2002 Research Fair Archive - Biology Abstracts
Characterization of Enterococcal Clinical Isolates
We are concentrating our studies on E. faecalis associated with urinary bladder infections. Our twenty-three isolates were obtained from urinary infections occurring at St. Francis Hospital in Hartford, CT. Several characterizing tests were performed with each strain to confirm the identity of the isolates. The tests were a simple gram stain; bile-esculin reduction; fermentation of mannitol salt; reduction of tellurite; growth at 45 C; and visual inspection of morphology and colony formation. The results of the characterization tests conclude that our strains are E. faecalis.
A Botanical Inventory of Dimple Dell Park
There has never been a systematic, botanical inventory conducted for this important nature park. The purpose of this project is to conduct such a survey by generating a detailed record and systematic growing season collection of native plants. In doing so, we hope to lay the groundwork for future research efforts involving Dimple Dell. This study will also help provide valuable information for use in the management and interpretive programming of the Park.
The mounted specimens collected in triplicate will be deposited in the appropriate herbaria at Westminster College, Utah Museum of Natural History at the University of Utah, and at Dimple Dell Regional Park Headquarters.
During the summer of 2001, we located, collected, and pressed 574 plant specimens consisting of 170 different species. We estimate that there are about 80 more plant species in Dimple Dell that need collecting and mounting. This leads us to conclude that the park contains at least 22% of all the wild plant species found in the Central Wasatch Area (Salt Lake and Davis Counties). These estimations further highlight the ecological and educational importance of the park.
Plasmid Isolation From E. Faecalis
Enterococci are common nosocomial pathogens that cause urinary bladder, endocarditis, and wound infections. E. faecalis causes the majority of these infections. One of the characteristics of E. faecalis that makes it hard to treat is its ability to transfer plasmids. Plasmids are small pieces of genomic material (DNA) that perhaps aid the bacteria in becoming highly adherent to mammalian cells, or resistant to antibiotics. A method of plasmid isolation from enterococci was developed from several sources and plasmids isolated from the 23 clinical E. faecalis strains. Plasmids ranged in size from 5-28 kb, however higher molecular weight plasmids (above 35 kb) were not found. A comparison of plasmid profile and adherence to HTB-5 (human bladder cells) cells was initiated. Currently studies are underway to develop a better protocol for extracting larger plasmids, as well as purifying all plasmids in greater quantity.
DNA Probe Adherence Assays
Antibiotic resistance of Enterococcus faecalis strains is on the rise and therefore, alternative means of combating infections must be investigated. We hope to utilize anti-sense technology to block synthesis of enterococcal surface proteins which allow it to adhere to mammalian cells. Initial investigation of these proteins involves determining levels of adherence of E. faecalis isolates through an in situ adherence assay with HTB-5 (urinary bladder carcinoma) cells. We have recently employed the use of PNA probes in order to detect and quantify bacterial adherence. This probe (Boston Probes) hybridizes to a ribosomal RNA sequence specific only to eubacterial cells. Initial experiments using the probe show high levels of target specificity. They also corroborate previous data concluding that enterococcal adherence is specific to HTB-5 cells and will not occur on MDCK (Madin-Darby Canine Kidney) cells. However, as only fluorescein-tagged bacteria and DAPI-stained nucleic acids can be visualized using fluorescence microscopy, the issue of mammalian cell boundary identification for determining adherence has been raised. We are currently working to remedy this by employing a modified Wright-Giemsa counterstain. Comparison of the brightfield and fluorescent images should then allow for a more definitive determination of adherence levels.
Characterization of Pheromone-Induced Aggregation
Enterococcus faecalis, has a unique sex pheromone mating system in which a plasmid free strain produces a peptide pheromone that attracts a plasmid carrying strain. After the cell is signaled, the two strains "clump" together via a proteinous "aggregation substance," which allows them to bind to each other and transfer plasmid DNA. The experiments performed to date have (i) determined if "aggregation" occurs between various strains; (ii) determined if the clumping is in fact "aggregation substance;" and (iii) determined the composition/ size of the "aggregation substance." To determine if the strains "aggregated," random pairs of strains were introduced into Tryptic Soy Broth and incubated overnight at 37° C. Four groups that produced the most "aggregate" were used in further studies. To test the size and composition of the "aggregation substance" a media with minimal amounts of protein was used so that the excess protein would not interfere with the separation of the aggregate. After a suitable media was found, SDS-PAGE was used to confirm the presence of "aggregation substance." These efforts at cell mating may be a useful tool in studies of alternative treatments of enterococci.