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Oral Presentations |
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Poster Presentations |
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# Jaime Zlamal, Central Michigan University (picture)(award) |
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*Best Oral or Poster Presentation, Graduate
# Best Oral or Poster Presentation, Undergraduate |
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RcaE regulates cell and filament morphology in the cyanobacterium Fremyella diplosiphon
*JULIANA R. BORDOWITZ1,2 and BERONDA MONTGOMERY-KAGURI1-3
1. MSU-DOE Plant Research Laboratory, East Lansing, MI 48824-1312 2. Cellular and Molecular Biology Program, MSU, East Lansing, MI 48824-4320 3. Department of Biochemistry and Molecular Biology, MSU, East Lansing, MI 48824
Fremyella diplosiphon is a freshwater filamentous cyanobacterium that possesses the ability to sense and adapt to changes in ambient light. In a process called complementary chromatic adaptation (CCA), the cyanobacterium enhances its photosynthesis by altering the phycobiliprotein composition of its light-harvesting antennae. RcaE, a phytochrome-class photoreceptor, is required in order for CCA to occur2. Over 30 years ago morphological differences were observed in the wild-type UTEX481 strain in response to red and green light1. We have characterized this photomorphogenic response using microscopic and biochemical analyses. Our results show that both UTEX481 and SF33, a shortened-filament strain with normal CCA pigmentation responses, maintain distinct RL and GL morphologies. Additional analyses of an RcaE null mutant strain (FdBk14) indicate that RcaE regulates filament length and cell shape in F. diplosiphon in response to red and green light. Furthermore, RcaE regulation of light-dependent morphology is photoreversible and, at least for RL, is mediated via the Rca signaling system.
This poster was presented at the “Sensory Transduction in Microorganisms” Gordon Research Conference in January, 2008.
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Comparisons of Antibiotic Resistance Patterns of Bacterial Isolates from the Kalamazoo River
Elizabeth A. Weage* and Ola A. Olapade, Albion College, Albion, MI 49224
Coliforms are groups of gram-negative bacterial populations that are present in the intestinal tracts of humans and animals. Therefore the presence and quantity of these bacterial populations are commonly utilized during the monitoring of water quality and also dependent on as indicators of fecal contamination of aquatic environments. Wastewater from agricultural facilities, pharmaceutical plants, hospitals, and human waste treatment plants have been linked to increased levels of antibiotic resistant and virulent organisms into aquatic systems (e.g., Muñoz-Aguayo 2007), due in part to increased usage of various antibiotics and their improper disposal thereafter causing serious public health concerns. Recently, there have been several reported cases of the methycillin-resistant Staphylococcus aureus [MRSA] associated with wounds and several other nosocomial infections. In this study therefore, the resistant patterns amongst several coliform isolates from two sites along the Kalamazoo River in Albion, MI, one site located upstream and the other downstream of the sewage treatment plant, were examined in response to various antibiotics including ampicilin, tetracycline, bacitracin, rifampin, and neomycin using the standard Kirby Bauer disk assay approach. The results obtained indicated that nearly all the isolated species from both sites were completely resistant to bacitracin based on an average zone of inhibition of 0.07cm, whereas the levels of susceptibility were variable in the presence of the remaining antibiotics examined.
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The Abundance and Compositional Variations of Surface-Associated Bacterial Populations in Teeth Plaque Assemblages in Response to Food Consumption
Liz E.White* and Ola A. Olapade, Albion College, Albion, MI 49224
Surface-associated bacterial populations (i.e., biofilms) are groups of immobilized microbial communities found commonly attached to various surfaces including rocks, human oral cavity, and catheter. The presence, abundance, and activities of these biofilm communities, comprising diverse microbial groups (i.e., bacteria, algae, and fungi) are of significant environmental and public health importance. Tooth decay is characterized by the formation of a sticky, colorless mass of bacterial cells referred to as plaque found either on the surface of teeth, near the gum line, or in spaces between teeth. One of the major factors in the development of dental plaque is the type of food consumed that may induce bacterial development and proliferation. Although some earlier studies have documented the importance of biofilm microbial communities in tooth decay as a result of consumption of food products containing high starch contents, to the best of our knowledge, no study is yet to examine the changes in the microbial community composition in biofilms to dietary change over a period of time. In this study, we used a combination of qualitative (API, Biomreux) and quantitative (viable counts and DAPI staining) approaches to examine bacterial populations on teeth temporally for about 5 weeks. The results obtained from examining total bacterial counts after brunch (mean value = 3.55 X 105/mL) and dinner (mean value = 3.79 X 105/mL) meals were not significantly different. Bacterial species belonging to the Streptococcus and Staphylococus genera predominated among the microbial communities examined. Overall, the results suggest that fairly high and nutritionally fastidious bacterial populations might have been supported by the various food products consumed during the period of the study.
