NC ASM 2015
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North Carolina American Society for Microbiology
2015 Meeting
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O c t o b e r 3 , 2 0 1 5
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NC ASM 2015
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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 Darnell
Systematic 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
10:30
Poster session 1 (Even-numbered poster should be attended)
Coffee break Sponsored by Alpha Aesar
Vendors/Sponsors
Session 2 : James Brown, Chair
11:15
Stephanie Mathews
Degradation 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 Cashay
Response 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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NC ASM 2015
Schedule
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?
2:30
Poster 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
3:45
Intermission
Coffee break Vendors/Sponsors
Awards committee meetings
4:00
Briana Burton
ASM Branch Lecture
NVS Influenza Vaccines
Rules of the Road: Motor Proteins that Mediate Chromosome
Segregation
Postscript
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5:00
Wrennie Edwards
Concluding remarks
Awards
5:30
Jim Brown
Business meeting
Officer election
6:00
Reception
8:00
Adjournment
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Abstracts (talks)
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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 production 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-muricholate, and hyodeoxycholate, which can inhibit spore germination and growth at physiological concentrations. 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 colonization 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 cholerae.
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-repressed 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 tuberculosis. M. tuberculosis ∆secA2 mutants are attenuated in macrophages and mice. SecA2 is an ATPase 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 secA2KR 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) suppressor. 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 lacking satS which we are using to study its role in export and its contribution to the virulence of M. tuberculosis.
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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 specialists, 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.
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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 nosocomial infections, we have envisioned employing textile products with antimicrobial properties to prevent 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 oxygen (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 impressive 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 diameter 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 production. The fuels and chemicals produced from lignocellulose are products of polysaccharide degradation; 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 examined 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 fermentation 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-related 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 recombinant 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 microscopy. This research shows HuNoVs bind to gastrointestinal bacteria dependent on: viral strain, bacterial 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 modifications to the TilS gene lead to increased planktonic growth for B. cenocepacia. The TilS gene is highly conserved among bacteria and is responsible for post-transcriptional modification of the wobble position 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 decoding a mRNA AUA (instead of AUG) codon. Loss of the TilS gene can impair the cell’s ability to translate 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 expression 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 cognate 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 increase 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 oligotrophic environments, where carbon and other nutrients are limited. Within these ecosystems, manganese(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 stimulated in situ with organic acids. Additionally, to examine potential long-term effects of anthropogenic nutrients on microbial communities associated with Mn(III/IV) oxide deposits in cave systems, Mn(II)-oxidizing 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 βdiversity 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 suggest 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. ANOVA tests show significant differences (P-value < 0.05) in phyla-level to species-level abundance observed before and after incubations. Taken together, these data demonstrate that exogenous carbon
sources impact manganese cycling and bacterial community assemblages and suggest that anthropogenic 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 EFFUSION 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 proliferation 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 MLN0128, have a reduced volume of effusion and lower levels of IL-6. Our in vitro and in vivo results suggested 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 foodborne 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. Salmonella 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 parameters of the poultry host, such as body temperature, have a role in modulating expression of virulence.
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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 probiotic 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, probiotic cultures in non-pasteurized, refrigerated, acidified cucumber products. Acidified cucumbers
were inoculated to approximately 10^8 CFU/mL with Lactobacillus plantarum or Pediococcus acidilactici 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 preadaptation in cucumber juice alone, with pH adjustments, and/or with the osmoregulator glycine betaine, and sodium benzoate concentration on survival was observed in either 8 oz pickle jars, a cucumber 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 inoculation improved survival, observing cell death of less than 2 log10(cfu/mL) and 3 log10(cfu/mL), respectively. Sodium benzoate, a common pickled cucumber preservative, was equilibrated in the cucumber 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 differences 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, refrigerated 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 citizenry 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, including 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 bioresponse 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
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Heidi Trusheim
Product Steward for Flu Cell Culture
NVS Influenza Vaccines
Currently, the majority of the influenza vaccines distributed globally derive from manufacture processes first developed in the 1940s and 1950s and is based on a technology which relies on replicating the influenza viruses in embryonated hens’ eggs. This production platform depends on a continuous 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 technology 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 propagate 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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ASM Branch Lecture
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Briana Burton
Harvard University
Rules of the Road: Motor Proteins that Mediate Chromosome Segregation
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 membrane barriers. Her group combines state-of-the-art biochemistry and imaging approaches with
classical microbiological techniques to explore the action of membrane-associated DNA transporters involved in DNA uptake and chromosome segregation and in protein secretion.
