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NC ASM 2015 North Carolina American Society for Microbiology 2 0 1 5 M e e t i n g O c t o b e r 3 , 2 0 1 5 Page 1
NC ASM 2015
! !North Carolina American Society for Microbiology
2 0 1 5 M e e t i n g
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O c t o b e r 3 , 2 0 1 5 Page !1
NC ASM 2015
!!!Meeting Sponsors !!
The American Society for Microbiology
North Carolina State University
East Carolina University
VWR International Alpha Aesar Sierra Nevada Brewing Co.
North Carolina Academy of Science
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!Notes !!
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Schedule
Preliminaries
8:00
Registration Poster and talk set-up Breakfast/Coffee break Award Committees meeting/organization Vendors/Sponsors set-up
9:00 Wrennie Edwards Welcome Introductory comments
Session 1 : Wrennie Edwards, Chair
9:15 Casey Theroit Secondary bile acids shape colonization resistance against Clostridium difficile in the large intestine
9:30 Michael Taveirne A new Function for a Classic Regulator: The Zinc and Oxidative Stress Regulator (ZOR) in Vibrio cholerae.
9:45 Brittany Miller A novel component of the specialized SecA2 protein export pathway in Mycobacterium tuberculosis
10:00 Cynthia DarnellSystematic deletion analysis of transcription factors and their responses to environmental stress in the halophilic archaeon Halobacterium salinarum
10:15 Sarah Stanley Preventing Nosocomial Infections: Antimicrobial Photodynamic Textiles
10:30Poster session 1 (Even-numbered poster should be attended) Coffee break Sponsored by Alpha Aesar Vendors/Sponsors
Session 2 : James Brown, Chair
11:15 Stephanie MathewsDegradation of Lignin and Lignin-Related Compounds by Paenibacillus glucanolyticus
11:30 Erin Almand Binding of Human Norovirus to Fecally Isolated Bacteria
11:45 Marc Muraski Cloning and characterization of tRNA(Ile) Lysidine Synthetase from Burkholderia cenocepacia
12:00 Mara CashayResponse of bacterial Mn(II)-oxidizing consortia to exogenous carbon sources in caves in the southern Appalachian Mountains
12:15 Lunch Poster Session 2 (unattended)
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Session 3 : Art Frampton, Chair
1:30 Carolina Caro-Vegas The novel drug MLN0128, a dual mTORC1/2 inhibitor suppresses proliferation of primary effusion lymphoma cell lines
1:45 Bryan Troxell It's Getting Hot in Here: Temperature-dependent Gene Regulation in Salmonella
2:00 Sarah Cauley Survival of Probiotic Lactobacillus plantarum and Pediococcus acidilactici in Refrigerated, Acidified Cucumbers
2:15 Melanie Lee-Brown What do student-generated, authentic research and bioterrorism have in common?
2:30Poster session 3 (Odd-numbered poster should be attended)Coffee break Vendors/Sponsors
Plenary session : Wrennie Edwards and Ece Karatan, Chairs
3:15 Heidi Trusheim NC Invitational Lecture NVS Influenza Vaccines
3:45
Intermission Coffee break Vendors/Sponsors Awards committee meetings
4:00 Briana BurtonASM Branch Lecture
Rules of the Road: Motor Proteins that Mediate Chromosome Segregation
Postscript
5:00 Wrennie Edwards Concluding remarks Awards
5:30 Jim Brown Business meeting Officer election
6:00 Reception
8:00 Adjournment
Schedule
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!Abstracts (talks) !! !1.1) C.M. Theriot, A.A. Bowman, and V.B. Young
Secondary bile acids shape colonization resistance against Clostridium difficile in the large intestine
Members of the gut microbiota are responsible for converting primary bile acids into secondary bile acids, which are known to inhibit C. difficile growth in vitro. Based on this our hypothesis is the pro-duction of secondary bile acids by the gut microbiota contributes to colonization resistance against C. difficile. Using 16S rRNA gene sequencing and a targeted bile acid LC-MS assay, we sought to define the relationship between the gastrointestinal microbiota, bile acids and how this alters susceptibility to C. difficile colonization. Here we show that specific classes of antibiotics significantly alter the structure of the gut microbiota, but more importantly the production of secondary bile acids. Susceptibility to C. difficile was seen in the small intestine before and after antibiotics due to the presence of primary bile acids, which act as germinants of C. difficile spores. Resistance to C. difficile in the large intestine was due to the presence of secondary bile acids: deoxycholate, lithocholate, ursodeoxycholate, ω-muri-cholate, and hyodeoxycholate, which can inhibit spore germination and growth at physiological concen-trations. This is the first study to identify the bacteria in the gut that are driving secondary bile acid production and in turn how physiological concentrations of secondary bile acids are able to suppress C. difficile spore germination and growth. Understanding how the gut microbiota regulates bile acids in both the small and large intestine will be important for designing future therapies to restore coloniza-tion resistance against C. difficile and for other metabolic disorders including obesity and diabetes.
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1.2) Michael E. Taveirne, Drew T. Dunham, and Victor J. DiRita
A new Function for a Classic Regulator: The Zinc and Oxidative Stress Regulator (ZOR) in Vibrio choler-ae.
Metal acquisition is a vital process required for bacterial survival. Transition metals, including zinc, iron, molybdenum and tungsten, are important structural and enzymatic cofactors of many proteins and enzymes. In many bacteria, zinc is transported into the cell via a high-affinity ABC-type transporter encoded by znuACB, with the transcriptional repressor ZUR regulating expression of this locus. Under zinc replete conditions, ZUR binds the znuA promoter repressing transcription, while gene expression is de-repressed under zinc limiting conditions. Vibrio cholerae, an important human pathogen, encodes an uncharacterized zinc transport system (znuACB). We set out to understand the role of zinc transport in bacterial growth and colonization, and to understand how this locus is regulated; given that V. cholerae does not encode an annotated ZUR transcriptional regulator. Utilizing transcriptional reporter strains, we determined that znuA and znuC are repressed under zinc replete conditions and de-re-pressed under zinc limiting conditions by a ZUR-like protein. In addition to regulating zinc transport, we determined that this ZUR-like protein also regulates transcription of the katG catalase, and that overexpression of this gene results in increased sensitivity to hydrogen peroxide. Therefore we propose to rename this protein ZOR, for the Zinc Oxidative stress Regulator. Furthermore, we show that the znuACB transport system is required for growth in zinc limiting growth conditions and is essential for colonization in an infant mouse model. We also show that overexpression of ZOR results in decreased in vivo colonization, demonstrating an important coordinated regulatory mechanism of zinc transport and the oxidative stress response.
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1.3) Brittany K. Miller, Lauren S. Ligon, Nathan W. Rigel, and Miriam Braunstein
A novel component of the specialized SecA2 protein export pathway in Mycobacterium tuberculosis
Bacteria have a variety of general and specialized systems to export proteins from the cytoplasm to extra-cytoplasmic spaces. One specialized pathway, denoted the SecA2-dependent export pathway, exports a subset of proteins important for virulence in several pathogens, including Mycobacterium tu-berculosis. M. tuberculosis ∆secA2 mutants are attenuated in macrophages and mice. SecA2 is an ATP-ase that provides the energy to export a subset of proteins across the cytoplasmic membrane. Our data thus far support a model where SecA2 uses the canonical SecYEG translocase to transport proteins. In the non-pathogenic model Mycobacterium smegmatis, a mutation in the ATP binding region, referred to as SecA2KR, renders SecA2 nonfunctional and dominant negative. We predict that SecA2KR is dominant negative because it is locked in a complex with SecY. Mutations arise spontaneously that suppress se-cA2KR phenotypes. By identifying extragenic suppressor mutations of secA2KR, our goal is to identify proteins that interact with SecA2. Whole-genome sequencing of six extragenic suppressors revealed unique mutations affecting the msmeg1684 gene, which we have renamed satS for secA2 (two) suppres-sor. SatS is a hypothetical protein with no informative homology to known proteins. We constructed a satS mutant and showed that it suppresses the SecA2KR phenotype. Additionally, we showed that we can complement the suppressor phenotype of a satS mutant by expressing either satS(msmeg) or satS(mtb). We hypothesize that SatS works with SecA2 to export substrates, and that in its absence the dominant negative secA2KR phenotypes are suppressed because SecA2KR is no longer recruited to SecY. Like secA2, satS is predicted to be an essential gene for survival in macrophages and mice. Our current work is focused on elucidating the function of SatS. We have constructed mutant M. tuberculosis lack-ing satS which we are using to study its role in export and its contribution to the virulence of M. tuber-culosis.
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1.4) Cynthia L. Darnell, Jordan G. Gulli, and Amy K. Schmid
Systematic deletion analysis of transcription factors and their responses to environmental stress in the halophilic archaeon Halobacterium salinarum
Microorganisms experience stress throughout their lifespans. Extremophiles are stress response special-ists, surviving in environments at the limits of life. The hypersaline-adapted archaea, or halophiles, colonize salt lakes, where salt concentrations can reach saturation, and survive within halite crystals for extended periods of time when the salt lakes evaporate completely. Halophiles also experience fluctuations in temperature and oxygen levels, as well as intense radiation. To deal with this barrage of stresses, halophiles have evolved a complex gene regulatory network (GRN). GRNs are comprised of the cell’s transcription factors (TFs) and their gene targets. This interconnected network has been studied extensively in the halophile Halobacterium salinarum and lead to the generation of a global GRN model [1]. However, it remains unclear how such regulation of gene expression impacts cellular physiology and phenotypes. To determine these roles in H. salinarum, we generated a mutant library of 27 TF deletion strains to test under standard growth and environmentally relevant stresses of heat and ROS (paraquat). We found while few TFs are required for normal growth under standard conditions and heat shock, many TFs were impaired in growth under oxidative stress. This finding validates the predicted GRN and reflects the harsh environments where halophiles thrive.
!1. Bonneau R, Facciotti MT, Reiss DJ, Schmid AK, Pan M, et al. (2007) A predictive model for transcriptional control of physiology in a free living cell. Cell 131: 1354-1365."
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1.5) Sarah L. Stanley
Preventing Nosocomial Infections: Antimicrobial Photodynamic Textiles
According to the CDC, 5-10% of all patients admitted into a hospital will acquire a new infection from exposure to local pathogens, resulting in approximately 1.7 million healthcare-associated infections in the United States each year. Such infections are the sixth leading cause of death in America, as well as a $30-45 billion burden on the healthcare system. In order to reduce the incidence rates of such noso-comial infections, we have envisioned employing textile products with antimicrobial properties to pre-vent pathogens from transmitting to immunocompromised patients in hospitals and in other high-risk environments. As our chosen method of sterilization, we have focused on antimicrobial photodynamic inactivation (aPDI), which employs a non-toxic photosensitizer, visible light, and ambient molecular oxygen to inactivate microbial pathogens. Upon illumination, the photosensitizer generates singlet oxy-gen (1O2), a highly reactive species that has been shown to cause non-specific cell damage, which is thought to prevent the evolution of drug resistance. In addition, singlet oxygen rapidly decays back to its triplet ground state if unreacted, so it is thought to be environmentally benign.
Via electrospinning, we have embedded a tricationic photosensitizer (Por+) into polyacrylonitrile (PAN) to produce PAN-Por+, a photodynamically-active nonwoven textile material. After washing to remove any photosensitizer remaining on the surface, the textile exhibited a broad spectrum of antimicrobial efficacy. Under reasonable illumination conditions (400-700 nm, 30 min), PAN-Por+ demonstrates ~6 log units (99.9999+%) of inactivation with Escherichia coli, Acinetobacter baumannii, vancomycin resistant Enterococcus faecium (VRE) and Staphylococcus aureus , and ~5 log units (99.999+%) of inactivation with Klebsiella pneumoniae. Initial results against vesicular stomatitis virus also demonstrate an im-pressive 7+ log units of viral inactivation. The results of these and other investigations, including our control experiments and efforts to increase the durability of the material by increasing the fiber diam-eter or altering the thickness of the textile, will be discussed.
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2.1) Mathews, Stephanie L., Grunden, Amy M., and Pawlak, Joel
Degradation of Lignin and Lignin-Related Compounds by Paenibacillus glucanolyticus
Lignocellulose is an abundant renewable carbon source that has been used for fuel and chemical pro-duction. The fuels and chemicals produced from lignocellulose are products of polysaccharide degrada-tion; however, these processes do not use the remaining constituent of lignocellulose: lignin. Lignin is a recalcitrant amorphous aromatic compound. Paenibacillus glucanolyticus SLM1, a facultative anaerobe that grows optimally at pH 9, was isolated from pulp mill waste. Initial characterization suggested this bacterium may be able to degrade lignin. The ability of P. glucanolyticus SLM1 and the type strain P. glucanolyticus 5162 to degrade lignocellulose, lignin and, aromatic lignin-related compounds was ex-amined using growth studies, dye degradation assays, GC-MS and GPC. The ability of these strains to grow on a variety of lignin and monomeric lignin compounds, degrade aromatic dyes, produce fermen-tation products as a result of growth on lignin as the sole carbon source, and decrease the dry and molecular weight of lignin show that both strains of P. glucanolyticus can degrade aromatic lignin-re-lated compounds under aerobic and anaerobic conditions as well as degrade polymeric lignin under anaerobic conditions. However, only P. glucanolyticus SLM1 can also degrade polymeric lignin under aerobic conditions. Analysis of the genomic sequencing of P. glucanolyticus SLM1 and P. glucanolyticus 5162 identified candidate enzymes involved in lignin degradation. Future work will focus on the re-combinant expression and characterization of enzymes from P. glucanolyticus strains responsible for lignin degradation.
