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The TLC plates were overlaid with S. aureus to identify compounds with antibiotic activity. A disk diffusion assay was used to confirm the TLC’s result. Species identification was done by 16S rRNA gene sequencing analysis. The active compound produced by isolate AC1359 was obtained from ethyl acetate extraction. The active compound from AC1359 was purified by using flash chromatography with a methanol-dichloromethane gradient. The 16S rRNA gene analysis showed that AC1359 has a 100% identity to Streptomyces bacillaris. AC1359 has been found to produce an active compound that acts as an antibiotic against S. aureus. Further investigation is necessary to understand the structure of the active compound produced by isolate AC1359.
ANTIMICROBIAL EFFECTS OF MIMULUS AURANTIACUSJackson Womck, Christine Case.
Skyline College, San Bruno, CA.
The rising prevalence of antibiotic-resistant infections has led to a public health crisis. New antimicrobial compounds are needed to combat resistant pathogens. Plants used by traditional healers may provide these new antibiotics.
Historically, the California Miwok used Mimulus aurantiacus leaves to treat skin infections. Our aim is to explore the antibacterial properties of M. aurantiacus. Ethanolic extracts of plant leaves were prepared by grinding leaves in 70% ethanol (400 mg/mL). This extract was tested against Micrococcus luteus (Gram-positive) and Escherichia coli (Gram-negative) bacteria in agar-diffusion assays. Our results suggest that M. aurantiacus contains effective antibacterial agents. The leaf extract inhibits growth of both bacteria. We are investigating the effects of root and flower extracts on the growth of Salmonella enterica and methicillin-resistant Staphylococcus aureus. The minimum inhibitory concentration and method of action will be determined. These are valuable findings as they may lead to the development of novel antimicrobials to which bacteria do not show resistance.
ANALYSIS OF THE EFFECTS OF BIOFILM FORMATION OF THE GASTROINTESTINAL PATHOGEN
CRONOBACTER BY PROBIOTIC SPECIES OF LACTOBACILLUSJessica Mesa, Maia Bland, Ana Vallor.
University of the Incarnate Word, San Antonio, TX.
Lactobacillus have been isolated and found to be beneficial in the maintenance of intestinal health by the production of antimicrobial substances and adherence to mucosal epithelial cell surfaces. Loss of Lactobacilli may result in dysbiosis in some cases and lead to increased infection rates of opportunistic pathogens and allergies in susceptible hosts. Cronobacter sakazakii has been identified as an emerging intestinal pathogen in infants causing bacteremia and necrotizing entercolitis. Fatality rates in neonates with invasive Cronobacter infections range between 40 and 80%. C. sakazakii has been shown to produce microbial biofilms in order to promote and sustain colonization.
Previous work in this laboratory has resulted in isolation and preliminary characterization of lactobacilli outer membrane S-layer proteins which may play a role in inter-species aggregation. Further, lactobacilli were found to form biofilms in vitro. This study characterized the effects of incorporation of Lactobacilli gasseri and Lactobacilli jensenii cells and S-layer proteins on C. sakazakii biofilm formation. Coincubation of Cronobacter with each Lactobacilli species was performed in a dose-dependent manner to assess the effects on Cronobacter biofilms as monitored by light microscopy and a metabolic, semiquantitative colorimetric assay. Initial results demonstrated that levels of Cronobacter-lactobacilli biofilms were lower as compared to C. sakazakii-only biofilm, and the difference in biofilm inhibition was more pronounced at subsequent time points. Studies are ongoing to assess the effects of Lactobacilli S-layer proteins on C. sakazakii biofilms. Future studies will also entail further characterization of the possible roles of other antimicrobial Lactobacilli factors (i.e., lactic acid production, bacteriocins) on C. sakazakii biofilm inhibition.
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BACTERIAL CONTENT ALONG THE SALINITY GRADIENT IN RIO GRANDE DE MANATÍ, PUERTO RICOAlexis Sanchez-Perez1, José R. Pérez-Jiménez2, José Lafontaine-Serrano1, Diana L. Laureano-Córdova3.
Universidad del Turabo, Gurabo, PR, 2Puerto Rico Institute for Microbial Ecology Research, Universidad del Turabo, 1 Gurabo, PR, 3Interdisciplinary Research Institute, Universidad del Turabo, Gurabo, PR.