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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 outstanding 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.
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Poster presentations
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1 Reza Tahergorabi
Antibacterial and Physico-Mechanical Properties of Sweet Potato
Starch Nanocomposite Films Enriched with Thyme Essential Oil
Saeed Hayek
Sweet potatoes as a basic component in developing a medium for
the cultivation of lactobacilli
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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'Campo
Effect 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 Jesus
Investigating 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 Aslett
Development 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
Eleanor Rodriguez20 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
Danielle M.
25 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. Khatibi
Elucidating 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
Ahmed E.
31 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. Sobe
Conserved binding pocket residues in the periplasmic binding
protein, NspS, influence polyamine signaling and biofilm formation
in Vibrio cholerae
Zachary P.
36 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 Dalton
Microbiology 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)
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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 inexpensive 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 vehicles for incorporating a wide variety of additives such as antimicrobial agents. The effect of these additives 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 bacterial growth around the film cuts. Films were effective against E. coli K-12 and Salmonella Typhimurium. The results showed that incorporating MMT into SPS film greatly enhanced (P<0.05) the physicomechanical 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. "
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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 average 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 lactobacilli 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 potato 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 phosphatase, and phytase activity respectively than MRS. The addition of metal ions to SPM caused an additional 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.
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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 respectively, 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 Shahbazi, 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 (bifidobacteria) 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 findings 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 microbiota in human gut.
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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 consumption 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. Because 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 lactase (β-galactosidase) activity of yogurt products available in local stores in Greensboro, NC. We evaluated 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."
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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 somewhat 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. sporogenes).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 directions will be to redesign the primers using known bioinformatics tools that will allow strong hybridization 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 proteins 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, collagen-binding domain, and fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered a prerequisite 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.
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9. Jenessa A. Winston, Allison Bowman, and Casey M. Theriot
Secondary bile acid deoxycholic acid inhibits Clostridium difficile growth and alters the gastrointestinal microbiota
Antibiotics lead to shifts in the gastrointestinal microbiota and metabolome resulting in a loss of colonization resistance against Clostridium difficile. Susceptibility to C. difficile infection (CDI) is associated 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 supplemented 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 compared to no DCA (p=0.0002 and p=0.0044). DCA altered the gut microbial community in a dose dependent manner resulting in an increase in relative abundance of Bacteroidetes, consisting largely of Porphyromonadaceae 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 evaluating other secondary bile acids, ursodeoxycholic acid (UDCA) and lithocholic acid (LCA), that populate 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 including 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 regulated. Observation of spreading colonies on agar plates suggests that gliding may be regulated by nutrient 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 sequences to the F. johnsoniae genome in order to determine the site of transposon insertion. The interrupted genes included: fjoh_1234 that encodes NADH dehydrogenase subunit M, fjoh_1262 that encodes a putative alpha,alpha-trehalase enzyme, and fjoh_1623 that encodes a putative metallophosphoesterase. These results support our hypothesis that metabolism, and perhaps second messengers
such as cyclic AMP, play a role in regulating gliding motility.
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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 human body have been causally associated with a multitude of diseases. However, the intestinal microbiota 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 feasible 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 experiments, 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 experiments 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 characterize interactions between species. Together, our integrated computational and experimental pipeline provides an in vitro parallel for in vivo mouse-ASF studies as well as a well-characterized, controlled 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 luminescens which lives in the nematode gut. Heterorhabditis bacteriophora has a wide range of susceptible 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.