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Colonization of Germfree Mice with Probiotic Bacteria to Create a Mouse Model of the Human Gastrointestinal Tract
*Sreelatha Ponnaluri, Andrea Abbas and Daniel Clemans, Eastern Michigan University, Canton, MI 48187
The Altered Shaedler Flora (ASF), which consists of eight anaerobic bacteria, were made to colonize germfree mice with a defined microbiota. These eight bacteria were known to not only provide the host with essential nutrients but to also colonize mucosal niches, which help protect the host from microbial pathogens. Therefore, the purpose of the ASF strains in mice were to create a model of the human gastrointestinal (GI) tract microbiota. However, with more research coming in, other key players in the GI tract of humans have come into the limelight. These other strains include Enterococcus faecalis, Bifobacteria, Bacteroides, Escherichia coli, Enterobacter spp., Klebsiella pneumonia, Methanobrevibacter smithii, and Candida albicans. Hence, the main goal of this study was to incorporate these new microbes with the ASF organisms into the GI tract of germfree mice to create a more complex defined microbiota for research purposes. Molecular techniques, such as Terminal Restriction Fragment Length Polymorphisms and Quantitative Real Time PCR, were performed on the isolated 16S rDNA from the GI tract organ contents of colonized germfree mice and were compared to culture data of the mouse gut contents. Preliminary culture data showed that the microbiota demonstrated high levels of colonization within the first three weeks and then leveled off, at about 7 weeks. This study not only provides a model of a more complex defined microbiota but also provides the molecular tools and information needed to measure changes in the microbiota after manipulation of any sort.
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DNA Sequencing of gyrA is a Rapid and Specific Assay for Identification of Arcobacter butzleri Isolates from the Environment
*Arun K. Nayak1, David L. Wilson3,5, John E. Linz3,4,5, and Joan B. Rose1,2, Departments of Fisheries and Wildlife1, Crop and Soil Science2, Food Science3, Microbiology and Molecular Genetics4, and the National Food Safety and Toxicology Center5, Michigan State University, East Lansing, MI 48824, USA
Arcobacter is an emerging human pathogen, which belongs to epsilon proteobacteria. Its presence in groundwater and commercial livestock populations suggests its potential for foodborne and waterborne associated disease. Because of the similarity between Campylobacter and Arcobacter, efficient diagnostics have proved challenging. During the investigation of an outbreak on South Bass Island in the summer of 2004, we recovered Campylobacter-like isolates from the island’s groundwater in 5 of 16 wells sampled (31%). Thirty four A. butzleri isolates recovered from human patients in the surrounding geographic area were also acquired to assess the relationship between the South Bass Island isolates and A. butzleri of public health concern. Genetic analyses were performed on the recovered cultures for further validation of the Arcobacter identification. A rapid PCR-RFLP(Restriction Fragment Length Polymorphism) assay, identified the isolates as Arcobacter butzleri and Arcobacter-like bacteria. Subsequent 16S rDNA and gyrA sequencing analysis supported the Arcobacter butzleri identification for specific isolates but both the gyrA and 16S rDNA sequence demonstrated the Arcobacter-like organisms belonged to a different bacterial Family. The analysis of gyrA sequences gives the better discrimination of Arcobacter species compared to PCR RFLP assay and is more efficient and cost effective than 16s rDNA sequencing. Phylogenetic analysis of a 300 bp gyrA fragment from each isolate was performed and sequence pair uniqueness confirmed as 97% to 100% of gyrA gene sequence identity of A. butzleri between environmental and human isolates. Our study illustrated that the gyrA phylogenetic analysis is a relatively efficient sequencing method for identification of species within the Campylobacteracae family and should prove a useful molecular tool in future studies in the surveillance of Campylobacter-like organisms. To our knowledge this is the first report establishing sequence level similarity between environmental and clinical isolates of Arcobacter spp. Arcobacter should be assumed to be a potential cause of waterborne disease.