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2.2) Erin Almand, Matthew Moore, and Lee-Ann Jaykus
Binding of Human Norovirus to Fecally Isolated Bacteria
Human norovirus (HuNoV) is the leading cause of foodborne gastroenteritis worldwide; however, lack of a cultivable strain severely hinders research into this ubiquitous pathogen. Recent studies suggest in vitro cultivation of HuNoV might depend upon bacterial cofactor(s), although their importance remains unknown. This study seeks to characterize the binding affinity of select bacterial isolates (3 ATCC strains; 5 fecal isolates obtained from HuNoV-positive stool samples) to representative HuNoV strains. We exposed anaerobically grown bacteria to GII.4 New Orleans, GII.4 Sydney, GI.6, or the Tulane virus surrogate. After two hours, we determined capture efficiency via RNA extraction followed by RT-qPCR. Turnip crinkle virus, a plant virus with similar size and structure to HuNoV, served as a negative control. Additional studies examined the bacteria for HuNoV receptors: AB, B, LeA, LeB, LeY and H histo-blood group antigens (HBGAs). We then visualized HuNoV binding to bacteria using transmission electron mi-croscopy. This research shows HuNoVs bind to gastrointestinal bacteria dependent on: viral strain, bac-terial strain and bacterial growth conditions. This interaction targets HBGA-like moieties located on the bacterial surface and the pili. These data have relevance in efforts to cultivate HuNoV and for methods to concentrate and purify HuNoV for downstream detection.
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2.3) Marc Muraski, Emil Nilsson and Rebecca Alexander
Cloning and characterization of tRNAIle Lysidine Synthetase from Burkholderia cenocepacia
Burkholderia cenocepacia is an opportunistic pathogen and one of those responsible for infections in the pulmonary system of immunocompromised cystic fibrosis patients. Of particular interest is the gene that encodes tRNAIle lysidine synthetase (TilS); prior work by our collaborator suggests that small modi-fications to the TilS gene lead to increased planktonic growth for B. cenocepacia. The TilS gene is high-ly conserved among bacteria and is responsible for post-transcriptional modification of the wobble po-sition cytosine (C34) in the tRNAIle2 anticodon. This chemical conversion switches the CAU anticodon to LAU where L is lysidine, enabling both aminoacylation with isoleucine (instead of methionine) and de-coding a mRNA AUA (instead of AUG) codon. Loss of the TilS gene can impair the cell’s ability to trans-late the AUA codon efficiently, leading to fitness loss. The purpose of this research was to clone TilS and methionyl tRNA synthetase (MetRS) from B. cenocepacia. The genes were ligated into a pET28a ex-pression vector and transformed into a BL21 E. coli host for overexpression and isolation of the protein via nickel affinity chromatography. Isolation of these two proteins allows for kinetic analysis using B. cenocepacia tRNAMet to identify a baseline for substrate specificity. The MetRS protein was verified through SDS-PAGE and an activity assay in which the MetRS enzyme was able to aminoacylate its cog-nate tRNAMet. Modifying the TilS gene and performing additional kinetic tests will provide insight into the evolutionary advantages of single nucleotide polymorphisms and their correlation to observed in-crease in bacterial fitness.
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2.4) Mara L. Cashay, Sarah K. Carmichael, Bryan T. Zorn, and Suzanna L. Brauer
Response of bacterial Mn(II)-oxidizing consortia to exogenous carbon sources in caves in the southern Appalachian Mountains
Cave and karst ecosystems in the southern Appalachian Mountains are generally considered to be olig-otrophic environments, where carbon and other nutrients are limited. Within these ecosystems, man-ganese(MnII)-oxidizing microbes have been cultured from sites exposed to exogenous nutrient sources including sewage, socks, electrical tape, fireworks and batteries, contributing to the hypothesis that Mn(II)-oxidation can be stimulated by nutrient input. To determine if the microbial communities would respond to exogenous carbon sources in a reproducible manner, bacterial Mn(II) oxidation was stimulat-ed in situ with organic acids. Additionally, to examine potential long-term effects of anthropogenic nu-trients on microbial communities associated with Mn(III/IV) oxide deposits in cave systems, Mn(II)-oxi-dizing bacterial communities from a historically polluted cave, Carter Saltpeter Cave (CSPC), and two ‘pristine’ caves, Daniel Boone Caverns (DBC) and Rye Cove-Cox Ridge (RCCR), were compared.
Preliminary results using Bray-Curtis and Jaccard indices with either NMDS or RDA plots suggest that β-diversity of bacterial and archaeal communities in CSPC and DBC were not significantly different, while communities in CSPC and RCCR were significantly different (P-values < 0.05). β-diversity analyses using PERMANOVA suggests that microbial communities prior to carbon addition were significantly different (P-value < 0.05) when compared to microbial communities after Mn(II) oxidation was stimulated, at least at the Watermark test site in CSPC. α-diversity analyses based on Shannon’s Diversity Index sug-gest that bacterial communities before succinic acid carbon addition were more evenly distributed when compared to the communities after carbon addition at Watermark and Lower Ledge in DBC. ANO-VA tests show significant differences (P-value < 0.05) in phyla-level to species-level abundance ob-served before and after incubations. Taken together, these data demonstrate that exogenous carbon sources impact manganese cycling and bacterial community assemblages and suggest that anthro-pogenic impacts such as sewage/wastewater dumping may alter bacterial microbial ecology within cave and karst ecosystems.
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3.1) Carolina Caro-Vegas, Dirk P. Dittmer
THE NOVEL DRUG MLN0128, A DUAL mTORC1/2 INHIBITOR SUPPRESSES PROLIFERATION OF PRIMARY EF-FUSION LYMPHOMA CELL LINES
MLN0128 (INK128) is a novel ATP-competitive inhibitor of mTOR kinase, which in contrast to rapamycin inhibits both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). MLN0128 has great potential as a chemotherapeutic drug for primary effusion lymphomas (PEL), because this type of cancer heavily relies on mTOR signaling. Currently, there is no established treatment for PEL and its median survival time is shorter than 6 months. In the current study, we tested the efficaciousness of MLN0128 against PEL in vitro and in vitro. Our results demonstrated that MLN0128 has a greater effect on inhibiting pro-liferation and inducing cell death in BC-1 and BC-3 than rapamycin. Further, colony formation assays confirmed MLN0128 superior inhibition of PEL proliferation. Mice injected with PEL luciferase producing cells, prove to be a good model to study this disease overtime. Mice treated with 0.3 mg/Kg of ML-N0128, have a reduced volume of effusion and lower levels of IL-6. Our in vitro and in vivo results sug-gested that MLN0128 might offer a new approach to the treatment of PEL and Kaposi Sarcoma. Further investigation is needed to define the mechanism of MLN0128 in the mTOR pathway.
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3.2) Bryan Troxell, Nicholas Petri, Caitlyn Daron, Rafaela Pereira, Mary Mendoza, Hosni M. Hassan, and Matthew D. Koci
It's Getting Hot in Here: Temperature-dependent Gene Regulation in Salmonella
Salmonella enterica serovars Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) are food-borne pathogens, and outbreaks are often associated with poultry products. Chickens are typically asymptomatic when colonized by these serovars; however, the factors contributing to this observation are uncharacterized. Whereas symptomatic mammals have a body temperature between 37°C and 39°C, chickens have a body temperature of 41°C to 42°C. Here, in vivo experiments using chicks demonstrated that the numbers of viable S. Typhimurium or S. Enteritidis bacteria within the liver and spleen organ sites were ≥ 4 orders of magnitude lower than within the ceca. When similar doses of S. Typhimurium or S. Enteritidis were given to C3H/HeN mice, the ratio of intestinal concentration to the liver/spleen concentration was 1:1. In the avian host, this suggested poor survival within these tissues or a reduced capacity to traverse the host epithelial layer and reach liver/spleen sites or both. Sal-monella Pathogenicity Island 1 (SPI-1) promotes localization to the liver/spleen tissues through invasion of the epithelial cell layer. Following in vitro growth at 42°C, SPI-1 genes sipC, invF, and hilA and the SPI-1 activator rtsA were downregulated compared to expression at 37°C. Overexpression of the hilA activators fur, fliZ, and hilD was capable of inducing hilA-lacZ at 37°C, but not at 42°C despite the presence of similar levels of protein at the two temperatures. In contrast, overexpression of either hilC or rtsA was capable of inducing hilA and sipC at 42°C. These data indicate that physiological pa-rameters of the poultry host, such as body temperature, have a role in modulating expression of viru-lence.
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3.3) Sarah Cauley1, Ilenys M. Pérez-Díaz2, Suzanne Johanningsmeier2
Affiliation: 1Department of Food, Bioprocessing and Nutrition Sciences, NC State University, Raleigh, NC; 2USDA-ARS Food Science Research Unit, Raleigh, NC"
Survival of Probiotic Lactobacillus plantarum and Pediococcus acidilactici in Refrigerated, Acidified Cucumbers
An acidified, refrigerated cucumber product containing high concentrations (10^9 cfu/serving) of pro-biotic cultures offers a non-dairy, low calorie, health-promoting product. This study aims to develop methods to achieve long-term (months), high survival rates of commercially available, lyophilized, pro-biotic cultures in non-pasteurized, refrigerated, acidified cucumber products. Acidified cucumbers were inoculated to approximately 10^8 CFU/mL with Lactobacillus plantarum or Pediococcus acidilac-tici cultures obtained from a variety of sources. Microbial counts of the pickles were determined for the cover brine and cucumber separately to assess survival as a function of time. The influence of pre-adaptation in cucumber juice alone, with pH adjustments, and/or with the osmoregulator glycine be-taine, and sodium benzoate concentration on survival was observed in either 8 oz pickle jars, a cucum-ber juice model system, or brine. Given that the cultures were inoculated immediately after brining the cucumbers, the equilibration and stability of brine and cucumber components was determined by HPLC analysis of organic acids and sugars. Survival of probiotics inoculated as lyophilized culture into acidified cucumbers resulted in cell death of 4 log10(cful/mL) after 1 month (4ºC). Incubation (1.5h) in cucumber juice alone and cucumber juice adjusted to pH 4.0 (HCl) plus glycine betaine prior to inocu-lation improved survival, observing cell death of less than 2 log10(cfu/mL) and 3 log10(cfu/mL), re-spectively. Sodium benzoate, a common pickled cucumber preservative, was equilibrated in the cu-cumber slices after about 2 hours of brining. Sodium benzoate (0-9 mM) had no effect on the survival of the probiotic cultures after a 2.5 h exposure at 4 ºC (p=0.1389). However, the strains and pH differ-ences due to sodium benzoate concentration were significant in an ANOVA modeling survival in log10(cfu/mL) (R2=0.94). Experimentation to date demonstrates that production of acidified, refriger-ated probiotic pickles, by inoculation to a high initial cell concentration, requires pre-adaptation in cucumber juice.
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3.4) Melanie Lee-Brown and Michele K.H. Malotky
What do student-generated, authentic research and bioterrorism have in common?
Teaching the next generation about bioterrorism and biodefense is critical to building an informed citi-zenry and part of the ethos of Guilford College; to develop critical thinkers who are able and willing to tackle real world issues. BIOL 291- Introduction to Scientific Inquiry: Bioterrorism, is an undergraduate ‘science by immersion’ course where students model discipline-specific modes of communication, in-cluding reading, writing and oral presentations. The students learn the value of communication as they work together to overcome the challenges, frustrations and exhilarations inherent to research. This course addresses the difficult concepts of government sovereignty, ethics, the difference between bioreadiness and biodefense, and the fine difference between the mindset of bioterrorists and biore-sponse personnel. As part of their course work, the students work in small groups to develop a plausible dissemination method, vehicle or exploitable weakness using Bacillus thuringienesis as the model for B. anthracis. In this authentic, student-generated research, students are responsible for experimental design, execution, data analysis and reporting. At the end of the semester the research groups present their findings to the Director of the Guilford County Regional Response Team to link their research to the biodefense community.
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NC Invited Lecture !Heidi Trusheim !Product Steward for Flu Cell Culture !NVS Influenza Vaccines !
