics. Increasing the number of people with an education in biomedical science will support these needs and expectations as well as address needs for an educated populace. Kevin Howlett, the chairman of Confederated Salish and Kootenai Tribes (CSKT) Tribal Health and Human Services, has presented to tribal council an overwhelming need for over 200 health care professionals in the next six years (personal communication). This provides a significant career opportunity for those within the reservation who would like to stay in their community and an incentive that further strengthens our proposal to serve similar regions across Montana. The mission of Salish Kootenai College is dedicated to providing such an education. One of the goals of Salish Kootenai College is, “Provide a learning environment in which students develop skills in effective communication, critical thinking, cultural understanding, and citizenship.” The President’s Report provides additional understanding into the meaning of cultural understanding at SKC, “Cultural understanding is important amongst all people on our Reservation, but it goes beyond the borders of the Reservation. People in today’s world need to understand and appreciate different cultures within our society. Each program imbeds cultural understanding into its course offerings.” SKC has many efforts addressing cultural competence of faculty, sharing with other programs, developing appropriate teaching methods, as well as the focus on developing cultural competence in students. SKC (2006) recently created the Department of Biological and Chemical Sciences and, through various outside grants and support including INBRE, has developed the Cellular and Molecular Biological Laboratory (CMBL), a modern equipped educational laboratory currently with Tribal students working on projects within the fields of Microbiology, Genetics, Cell Biology and Biochemistry. This proposal seeks to apply and extend these capabilities by studying the health related hazards of non-point pollution in Flathead Lake, the largest natural freshwater lake in the western United States, and a significant cultural site and economic generator for the Confederated Salish and Kootenai Tribes. Additionally, the high quality of the lake exemplifies the quality of life present within the Flathead Valley and the state of Montana. This proposal seeks to enable students
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Harmsen, Allen (Investigator Dilts)
and faculty to obtain meaningful scientific data which will advance the art and knowledge of identifying nonpoint source pollution by the development of quantitative methodologies for fecal coliforms as well as the genetic profile and origin of associated pathogens present in the lake; particularly within popular swimming areas within the Reservation during the months when the lake is utilized by large numbers of recreational users. The results of this research will address the unmet need of assessing the health hazards under current conditions, as well as obtain data that can serve as a reference as the area continues to be developed. Furthermore, it is anticipated that these data will also allow SKC, through presentations and publication of the results in relevant peer reviewed journals, to advance the knowledge of source tracking microbiological contaminants. Developing these scientific methods and obtaining these data will provide for student research projects and internships, both during the school year as well as the summer months, enhance curriculum, as well as provide a bridge from the college into the reservation community by demonstrating the application of scientific methodology that can aid in the preservation of the environmental and cultural heritage of the region. It is believed that these data, student studies and the dissemination of the information will lead to further understanding the dynamics of Flathead Lake in regards to the influence of development and increased usage on this resource, as well as, assess the public health risk to those recreating in or otherwise utilizing Flathead Lake or similar water resources. Modern science has led to the identification of numerous water borne pathogens, potentially harmful bacteria and viruses, such as Vibrio cholera, Giardia lamblia and Hepatitis A. The vast majority of these pathogens are associated with fecal matter, though the pathogens themselves are not usually the most predominant species within the fecal material. Thus, evaluating the health risk from fecal contamination poses a problem due to the dilution of pathogens and the resulting extremely low concentration that may occur in the plume of water emanating from the source. This is the expected scenario for Flathead Lake which has extensive volume but the areas of highest human contact occur along the shoreline. Additionally, the sources of the fecal pollution might also be diffuse, further complicating detection of the plume itself. These diffuse sources which may be difficult to detect but have an impact on the resource in aggregate are called non-point source pollution. To detect non-point fecal pollution, and assess potential health risk, the concept of indicator organisms has developed. Indicator organisms are the predominant microbiological organisms present within the gut of warm blooded animals, are generally more persistent in the environment and easier to quantify, and shed into the environment upon defecation. The majority of indicator organisms belong to the group of bacteria referred to as coliforms, which are defined as facultative anaerobic gram negative non-spore forming rods that ferment lactose with gas formation, within the taxonomic family Enterobacteriaceae; of these, Escherichia coli is the predominant species. Other indicator organisms have been proposed for environmental samples including; fecal streptococci, enterococci, Clostridium perfringens and Aeromonas. However, because of its known association with fecal matter and its prevalence within the intestinal tract, the predominant indicator of healthfulness in recreational waters is E. coli. Therefore, initial studies in this proposal will focus on the prevalence of E. coli in Flathead Lake and the surrounding watershed. Subsequent studies in this proposal may initiate investigations into the presence of pathogenic organisms within areas of fecal contamination detected by E. coli, as well as correlate the results of the E. coli studies with those of other proposed indicator organisms. Traditional detection of coliforms and subsequently E. coli is dependent on the presence of two key enzymes, -D-galactosidase and -glucuronidase respectively. There are a variety of methodologies that detect the products of these enzymes, as well as other biochemical assays to determine unequivocally the presence of coliforms. Biochemical