Studies of microbial communities from aquatic ecosystems provide important insights into relations between various aspects of ecosystem functioning and changes in biodiversity. The differences between marine and freshwater bacterial populations have always been intriguing. Our objective is to assess the aquatic bacterial communities in response to changes in salinity. Water samples from river, estuary, and ocean along Río Grande de Manatí were cultivated on tryptic soy broth (0.5X) made with site water, and alternative salinity conditions. The resulting microbiota were diluted and cultivated on contrasting saline conditions. Bacteria from the river sample grew on riverine- and marine-based media to 10,000,000 cells/mL. A similar amount was found for the estuary sample under native conditions. However, estuarine bacteria increased in number under marine conditions (100,000,000 cells/mL). Still, marine bacteria were found at lower concentrations (approximately 1,000,000 cells/mL). The growth in plated media did not show evident antimicrobial activity. Bacterial populations seem to survive the effects of changes in salinity along the river system examined. However, the bacterial content may have changed as the fittest strains cope with the environmental stress of salt concentration. Modern molecular techniques based on DNA will be applied to overcome the limitations of traditional cultivation methods to assess microbial diversity. Additionally, isolates will be tested for biotechnological applications.
CONTINUED EMERGENCE OF H5N1: THE URGENCY FOR NOVEL INHIBITORSVelid Seferovic, Emily Rumschlag-Booms, Mairead O’Connor-Maleney.
Northeastern Illinois University, Chicago, IL.
H5N1 influenza, more commonly known as the bird flu, is a reemerging virus that continues to surface in avian and human populations around the world. The reemergence of such highly pathogenic influenza viruses poses a serious threat as their resistance to current therapeutics continues to increase. The purpose of our research is to identify novel H5N1 influenza entry inhibitors. Discovering new viral countermeasures would provide a great deal of potential to create new therapeutics that can be circulated to prevent possible viral outbreaks in the future. In our study, 3,000 small drug-like molecules were screened against an H5N1 pseudovirus carrying a luciferase reporter gene using a high-throughput screening platform. A549 human lung epithelial cells were infected with H5N1 pseudovirus and incubated with each compound. Forty-eight hours post-infection, luciferase levels were assayed as an indirect measure of viral entry. Lead compounds were chosen based on their ability to decrease H5N1 pseudovirus entry, indicated by decreased luciferase levels when compared to the controls. We predict that the lead compounds will prevent entry by inhibiting either the influenza glycoprotein hemagglutinin, which mediates entry, or its receptor sialic acid, which is present on the host cell surface.
EXPRESSION OF THE IMMUNE MEDIATOR GALECTIN-9 IN A MURINE MODEL OF NEUROCYSTICERCOSISLuis Munoz, Pramod Mishra, Judy Teale.
University of Texas at San Antonio, San Antonio, TX.
Neurocysticercosis (NCC) is the most common parasitic disease of the central nervous system (CNS) caused by the metacestode stage of the parasite Taenia solium. NCC is characterized as having an initial prolonged asymptomatic stage followed by a range of symptoms associated with the immune response towards the degenerating parasite.
The metacestode harbors antigenic material rich in glycan moieties recognized by C-type lectin receptors (CLRs).
Galectin-9 (gal9) is a lectin receptor that recognizes β-galactoside residues, carbohydrate moieties that are known to be abundant in helminths. Using a murine model of NCC (infecting with the related parasite Mesocestoides corti), our goal was to determine potential infection-induced changes in the expression of gal9 in the CNS. To this end, we compared brain sections at 3 days (3dpi), 1 week (1wkpi), and 2 weeks post-infection mice (2wkpi) as well as mock-infected mice. We used immunofluorescence staining to determine changes in gal9 expression as well as to characterize cell types expressing gal9. We found that there is an elevated expression of gal9 in the 2wkpi mice compared to earlier time points and mock-infected mice. Double immunofluorescence staining with GFAP, an astrocyte-specific marker, showed co-localization with gal9 indicating that astrocytes express gal9 during NCC. Other cell types appear to express gal9 as well. In the future, we plan to characterize the distribution pattern of gal9 during
IDENTIFICATION OF TRANSCRIPTIONAL REGULATORS OF RBMA IN VIBRIO CHOLERAEDavid Cruz, Fitnat Yildiz, Jiunn Fong.
University of California, Santa Cruz, Santa Cruz, CA.