ASM Branch Lecture !Briana Burton !Harvard University !Rules of the Road: Motor Proteins that Mediate Chromosome Segregation
Dr. Burton’s plenary lecture is supported by the ASM Branch Lectureship Program. The ASMBL program, formerly known as the Waksman Foundation for Microbiology Lectures Program, allows ASM branches to secure outstand-ing lecturers for their scientific meetings. The program has been operating for over 40 years, and lecturers continue to enhance scientific meetings at the local level.
Dr. Briana Burton is Associate Professor in Molecular and Cellular Biology at Harvard University. The research in her laboratory has provided several key advances in understanding biochemical mechanisms underlying the physically challenging problem of macromolecule transport at mem-brane barriers. Her group combines state-of-the-art biochemistry and imaging approaches with classical microbiological techniques to explore the action of membrane-associated DNA trans-porters involved in DNA uptake and chromosome segregation and in protein secretion. !
Currently, the majority of the influenza vaccines distributed globally derive from manufacture pro-cesses first developed in the 1940s and 1950s and is based on a technology which relies on replicat-ing the influenza viruses in embryonated hens’ eggs. This production platform depends on a contin-uous supply of eggs which can become problematic in case of a pandemic outbreak because of a sudden reduction in egg supply. It has also shown that human influenza viruses isolated and grown in eggs can acquire amino acid changes in the hemagglutinin protein, the antigen in the vaccine, which alters the immunogenic property of this important protein. More recently, cell culture based tech-nology became available as a method for the production of influenza vaccines in addition to eggs, which presents a number of benefits and also enables a more rapid response to the increasing worldwide demand of influenza vaccines. In 2012 Novartis licensed Flucelvax™, a trivalent vaccine manufactured on a platform utilizing the canine derived continuous cell line MDCK33016PF to prop-agate the influenza virus, for distribution in the US. The presentation reviews the cell culture based process developed by Novartis for the manufacture of seasonal and pandemic influenza vaccines. The talk also provides an insight into a collaboration with the World Health Organization to support the effort to overcome challenges in the provision of reference viruses used in the initiation of the influenza vaccine production. !
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Poster presentations !1 Reza Tahergorabi Antibacterial and Physico-Mechanical Properties of Sweet Potato
Starch Nanocomposite Films Enriched with Thyme Essential Oil
2 Saeed Hayek Sweet potatoes as a basic component in developing a medium for the cultivation of lactobacilli
3 Rabin Gyawali Enzymatic activity of Lactobacillus reuteri ATCC 55730 in a sweet potato base medium and in the presence of metal ions
4 Amira A. Ayad Use of date palm (Phoenix Dactylifera) to develop a medium for cultivation of lactic acid bacteria
5 Timitayo O. Obanla Exposure to aspirin changes mortality and functionality of human microbiota
6 William R. Rowe Determination of lactase activity of commercial yogurt products
7 Nicole Clark The Investigation of the Transcription Regulator GerE in Clostridium Species
8 Jeffrey Hymes Functional analysis of an S-layer associated fibronectin-binding protein in Lactobacillus acidophilus NCFM
9 Jenessa A. Winston Secondary bile acid deoxycholic acid inhibits Clostridium difficile growth and alters the gastrointestinal microbiota
10 Whitney G Bond Determining Genes Involved in Regulation of Gliding Motility using Mariner Transposon Mutagenesis
11 Biggs MB Systems Biology of a Complete, Defined Gut Microbiome
12 Mena Issa Heterorhabditis bacteriophora: Ecofriendly Biological Control Agent
13 Elizabeth Gerdes Photorhabdus luminescens: Virulent Properties and Agricultural Applications
14 Jesee O'CampoEffect of Environmental Factors on Growth Kinetics of Photorhabdus luminescens Phase-I cells using a 2 L Sartorius Stedim Biostat® A+ Batch Fermentation System.
15 Robin Brice Microporous and mesoporous carbons do not inhibit growth of Escherichia coli
16 Algenis De JesusInvestigating the role of endoplasmic reticulum aminopeptidase 1 (ERAP1) in pathogenesis of spondyloarthritis in HLA-B27 transgenic rats
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17 Dakota Goad The effect of oncolytic vesicular stomatitis virus on cancer metastases
18 Denise AslettDevelopment of a Microbe-Derived, Synthetic Condensed Reverse TCA Cycle to Improve Carbon Fixation in the Seed Oil Crop Camelina sativa
19 Joshua T. Martin Deletion of an ExbB/ExbD proton channel in Flavobacterium johnsoniae confers erythromycin resistance
20 Eleanor Rodriguez-Rassi
Glycoside hydrolases are required for virulence of Agrobacterium tumefaciens
21 Elizabeth A. Villa Characterization of the norspermidine/spermidine ABC-type transporter PotABCD1 in Vibrio cholerae
22 Brian E. Moy Investigation of protein glycosylation on gliding motility in Flavobacterium johnsoniae
23 Jenny Hayden Mycobacterium smegmatis metabolism is regulated by lysine acetylation
24 Justin T. Groody Twin Arginine Translocation is Essential Flavobacterium johnsoniae
25 Danielle M. D'Angelo
Isolation, Characterization and Antibiotic Susceptibility of Staphylococcus Species on Surfaces in a University Weightlifting Facility
26 Salma El-Behaedi Modification of a Suicide Vector for Rapid Screening in an Allelic Exchange System
27 Austin Harbison A Novel Facultatively Anaerobic, Fermentative Bacterium in the Hyphomicrobiaceae
28 Isabel Campos Generation of a DNA Polymerase Knockout in Equine Herpesvirus-1
29 Piyum A. KhatibiElucidating the role of chemotaxis during lignocellulose deconstruction by the extremely thermophilic bacterium Caldicellulosiruptor bescii
30 Emily A. Stout Native Cleavage Escape Mechanisms in a Type II CRISPR-Cas System
31 Ahmed E. Elhassanny
The Brucella MucR has a direct role in regulating the expression of the genes encoding the ferrous iron specific transporter FtrABCD in response to acidic pH
32 Hannah Wapshott Improving algal biofuel processing with the use of a thermostable fatty acid decarboxylase
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33 Lauren Warzecho Antibiotic-producing bacteria finds home in Cape Fear River
34 Callan Corcoran Investigating regulatory binding motifs for archaeal histone protein HpyA
35 Richard C. SobeConserved binding pocket residues in the periplasmic binding protein, NspS, influence polyamine signaling and biofilm formation in Vibrio cholerae
36 Zachary P. Johannesson
Staphylococcus aureus Carriage in the Nose and Throat of Undergraduate Nursing Students at UNC-Wilmington
37 Joshua Boggs Identifying Virulence Factors in Cryptococcus neoformans
38 Adam Groth Isolation of Paenibacillus larvae and Bacteriophages from North Carolina Honey Bees
39 Leslee DaltonMicrobiology for a Microbrewery: Establishing a Working Microbiology Lab for the Management and Propagation of Yeast and the Monitoring of Bacterial Contamination
40 Lori Roberts Antimicrobial activity of Goldenseal (Hydrastis canadensis) against opportunistic, potentially pathogenic bacteria
41 Kaitlyn G. Jackson Experiencing Transformative Education through Applied Learning (ETEAL): Development of a Free STI Test for College Students
42 Layth Awartani A Survey of the Bacterial Microflora Present in the Feces of Loggerhead Shrike (Lanius ludovicianus) Nestlings
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Abstracts (posters) !! !1. Reza Tahergorabi, Aseel Isaa, Salam A. Ibrahim
Antibacterial and Physico-Mechanical Properties of Sweet Potato Starch Nanocomposite Films Enriched with Thyme Essential Oil
Background The main types of plastics that are currently used in all applications are derived from non-renewable petroleum resources, and give non-biodegradable plastic materials. The volume of plastics discarded annually creates a substantial waste disposal issue. Sweet potato (Ipomoea batatas Lam) is an inexpen-sive and readily available vegetable. However, sweet potato does not have the mechanical and barrier properties matching those of plastics. By far the most promising nanoscale fillers for biodegradable packaging are montmorillonite (MMT) nanoclays. Furthermore, biodegradable films are excellent vehi-cles for incorporating a wide variety of additives such as antimicrobial agents. The effect of these addi-tives may result in improvement of food quality and safety.
Method Sweet potato starch (SPS) nanocomposite films containing thyme essential oil (TEO) at 0.5, 1 and 1.5% v/v were prepared to examine their antibacterial, physical and mechanical properties. MMT nanoclay weight percent relative to SPS was constant at 3%.
Results Incorporation of TEO at higher than 0.5% v/v exhibited a clear inhibitory zone by the absence of bacte-rial growth around the film cuts. Films were effective against E. coli K-12 and Salmonella Typhimuri-um. The results showed that incorporating MMT into SPS film greatly enhanced (P<0.05) the physico-mechanical properties and TEO improved tristimulus color values (P<0.05). These improvements could be related to the MMT exfoliation and good interaction between SPS and MMT in the presence of TEO.
Conclusions The results of this study showed that TEO have a good potential for use with SPS and MMT nanoclay to make antimicrobial films and coatings for various food applications. "
!! !2. Saeed Hayek, Rabin Gyawali, Reza Tahergorabi, Valerie L. Giddings, Aboghasem Shahbazi, Salam A. Ibrahim
Sweet potatoes as a basic component in developing a medium for the cultivation of lactobacilli
Lactobacilli are among the most important group of bacteria in applied microbiology. This study aimed to determine the growth of Lactobacillus in a sweet potato base medium. Extract from baked sweet potatoes was used to form a sweet potato medium (SPMs). To prepare SPM, fresh sweet potato was baked (400˚C, 1h), peeled and blended with distilled water at a ratio 2:1. Samples were centrifuged (7800×g for 10 min) and supernatant was collected. Lactobacilli MRS was used as a control medium. Lactobacilli strains were individually inoculated into batches of MRS and SPMs at an average of 2.34±0.29 log CFU/mL. Samples were incubated at 37ºC for 48h and bacterial growth was monitored using turbidity (OD at 610 nm), bacterial population, and pH values. Our results showed similar growth patterns for lactobacilli strains growing in MRS, SPM2, or SPM3. Lactobacilli strains populations reached averages of 10.59±0.27 and 10.72± 0.19 log CFU/mL in SPM2 and SPM3, respectively whereas the aver-age populations in MRS was 10.41±0.35 log CFU/mL. SPM2 and SPM3 maintained higher pH values throughout the incubation period compared to MRS. Sweet potato could partially replace expensive nitrogen sources in lactobacilli media and thus provide a low-cost medium that is suitable for lacto-bacilli growth.
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!3. Rabin Gyawali, Saeed Hayek, Reza Tahergorabi, Valerie L. Giddings, Aboghasem Shahbazi, Salam A. Ibrahim
Enzymatic activity of Lactobacillus reuteri ATCC 55730 in a sweet potato base medium and in the presence of metal ions
Lactobacillus reuteri is probiotic bacterium that inhabits the gut of humans and animals. Sweet potato is rich in many nutrients and North Carolina is the leading producer of sweet potatoes, 47.5% of the total production. The objective of this study was to determine the impact of metal ions in sweet pota-to based media (SPM) on the enzymatic activity of L. reuteri ATCC 55730. To prepare SPM, fresh sweet potato was baked (400˚C, 1h), peeled and blended with distilled water at a ratio 2:1. Samples were centrifuged (7800×g for 10 min) and supernatant was collected. SPM was then supplemented with 4 g/L of each beef extract, yeast extract, and proteose peptone #3 to form the SPM. Enzymatic activity of L. reuteri ATCC 55730 (α–glucosidase, β-glucosidase, acid phosphatase, and phytase) was determined spectrophotometrically using corresponding substrate. Standard MRS was used as control. Our results showed similar growth patterns for L. reuteri ATCC 55730 growing in SPM and MRS. Interestingly, L. reuteri ATCC 55730 grown in SPM showed 103.5, 74.5, and 43.6 % increase in β-glucosidase, acid phos-phatase, and phytase activity respectively than MRS. The addition of metal ions to SPM caused an ad-ditional increase in the tested enzymes. The increase in β-glucosidase due to the addition of Mn2+, Na+, Mg2+ and Ca2+ ranged between 167 and 208 %. Phytase was increased by 69 and 62 % in the presence of Mn2+ and Ca2+ respectively. These findings indicated that sweet potato can improve the enzymatic activity of L. reuteri ATCC 55730 and the addition of selected metal ions may produce an enhanced level of enzymes. Sweet potatoes could be used with probiotic bacteria such as L. reuteri to improve the health conditions of the host.