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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 Enterobacteriaceae. It is part of a symbiotic relationship with and resides in the gut of the entomopathogenic nematode, Heterorhabditis bacteriophora. Photorhabdus luminescens produces many virulence factors,
toxin complexes and antimicrobial compounds allowing it to kill insect hosts while simultaneously protecting itself from other bacteria. Due to its virulent properties, the Photorhabdus luminescens-Heterorhabditis 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, nontarget insects, plants, and the environment. The entomopathogenic nematode is exempt from registration regulations in most countries.
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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. luminescens 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 Engineering 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 purpose 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 carbons in broth culture overnight. Growth in the presence of the porous carbons was enumerated by serial 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 appears to be a trend towards an enhancement of microbial growth in the presence of the porous carbons. 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."
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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, National Institutes of Health, Bethesda, Maryland 20892
Investigating the role of endoplasmic reticulum aminopeptidase 1 (ERAP1) in pathogenesis of spondyloarthritis in HLA-B27 transgenic rats
HLA-B27 (B27) confers a strong predisposition to spondyloarthritis (SpA), and accounts for approximately 23% of overall heritability of ankylosing spondylitis (AS). Rats transgenic for B27 and human beta-2microglobulin (B27-TG) develop SpA-like disease, while rats transgenic for HLA-B7 and human beta-2microglobulin (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 individuals, 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-nucleotides. This created a frame-shift resulting in 3 in-frame stop codons in the first exon. ERAP1 protein 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 B27TG 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 manifestations 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 histological 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 formation 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 invadopodia 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 infected 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 invadopodia formation by VSV is separable from its ability to decrease proteolytic degradation of the extracellular matrix. Ongoing studies will further determine the mechanisms by which VSV alters oncogenic invasion.
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18. Denise Aslett, Caroline Smith, Mikyoung Ji, Kai Li, Xuili Lin, Christophe La Hovary, Jyoti Kajla, Jennifer 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 Fixation in the Seed Oil Crop Camelina sativa
With the rising cost and diminishing quantities of fossil fuels, the generation of sustainable, cost-effective, 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 implementation, 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 biofuel from Camelina alone is unlikely to be sufficiently cost effective to compete with current petroleum-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 composed 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 increase 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 transient 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 resistance
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 outer membrane. The genes encoding the proton channels and receptors are often adjacent to one another 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, fjoh_0491-0493 and fjoh_2763-2764. To test this, we deleted the putative ExbB/ExbD proton channels using an allelic exchange system. Primers were designed with engineered restriction sites, and the regions 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 fjoh_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 gliding 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.
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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 transforms 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. However, the T-pilus is too large to penetrate the plant cell wall easily. In order to examine possible mechanisms that would allow the T-pilus to reach the plant cell membrane, we investigated the role of enzymes that might loosen the plant cell wall. The CAZY web site identifies 47 putative glycoside hydrolases 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 digestion 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 βgalactosidase 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 encountered 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 hydrocarbon 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 Chromatography, we show that PotA, PotB, and PotC are all required for uptake of both norspermidine and spermidine. 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 establishes PotABCD1 as the first norspermidine transporter ever reported in any species, but also further
elucidates the role polyamines play in V. cholerae biofilm formation.
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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 transport 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 amplified 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 bacteriophage assays failed to detect phenotypic differences between wild-type and mutant cells. These results 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 motility 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 rhamnosecontaining 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 often 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 acetyltransferases, 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.
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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 downstream 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 suggesting 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 student 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 textures, were sampled on three different occasions throughout the Fall 2013 semester to identify the various 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 evaluated for efficiency on identifying Staphylococcus species, consisting of the 16S-23S rDNA intergenicspacer 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 examined inducible-clindamycin resistance, but only a few isolates revealed it. Of the many Staphylococcus species obtained and identified from the campus training facility, they accumulated on rubber textured 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 bacteria, resistance and infections spread between athletes.
