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Sabbatical Leave Report
Eric T. Gillock, Ph.D.
Professor, Department of Biological Sciences
College of Health and Life Sciences
Fall 2014
Since my initial appointment at FHSU, research in my lab has been largely focused on
isolating and characterizing antibiotic-resistant microbes from a variety of sources. My
students and I have published several peer-reviewed manuscripts on these
microorganisms isolated from such sources as residential soils, feedlots, streams, and
sewage treatment plants. During this time there have been several areas of research I
have wanted to pursue but have not had the time to do so, due to teaching, advising,
committee work, and other typical faculty duties. Part of the reason for wishing to
explore these other lines of research has been to examine the feasibility of taking my
laboratory in a new direction, and the to evaluate the “proof of concept” of several lines
of research and the adaptability of these lines of research for possible undergraduate
and graduate student projects.
My Ph.D. research dealt with animal virology, specifically investigating the capsid
proteins of a cancer-causing virus called murine polyomavirus. My post-doctoral work
was in the field of plant virology, specifically studying the mechanisms by which plant
viruses move from cell to cell in infected plants. When I first came to FHSU, the facilities
were inadequate and the costs were prohibitive for doing detailed virology research. It
was for this reason I began to examine antibiotic resistance in environmental bacteria
as an area of investigation. Bacteria generally are much easier and less expensive to
work with than viruses and are easily adaptable to both undergraduate and graduate
student research. In addition, the facilities in the department at the time were ideal for
working with bacteria.
During my sabbatical leave, I evaluated four lines of research, with special
consideration on the feasibility of adapting these to future undergraduate and graduate
projects. These lines of research were as follows:
1. Bioprospecting for antibiotic-producing microbes
Currently there is a global health crisis due to infections caused by antibiotic-resistant
bacteria. The Centers for Disease Control (CDC) estimates that at least two million such
infections occur per year in the United States, causing around 23,000 deaths annually.
There are many reasons for this increase, such as pharmaceutical companies
suspending antibiotic research and development due to lack of profitability and the
misuse and overuse of antibiotics in the medical, veterinary and livestock production
industries. Most antibiotics used today were initially discovered as products made by
soil microbes. The soil provides a nearly limitless supply of microorganisms for study,
with the vast majority being either overlooked or unculturable. The two main genera of
soil bacteria that produce antibiotics are Streptomyces and Bacillus. Historically, genus
Bacillus has not been as thoroughly investigated for antibiotic production as the
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Streptomyces organisms. For this reason, I chose to examine soil Bacillus organisms
for this phase of my sabbatical research. To do this, I adapted and modified several
existing published protocols for specifically isolating these microbes from the soil. Once
they were isolated, I adapted an existing published method for assaying these
organisms for the ability to produce antibiotics effective against the problematic
bacterium Staphylococcus aureus. I found the methods used to be relatively easy to do
and easy to scale up, allowing many soil samples to be screened for antibioticproducing Bacillus bacteria. Using these methods I was able to isolate several species
of soil bacteria that look initially promising for future antibiotic development.
Conclusion:
Due to the relatively low cost and user-friendly nature of these assays, this line of
research would be well suited for future undergraduate and graduate students. The
protocols could be easily adapted to examining a wide range of soil samples for
antibiotics against a wide range of target microbes.
2. Bacteriophage isolation for viruses infecting Enterobacter cloacae
As mentioned above we are currently facing a global crisis in untreatable antibioticresistant bacterial infections. One possible solution to the problem is to isolate more
antibiotics. Another lesser-known solution is to use viruses that specifically attack and
kill the bacteria, while leaving human cells untouched. Viruses that infect and kill
bacteria preferentially are called bacteriophages. Using these viruses to kill infectious
bacteria is called bacteriophage therapy. Bacteriophage therapy has been used since
the early 1920’s; mostly in former Soviet bloc countries. It was briefly used in the United
States in the 1930’s but was largely abandoned after the discovery and widespread use
of penicillin and other antibiotics. With the worldwide increase in antibiotic-resistant
bacterial infections in the past few years, bacteriophage therapy is being considered
again in western countries as a possible solution to this crisis.