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Evaluation of Bacteroides, a new alternative indicator for fecal contamination
*Sangeetha Srinivasan, Marc P. Verhougstreate & J.B. Rose Department of Crops and Soil Sciences, Michigan State University, East Lansing, MI, USA.
Identification of the source of the fecal contamination is necessary to develop proper management strategies inorder to minimize and control fecal contamination. Researchers have recently evaluated a group of anaerobic bacteria, Bacteroides-Prevotella, that employ unique markers capable of detecting fecal contamination. Bacteroides are strict anaerobes which make cultivation difficult and therefore, have to depend on molecular methods. Primers of this MST assay differentiate between human and cow feces because they target the 16SRNA region. Our goals are to determine the sensitivity of the Bacteroides assay, to evaluate this tool in diverse watersheds of Michigan, and to examine the statistical relationship between Bacteroides and the human/zoonotic pathogens. Since this assay relies solely on DNA and no cultivation based results are available, the stability of these markers needs to be evaluated and compared to that of the conventional indicators. This assay is currently being used to analyze water samples from sites potentially impacted by septic tanks, CSOs, animal manure. We have compared the results with other human fecal markers/indicators such as the Enterococci esp gene, E. coli, Clostridium perfringens, Coliphage, adenovirus, and Enterococci. Our analyses indicate cow influences are more prevalent than human influences in rural watersheds of Michigan. Conversely, human markers dominate samples from urban systems with mixed land-use. The detection limit of this method is also being evaluated by seeding 1L of sterile distilled water with various dilutions of sewage. Preliminary results indicate that the human signal is present with 1µl of sewage per liter of water. Our future goal is to conduct a similar study with manure samples to evaluate the bovine marker assay.
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Multiplex PCR Primers for the Detection of Polycyclic Aromatic Hydrocarbon-Degrading Genes
*Michael J. Pressler and Gregory M. Colores, Biology Department, Central Michigan University, Mount Pleasant, MI 48859
As part of a larger study using Michigan native plants to remediate polycyclic aromatic hydrocarbon (PAH) impacted soils, we set out to develop a set of multiplex PCR primers to determine what catabolic genes are enriched in the rhizosphere's of different plant species. Our criteria for these primers were that they 1) target PAH-degrading genes from a diverse array of bacteria, 2) yield PCR products that can be separated easily and 3) contain a minimal number of degeneracies so that they would be suitable for use in more detailed analysis using denaturing gradient gel electrophoresis. We produced several alignments using over 100 PAH dioxygenase gene fragments and clones obtained from GenBank. To limit primer degeneracy to one primer per set we developed 6 different primers that yield PCR products ranging between 460-993 bps and were theoretically suitable for multiplex reactions. Primers were tested on individual and mixtures of PAH-degrading isolates and the PCR products sequenced to verify their specificity. Once we obtained positive results for each primer set the primers were tested on DNA extracted from a number of rhizosphere soil samples. Using these primer pairs we retrieved PCR products from 5 of the sets giving us a rapid view of dioxygenase diversity within the rhizosphere's of the plants used in this study. Overall, the protocols developed provide a screening method to rapidly assess the functional genetic diversity of PAH-degrading bacteria and may also be used in screening environmental samples for bioremediation applications.