!!!! !4. Amira A. Ayad, Deiaa Gad El-Rab, Aboghasem Shahbazi, and Salam. A. Ibrahim
Use of date palm (Phoenix Dactylifera) to develop a medium for cultivation of lactic acid bacteria
This study investigated the use of date palm (Phoenix dactylifera) as a basic component to develop a medium for cultivation of lactic acid bacteria (LAB). Date palm was pressed, blended with DDW at ratio 1:2 (w/v), and centrifuged. The collected supernatant was autoclaved at 110°C for 15 min to obtain date palm extract (DPE). The (DPE) was mixed with buffer solution (Tween 80 1mL, L-Cysteine.HCL (1g), Na2HPO4 (2g), NH4C6H5O7 (2g) C₂H₃NaO₂ (5g), CaCl₂ (0.15g), K2HPO4 (2g), MgSO4.7H2O (0.2g) and MnSO4.5H2O (0.05g)) to form a date palm medium (DPM). Lactobacilli MRS was used as a control medium. Three Lactobacillus reuteri (DSM 20016, CF2-7F, and DSM 17938) strains containing an average of 3.34±0.29 log CFU/mL were individually inoculated into batches of MRS, and DPMs. Inoculated media were incubated at 37ºC for 18h. The bacterial growth was monitored using turbidity (OD at 610 nm) and bacterial population (log CFU/mL). Our results showed that bacterial cells continued to grow in both DPM and MRS to reached an average of 0.7- 0.9 (610 nm) within 18h at 37°C. The population of LAB strains (DSM 20016, CF2-7F, and DSM 17938) reached averages of 7.98±0.1, 7.79±0.9, and 8.1±0.6 re-spectively, while the average population in MRS was 9.45±0.1, 8.99±0.9, and 8.85±0.8. These results demonstrated that DPM could be a suitable medium for the growth and high cell mass of LAB and thus should be used as an alternative low cost medium.
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5. Timitayo O. Obanla, Reza Tahergorabi, Desarae N. Johnson, Valerie L. Giddings, Aboghasem Shah-bazi, Salam A. Ibrahim
Exposure to aspirin changes mortality and functionality of human microbiota
The objective of this study was to examine the mortality and functionality of common probiotics (bifi-dobacteria) naturally present in human gut during exposure to aspirin. Batches of 10mL of MRS broth were mixed with one tablet of aspirin. Samples were then inoculated with active cultures of different bifidobacterial ( B. breve, B. longum, B. infantis, B. bifidium and B. adolesenctis) strains to obtain an initial population of approximately 8 Log CFU/ml. Bacterial populations, β-galactosidase activity, and the protein expression were examined after exposure to aspirin. Our results showed a decrease in the population of bifidobacteria by an average of 4.3 ± 0.27 log CFU/ml. Activity of β-galactosidase was totally inhibited in the presence of aspirin. Aspirin also caused changes of protein expression. Our find-ings conclude that intake of aspirin may alter the mortality and functionality of probiotics. Therefore, regular intake of probiotic supplements and functional foods may help balance the beneficial microbio-ta in human gut.
! !6. William R. Rowe, Salam A. Ibrahim
Determination of lactase activity of commercial yogurt products
Nearly 75% of African Americans are affected by the common health issue related to dairy food con-sumption known as lactose intolerance. Lactose intolerance is the inability to break down adequate amounts of lactose commonly found in dairy products making it difficult for the body to digest. Be-cause of this occurrence, many African-Americans typically do not prefer or consume dairy products. Fortunately, good bacteria known as probiotics contain lactase (β-galactosidase), the active enzyme that is responsible for the breakdown of lactose. The objective of this study was to determine the lac-tase (β-galactosidase) activity of yogurt products available in local stores in Greensboro, NC. We evalu-ated the lactase activity using ortho-Nitrophynl-β-galactoside (ONPG) as the substrate for the reaction. Our results showed that some yogurt cultures had lactase activity ranging between 350 and 400 Miller Units. We also investigated the impact of several functional nutrients such as Vitamin C, Manganese, and 3 protein sources (yeast extract, beef extract, and proteose peptone # 3). In the presence of Mn2+ and Vitamin C, lactase activity increased in the range of 600-800 Miller Units. From this study, we can suggest that regularly consuming dairy products, could help alleviate symptoms of lactose intolerance amongst the African American population."
! !7. Nicole Clark and Dinene L. Crater
The Investigation of the Transcription Regulator GerE in Clostridium Species
Organisms of the Firmicutes Phyla including Bacillus and Clostridium are able to undergo a process called sporulation during a time distress. Sporulation in Bacillus cannot occur without the help of the transcription regulator GerE, a DNA-binding protein that controls gene transcription in the late stages of sporulation. Clostridium, however, is ancestral in comparison to Bacillus and sporulation is some-what different. The transcription regulator SpoIIID is required for sporulation, but we believe that there must be more than one transcription regulator in Clostridium. We hypothesize that Clostridium species have a GerE-like transcription regulator. GerE from Bacillus subtilis binds to DNA, so we used that knowledge to identify homologous proteins in Clostridium. We used Bacillus subtilis as a control to study four different Clostridium species (C. acetobutylicum, C.tetani, C. butyricum, and C. sporo-genes).We used a bioinformatics approach to conduct a genomic database search and found similar gene sequences amongst different Clostridium species. We then designed degenerative primers that were compatible to gerE; however, our PCR analysis using those primers was unsuccessful. Future di-rections will be to redesign the primers using known bioinformatics tools that will allow strong hy-bridization to gerE from related organisms.
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!!! !8. Jeffrey Hymes, Brant Johnson, Rodolphe Barrangou, and Todd Klaenhammer
Functional analysis of an S-layer associated fibronectin-binding protein in Lactobacillus acidophilus NCFM
Bacterial surface layers (S-layers) are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Additional pro-teins associated with or embedded within the S-layer have recently been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotics supplements. One putative S-layer associated protein (SLAP), LBA0191, was predicted to mediate fibronectin adhesion based on the in silico detection of a signal peptide cleavage site, colla-gen-binding domain, and fibronectin-binding domain. Fibronectin is a major component of the extracel-lular matrix of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered a prerequi-site trait for probiotic efficacy. To investigate the functional role of LBA0191 in L. acidophilus NCFM, an lba0191-deficient strain was constructed. Phenotypic characterization of the resulting mutant, NCK####, demonstrated that LBA0191, now designated FbpB, contributes to the ability of the cell to adhere to mucin and fibronectin in vitro.
!!!! !9. Jenessa A. Winston, Allison Bowman, and Casey M. Theriot
Secondary bile acid deoxycholic acid inhibits Clostridium difficile growth and alters the gastrointesti-nal microbiota
Antibiotics lead to shifts in the gastrointestinal microbiota and metabolome resulting in a loss of colo-nization resistance against Clostridium difficile. Susceptibility to C. difficile infection (CDI) is associat-ed with a significant decrease in secondary bile acids in the gut. Secondary bile acids inhibit C. difficile growth and can alter the gut microbiota, and the host response. Based on this, our hypothesis is that secondary bile acid deoxycholic acid (DCA) will inhibit C. difficile growth and alter the gut microbiota. Using an ex vivo approach, C. difficile was added to antibiotic treated mouse cecal content supple-mented with varying concentrations of DCA (0-1%) and allowed to incubate anaerobically for 24 hr at 37°C. C. difficile growth and 16S rRNA gene sequencing to evaluate changes in the microbiota were measured. Higher concentrations of DCA (0.1% and 1%) significantly inhibited C. difficile growth com-pared to no DCA (p=0.0002 and p=0.0044). DCA altered the gut microbial community in a dose depen-dent manner resulting in an increase in relative abundance of Bacteroidetes, consisting largely of Por-phyromonadaceae family members (Pearson’s coefficient, r=0.6581, p=0.02). Further in vivo studies to investigate the role of DCA in restoring colonization resistance against CDI are needed, as well as eval-uating other secondary bile acids, ursodeoxycholic acid (UDCA) and lithocholic acid (LCA), that popu-late the colon. Understanding the relationship between the gut microbiota and bile acids may lead to novel therapies able to rescue colonization resistance against C. difficile and other disease states in-cluding IBD, obesity, and type II diabetes.
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!!! !10. Whitney G Bond and Ryan R Rhodes
Determining Genes Involved in Regulation of Gliding Motility using Mariner Transposon Mutagenesis
Flavobacterium johnsoniae cells glide over a variety of solid surfaces including glass and agar. While the mechanism of gliding motility is extensively studied, little is known about how the system is regu-lated. Observation of spreading colonies on agar plates suggests that gliding may be regulated by nutri-ent availability, as cells readily spread on the nutrient deficient medium PY2, but fail to spread on the nutrient rich medium CYE. We hypothesized that interruption of genes important for regulating motility would result in spreading colonies on the nutrient rich medium. To test this, random mutagenesis was conducted using a mariner transposon. The plasmid pHimarEm1 was transferred to F. johnsoniae cells, and three mutants with spreading phenotypes were selected on CYE medium containing erythromycin and 1.5% agar. DNA was extracted from these mutants, digested with the restriction enzyme NsiI and circularized using T4 DNA ligase. The ligation mix was electroporated into E. coli strain EC100D ʎ pir, and the plasmid was extracted and sequenced. The BLAST algorithm was used to align plasmid DNA se-quences to the F. johnsoniae genome in order to determine the site of transposon insertion. The inter-rupted genes included: fjoh_1234 that encodes NADH dehydrogenase subunit M, fjoh_1262 that en-codes a putative alpha,alpha-trehalase enzyme, and fjoh_1623 that encodes a putative metallophos-phoesterase. These results support our hypothesis that metabolism, and perhaps second messengers such as cyclic AMP, play a role in regulating gliding motility.
!!! !11. Biggs MB, Medlock GL, Moutinho T, Swann J, Kolling G, Papin JA.
Systems Biology of a Complete, Defined Gut Microbiome
The composition and abundance of microbial species in various physiological niches in and on the hu-man body have been causally associated with a multitude of diseases. However, the intestinal micro-biota is difficult to study because most gut microbes are unculturable using standard microbiological techniques; furthermore, existing DNA-based approaches such as metagenomic or 16S sequencing are not well-equipped to identify the causal agent in host-microbe interactions. To overcome these issues and study the metabolic interactions between physiologically relevant microbes, we have developed an in vitro pipeline for culturing and studying the Altered Schaedler Flora (ASF). Mice colonized with the ASF develop a relatively normal immune system and as a defined group of 8 bacterial species it is fea-sible to study individual members alongside community function. We present the first characterization of colony and cellular morphology of ASF species and a comparative analysis between the ASF and wild murine microbiomes. We have further characterized the growth of ASF members individually and in coculture on defined media through the use of optical density-based and species-specific qPCR-based methods. To isolate metabolic interactions between species, we performed spent media growth exper-iments, in which a single species is grown in the filter-sterilized media that another species grew in. Metabolomics data has been collected for all media before and after inoculation in spent media exper-iments to determine compounds being consumed and produced, thereby revealing potential symbiosis or competition. We have developed genome-scale metabolic models (GEMs) for all 8 ASF species using a metabolic network reconstruction method that leverages the phylogenetic relationship between species. GEMs are being used alongside metabolomics data from spent media experiments to help char-acterize interactions between species. Together, our integrated computational and experimental pipe-line provides an in vitro parallel for in vivo mouse-ASF studies as well as a well-characterized, con-trolled testing ground for the development of community modeling methods.
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!! !12. Mena Issa, Neveen Issa, Elizabeth Gerdes, Devang Upadhyay and Dr. Len Holmes
Heterorhabditis bacteriophora: Ecofriendly Biological Control Agent
The entomopathogenic nematode Heterorhabditis bacteriophora is an environmentally safe alternative to chemical pesticides. It is half of a symbiotic relationship with the bacteria, Photorhabdus lumi-nescens which lives in the nematode gut. Heterorhabditis bacteriophora has a wide range of suscepti-ble insects, however it is safe to humans, non-target insects, and wildlife. This poster will be review of the current technology and applications of the beneficial nematodes in agriculture.
!!!! !13. Elizabeth Gerdes, Devang Upadhyay and Leonard D. Holmes
Photorhabdus luminescens: Virulent Properties and Agricultural Applications
Photorhabdus luminescens is a gram-negative, bioluminescent bacterium from the family Enterobacte-riaceae. It is part of a symbiotic relationship with and resides in the gut of the entomopathogenic ne-matode, Heterorhabditis bacteriophora. Photorhabdus luminescens produces many virulence factors, toxin complexes and antimicrobial compounds allowing it to kill insect hosts while simultaneously pro-tecting itself from other bacteria. Due to its virulent properties, the Photorhabdus luminescens-Het-erorhabditis bacteriophora relationship is a promising candidate for agricultural use as a biocontrol agent to infect a wide range of insect pests. It has been deemed safe towards humans, animals, non-target insects, plants, and the environment. The entomopathogenic nematode is exempt from registra-tion regulations in most countries.
!!!! !14. Jesee O'Campo, Ke’Anna K. Gulley, Devang Upadhyay and Leonard D. Holmes
Effect of Environmental Factors on Growth Kinetics of Photorhabdus luminescens Phase-I cells using a 2 L Sartorius Stedim Biostat® A+ Batch Fermentation System.