Bacteriophages are the most numerous biological entities on the planet, with
concentrations of up to one billion being present in a milliliter of seawater. They are
widely found in every environment that has been examined to date; it’s thought that up
to 1031 exist on planet Earth. They are relatively easy to isolate and grow in the
laboratory. Even though they are viruses, they rely on bacteria as hosts; therefore
standard techniques for growing bacteria are used for their propagation. As a proof of
concept for this line of my sabbatical research, I chose the bacterium Enterobacter
cloacae as my target. In hospitals, the virulent form of this organism can cause lifethreatening infections. Due to safety considerations I chose a non-virulent strain to work
with. Protocols for working with bacteriophages are well documented and extremely
numerous. I found it relatively easy to isolate several bacteriophages, from Big Creek on
the FHSU campus, with the capability to infect and kill Enterobacter cloacae.
Conclusion:
Due to the relative ease of isolating and working with bacteriophages through welldocumented protocols, and the ease and low cost of culturing bacteria with available
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equipment, these types of projects would be easily adaptable to undergraduate or
graduate research. It would give the student experience working with viruses that are
completely harmless to humans, while being able to contribute valuable knowledge to
the antibiotic-resistance problem.
3. Yeast anti-prion assays
According to the most widely accepted hypothesis, a prion is an infectious agent
composed only of a misfolded version of a normal cellular protein. Prions are thought to
be responsible for causing such diseases as Creutzfeldt-Jakob disease and Kuru in
humans, as well as a number of diseases in other animals such as bovine spongiform
encephalopathy (mad cow disease) in cattle, scrapie in sheep, and chronic wasting
disease in deer and elk. Obviously labs at FHSU are not adequately equipped to deal
with the difficulties and potential risks of working with the purified agents. Fortunately,
another model system exists in which safe, non-pathogenic prions can be found and
studied; baker’s yeast or Saccharomyces cerevisiae. Yeasts are easily grown and
manipulated with equipment available in the Department of Biological Sciences and are
ideal for student projects.
In humans and most animals, the outcomes of prion diseases are nearly universally
fatal. To date no treatment exists for these diseases. Research by the Blondel lab at
Université Paris Descartes, indicates that anti-prion molecules can be screened using a
yeast model, and that these compounds also show activity against prion infections in
cell culture as well as in mouse models. I’ve previously been in contact with Reed
Wickner of the National Institutes of Health, one of the discoverers of yeast prions. He
graciously sent me some of his yeast prion cell clones containing genetic markers that
allow detection of yeast prions. I spent the majority of my sabbatical leave laboratory
time in adapting the Wickner strains to the Blondel assays for yeast prion detection,
then using these modified protocols to test various natural product extracts for anti-prion
activity. The detection of activity with these assays was not ideal and rather difficult to
read, as the positive and negative control reactions are relatively faint. I also
corresponded with Dr. Blondel as well, and was interested to find his group also
experienced similar problems. With further manipulation of growth media and
conditions, the assays I worked on may prove valuable in the future.
Conclusion:
At this time, due to subtleties in reading the positive and negative controls, the assay in
its current form may prove too unwieldy and discouraging for undergraduate research.
Perfecting the assay could potentially be a suitable M.S. thesis project for a dedicated
graduate student. The yeast prion genes themselves are relatively easy to amplify and
clone by standard PCR-based methods, so a project dealing with gene variations in
yeast strains could be an appropriate project for undergraduate or graduate students.
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4. Cloning avian prion protein genes
Prion protein genes have been found in all vertebrate species examined so far. One
group of animals that has not received a large amount of attention regarding prions is
the birds. Previous work has shown that the prion genes are present in several bird
species, with the first being discovered in domestic chickens. In other birds assayed, the
prion protein genes appear to be highly conserved. As a proof of concept, I spent time
adapting the chicken prion gene detection methods to amplifying the prion gene in
turkeys. The PCR product did show some experimental variations which indicated that
the primers and cycling conditions need to be further optimized.
The prion genes of other animal groups, including reptiles and amphibians, have also
not been widely examined. Several published sequences are available in public
databases and could be used to devise consensus primers for amplification and cloning
of these genes.
Conclusion:
Due to the ease and versatility of PCR, combined with the relative lack of information
about avian, reptilian, and amphibian prions, this type of project would likely be
appropriate for an undergraduate or graduate student. Students would gain experience
with hand-on molecular biology laboratory techniques as well bioinformatics based
sequence analysis.
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