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Robust yeast for increased bio-ethanol production
*Sandra Allen, William Clark, and Steven Gorsich, Biology Department, Central Michigan University, Mount Pleasant, MI 48859
Due to economical and environmental concerns, there is a high demand for an alternative and renewable fuel source. Bio-ethanol is one alternative fuel that has been proposed and tested. Currently, bio-ethanol is produced from baker’s yeast, Saccharomyces cerevisiae, using cornstarch as a fermentation substrate. While this process is quite efficient, our nation lacks the amount of corn needed to produce the desired amount of bio-ethanol. As a solution to this problem it is imperative to develop new ways to ferment ethanol by using alternative substrates such as agricultural and woody biomass lignocellulose. These substrates have great potential, but are also problematic due to multiple inhibitors they produce during fermentation. We demonstrate that one of these inhibitors, furfural, induces cellular damage to membranes, DNA, and the cytoskeleton. Moreover, an engineered strain with improved tolerance lacks the same severity of cellular damage. This new engineered yeast strain offers promise in developing a strain robust enough for industrial fermentation of biomass lignocellulose.
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Thymidylate Kinase: A Novel Target for Antifungal Drug Discovery*
Cheryl L. Quinn1†, Daniel Everdeen2, John Herberg3, Jeanne Hagadorne3, Joseph Leone3, Eric Lull3, Joseph P. Martin3, Ginny Garlock and Paul Tomich.
*Based on work conducted at the former Pharmacia Corp of Kalamazoo, MI, now Pfizer, Inc. Current addresses: 1PharmOptima LLC, Portage, MI ; 2MRI, Ann Arbor MI ; 3Pfizer Inc., St. Louis, MO
†Communicating author contact: cheryl.l.quinn@gmail.com
Thymidylate kinase (TMK), the enzyme that catalyzes the conversion of dTMP to dTDP was investigated for potential as an antifungal target. As a critical enzyme required to provide the building blocks for DNA replication, we thought TMK was likely to be an essential gene in fungal pathogens. The enzyme activity is also amenable to high throughput screening assays to enable the search for inhibitors that could be used as starting templates for a drug discovery effort. In this report, we present data that the gene encodingTMK is essential in the predominant fungal pathogen, Candida albicans. We also identified the cDNA sequence of the Aspergillus fumigatus TMK gene . The amino acid sequences for the fungal genes have less homology to their human counterpart then does the target of the fungal specific azoles. This information predicts that specific inhibitors of fungal TMK enzymes over human TMK are possible. Finally, we cloned, overexpressed and purified the fungal and human TMK enzymes from heterologous E. coli strains, and used these proteins to develop screening assays and characterize kinetic parameters. A high throughput screen on 100,000 commercially acquired compounds identified several classes of compounds that were inhibitors of Aspergillus fumigatus TMK.
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Cloning and Recombination of a Tetracysteine Tagged ipaD into the Virulence Plasmid of Shigella flexneri
Martinson, David A.*, and Baxter, M.A. Grand Valley State University Allendale, MI 49401
Shigella flexneri is the cause of bacillary dysentery. The disease is mediated by the formation of a type III secretion system (TTSS) that is encoded on a large virulence plasmid. This secretion system allows for the transit of key effectors from the cytoplasm of the bacterium directly into the cytoplasm of the targeted host cell. These secreted effectors target the host cell’s actin cytoskeleton and directs the uptake of the bacterium into the cell. IpaD is an important protein that localizes to the tip of the TTSS. This protein is responsible for controlling when secretion occurs in response to the local environment and in directing the insertion of other key bacterial effectors into the targeted cells membrane. Utilizing a tetracysteine tag, it has been shown that IpaD is also secreted directly into the target cell cytoplasm where it appears to target the cell membrane between cells and other intracellular membrane networks. This evidence would suggest that IpaD may also have a role in cell to cell spread once an infection has occurred. Currently, the ipaD gene containing the tetracysteine tag resides on a multicopy plasmid. Past experience has shown that multicopy expression of many of the genes involved in virulence do not display physiologic localization and function at high expression levels. Therefore, it is necessary to move the tagged ipaD gene back into the single copy virulence plasmid. Genetic manipulation of the Shigella virulence plasmid is difficult due to environmental instability of the plasmid within the laboratory and to the low expression of endogenous recombinases. The focus of this project is to optimize the process of creating specific mutations within the virulence plasmid of Shigella flexneri as we replace wild type ipaD with our tetracysteine tagged ipaD.