Photorhabdus luminescens, a pigment-producing enteric bacterium and symbiont of Heterorhabditis bacteriophora, was studied in batch cultures to determine the specific growth rates and doubling times. The purpose of this research is to investigate the growth kinetics of Photorhabdus luminescens in a bioreactor containing complex growth media at different environmental conditions. Fermentation parameters were controlled over the course of the bacterial growth. The reactor parameters included pH, temperature, agitation and aeration. The specific growth rates and doubling times of P. lumi-nescens were determined. The study showed that the parameters of 1.0 vvm, pH 7.8, 28°C and 300 RPM had the highest specific growth rate 0.59 h-1 and the shortest doubling time 1.2 hr.
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! !15. Robin Brice1, Dipendu Saha2, Marcus Hunt1, Kristen Delaney Nguyen1
Fayetteville State University Department of Biological Science1, Widener University Chemical Engineer-ing Department2"
Microporous and mesoporous carbons do not inhibit growth of Escherichia coli
Mechanisms for the slow release of drugs are in demand. Recent studies reveal that porous carbons could be used to adsorb drugs for delivery. Previous studies demonstrated that micro and mesoporous carbons are not toxic to human cells and thus may be a vehicle for drug delivery in bacteria. The pur-pose of this study was to evaluate the toxicity of micro and mesoporous carbons in bacteria. Based on the results in human cell lines, we hypothesized that microporous and mesoporous carbons would also be nontoxic to bacteria. To this end, two mesoporous and two microporous carbons were evaluated for toxicity in Escherichia coli. E. coli were exposed to two microporous carbons and two mesoporous car-bons in broth culture overnight. Growth in the presence of the porous carbons was enumerated by ser-ial dilutions and plate counts so that only living bacteria were accounted for. Our results showed no significant reduction in bacterial growth in the presence of micro or mesoporous carbons. There ap-pears to be a trend towards an enhancement of microbial growth in the presence of the porous car-bons. Evaluating the samples by light microscopy showed that bacteria tend to accumulate around carbon particles. In conclusion, micro and mesoporous carbons do not show any evidence of toxicity to E. coli and might actually enhance their growth. Future studies will test the hypothesis that E.coli replicates more rapidly when in contact with the carbon samples.
Acknowledgment: This work was supported by the Fayetteville State University Research Initiative for Scientific Enhancement (FSU-RISE) Program."
! 16. Algenis De Jesus, Tri M. Tran, Sohee Hong, Tejpal Gill, Joshua Bennett, Robert A. Colbert. Pediatric Translational Research Branch, National Institute of Arthritis, Musculoskeletal, and Skin Diseases, Na-tional Institutes of Health, Bethesda, Maryland 20892
Investigating the role of endoplasmic reticulum aminopeptidase 1 (ERAP1) in pathogenesis of spondy-loarthritis in HLA-B27 transgenic rats
HLA-B27 (B27) confers a strong predisposition to spondyloarthritis (SpA), and accounts for approximate-ly 23% of overall heritability of ankylosing spondylitis (AS). Rats transgenic for B27 and human beta-2-microglobulin (B27-TG) develop SpA-like disease, while rats transgenic for HLA-B7 and human beta-2-microglobulin (B7-TG) remain disease free. Although the role of B27 in pathogenesis remains unclear, it may be related to its unusual tendency to misfold. Polymorphisms in ERAP1, which trims peptide cargo for HLA class I molecules in the ER, have been associated with AS in B27 positive and negative individu-als, although the influence on heritability is much smaller than for B27. The objective of this work is to determine how ERAP1 loss-of-function affects the development and severity of SpA-like disease in rats. We hypothesize that ERAP 1 loss of function may protect rats from SpA-like disease. To this end, the first exon of the rat ERAP1 gene was edited using TALEN technology resulting in a deletion of 29-nu-cleotides. This created a frame-shift resulting in 3 in-frame stop codons in the first exon. ERAP1 pro-tein expression was assessed by Western blotting, which revealed complete lack of detectable protein in ERAP1-/-, and about 50% expression in ERAP1+/- rats. ERAP1-/- or ERAP1+/- Sprague-Dawley (SD) rats (disease permissive background) were crossed with B27-TG Lewis rats to generate cohorts of B27-TG with ERAP1+/+, ERAP1+/-, and ERAP1-/- genotypes. B7-TG rats and WT rats with ERAP1+/+, +/- or -/- were used as controls. Rats are being observed for arthritis, fecal scores and other disease manifes-tations for up to 6 months of age. Based on upcoming results, we will elucidate whether loss of ERAP1 is protective or results in more frequent or severe spondyloarthritic disease. Additionally, the effects of loss of ERAP1 on the biology of HLA-B27 and inflammation will be assessed. Future studies will include flow cytommetry to quantify surface expression of folded and free heavy chains, immunoprecipitation and western blotting to analyze folded and free heavy chains within the cell, and preparation of histo-logical slides to view inflammation of rats at 6 months of age.
Acknowledgements: This work was supported by the National Institutes of Health. Recognition must also be given to the NIAMS Intramural Research Program and its chief Dr. Robert Walker."
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!! !17. Dakota Goad, G.T. Tabor, D.F. Seals and M. Ahmed
The effect of oncolytic vesicular stomatitis virus on cancer metastases
Vesicular stomatitis virus (VSV) is currently being investigated as a candidate oncolytic agent due to its capacity to kill cancer cells while exhibiting low virulence in vivo. My project involves investigating the impact of VSV on cancer cell invasion mechanisms. A main trigger for cancer metastasis is the forma-tion of actin-rich structures known as invadopodia which function to degrade the extracellular matrix. Tks-5 is a large scaffolding protein with an amino-terminal PX domain and five SH3 domains which was found to be expressed and localized to invadopodia in invasive human cancer cell lines and in tumor tissue. We hypothesize that treatment of cancerous fibroblasts cell lines with VSV will hinder in-vadopodia formation and decrease proteolytic activity due to the global ability of VSV to inhibit host gene expression in infected cells. Our results show that VSV increased invadopodia formation in infect-ed cancer cell lines at early times post-infection. This enhancement was correlated with an increase in the expression of Tks-5 but not with matrix degradation. These results suggest that the increase in in-vadopodia formation by VSV is separable from its ability to decrease proteolytic degradation of the ex-tracellular matrix. Ongoing studies will further determine the mechanisms by which VSV alters onco-genic invasion.
!!!!! !18. Denise Aslett, Caroline Smith, Mikyoung Ji, Kai Li, Xuili Lin, Christophe La Hovary, Jyoti Kajla, Jen-nifer Swift, Maria Rodriguez-Welsh, Hannah Wapshott, Benjamin Bobay, De-yu Xie, Heike Sederoff, Amy M. Grunden
Development of a Microbe-Derived, Synthetic Condensed Reverse TCA Cycle to Improve Carbon Fixa-tion in the Seed Oil Crop Camelina sativa
With the rising cost and diminishing quantities of fossil fuels, the generation of sustainable, cost-effec-tive, high quality biofuels is vital. While many plant based biofuel methods are capable of producing sustainable, high quality fuels, they are either not cost effective, lack proper infrastructure for imple-mentation, or utilize food crops. Camelina sativa is an ideal candidate for biofuel production as it is not a food crop, can be grown on marginal land, and has high seed oil content. However, producing bio-fuel from Camelina alone is unlikely to be sufficiently cost effective to compete with current pe-troleum-based fuels. One of the issues limiting cost effectiveness is the amount of usable biomass the plant is capable of producing that can be converted into fuel. To overcome this limitation, a synthetic carbon fixation cycle (SynCycle) has been generated for use in Camelina sativa. The SynCycle is com-posed of five microbial enzymes which form the shortest, energetically feasible reverse TCA cycle known. This SynCycle will scavenge carbon dioxide and bicarbonate from the chloroplast and generate glyoxylate. The glyoxylate will feed into an engineered photorespiratory bypass and be incorporated into the Calvin-Benson cycle. By increasing the amount of fixed carbon available to the plant, an in-crease in overall plant biomass and seed oil production will be possible. In vitro SynCycle activity has been demonstrated using LC-MS. In addition, SynCycle expression has been demonstrated through tran-sient tobacco expression and in transformed Camelina sativa lines.
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!!! !19. Joshua T. Martin and Ryan G. Rhodes
Deletion of an ExbB/ExbD proton channel in Flavobacterium johnsoniae confers erythromycin resis-tance
Flavobacterium johnsoniae is a Gram-negative, aerobic bacterium in the Bacteroidetes phylum and is motile via gliding motility; the process by which cells crawl rapidly over surfaces. Gliding motility in F. johnsoniae is mediated by the movement of cell surface filaments around the outside of the cell, a process predicted to be controlled by the Gld motor complex that draws energy from the proton motive force. In other bacteria, ExbB and ExbD form a proton channel that harvests energy from the proton motive force to drive the transport of specific substrates through TonB-dependent receptors in the out-er membrane. The genes encoding the proton channels and receptors are often adjacent to one an-other on the genome; however, recent work in Myxococcus xanthus identified an exbB/exbD pair that was not adjacent to a tonB-dependent receptor and was required for gliding motility in the organism. Inspection of the F. johnsoniae genome revealed three loci predicted to encode ExbB/ExbD proton channels that could potentially harvest the energy required for gliding motility: fjoh_0459-0460, fjo-h_0491-0493 and fjoh_2763-2764. To test this, we deleted the putative ExbB/ExbD proton channels us-ing an allelic exchange system. Primers were designed with engineered restriction sites, and the re-gions flanking each set of genes were amplified by PCR and cloned into the suicide vector pRR51. The deletion constructs were introduced into F. johnsoniae by tri-parental conjugation and deletion of fjo-h_0459-0460, fjoh_2763-2764, and fjoh_0491-0493 was determined by colony PCR. Mutant cells spread similar to wild-type cells on PY2 agar, indicating that these exbB/exbD genes are not required for glid-ing motility. Further phenotypic testing of the mutants revealed that the deletion of the ExbB/ExbD proton channel encoded by fjoh_0491-0493 conferred erythromycin resistant; suggesting this proton channel and the associated TonB may play a role in the uptake of erythromycin.
!! !20. Eleanor Rodriguez-Rassi, Stephanie L. Mathews, Haylea Hannah, Hillary Samagaio, and Ann G. Matthysse
Glycoside hydrolases are required for virulence of Agrobacterium tumefaciens
Agrobacterium tumefaciens is a rhizosphere bacterium which is capable of infecting wound sites on plants. The bacteria transfer a DNA segment, the T-DNA, to the plant cells in the wound using a type IV secretion system. The T-DNA becomes integrated into the host chromosomes and its expression trans-forms the host cell into a tumor cell resulting in the formation of crown tumors. DNA transfer requires the contact of the T-pilus (part of the Type IV secretion system) with the host cell membrane. Howev-er, the T-pilus is too large to penetrate the plant cell wall easily. In order to examine possible mecha-nisms that would allow the T-pilus to reach the plant cell membrane, we investigated the role of en-zymes that might loosen the plant cell wall. The CAZY web site identifies 47 putative glycoside hydro-lases and one polysaccharide lyase in the genome of A. tumefaciens strain C58. Twenty-four of the glycoside hydrolases had either known or highly probable functions unrelated to plant cell wall diges-tion and were not studied. Insertion mutations were made in the other 23 glycoside hydrolases and the lyase. Seven of the resulting mutants were found to be avirulent on Bryophyllum diagremontiana and tomato. One of these seven genes (Atu3104) appears to encode an arabinosylfuranosidase. Wild type A. tumefaciens can grow slowly using agarose as a carbon source. Mutations in either Atu3129 or Atu4560 render the bacteria unable to grow on agarose. Atu3129 appears to encode a protein with β-galactosidase activity. The enzymatic activities of the proteins encoded by Atu4560 and the other four glycoside hydrolases required for virulence are still unidentified. These results suggest that the genome of A. tumefaciens encodes genes for enzymes with the potential to digest the plant cell wall and that seven of these enzymes are involved in virulence.