techniques remain time and resource consuming, as well as ambiguous when attempting to identify the species, or source, of origin of the fecal pollution. Thus, without knowing the species of contamination, it is difficult to assess and mitigate the health risk as well as manage the resource. In this regard, molecular biological techniques, most notably the polymerase chain reaction (PCR), have been employed in order to enable the identification and genetic typing of the fecal coliform, and with a sufficiently large data base, indicate the specific source of the pollution (Versalovic et al., 1991; Versalovic et al., 1994). This is a very active area of research in applied microbiology (Healy et al., 2005). As described within the cited literature, there have been a number of approaches and varied success in applying molecular biology or a combination of molecular biology and biochemistry to this problem. It has been suggested that the ideal methodology, for the purposes of assessing recreational health risk, is one that is able to rapidly screen and
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identify the genotype present but not be so sensitive as to recognize generational mutations. Such generational mutations would not be a characteristic of the species shedding the bacteria and would require a data base of such as size as not to be practical, in obtaining an above average rate of correct classification (ARCC). Thus, genotyping methods have included plasmid analysis, restriction endonuclease analysis, multilocus enzyme electrophoresis (MLEE), multilocus sequence typing (MLST), pulsed field gel electrophoresis (PFGE), ribotyping, DNA sequencing, restriction fragment length polymorphisms, randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and repetitive sequencebased PCR (rep-PCR) (see Healy et al., 2005) . The technique of rep-PCR uses small oligonucleotide primers and a known repetitive DNA sequence expressed throughout the bacterial genome to identify the subspecies of bacteria and if needed, delineation the bacterial strain (Versalovic et al., 1991; deBrujin, 1992; Versalovic et al., 1994; Koeuth et al., 1995; Mohapatra et al., 2005). Standardization of rep-PCR allows for multi-laboratory analysis, creation and utilization of a single large data base, or library, thereby attaining the statistical resolution necessary for strain identification (Healy et al., 2005). The scientific goal of this project is to create a library of data and standardization of methodologies using rep-PCR that enable the assessment of non-point source pollution and health risk as well as the biological source, initially within the Flathead Lake drainage but with methodologies that can also be utilized in the seven major reservations of Montana as well as throughout the state. C. Educational Project Plan/Research Design and Methods Purpose: Increase enrollment of Tribal and Disadvantaged students in the biomedical science curriculum at Salish Kootenai College and the INBRE network through enhancing the research experiences available within the Cellular and Molecular Biology Laboratory as well as the Environmental Analytical Laboratory. This research will focus on Flathead Lake, a cultural centerpiece of the Flathead Reservation, and the assessment of health risk during recreational usage of the lake. Goal 1: Increase Enrollment in General Science at Salish Kootenai College Goal 2: Increase Enrollment of Tribal and Disadvantaged Students within the INBRE Network Goal 3: Increase the number of Tribal Students, SKC graduates as well as other Disadvantaged students applying for Professional Schools from the INBRE Network The Howard Hughes Medical Institute initiated a successful science learning study program based on “hands-on” learning and peer mentoring. Success being defined as an increase in the number of students interested in science, as well as students matriculating from the science related programs. SKC Biological and Chemical Science Department has been collaborating with Montana Tech in this on-going hands-on learning project, which is a bacterial phage discovery project. To date, 8 SKC students have participated in this program, with results consistent with the success defined above. Dr. Marisa Pedulla, at Montana Tech, first initiated this project in Montana, which has successfully involved over 1500 secondary students and resulted in the matriculation of “phage-hunters” into medical school, as well as matriculating graduate students in biomedical sciences. The key to this programs success is not only the hands-on science but peer mentoring, students teaching students. Initial outcomes of the phage project are initiating curiosity and a desire for discovery in a population where this is novel concept. This allows for furthering the outcomes to include an introduction to molecular biology and further study into scientific methodology leading to careers in biomedical science. Particularly significant, is that the peer mentoring and/or the identification of a new phage, brings a sense of accomplishment and enables the students to gain confidence to attempt new tasks. Consistent with these observations, at SKC, we have experienced that our tribal students are more apt to complete rigorous science courses, which are also prerequisites for any career in the biomedical sciences as well as health careers, when accompanied with “hands-on” research laboratory experiences. Research Experiences: Our source tracking project is based on the collaborative success of the phagehunting and is designed to address these key points: 1) easy entry into the scientific process, initiating curiosity and desire for discovery 2) peer mentoring with students teaching students 3) generation of meaningful data as well as a sense of accomplishment 4) open source learning such that students can pursue numerous avenues of interests of increasing complexity at their own pace with each individual project contributing to the overall
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success of the program, and 5) being part of a larger project and the scientific community at large. The source tracking program will be based on educational level modules designed to give participants “hands-on” experiences within both the SKC Cellular and Molecular Biology Laboratory (SKC-CMBL). Students will be counseled into the different modules based on their stated interests, aptitude and course work accomplished to date. Modules will be designed to attract students with different interests and at different levels in their career developments. The goal of this program is to matriculate students into 4 year degree programs resulting in a degree in a life science related field of study and/or enrollment into a professional career track, such as medicine, dentistry, pharmacy or biomedical research careers.