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Hydrogen Fermentation by Soil Anaerobes and their Prospective Role in Environmental Cleanup and Alternative Fuel Production
Michael D. Millican* and M. Aaron Baxter Ph.D. Grand Valley State University Allendale, MI
We are investigating hydrogen production through the fermentation of glucose under anaerobic conditions. Soil anaerobes, such as Clostridium spp., are known to produce hydrogen naturally through the fermentation of glucose to butyrate. In the butyrate pathway the hydrogen is released in the conversion of pyruvate to acetyl-CoA. Pyruvate is converted to acetyl-CoA through the action of the enzyme pyruvate-ferredoxin oxidorectuctase(PFO), in a redox reaction where the PFO removes electrons from the pyruvate to form acetyl-CoA and PFOreduced. PFOreduced then reduces four protons with the action of hydrogenase to release two molecules of hydrogen gas. There is a theoretical yield of 4 mol H2/mol glucose, but this is a theoretical yield and doesn’t account for the intermediates of the fermentation being used in other cellular processes.
The goal is to produce hydrogen gas from a heat treated soil inoculum. Once hydrogen gas is detected with the soil inoculum various conditions will be tested to observe if an increase in hydrogen yield can be achieved. It is possible to grow soil anaerobes on various organic acids, such as in decomposition, and produce hydrogen gas. This is promising in using waste water for the organisms to grow and produce hydrogen gas. Enviromentally this is very interesting because the organisms can use the contaminated water to grow, cleaning it in the process, and produce a clean energy source.
First a positive control was set up using Clostridium acetobutylicum. C. acetobutylicum is know to use butyrate pathway when growing on glucose, and is also known to be a common soil organism. The organism was grown in the Ward’s Fermentor in various conditions for a period of 48 hrs. The same CM3 media was used in all tests.
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Microbiology Exercises for Pre-College Education in Alternative Fuels
Graves, James, F.* University of Detroit Mercy, Detroit, MI 48221
Students need education in alternative energy sources to act as wise consumers and reduce dependence on fossil fuels. However, a requirement existed for educational activities to match State Curriculum Framework and National Education Science Standards. Also, restrictions on the types of activities that could take place were created as a result of conditions in schools. The content of exercises prepared included: linkages to education standards, narrative, science vocabulary, purpose, materials list, methods, results (tables, charts and graphs), questions to answer, references for reading and sources for materials. Students first become acquainted with the concept that microorganisms for the production of fuel exist in nature. This will take place by recovering microorganisms that are naturally found on fermentable fruit. The problem that only specific carbon sources can be used for the making of ethanol is to be uncovered through testing the ability of Saccharomyces cerevisiae to utilize various carbohydrates. Assessment will be accomplished by measuring the relative amount of carbon dioxide produced, as foam volume or balloon inflation time. Because an anaerobic environment is required for products like methane and butanol, students need to learn that the presence or absence of oxygen in the environment effects the physiology and growth of microorganisms. This is to take place through scoring for the formation of bubbles in the catalase test and measuring the depth of growth in agar stab tube cultures for selected nonpathogenic aerobic and facultative anaerobic bacteria. It is believed that these exercises may provide some practical activities for pre-college students to help learn about microorganisms and alternative fuels.
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The effect of the woody exotic plant autumn olive (Elaeagnus umbellata) on soil bacterial communities depends on the invasion context.
*Jaime Zlamal and Peter Kourtev, Dept. of Biology, Central Michigan University.
Invasive exotic plants are a major ecological problem and can cost millions of dollars a year per species in restoration efforts. We present a study on the influence of the woody exotic plant autumn olive (Elaeagnus umbellata, L.) on soils in two different invasion contexts: a forest edge and a forest interior. Autumn olive invasion did not alter the levels of nitrate in the soil, however, it had a significant effect on ammonium (and total N) amounts. The effect of the invasive differed in the two invasion scenarios: ammonium (and total N) significantly increased in invaded soils only in the forest interior. Autumn olive also affected the bacterial community in soils (as evidenced by DGGE of the 16S rRNA bacterial gene). Similarly, the effect depended on the context of the invasion. In the forest edge, the invasive plant appeared to enrich certain bacteria in soil. In the forest interior autumn olive invasion altered the bacterial communities, however, the change was not consistent between different autumn olive samples. Our results clearly indicate that autumn olive is altering the structure and function of microbial communities in the soil. It appears that the effect is context specific, which should be taken into account during restoration efforts.