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!! !21. Elizabeth A. Villa, Black Sanders, Richard Sobe, Alexandria Rutkovsky, and Ece Karatan
Characterization of the norspermidine/spermidine ABC-type transporter PotABCD1 in Vibrio cholerae
Vibrio cholerae, an intestinal pathogen and the causative agent of cholera, is capable of prolonged persistence in aquatic environments through formation of biofilms. In biofilm formation, cells undergo physiological changes in order to aggregate and become encased within a self-produced matrix. Biofilms can provide protection from various environmental stressors and unfavorable conditions, and have been indicated in V. cholerae pathogenesis by providing protection from the acidic conditions en-countered in the stomach. A variety of environmental signals have been shown to play a role in the switch from the planktonic cell state into the nonmotile biofilm. Polyamines, small, cationic hydrocar-bon molecules that are synthesized by virtually all cells, are one such group of signals. Many bacteria, including V. cholerae, are capable of uptake of polyamines from the environment, which subsequently affect biofilm formation. V. cholerae has a putative polyamine ABC-type transporter, PotABCD1. PotA is annotated as the ATPase, PotB and PotC as the transmembrane channel-forming proteins, and PotD1 as the periplasmic substrate-binding protein. Previously, our lab demonstrated a role of PotD1 in the uptake of the polyamines spermidine and norspermidine. In this work, we aim to characterize the role of the remaining components of the transport system in polyamine uptake, and subsequent effects on biofilm formation. Through cellular polyamine extraction and High Performance Liquid Chromatogra-phy, we show that PotA, PotB, and PotC are all required for uptake of both norspermidine and spermi-dine. Furthermore, deletion of each gene results in increased biofilm formation in V. cholerae. Our work indicates that the entire system must function as a whole to facilitate uptake of polyamines, which then mediate the biofilm phenotype through an unidentified mechanism. This work not only es-tablishes PotABCD1 as the first norspermidine transporter ever reported in any species, but also further elucidates the role polyamines play in V. cholerae biofilm formation.
!! !22. Brian E. Moy, Advisor: Ryan G. Rhodes
Investigation of protein glycosylation on gliding motility in Flavobacterium johnsoniae
Flavobacterium johnsoniae serves as a model organism for studying gliding motility in Bacteroidetes. Based on a phylum-wide 3 amino acid motif denoting a glycosylation site, D(S,T)(A, L, V, I, M, T), a number of proteins involved in gliding motility are predicted to be glycosylated. Specifically, a single glycosylation motif has been identified in eight proteins (GldB, GldC, GldI, GldL, SprA, SprT and RemA) and multiple glycosylation motifs have been identified in eight other proteins (GldD, GldG, GldH, GldJ, GldK, GldN, SprB and SprE). As well, many of these predicted glycoproteins have been identified as components of the Type IX secretion system, which has been shown to be essential for gliding motility. Genomic analysis identified a single flippase homologue, wzx (fjoh_2240), which is predicted to trans-port polysaccharides across the cytoplasmic membrane for glycosylation of proteins in the periplasm. In this study, an allelic exchange system was used to delete wzx to determine the role of glycosylation in motility. Primers were designed with engineered restriction sites, and regions flanking wzx were am-plified by PCR. The PCR products were cloned into the suicide vector pRR51 to generate pBEM02. Plasmid pBEM02 was introduced into wild-type F. johnsoniae by tri-parental conjugation to generate the wzx deletion mutant. Motility assays of colonies and individual cells, growth curves, and bacterio-phage assays failed to detect phenotypic differences between wild-type and mutant cells. These re-sults were unexpected as evidence in other bacteroidetes suggests glycosylation is important for growth and colonization. In addition, the presence of the conserved glycosylation motif in many motil-ity proteins suggests glycosylation may be important for gliding. Western-blotting and glycoprotein staining are being conducted to evaluate protein glycosylation in the absence of the wzx flippase. Also, we are deleting fjoh_0331-0333 and fjoh_0354 which are predicted to synthesize the rhamnose-containing core glycan.
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! !23. Jenny Hayden*, Abigail Nails*, Lanisha Brown†, Ellen Perkowski†, and Miriam Braunstein†
*Department of Biology, William Peace University, Raleigh, NC 27604 †Department of Microbiology & Immunology, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599-7290"
Mycobacterium smegmatis metabolism is regulated by lysine acetylation
Tuberculosis disease is caused by Mycobacterium tuberculosis and is a complex global health threat. While many metabolic processes important to M. tuberculosis pathogenesis are understood, regulation of these various processes remains unclear. We have been focusing on lysine acetylation as a possible metabolic regulator. Our studies show that lysine acetylation does impact mycobacterial metabolism, and we identify the mediators of lysine acetylation in Mycobacterium smegmatis, a soil bacterium of-ten used as a model for M. tuberculosis.
Lysine acetylation is a post-translational modification in which an acetyl group is covalently attached to lysine side chains. While not as well-studied as phosphorylation, lysine acetylation is likely to regulate a similarly large and functionally varied number of proteins. Lysine acetylation is mediated by acetyl-transferases, which add acetyl groups to lysine residues, and deacetylases, which remove the acetyl groups. One of the few well-studied acetylated bacterial proteins is acetyl-CoA synthetase (Acs). In model bacterial species, Acs is responsible for converting acetate to acetyl-CoA, and reversible lysine acetylation renders Acs non-functional. We used Acs as a tool to elucidate the mediators of lysine acetylation and deacetylation in mycobacteria. We confirmed that Acs is acetylated in Mycobacterium smegmatis cells and showed that Acs is required for acetate and propionate metabolism. By measuring acetate metabolism in various mutants, we identified that the lysine acetylation/deacetylation system in M. smegmatis cells is comprised of one acetyltransferase, PatA, and one deacetylase, SrtN. Now that we have defined the lysine acetylation regulatory circuit in mycobacterial cells, we can determine what other processes are regulated by lysine acetylation and examine how various cellular signals are integrated into the lysine acetylation pathway.
!! !24. Justin T. Groody, and Ryan G. Rhodes
Twin Arginine Translocation is Essential Flavobacterium johnsoniae.
Flavobacterium johnsoniae cells exhibit rapid gliding motility across certain surfaces. The mechanism for gliding motility in this organism is not fully understood, but protein secretion across the inner and outer membranes via the Sec-dependent and Type IX secretion systems is required. A third secretion system, the Twin-Arginine-Translocation (Tat) system secretes folded proteins across the inner cell membrane, and its role in gliding motility is unknown. This study aimed to investigate the role of Tat secretion in gliding motility by deleting tatC, a gene encoding a structural protein of the system. Primers were designed with engineered restriction sites to amplify the regions upstream and down-stream of tatC by PCR. The regions were cloned into the suicide vector pRR51, and restriction enzyme digest was used to confirm the insertions. The resulting suicide vector, pJG02, was introduced into F. johnsoniae by tri-parental conjugation, but attempts to isolate a tatC mutant were unsuccessful sug-gesting this mutation may be lethal. Experiments are ongoing to confirm that this secretion system is essential in F. johnsoniae.
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! !25. Danielle M. D'Angelo, Camry L. Wagner, Kristen T. Jernigan, and Kevin B. Kiser
Isolation, Characterization and Antibiotic Susceptibility of Staphylococcus Species on Surfaces in a University Weightlifting Facility
Staph infections can be easily spread in public places where people are constantly interacting and there can be extensive skin-to-fomite contact, such as in workout facilities. Benches and weights are potential vehicles for the spread of pathogenic species of Staphylococcus between the hundreds of stu-dent athletes sharing a weightlifting training facility. Normal flora Staphylococci may also be a threat by carrying antibiotic-resistant genes that can be transmitted to pathogenic species. Rubber and metal surfaces in the UNCW Hanover Hall weightlifting facility, further subdivided into smooth and rough tex-tures, were sampled on three different occasions throughout the Fall 2013 semester to identify the var-ious Staphylococcus species present. Salt-enriched cultures were spot-transferred to ChromAgar™ plates to differentiate between S. aureus and other species. Different methods were utilized and eval-uated for efficiency on identifying Staphylococcus species, consisting of the 16S-23S rDNA intergenic-spacer PCR analysis, RapID™ Staph Plus system and genus-specific DNA sequencing of 16S rRNA. Isolates of each Staphylococcus species were tested for antibiotic susceptibility by disk diffusion. Effectiveness of an assortment of antibiotics against the isolates, such as penicillin, tetracycline, and cefoxitin, were tested. Part of the disk diffusion test included the D test using clindamycin and erythromycin that ex-amined inducible-clindamycin resistance, but only a few isolates revealed it. Of the many Staphylococ-cus species obtained and identified from the campus training facility, they accumulated on rubber tex-tured surfaces with the most prominent species consisted of S. saprophyticus, S. cohnii, S. warneri, S. sciuri, and S. haemolyticus. Results of the antibiotic susceptibility testing showed resistance to some antibiotics in a variety of the species. Proper and frequent sanitation of all surfaces throughout the facility and maintained personal hygiene can assist in preventing accumulation of Staphylococcus bac-teria, resistance and infections spread between athletes.
!! !26. Salma El-Behaedi and Ryan G. Rhodes
Modification of a Suicide Vector for Rapid Screening in an Allelic Exchange System
The Gram-negative bacterium Flavobacterium johnsoniae exhibits rapid gliding motility over solid sur-faces. Genetic manipulation, including allelic exchange, is well characterized in this organism and has been important in identifying and characterizing genes involved in motility. While allelic exchange is relatively straightforward, we sought to improve the system through modification of the suicide vector, pRR51. The cloning steps required for allelic exchange include amplifying the regions upstream and downstream of the site of interest and sequentially cloning these regions into pRR51. Following each cloning step a colony PCR must be performed to confirm insertion of the PCR fragment, a time-consum-ing process that would benefit from an alternative approach. With blue-white screening, identifying colonies carrying recombinant plasmids is relatively easy as colonies carrying plasmids with inserts that interrupt the lacZ gene exhibit a white hue, while colonies carrying plasmids without inserts exhibit a blue color. Therefore, a strategy was devised to modify the pRR51 plasmid for blue-white screening. First, the multiple cloning site (MCS) of pRR51 was removed by digesting with BamHI and SphI, blunting the ends and ligating to generate pSEE01. We then PCR amplified the lacZ alpha gene and associated MCS from pUC18 using primers with engineered AflII restriction sites, and cloned the PCR fragment into the AflII site of pSEE01 to generate pSEE03. Colonies carrying pSEE03 were blue when plated on a medium containing ampicillin and X-gal, indicating successful insertion of the pUC18 lacZ alpha gene into the modified pRR51. Additionally, pSEE03 was isolated and confirmed by restriction digest. This modified vector increases the number of restrictions sites in the MCS and reduces the number of colony PCR reactions as colonies containing empty vectors can be visually identified.
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! !27. Austin Harbison and SL Bräuer
A Novel Facultatively Anaerobic, Fermentative Bacterium in the Hyphomicrobiaceae
Peat-forming wetlands are one of the largest natural sources of atmospheric methane and as such are important contributors to global climate change. In these anaerobic environments, plant material is slowly decomposed by bacteria who then release H2, CO2, acetate and other methane precursors. Lit-tle is known about the microbes involved in these processes, and a large proportion of the organisms are novel and uncultured. The aim of this project was to culture and identify anaerobic decomposers in local peat bogs and characterize their physiological properties. Peat samples were taken from a rela-tively acidic (pH 5.0) Sphagnum dominated bog located in Pineola, NC and were anaerobically cultured in an N2 or N2/CO2-rich headspace with glycerol or casamino acids as carbon source. A primary culture of interest, CS4, exhibited marked growth on glycerol with a N2 headspace and was diluted to extinc-tion and streaked for isolation in order to obtain a pure, anexic culture. The CS4 culture was observed microscopically and found to contain small curved rods approximately 0.3 µm by 0.5 to 1 µm occurring in clumps. Phylogenetic analysis revealed low identity (~94%) to cultured strains including Rhizomicro-bium palustre and Rhizomicrobium electricum described in 2010 and 2011, respectively. This organism represents a novel species, and most likely, a novel genus within the Hyphomicrobiaceae family of the Alphaproteobacterial class. Based on this analysis the name Candidatus ‘Microrhizomicrobium pineolum strain CS4’ has been proposed. Analysis of the microbial diversity of the Pineola site was also conducted via TA cloning and Sanger sequencing. A total of 95 sequences (~291 bp) were analyzed with 81.1% cor-responding to the Bacteria domain, 15.8% corresponding to the Archaea domain and 3.2% reported as unclassified. Illumina sequencing has also been completed to capture a greater depth of sequences and compare any differences in relative abundances between Illumina analysis and TA cloning.
!!! !28. Campos, I., and Frampton A.
Generation of a DNA Polymerase Knockout in Equine Herpesvirus-1
Equine herpesvirus type 1 (EHV-1) is a recurring viral infection that causes respiratory distress and can lead to abortion, and neurologic disease. The neurologic manifestation of EHV-1 is termed equine her-pesvirus myeloencephalopathy (EHM). In recent years, there has been an increase in EHM cases in the United States prompting the USDA to label EHM as an emerging disease (1). Currently, EHV-1 vaccines that are commercially available only provide modest protection against respiratory infection and abor-tion but none have been shown to be effective against EHM (2, 3). The goal of this study is to develop a new, modified live vaccine that will stimulate long-term, protective, cell-mediated and humoral immu-nity against EHV-1. To generate this vaccine, the essential viral DNA polymerase gene (DNA Pol) in the highly neurovirulent EHV-1 strain T953 (Ohio 2003) will be rendered non-functional through insertion of a LacZ reporter gene (5). We hypothesize that this vaccine will be able to stimulate both arms of the adaptive immune system due to its ability to initiate infection and express the immunodominat imme-diate early gene. In addition, we propose that this vaccine will be safe due to its inability to replicate viral DNA and produce progeny virions post-vaccination. To date, the DNA Pol knockout plasmid and the DNA Pol complementing cell line have been generated. Work is currently underway to create the DNA Pol knockout virus which will then be tested for its ability to stimulate a protective immune response against EHV-1.