In Vibrio cholerae, biofilm formation enhances environmental survival and host colonization. Biofilms are a sessile community of organisms enclosed in a three-dimensional matrix. The formation of this matrix directly depends on the production of exopolysaccharides (VPS) and structural extracellular proteins (RbmA, RbmC, and Bap1). We focused on identifying transcriptional regulators of rbmA. RbmA is required for retention of the daughter cell after cellular division; it is necessary for proper stability and architecture of biofilms. Little is known about the transcriptional regulation of rbmA in the formation of biofilms. Our objective was to randomly suppress the expression of genes of V.cholerae A1552 harboring a pBBR rbmAp-lux reporter construct, by disrupting their sequence with a transposition insertion. We evaluated a mariner-based transposon for its ability to generate insertion mutations with increase, decrease, or abolished rbmAp-lux expression. Using the mariner derivative transposon, we screened 25,000 insertion mutants and identified 236 candidates that may represent transcriptional regulators of rbmA. Therefore, this experimental approach can be used to comprehensively characterize the transcriptional regulation of genes encoding exoproteins involved in biofilm formation for the study of Vibrio cholerae pathogenesis.
SULFIDOGENIC COMMUNITY ALONG LIFE ZONE AT EL YUNQUE RAIN FOREST IN PUERTO RICOEdaris Rodriguez Izquierdo, Diana Liz Laureano-Córdova, José R. Pérez-Jiménez.
Universidad del Turabo, Gurabo, PR.
Sulfate-reducing bacteria (SRB), prevailing in anoxic sites, have been detected on terrestrial habitats with limited perspectives on diversity and distribution. Five life zones in El Yunque tropical rain forest (tabonuco, palm, elfin, dry, and colorado forest) are developed in an elevation gradient subjected to natural disturbances and contrasting physicochemical conditions. Diverse SRB must prevail across life zones. Our goal is to describe the structure and distribution of SRB throughout life zones in the rain forest. Soil samples were collected during June and December 2005 from plots at 2 depths (0 - 5 cm and 5 - 10 cm). Total genomic DNA was extracted for amplification of the dissimilatory sulfite reductase gene (dsrAB) and terminal restriction fragment length polymorphisms (TRFLP) analysis.
A diverse sulfidogenic community is comprised by 451 TRF (representing 371 phylotypes) among the set of samples analyzed. SRB were more predominant for June and the deeper layer (5 - 10 cm) than December and top soil (0 - 5 cm). The dsrAB cloned from the elfin forest were phylogenetically diverse, with closest similarity to Desulfobacca acetoxidans and Desulfomonile tiedjei, and minimal similarity to those form mangroves (~2%). Most of the clones comprised novel lineages within the dsr-based phylogeny. Phylogeographic data for SRB is more related to vegetation type than precipitation, pH, and temperature. The sulfidogenic communities across the elevation gradient are rich, with diverse distributional patterns, and seem to thrive within the saturated soils of the forests with a potential role on decomposing complex substances from decaying trees.
IDENTIFICATION OF POTENTIAL LMP1-BINDING PROTEINS FOR SIGNALING OF EPSTEIN-BARR VIRUSASSOCIATED DISEASES USING BIMOLECULAR FLUORESCENCE COMPLEMENTATIONMaria Sanchez, David Everly.
Rosalind Franklin University of Medicine and Science, North Chicago, IL.
The Epstein-Barr virus (EBV) is a herpes virus which has been associated with many different types of diseases. EBV has been connected to Burkitt’s lymphoma, Hodgkin’s lymphoma, gastric carcinoma, and nasopharyngeal carcinoma.
EBV has also been connected to various autoimmune diseases including multiple sclerosis, lupus, rheumatoid arthritis, and Sjögren’s syndrome. Understanding the association of EBV to multiple diseases is of particular interest to health professionals. It has been determined that a vital component of EBV infection and EBV-associated diseases lies within the signaling of the viral oncogene, latent membrane protein 1 (LMP1). In a previous study, several proteins were identified as potential LMP1 binding proteins. The focus of this study is to determine if bimolecular fluorescence complementation (BiFC) between candidate proteins and LMP1 is affected by LMP1-signaling mutants. It is known that tumor necrosis factor receptor associated factors (TRAFs) are required for LMP1 signaling. BiFC between TRAFs
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and LMP1-signaling mutants has been shown to go down. Thus, our hypothesis is that if the identified LMP1 binding proteins are needed for LMP1 signaling, BiFC between LMP1 mutants and the candidate proteins will decrease.