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Development of Methods for Rapid Assessment of Influenza Virus in Avian Environmental Samples
E.S. Clark and E.W. Alm Central Michigan University, Mount Pleasant, MI, 48858
Migratory waterfowl infected with Avian Influenza Virus (AIV) shed high titers of virus in their feces, and AIV shed into the environment can persist for substantial periods of time under certain conditions. Surface waters contaminated by infected birds are a major route of transmission within bird populations, and may act as a seasonal reservoir for the virus. Human use of recreational areas contaminated by infected birds raise concerns of potential human transmission. Therefore monitoring potentially impacted sites should be considered and methods for rapid assessment of AIV in environmental samples developed. The objective of this study was to develop and optimize viral RNA collection and extraction protocols specific for environmental samples for detection using real-time RT-PCR (rRT-PCR). Droppings from waterfowl were collected at migratory stopovers and human recreational sites along the west coast of Lake Huron. Viral RNA was extracted from samples and then subjected to rRT-PCR using a previously developed Influenza A matrix gene assay specific for all sixteen (H1-H16) influenza A hemagglutinin (HA) subtypes. A novel method of extraction from fecal samples was developed to reduce PCR inhibition and improve sensitivity of detection. Samples were stored in viral transport media (VTM), vortexed, and centrifuged. The resulting supernatant was analyzed in downstream applications with improved detection from samples only vortexed. Preliminary processing has indicated Influenza A virus in 23 of 124 bird droppings. Feces collected during the Fall migration period contained more type A Influenza positive samples than feces collected during other times of year. These results indicate that environmental sampling could be a cost effective system method of monitoring circulating strains of Influenza within bird population.
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The Ribosomal Database Project
J. R. Cole (colej@msu.edu), Q. Wang, B. Chai, *E. Cardenas, R. J. Farris, A. S. Kulam-Syed-Mohideen, D. M. McGarrell, J. A. Fish, G. M. Garrity, and J. M. Tiedje Center for Microbial Ecology, Michigan State University, East Lansing, Michigan
Through its website (http://rdp.cme.msu.edu/), The Ribosomal Database Project II (RDP) offers aligned and annotated rRNA sequence data and analysis service to the research community. These services help researchers with the discovery and characterization of microbes important to bioenergy production, biogeochemical cycles, and bioremediation.
New Genome Browser: allows users to browse information, including rRNA sequences, from DOE and other bacterial genome projects, including information about the organism, whether the sequenced strain is recognized as the type strain for the species, and additional links and information provided by the Genomic Standards Consortium (http://darwin.nox.ac.uk/gsc/).
New Taxomatic Visualization Tool: This interactive tool allows researchers to choose subsets of RDP data and view a distance-based "heatmap" comparison of the user-chosen sequences by zooming out on the map to gain an overview of the entire data set, or zooming in to examine specific regions. Taxonomic boundaries can be interactively displayed on the heatmap by manipulating a hierarchy of taxonomic information or by "mousing over" corresponding regions of the heatmap.
Coming Soon: The RDP is building a Pyrosequencing Pipeline to automate the processing of large data-sets and provide researchers with the most common ecological metrics, along with the ability to download the processed data in formats suitable for common ecological and statistical packages. In addition, we are developing new tools to align, cluster, dereplicate and simplify the compute-intensive analysis of such large sequencing libraries.
The RDP taxonomy has been updated to reflect changes in release 7.7 of The Taxonomic Outline of Bacteria and Archea (http://www.taxonomicoutline.org/), with the addition of representatives for non-aligned phyla.