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! !29. Piyum A. Khatibi, Jeffrey V. Zurawski, Jonathan M. Conway, Laura L. Lee, and Robert M. Kelly
Elucidating the role of chemotaxis during lignocellulose deconstruction by the extremely thermophilic bacterium Caldicellulosiruptor bescii.
The primary feedstock for biofuel production in the U.S is currently glucose from starch. However, to become energy independent and to meet future production demands, lignocellulosic substrates will need to be utilized. One of the challenges facing the fuel ethanol industry is the lack of a robust mi-crobial catalyst capable of degrading lignocellulosic feedstocks. Caldicellulosiruptor species are ex-tremely thermophilic bacteria that have the unique ability to degrade unpretreated lignocellulosic sub-strates and ferment both C5 and C6 sugars. This makes Caldicellulosiruptor an excellent candidate for producing second-generation biofuels using a consolidated bioprocessing (CBP) strategy, however, increasing the amount of plant biomass solubilized by Caldicellulosiruptor remains a key challenge. In this study, genome-wide transcriptomic analysis revealed that chemotaxis and cell motility genes were highly expressed when Caldicellulosiruptor bescii was grown on Avicel and switchgrass, suggesting an important role for these microbial behaviors in biomass solubilization. Thus, the specific aim of this work is to determine whether chemotaxis and cell motility are critical to plant biomass degradation and to ultimately improve biomass degradation through genetic engineering. To evaluate the role of chemotaxis and motility in C. bescii, the chemotactic genes CheY (response regulator) and CheB (methylesterase) were chosen for targeted deletion in C. bescii strain JWCB018. While much of the focus on improving degradation of lignocellulosic feedstocks has been on plant-microbe interactions, plant deconstruction enzymes, and altering metabolic pathways, this study takes a unique look at key microbial behaviors that have largely been ignored.
!!! !30. Emily A. Stout and R. Barrangou
Native Cleavage Escape Mechanisms in a Type II CRISPR-Cas System
CRISPR repeat-spacer arrays (RSA) together with the CRISPR-associated proteins (Cas) form adaptive immune systems in bacteria and archaea that vaccinate the cell against mobile genetic elements (MGE). Persevering MGEs often escape cleavage via CRISPR-Cas due to mutations that occur in se-quences essential to the CRISPR interference process. These escape processes were examined using a plasmid interference model in two strains of Lactobacillus gasseri containing a Type II CRISPR-Cas sys-tem. In this study, plasmids containing a protospacer, a validated PAM, and an antibiotic resistance gene were transformed into strains JV-V03 and NCK1342. The protospacer-PAM combination on each plasmid was targeted by the genomically-encoded CRISPR-Cas elements of each strain. If the plasmid underwent CRISPR-Cas mediated double-stranded cleavage, it lost its ability to replicate, causing the organism to lose its antibiotic resistance. Mutants able to escape plasmid cleavage via CRISPR targeting were recovered via selective media. As CRISPR escape is an elusive phenomenon, the mechanism of survival in the escapees was investigated to better understand this biological process. No mutations were observed in the endonuclease regions of Cas9 or in the protospacer-PAM region of the plasmid, but mutations in the RSA were a major pattern of escape in both strains, appearing in every indepen-dent replication. Analysis of sequence mutations revealed polarized excisions from the leader end that always included the targeting spacer. This demonstrates that mutations and deletions can occur throughout the RSA when selective pressure is applied, despite the previously observed evolutionary bias for excisions and mutations to occur at the ancestral end of the RSA. Overall, these findings sug-gest that native escape mechanisms via alterations to the RSA are a key mechanism whereby cells adapt to maintain uptake of beneficial plasmids and that escape mutation patterns may vary in fre-quency and diversity by strain.
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!!! !31. R.M. Roop and Ahmed E. Elhassanny
The Brucella MucR has a direct role in regulating the expression of the genes encoding the ferrous iron specific transporter FtrABCD in response to acidic pH
Brucella strains cause abortion and infertility in their natural animal hosts resulting in extensive eco-nomic losses. Humans can also acquire a chronic, debilitating febrile illness known as brucellosis, as the result of contact with infected animals or their products. Recently, we identified FtrABCD, a fer-rous iron (Fe2+) transporter that is essential for the virulence of Brucella abortus 2308 in mice. The iron response regulator (Irr), the predominant iron-responsive regulator in Brucella and the other al-pha-proteobacteria, activates the expression of the ftrABCD operon in response to iron deprivation. The expression of these genes is also activated by exposure to acidic pH. This acid-responsive expres-sion of the ftr locus is important because it potentially allows the brucellae to fine-tune the expression of their iron acquisition genes to adapt to the acidic environment they encounter in the phagosomal compartment of host macrophages, where Fe2+ is thought to be a biological relevant iron source. The induction of expression of the ftr locus in response to acidic pH is retained in an isogenic irr mutant, indicating that the iron- and pH-responsive expression of these genes are independently regulated. Phenotypic analysis of mutants and electrophoretic mobility shift assays have identified the transcrip-tional regulator MucR as a potential candidate for the acid-responsive regulator of ftrABCD expression in B. abortus 2308. MucR is a zinc finger protein that coordinates the transcription of numerous genes in the α-proteobacteria required for the successful endosymbiotic and pathogenic interactions of these bacteria with their animal and plant hosts. Within Brucella strains specifically, MucR-regulated genes play important roles in maintaining cell envelope integrity, polysaccharide biosynthesis and iron ho-meostasis. Current studies are aimed at better defining the role that MucR plays in the acid-responsive regulation of the Brucella ftrABCD operon.
!! !32. Hannah Wapshott, Vijayakumar Somalinga, Amy Grunden
Improving algal biofuel processing with the use of a thermostable fatty acid decarboxylase
A challenge in the biofuels industry is development of an economically feasible approach for processing alternative fuels, such as algal biofuels, to compete with the cost of industrial scale processing of fossil fuels. Many current approaches focus on identifying algae with desired attributes, modifying the algal strains to enhance fuel molecule production, or otherwise improving fuel processing infrastructure. For this research, a potential fatty acid decarboxylase enzyme from the bacterium Bacillus methanolicus will be purified. This decarboxylase will be evaluated as a catalyst for the reaction that converts fatty acids from algal biomass into long-chain alkanes that are very desirable for fuel production. Bacillus methanolicus is typically grown around 50°C, though it can tolerate temperatures up to 60°C. This bac-terium’s adaption to higher temperatures makes it more likely to have enzymes also active at higher temperatures. Since this enzyme will be expressed in algae, and will likely interfere with algal lipid metabolism, it is ideal the recombinant fatty acid decarboxylase remain inactive at the cultivation temperature of the algae (27°C). Once the algal culture is grown and the cells harvested, a modest temperature increase should allow activation of the enzyme for further fuel processing. The current process of converting algal fatty acids to long chain alkanes involves extreme pressure, high tempera-tures, and an expensive catalyst. By using a recombinant fatty acid decarboxylase enzyme, these high energy costs and steps involving the expensive catalyst can be completely bypassed. This will signifi-cantly improve the economic cost of processing algal biomass for fuel, making way for an infrastructure that will provide more environmental and human-friendly energy. It is hypothesized that the transfor-mation of his fatty acid decarboxylase into the alga Dunaliella viridis will successfully allow the con-version of Dunaliella viridis triacylglycerides into long chain hydrocarbons once algal cultures have been grown and lysed for biomass processing.
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!!! !33. Lauren Warzecho, Kevin Kiser
Antibiotic-producing bacteria finds home in Cape Fear River
Antibiotic resistance is a major clinical obstacle in modern medicine. In attempt to combat this prob-lem, students from across the globe are searching their local environments for novel antibiotic-produc-ing microbes. The Cape Fear River is a unique environment to Wilmington, NC and was used to collect sediment samples for this project. Sediment samples collected at River Road Park along the Cape Fear River were combined with water, serially diluted, and cultured onto Actinomycete Isolation Agar (AIA). After incubation, cultures were overlayed with a Tryptic Soy Broth (TSB) soft agar containing Staphylo-coccus epidermidis. Two colonies measuring zones of inhibition were then isolated onto Tryptic Soy Agar (TSA) and incubated. To confirm the antibiotic activity of the Cape Fear River Sediment (CFRS) isolates, a cross-streak test was performed against the ESKAPE pathogens. Gram staining, BIOLOG, and 16S rRNA sequencing techniques were then used to identify the species of each unknown isolate. The cross-streak test revealed that both isolates showed antibiotic activity against a variety of bacteria. Observations on physical appearance suggested that both CFRS isolates might be the same species. Both CFRS isolates were found to be Gram-negative bacilli through gram staining and identified as Bor-datella trematum with the BIOLOG assay. Sequencing of the 16S rRNA gene revealed the species of each CFRS isolate to be Alcaligenes aquatilis, a species similar to Bortadella trematum. No previous reports suggest that Alcaligenes aquatilis has antibiotic activity, indicating that this may be a novel finding.
!!!! !34. Callan Corcoran, Keely Dulmage, and Amy K. Schmid
Investigating regulatory binding motifs for archaeal histone protein HpyA
Histones package DNA in eukaryotes and regulate gene expression. Archaea encode histone proteins homologous to those of eukaryotes. However, it remains unclear how histones function in archaea. Gene expression data suggest that histones in halophilic archaea may function as transcription factors rather than DNA packaging proteins. In this analysis, we sought to identify a histone DNA regulatory sequence in Halobacterium salinarum. This was done by searching for consensus sequences upstream of those genes differentially regulated by the archaeal histone protein HpyA. The resultant motif was compared to preferred motifs of archaeal histones as well as binding sites of eukaryotic nuclear tran-scription factors in an effort to elucidate the function of HpyA. No specific binding motif was identified using sequence analysis. Experiments are currently underway to directly determine a common regula-tory binding motif for HpyA using chromatin immunoprecipitation.
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!!! !35. Richard C. Sobe and Ece Karatan
Conserved binding pocket residues in the periplasmic binding protein, NspS, influence polyamine sig-naling and biofilm formation in Vibrio cholerae
Vibrio cholerae, a robust biofilm former, inhabits various aquatic ecosystems globally and causes the severe diarrheal disease, cholera. The biofilm lifestyle plays a major role in the V. cholerae infectious cycle by enhancing environmental persistence in interepidemic periods and shielding members of the biofilm from the acidic pH of the stomach during infection. Subsequent passage into the small intestine induces reversion from the biofilm lifestyle to the free-swimming lifestyle necessary for virulence. Two polyamines, norspermidine and spermidine, have opposite effects on V. cholerae biofilm formation. Norspermidine, is self-generated and enhances V. cholerae biofilm formation. Spermidine, is produced by both the members of the gut microbiota and the human host, and diminishes biofilm formation in vitro. These effects are abolished in mutants lacking either component of a two-protein signaling sys-tem consisting of the periplasmic binding protein, NspS, and the transmembrane phosphodiesterase, MbaA. Interestingly, mutation of several NspS binding cleft amino acids predicted to play a role in polyamine binding result in severe defects in biofilm formation. The goal of this project was to investi-gate sensitivity of altered NspS proteins to exogenous polyamines. In media devoid of polyamines, one mutant formed robust biofilms comparable to wild type while two others formed low biofilms. The low biofilm formers did not respond to spermidine but showed biofilm phenotypes comparable to that of wild type at high concentrations of norspermidine suggesting, that these residues are required for spermidine but not norspermidine signaling. In contrast, the high biofilm former did not respond to ei-ther polyamine. The mutations affected transcription of the genes involved in synthesis of the biofilm polysaccharides similarly, corroborating the biofilm data. Our results indicate some amino acids in the ligand binding pocket of NspS might be utilized for sensing both norspermidine and spermidine while others may be responsible for differentiating between these polyamines.
!! !36. Zachary P. Johannesson, Ashton L. Honeycutt, Matthew C. Mason, Jaime F. Randise, Ashley E. Van-Derlip, and Kevin B. Kiser.