Module Sampling waters for the presence of coliform bacteria, and subsequent isolation, culturing and identification PCR and genetic analysis of the coliform DNA, analysis of cultures by biochemical assays Data Base management and Statistical analysis of data
Science Introduction Microbiology, microscopy
biochemistry, cell biology, molecular biology, microbiology
Bioinformatics, Computer Science, all branches of science
Biomedical and Health Careers Microbiologist, Biology, Environmental Science, Public Health Biochemist, Laboratory Technicians, Geneticists, unlimited Mathematics, Statistics, Bioinformatics, Proteonomics
Measurable Outcomes: Number of students enrolled in General Science; Number of students participating in the research programs at SKC; Number of Native American students enrolled in preprofessional health care career programs; number of A.S., B.S. or higher degrees awarded to participants; Student Presentations at Scientific Meetings; Number of students entering professional programs. Curriculum Development: In addition to enhancing the existing curriculum with the research based learning, it is anticipated that students’ interest in molecular biology will allow for the development of two new courses, molecular microbiology and immunology as well as an expanded human genetics course. These courses will be designed as sophomore or upper level undergraduate courses. Upper level courses will have as a pre-requisite our newly developed (2007-2008) sophomore series in Cellular and Molecular Biology. It is anticipated that the Cellular and Molecular Biology classes will incorporate both phage-hunting and source tracking techniques into their laboratory components. The addition of these new molecular based courses, as well as the existing full year Organic Chemistry sequence, enables students at SKC to fulfill the prerequisites for entry into professional medical degree programs at both University of Montana (Pharmacy) and Montana State University (WWAMI Medical Program), though our degree program remains a Associates Degree. The additional courses from this proposal, as well as those developed under SKC’s current NSF-TCUP Program, will allow students to achieve further instruction and understanding of life processes based on molecular interactions thereby increasing the likelihood of successful completion of the chosen professional or degree programs. It is further anticipated that the Department of Biological and Chemical Sciences will make significant progress towards a successful application for certification of a 4-year Bachelor Degree program by the end of this two year proposal. Expected Outcomes: The Department of Biological and Chemical Sciences, created in 2006, has thus far matriculated 4 students. These students were previously enrolled in SKC’s general science A.S. program prior to the formation of the Department. Additionally, one of these students went to pharmacy school in fall 2007, but subsequently returned and entered the B.S. degree program in Environmental Science. An additional tribal student took part of our new curriculum in 2007-2008 and has been accepted into Pharmacy school in fall 2008. We would anticipate in this proposal to increase the A.S. degrees
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awarded to 10 students with the objective to have at least 6-12 new Tribal students annually. We also anticipate 1-3 applicants to professional programs at the completion of this proposal. Sampling and Bacteria Culture Identification: Consistent with increased usage leading to increased contamination, preliminary studies of water quality and beaches by SKC students has revealed a significant increase in coliform concentration during the late summer months in Flathead Lake. We are presently working to extend these observations and increase the number of samples in order to perform statistical analysis. These preliminary observations will be extended into the first year of this proposal by student interns. Students will collect 200-250 mls of lake water and quantitiate total coliform, E. coli and non E. coli coliform using technique and methods consistent with accepted protocol (Standard Methods, 2000). Water samples will be filtered by vacuum through a 0.45 micron filter which will be cultured using Coliblue® (Hach Co., USA). The non-E. coli coliforms will stain red, E. coli will produce an additional blue and colonies will be counted following incubation at 37oC for 20-24 hrs and reported as CFU/100 ml. The E. coli will be collected and grown on nutrient agar (Luria-Bertani, LB), isolated, and re-cultured on a second plate. Isolated cultures will be confirmed by Kovac’s reagent in the Indole test. Additionally, selected E. coli cultures will be confirmed by PCR for