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Fecal Indicator Bacteria at Beaches Show Reduced Susceptibility to Antibiotics
*David Sadler, Erin Zacharias, Dan Zimbler, and Elizabeth W. Alm Central Michigan University, Mount Pleasant MI, 48859
Bacterial antibiotic resistance is a global healthcare concern and the roles of environmental reservoirs in antibiotic resistance are being investigated. This study was undertaken to examine antibiotic resistance among fecal indicator bacteria isolated from recreational beaches. Swimming water and beach sand were collected from six beaches along the west coast of Lake Huron. A disk diffusion assay for antibiotic susceptibility was performed on 177 E. coli and 154 enterococci. Beach enterococci exhibited greater resistance than did E. coli. 136 (88.3%) enterococci demonstrated reduced susceptibility to at least one antibiotic, whereas 61 (34.5%) E. coli demonstrated reduced susceptibility. The greatest reductions in susceptibility for E. coli were to ticarcillin (92.6% susceptible) and ampicillin (76.8%). The greatest reductions in susceptibility for enterococci were to vancomycin (72% susceptible) and rifampin (44.8%). Furthermore, 10 E. coli isolates and 34 enterococci isolates had patterns of susceptibility consistent with a definition of “multiple drug resistance”. Therefore minimum inhibitory concentrations (MIC) to select antibiotics were determined for these 44 isolates. For 7 of 10 E. coli isolates the ampicillin MIC was ≥256 µg/ml, with 2 isolates also showing resistance to cefoxitin. Five E. coli isolates had an MIC of ≥256 µg/ml for trimethoprim/sulfamethoxazole; all but one of these isolates was also resistant to ampicillin. Of the 34 enterococci isolates, 10 had MIC to vancomycin ≥256 µg/ml and 2 had MIC to ampicillin ≥256 µg/ml; one of which was also resistant to vancomycin. The results of this study indicate that antibiotic resistance is widespread among fecal indicator bacteria isolated at recreational beaches. These bacteria may represent an environmental reservoir of antibiotic resistance.
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Characterization of the Developmental Defect of Copine A Knockout Cells in Dicytostelium
Tasha S. Smith* and Damer, Cynthia K. Central Michigan University, Mount Pleasant, MI 48859
Copines are a family of calcium-dependent membrane-binding proteins that are found in numerous eukaryotic organisms. We are studying the function of copines in the model organism, Dictyostelium. When placed in starvation conditions, wild type Dictyostelium cells will develop into multicellular fruiting bodies consisting of stalk and spore cells. However in cells lacking copine A (cpnA- cells), development becomes arrested; cells form finger-like structures instead of fruiting bodies. We are using various molecular and microscopy techniques to determine the exact role cpnA has in Dictyostelium development. We found that if cpnA- cells are allowed to develop in reduced calcium conditions, they are able to form fruiting bodies, but with shorter stalk structures. We also found that mixing a small percentage of wild-type cells with cpnA- cells leads to rescue of the developmental defect. Our results suggest that cpnA may have a role in secreting signaling molecules during development. In addition, our results suggest that cpnA’s regulation by calcium is important to its role in development.
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Localization and Effect of YopE in Saccharomyces cerevisiae
Veronica Garcia-Bayo* and John R. Geiser, Western Michigan University, Kalamazoo, MI 49008.
Objective: To identify the cellular targets and mechanisms that YopE uses when it is injected into cells by type III Secretion System. Our goal is to identify a way to combat plague. We have undertaken this study in Saccharomyces cerevisiae where we have demonstrated that YopE is lethal.
Methods: Spontaneous suppressors of YopE induced lethality. A vector expressing YopE under the control of the inducible GAL1 promoter was constructed and transformed into diploid WT yeast. Yeast transformants of this vector die upon addition of galactose. 23 independent cultures were grown under non-restrictive conditions and spread at the concentration of 108 cells on galactose containing plates. Colonies that arose spontaneously on galactose containing plates were selected for further study. Some of the assays that have being preformed is the localization with rhodamine-palladine of the acting in presence of YopE over 5 Hr in yeast. A second assay studied is the interaction of the Rho proteins in Galactose induction in order to see protein interaction.
Results: From the original 23 independent cultures, 70 potential spontaneous suppressors have been identified. We are in the process of confirming and characterizing these suppressors. Conclusions: We have identified genes that serve to suppress the lethal phenotype of YopE in yeast. We expect these genes to identify physical targets of YopE as well as genes that involved in a genetic pathway that allows YopE lethality. YopE interacts with five out of six Rho proteins in yeast. There is not obvious results of direct depolymerization of actin in presence of YopE.
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