Staphylococcus aureus Carriage in the Nose and Throat of Undergraduate Nursing Students at UNC-Wilmington
Methicillin-resistant Staphylococcus aureus (MRSA) carriage in patients is a risk factor for infections in hospitals. Approximately 30% of healthy individuals carry S. aureus in the nose; however, only a small percentage of those isolates are MRSA. Healthcare workers may serve as transient carriers of S. aureus on skin and clothing and may pose a transmission risk to patients. Whether healthcare workers acquire nose and/or throat carriage when repeatedly exposed to MRSA patients has not been clearly deter-mined. To examine the role of clinical exposure in MRSA carriage, UNCW nursing students were tested over the past five semesters for nose and throat carriage. After students filled out consent forms and questionnaires, swab samples from their nose and throat were cultured on CHROMagar Staph aureus plates. Once isolated and identified, each S. aureus isolate was tested for antibiotic sensitivity by disk diffusion. MRSA was characterized by cefoxitin-resistance. Across five semesters, the S. aureus car-riage rates were 52% (36% nose/31% throat), 43% (23% nose/29% throat), 41% (24% nose/31% throat), 62% (46% nose/34% throat), and 50% (22% nose/36% throat). This study highlights the importance of testing the throat, as well as nose, for S. aureus colonization, where nineteen percent of subjects were exclusive throat carriers. Only six students (1%) tested positive for MRSA. This low rate may have oc-curred because students were well informed about protecting themselves against MRSA or newly admit-ted nursing students had not yet been exposed to MRSA. A longitudinal study is currently underway to track students from the semester prior to starting the nursing program, through their clinical experi-ence, until graduation.
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!! !37. Joshua Boggs, Indrani Bose
Identifying Virulence Factors in Cryptococcus neoformans
The basidiomycetous yeast Cryptococcus neoformans is an opportunistic pathogen that is responsible for the most common fungal infection of the central nervous system, cryptococcal meningoencephali-tis. This is a signal disease of AIDS patients and can be found less commonly in patients with other af-flictions. The most important virulence factors of this organism include melanin production, the pres-ence of a polysaccharide capsule, and the ability to grow at high temperatures. The goal of this project was to identify novel genes that might be required for production of melanin in this yeast.
Using an RNAi library that was developed using 2kb fragments of cryptococcal DNA, genes were si-lenced randomly in this fungus and then screened to determine if they affect melanin production. Transformants that showed altered production of melanin when compared to the wild type strain on niger seed agar media were analyzed further. DNA fragments responsible for the silencing were ampli-fied by PCR, and sequenced to determine the identity of the candidate gene. Identification of unknown genes that affect this phenotype can potentially provide additional information about the pathways that lead to the pathogenicity of C. neoformans.
To date a total of 30 gene fragments have been sequenced and analyzed. One of the genes that has been identified multiple times is CnLAC1. This gene codes for the enzyme laccase and is known to be crucial to the production of melanin in C. neoformans indicating that the RNAi screen for altered melanization is identifying genes in the pathway. Many other novel genes have also been identified in this screen and we are currently in the process of confirming the results.
!!! !38. Adam Groth and Eric S. Miller
Isolation of Paenibacillus larvae and Bacteriophages from North Carolina Honey Bees
Paenibacillus larvae causes American Foulbrood (AFB) disease in honey bees. AFB, which affects the honey bee larval stage, may be a factor influencing the recent decline in honey bee health and num-bers. The symptoms of the disease are the death of the bee larvae, resulting in a gluey mass filled with P. larvae spores. These spores can then be spread throughout the hive and to neighboring hives. Be-cause P. larvae forms spores the most common way to completely eliminate a seriously infected hive, is to seal the bees inside and burn the entire hive. Antibiotics have been used historically to slow the spread of P. larvae but concerns about their presence in consumer honey has led to research into alter-native treatments, bacteriophage therapy being one. Despite the destructive nature of the disease, relatively little is known about Paenibacillus larvae and its associated phages. Current ATCC lab strains were isolated 20 years ago, and have not been tested for continued infectivity. A new isolate in Ger-many (strain DSM 25430) was recently described and its fully sequenced genome deposited in GenBank. However, the strain is not available for distribution outside of the country. We are interested in the occurrence, distribution and properties of P. larvae and P. larvae phages among honey bee hives in North Carolina. Frames from AFB diseased hives in North Carolina were used as starting material for enrichment and isolation of new P. larvae strains. Using an approach to be presented, four new P. lar-vae isolates have been obtained from two separate locations in NC. These strains were then used to isolate new plaque-forming bacteriophages. Phages have been isolated from three different locations on two different hosts of Paenibacillus larvae.
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!! !39. Leslee Dalton, Alice Lee
Microbiology for a Microbrewery: Establishing a Working Microbiology Lab for the Management and Propagation of Yeast and the Monitoring of Bacterial Contamination
Several ingredients are required to produce a good beer, yet no single ingredient is as crucial to the brewing process as healthy yeast. Without yeast there is no fermentation and without fermentation there is no transformation from a sweet, malty wort to the complex, intoxicating libation that is beer. The brewing of beer dates back thousands of years with the oldest known barley based beer dating back to 3400 B.C. It was long postulated that fermentation was simply a spontaneous, non-biological reaction that occurred after assembling all the ingredients and then allowing them to sit. This idea per-sisted until scientists like Louis Pasteur applied quantitative methods for determining that yeast cells were the force behind fermentation. It is important to note that yeast contribute more than just ethanol during fermentation. Yeast produce many other metabolites, such as esters and diacetyl, that contribute heavily to the overall flavor profile. With an ever present consumer demand for consistent tasting beer the health of yeast is of utmost importance to breweries. Unfortunately for brewers, brewing yeasts are not the only microbes that impact the flavor and quality of beer. Beer is naturally an environment that would kill most bacteria and "wild" yeast species, yet some not only survive but thrive. Some bacteria such as Lactobacilli spp. and wild yeasts such as Brettanomyces spp. are actually necessary for certain beer styles but most times these microbes are considered unwelcome guests. A microbial contaminant will create off flavors and ruin the best of recipes which usually leads to a ma-jor loss in profits for the brewery. Most large breweries have in-house microbiology labs with experi-enced staff available for yeast propagation and troubleshooting as well as the identification of poten-tial bacterial contaminants. Few small-scale breweries can afford such a luxury, yet Bombshell Beer Company of Holly Springs recognized the importance of healthy yeast to their rapidly expanding small business. The goal of this project is to establish a working microbiology lab for the purpose of yeast propagation and monitoring as well as screening for bacterial contamination at a variety of production stages. Diligence with aseptic technique and attention to possible sources of contamination have shown to be the most important aspect of establishing a work flow in a brand new lab. In regards to propaga-tion, media formulation and proper growth conditions have proved to be a critical step in ensuring a cell count target is reached without impacting overall quality. The introduction of cell counts and via-bility testing during all stages of production has resulted in improved monitoring of fermentation pro-gression and a better understanding of the importance of yeast health. Microbial contamination screen-ing has consisted of testing lab propagated yeast slurries, yeast slurries re-pitched from completed batches to fresh batches, and final product samples. Future project goals include establishing lab pro-cedure guides and protocols, establishing a schedule for contamination screening, and creating a stan-dard operating procedure for managing contamination events.
! !40. Lori Roberts and Melanie Lee-Brown.
Antimicrobial activity of Goldenseal (Hydrastis canadensis) against opportunistic, potentially path-ogenic bacteria
The rise of antibiotic resistance and multi-drug resistant (MDR) bacteria is a major health concern worldwide. Most large pharmaceutical companies no longer prioritize the discovery of new antimicro-bials, and so the exploration for new targets or compounds has fallen primarily to small labs and biotech start-ups. Of interest to many labs are plant-derived compounds based on alternative medi-cines. In this study Goldenseal (Hydrastis caradensis) crude extract was tested because of its known antimicrobial properties. Previous studies reported that Gram-positive organisms are more sensitive to the antimicrobial effects of Goldenseal than Gram-negative bacteria, however these studies used lim-ited numbers of Gram-negative organisms and only Staphylococcus species representing the Gram-posi-tive bacteria. The aim of this study is a comprehensive analysis of the antimicrobial effect of Gold-enseal on a wide range of culturable, aerobic species that span Proteobacteria, Firmicutes and Acti-nobacteria (which include the preponderance of important human pathogens).
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!! !41. Kaitlyn G. Jackson, Justine M. Celli, Jessica L. Cooper, Elizabeth A. Brady, Keri A. McKenna, Can-dice Passerella, and Kevin B. Kiser
Experiencing Transformative Education through Applied Learning (ETEAL): Development of a Free STI Test for College Students
UNCW Biology majors are required to participate in applied learning experiences, including mentored research projects and service learning. Experiencing Transformative Education through Applied Learn-ing (ETEAL) initiative funded an innovative proposal with three complementary objectives: 1) Research – develop an inexpensive test for STIs and assess infection rates on campus; 2) Service – raise campus awareness of STIs and the importance of testing; and 3) Student Learning – use applied learning to de-velop critical thinking, communication, organizational, and teamwork skills. In 2011, 1.4 million chlamydia infections and 300,000 gonorrhea infections were reported in the U.S. These infections are caused by bacteria Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), respectively. Young women (ages 15-24) are the most susceptible to these infections which can lead to pelvic inflammatory disease (PID) and infertility. In addition, individuals who are infected are two to five times more sus-ceptible to acquiring HIV if they are exposed to the virus. Regular testing is imperative due to the high rate of asymptomatic cases of these infections. Studies have shown that only about 50% of men and 25% of women develop symptoms from chlamydia and 90% of men and 20% of women develop symptoms from gonorrhea.Because the majority of UNCW students are young and sexually active, this research was critical to understanding the dangers associated with contracting chlamydia and gonorrhea and how to minimize the risk of transmission. This study also aimed to increase awareness of chlamydia and gonorrhea and to create a free testing service that students could utilize. To reinforce the impact of combining microbiological research and service learning on their educational development and future careers in science and/or medicine, ETEAL-supported students reflected on each semester’s experi-ences.
!! !42. Layth Awartani, Dr. Christine Stracey, and Dr. Melanie Lee-Brown
A Survey of the Bacterial Microflora Present in the Feces of Loggerhead Shrike (Lanius ludovicianus) Nestlings
There are few studies on non-domesticated avian gut flora and even less research on the microbial populations of nestling fecal sacs. Of the few studies, most focus on zoonotic bacteria or the health of agriculturally important domestic avian species. This study examined the culturable gut microbes from the fecal sacs of 9 and 11 day-old Loggerhead Shrike (Lanius ludovicianus) nestlings. Loggerhead Shrikes are migratory passerines (perching birds) and top-level predators. Their diet consists of arthro-pods, amphibians, reptiles, small mammals, and other birds. Their populations have fallen sharply over the past half-century and they are now classified as a Tier I at-risk species in the U.S. and critically endangered in Canada. Fecal sacs were collected from four different nests over a four-month period in Antelope Island State Park Salt Lake, Utah summer 2014. Collectively, we hypothesized that fecal sac microflora flora composition, species richness, and overall abundance should increase with nestling age. In addition, we investigated whether geographic and temporal differences on the island govern any variation in the culturable gut microbes. We tested our predictions by means of culturing and char-acterizing bacteria using molecular and biochemical approaches. After screening 98 isolates from 23 fecal sac samples, our methods allowed us to identify the presence of 13 culturable Gram-Positive bac-terial species. The isolation of only Gram-Positive bacteria is consistent with published reports docu-menting the normal gut flora composition of omnivorous passerine species. Variation and abundance of culturable gut microbes increased with nestling age. Cultured fecal sac microbes varied both geograph-ically and with the age of the nestling.
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Presentation Awards
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The Mary Poston Award was established to recognize the best paper given by a student at meetings of the NC Branch of the ASM. Mary Pos-ton was a longtime employee of Duke University who contributed much to the NC Branch and she was held in high esteem both by her col-leagues and by medical students. She contributed much to the NC Branch, including service as Branch Secretary-Treasurer from 1950 until her death in 1961. Many letters of appreciation have been written over the years by student recipients of the Mary Poston Award, commenting on the confidence the award gave them and on the importance of the competition for the award as part of their graduate training.
The Thoyd Melton Award was established to recognize an outstanding oral presentation by a graduate student. At the time of his premature death on Nov. 22, 2000, Thoyd Melton was Associate Vice Chancellor for Academic Affairs and Dean of graduate studies at N.C. A&T State Uni-versity. Prior to this position, Dr. Melton was a member of NC State Uni-versity's Department of Microbiology and an Associate Dean of the Grad-uate School. Dr. Melton was very active in research and particularly in graduate education. In 1999, he received the William A. Hinton Re-search Training Award from ASM. This award honors an individual who has made significant contributions toward fostering the research train-ing of underrepresented minorities in microbiology.
The Best Poster award is open to anyone presenting a poster at the NC ASM meeting. .
The Paul Phibbs Award is awarded for the best presentation by an un-dergraduate student at NC ASM Branch meetings.
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!Meeting Sponsors
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!!!Meeting Organization Committee / NC ASM Officers
The American Society for Microbiology
North Carolina State University
East Carolina University
VWR International Alpha Aesar Sierra Nevada Brewing Co.
North Carolina Academy of Science
Wrennie Edwards President-elect NVS Influenza Vaccines
Ece Karatan President Appalachian State University
Eric Anderson Past-president East Carolina University
Jim Brown Secretary NC State University
Wrennie Edwards Treasurer NVS Influenza Vaccines
Ece Karatan Councilor Appalachian State University
Ed Swords Alternate Councilor Wake Forest University
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