B-glucoronidase gene uidA (Venkateswaran et al., 1996). Confirmed E. coli cultures will be harvested, placed into 30% buffered glycerol, and frozen at -70oC and further analyzed by rep-PCR. Completion of the first module in bacteria identification and quantification will matriculate into the rep-PCR analysis along with the mentoring duties. Rep-PCR and Genetic Analysis of coliform DNA: As described in the scientific background, numerous molecular biological techniques have been employed to date to identify strains of bacteria (Versalovic et al., 1994). Of these, rep-PCR using Box-A1R primer, appears to lend itself to standardization, produces consistent intra-laboratory results, and can be used on multiple species and sources to identify bacterial strains (Koeuth et al., 1995; Healy et al., 2005). We have recently initiated the collection of fecal samples from the Flathead Lake Drainage and sent them to the MSU Department of Microbiology in order to isolate E. coli , develop standardize methodologies for BOX-A1R rep-PCR and compile DNA profiles attained using Box-A1R into a library. Additionally, the project PI recently collaborated in a workshop with water quality monitoring students at Little Bighorn College that produced strain differences with Box-A1R using rep-PCR. Initial data and trial of the first proposed module has occurred as well when two SKC students successfully collected water and fecal samples this past quarter. E. coli isolates from these samples are awaiting rep-PCR analysis at SKC. In this regard, the CMBL recently purchased an Agilent 2100 Bioanalyzer for performing microfluidics, Lab-on-a-chip, and analysis of the E. coli rep-PCR products. This proposal contains a request to upgrade the analysis of data obtained on the Bioanalyzer by obtaining a software package and computing system designed for DNA profile analysis. This is necessary because of the large sample size, 300–3,000, that are needed in the statistical analysis of these DNA profiles for strain specific pattern recognition (Dickerson et al., 2007). It is anticipated that rep-PCR will occur during the duration of this project, and samples will continually be added to the library in order to increase sample size and thereby increase the statistical power and AARC of the E. coli strains. These data will be collected by second year students. If enough E. coli are isolated such that strain identification can be routinely performed with greater than 95% confidence, than the applied techniques will focus on other proposed indicator coliforms such as staphylococci or enterococci, thereby adding further critical assessment of the health risk in recreational usage of Flathead Lake (Dickerson et al., 2007). If a library of sufficient size is attained and AARC of 85% for human E. coli strains is not possible with greater than 90% confidence using BOX-A1R profiles alone, than SKC will pursue adding additional DNA profiles to the analysis, particularly ERIC2 or (GTG)5 to increase resolution and confidence to achieve AARC greater than 85% (Johnson & Johnson, 2000; Casarez et al., 2007). However, the complexity may not be needed or may add too much sensitivity thereby producing the unwanted effect of detecting generational polymorphisms and further complicating the analysis. Once a library of sufficient size is obtained, it is proposed that we expand our capabilities for more rigorous statistical analysis. Data Base Management and Statistical Analysis As the information obtained during the course of the proposed studies are non-parametric, the analysis of this data will require the purchase of image analysis and pattern recognition software, in addition to those present
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in the software of the Agilent 2100 Bioanalyzer. This information, the management of the data present in the library and the continued analysis of samples collected will create opportunities for student internships in data base management, applied mathematics, statistics computer technology and networks which are consistent with entering the biomedical fields of bioinformatics, particularly genomics and Proteonomics. While these are not currently career paths as defined in the objectives and goals of this project, it is anticipated that this will be an unmet need in the near future considering the growth in the field of Bioinformatics. Measured Outcomes: Statistical Analysis of Coliforms and potential Health Hazard. Number of DNA profiles and species isolates. Dissemination of data in presentations and publication. Expected Outcomes: There will be sufficient data for statistical analysis of the presence or absence of fecal coliforms at the beaches on Flathead Lake in the first year of this proposal. These data will be presented by a