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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 surfaces. 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-consuming 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. Little 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 relatively 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 extinction 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 Rhizomicrobium 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% corresponding 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.
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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 herpesvirus 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 abortion 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 immunity 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 immediate 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 microbial catalyst capable of degrading lignocellulosic feedstocks. Caldicellulosiruptor species are extremely thermophilic bacteria that have the unique ability to degrade unpretreated lignocellulosic substrates 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.
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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 sequences 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 system. 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 independent 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 suggest 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 frequency 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 economic 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 ferrous 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 alpha-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 expression 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 transcriptional 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 homeostasis. Current studies are aimed at better defining the role that MucR plays in the acid-responsive
regulation of the Brucella ftrABCD operon.
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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 bacterium’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 temperatures, 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 significantly 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 transformation of his fatty acid decarboxylase into the alga Dunaliella viridis will successfully allow the conversion 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 problem, students from across the globe are searching their local environments for novel antibiotic-producing 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 Staphylococcus 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 Bordatella 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.
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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 transcription 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 regulatory 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 signaling 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 system 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 investigate 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 either 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.
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36. Zachary P. Johannesson, Ashton L. Honeycutt, Matthew C. Mason, Jaime F. Randise, Ashley E. VanDerlip, and Kevin B. Kiser.
Staphylococcus aureus Carriage in the Nose and Throat of Undergraduate Nursing Students at UNCWilmington
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 determined. 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 carriage 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 occurred because students were well informed about protecting themselves against MRSA or newly admitted 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 experience, 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 meningoencephalitis. This is a signal disease of AIDS patients and can be found less commonly in patients with other afflictions. The most important virulence factors of this organism include melanin production, the presence 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 silenced 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 amplified 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.
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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 numbers. 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. Because 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 alternative 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 Germany (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. larvae 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 persisted 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 major loss in profits for the brewery. Most large breweries have in-house microbiology labs with experienced staff available for yeast propagation and troubleshooting as well as the identification of potential 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 propagation, 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 viability testing during all stages of production has resulted in improved monitoring of fermentation progression and a better understanding of the importance of yeast health. Microbial contamination screening 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 procedure guides and protocols, establishing a schedule for contamination screening, and creating a standard operating procedure for managing contamination events.
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40. Lori Roberts and Melanie Lee-Brown.
Antimicrobial activity of Goldenseal (Hydrastis canadensis) against opportunistic, potentially pathogenic 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 antimicrobials, 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 medicines. 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 limited numbers of Gram-negative organisms and only Staphylococcus species representing the Gram-positive bacteria. The aim of this study is a comprehensive analysis of the antimicrobial effect of Goldenseal on a wide range of culturable, aerobic species that span Proteobacteria, Firmicutes and Actinobacteria (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, Candice 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 Learning (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 develop 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 susceptible 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 experiences.
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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 arthropods, 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 characterizing 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 bacterial species. The isolation of only Gram-Positive bacteria is consistent with published reports documenting 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 geographically and with the age of the nestling.
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Presentation Awards
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 Poston was a longtime employee of Duke University who contributed much
to the NC Branch and she was held in high esteem both by her colleagues 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 University. Prior to this position, Dr. Melton was a member of NC State University's Department of Microbiology and an Associate Dean of the Graduate School. Dr. Melton was very active in research and particularly in
graduate education. In 1999, he received the William A. Hinton Research Training Award from ASM. This award honors an individual who
has made significant contributions toward fostering the research training of underrepresented minorities in microbiology.
The Best Poster award is open to anyone presenting a poster at the NC
ASM meeting.
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The Paul Phibbs Award is awarded for the best presentation by an undergraduate student at NC ASM Branch meetings.
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Meeting Sponsors
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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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Meeting Organization Committee / NC ASM Officers
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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