student at a scientific meeting following the summer internship in each year of this proposal. It is anticipated that initial results will be presented within the Reservation Community, either on a website or through public meetings. Community Involvement: We are proposing to work with the Flathead Lakers, a long standing non-profit organization with over 1000 Tribal and non-Tribal members working to maintain water quality (www.flatheadlakers.org), in obtaining and disseminating the data and the results in these studies to the communities within the Flathead Lake Watershed. It is anticipated that this collaboration with a community based organization will generate more interest in science education both at SKC as well as in secondary education within the community. Particularly, it is hoped that this will reach into the schools that reside in and around the reservation enabling further recruitment into the career paths within the Department of Biological and Chemical Sciences. The initial module in this proposal, collecting and identifying coliform bacteria, is applicable to secondary education and could be a second outreach project, in addition to phage hunting, for the high schools or summer science camps that involve youths from grades 7-12 including SKC’s UPWARD Bound Program. Measured Outcomes: Presentations and Reports Expected Results: As results become available, the PI will present the information at meetings of the Flathead Lakers. D. Timeline Activity/Format - Content Performance Indicator Person Responsible Curriculum Development – Courses in Immunology and Molecular Microbiology SKC Catalog listing & PI – Roger Dilts, Ph.D. Department website description & offering Research Modules – Coliform identification and isolation, Box-1AR rep-PCR and DNA profile library, Statistical Analysis, Chemical Analysis Student progression and PI – Roger Dilts, Ph.D. participation. Development of peer to peer mentoring Community and Secondary Outreach – Public meetings and educational seminars. Demonstrations to students in secondary schools. Posting of results on website www.flatheadlakers.org for public access Timeline Years 1-2
Academic Years 1-2 and summer internships
Year 2
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E. Human Subjects None F. Vertebrate Animals None G. References Casarez EA, Pillai SD, Mott JB, Vargas M, Dean KE, Di Giovanni GD. (2007) Direct comparison of four bacterial source tracking methods and use of composite data sets. J. Appl. Microbiol. 103(2):350-64. De Bruijn FJ (1992) Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl. Environ. Microbiol. 58:2180-2187. Dombek PE, Johnson LK, Zimmerley ST, Sadowsky MJ. (2000) Use of repetitive DNA sequences and the PCR to differentiate Escherichia coli isolates from human and animal sources. Appl. Environ. Microbiol. 66:2572-2577. Dickerson JW Jr, Crozier JB, Hagedorn C, Hassall A. (2007) Assessment of the 16S-23S rDNA intergenic spacer region in Enterococcus spp. for microbial source tracking. J. Environ. Qual. 36(6):1661-9. Healy M, Huong J, Bittner T, Lising M, Frye S, Raza S, Schrock R, Manry J, Renwick A, Nieto R, Woods C, Versalovic J, Lupski JR. (2005) Microbial DNA typing by automated repetitive-sequenced-based PCR J. Clin. Microbiol. 43(1):199-207. Johnson JR and O’Bryan TT. (2000) Improved repetitive-element PCR fingerprinting for resolving pathogenic and nonpathogenic phyligenetic groups with Escherichia coli. Clin. Diagn. Lab. Immunol. 7(2):265-273. Koeuth T, Versalovic J, Lupski JR. (1995) Differential subsequence conservation of interspersed repetitive Streptococcus pneumoniae BOX elements in diverse bacteria. Genome Res. 5:408-18. McLellan SL, Daniels AD, Salmore AK. (2003) Genetic characterization of Escherichia coli populations from host sources of fecal pollution by using DNA fingerprinting. Appl. Environ. Microbiol. 69(5):2587-2594. Mohapatra BR, Broersma K, Mazumder A. (2007) Comparison of five rep-PCR genomic fingerprinting methods for differentiation of fecal Escherichia coli from humans, poultry and wild birds. FEMS Microbiol. Lett. 277:98-106. Seurinck S, Verstraete W, Siciliano SD. (2003) Use of 16S-23S rRNA intergenic spacer region PCR and repetitive extragenic palindromic PCR analyses of Escherichia coli isolates to identify nonpoint fecal sources. Appl. Environ. Microbiol. 69(8):4942-50. Venkateswaran K, Murakoshi A, Satake M. (1996) Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in Foods. Appl. Environ. Microbiol. 62(7):22362243. Versalovic J, Koeuth T, Lupski JR. (1991) Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19:6923-6831. Versalovic J, Schneider M, deBrujin FJ, Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol. Cell. Biol. 5:25-40.
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