1. EHSC 3910 Report
Internship in EHSC with Dr. Lipp
Thisreportoutlinesthe course EHSC3910 underDr. Erin Lipp
and ledbyKeri Lydonin the analysisof Vibriovulnificus
resistance totriclosan.Thisreportincludesanoverview of
the course as well asbackgroundon the bacteriumof focusin
reference topotentialpublichealthrisksassociatedwith
climate change.
AJ Shockley
5/1/2015

2. Spring semester 2015, I participated in an internship with UGA EHS under Dr. Lipp. Keri Lydon
was my instructor and I worked in Dr. Lipp’s lab under a grant provided to EHS. I received 3 credit hours
for this internship and worked in the lab an average of 10 hours a week. Under the instruction of doctoral
student Keri Lydon I was trained to perform a series of lab techniques from making media and lab upkeep
to polymerase chain reaction (PCR). I assisted Lydon in her dissertation research,the predominate focus
of which is vibrio vulnificus.
V. vulnificus is a bacterium belonging to the same genus as cholera and V. parahaemolyticus.This
bacterium is a halophile and is found most commonly in warm salty water,which means their highest
concentrations coincide with summer months. V. vulnificus is a gram negative motile curved rod.1
Like
other gram negative bacteria V. vulnificus is surrounded by a thin layer of Type IV pilli containing
petidoglycan and an inner cell wall. Both the
pilli and capsule are necessary for virulence
because the bacterium utilizes these to adhere
to epithelial cells in the human body and
produce biofilms. Though more rare,gram
negative bacteria are more difficult to treat
because the outer membrane contains
compounds that serve to shield the bacterium to damage sustained from antibiotics.1
There are three
biotypes of V. vulnificus two of which are associated with disease in humans.1
Type I and III cause
gastrointestinal infection and wound infection respectively. Type II is associated with illness and death in
eels.1
V. vulnificus can enter the body via ingestion of contaminated seafood such as salmon or oysters
(Type I).2
Infection of the gastrointestinal tract results in abdominal pain, diarrhea and vomiting. This
1 http://textbookofbacteriology.net/V.vulnificus_2.html
2 http://www.cdc.gov/vibrio/vibriov.html

3. bacterium can also enter the body through open wounds and infect the bloodstream resulting in the
development of necrotizing fasciitis (Type III). Necrotizing fasciitis or flesh eating bacteria infection
destroys flat layers of connective tissues known as fascia when the bacteria enter the bloodstream.3
This
infection is caused by multiple bacteria including Streptococcus, E. coli and Clostridium and often
requires surgery in conjunction with intravenous administration of antibiotics.3
Some infections like
Group A Strep are less severe than a V. vulnificus infection, which results in rapid necrosis and septicemia
requiring immediate medical attention.3
Systemic infections from V. vulnificus are highly dangerous and
50% fatalto its sufferers. The immunocompromised such as children, the elderly, and people with
existing disease especially of the liver and kidneys are more susceptible to fatal contraction of the disease.
Though contraction of V. vulnificus wound infections are rare a rise in the bacterium and
subsequent infections have been reported over the past decade. The CDC reports about 400 vibrio
infections a year 90 of which are V. vulnificus.4
Also because of their shared symptomatology with
other infections vibrio infections often go
unreported so an absolute number of cases is
difficult to pin down.
Vibrio bacteria have become a growing
concern in connection to climate change because
of their mesophilic nature. These bacteria not only
thrive during summer months but exponential
growth of the vibrio genus has also been observed
over the past decade due to the warming of surface waters. Since the highest growth rates of vibrio
coincide with months where tourism to coastal regions of the United States reaches its peak there are
3 http://www.cdc.gov/features/necrotizingfasciitis/
4 http://emergency.cdc.gov/disasters/vibriovulnificus.asp

4. major public health concerns with the growth of vibrio concentrations especially in coastal sub-tidal
regions of the country.
Global warming is unequivocal and caused by anthropogenic CO2 emissions. Green house gases
like CO2,CH4 and water vapor trap infrared radiation within the Earth’s atmosphere causing a reduction
in heat loss from the mixing layer of the ocean resulting in overall heating of the ocean.5
90% of the
Earth’s excess heat is stored in the ocean which contributes to the growth of mesophilc bacteria like
vibrio in coastal, riverine and estaurine areas.6
Though the bacterium’s response to warmer water
temperatures between 25-40℃ is understood responses to pH changes require more in depth research
since ocean acidification is also a growing issue.6
Keri Lydon’s work focused on analyzing triclosan resistance as a potential health risk in the
promotion of vibrio growth. Triclosan is an antimicrobial compound found in many soaps, detergents,
cleaners,and deodorants. 7
These products are washed into local sewage systems which are deposited into
surface waters in coastaland riverine systems. Tricolsan is toxic to bacteria like vibrio and inhibits the
enzyme ACP reductase which is essential to fatty acid biosynthetic pathways used to build cell walls.7
Increased urbanization poses a major risk to public health by raising vibrio exposure to triclosan. It has
already been observed that tricolsan acts a selective pressure factor in causing bacterial resistance.
This research took place in a series of steps. The first month of the internship was dedicated to
instructing me on proper lab techniques and making media for Keri’s research as wellas her classes and
other doctoral students. I learned how to make a variety of media both agar and broth and how to work
the autoclave.
5 http://judithcurry.com/2014/05/21/mechanisms-for-warming-of-the-oceans/
6 http://link.springer.com/article/10.1007%2Fs00248-012-0163-2
7 http://pubs.acs.org/doi/ipdf/10.1021/es401919k

5. Using the marine broth I made 15mL plates in order to isolate vibrio samples Keri collected at the
Keys the summer before. The samples were gathered in .5mL tubes numbered 1-416 in matte board
boxes. A previous intern had already begun separating the isolates so I started at tube #170 working my
way up to #416. After making the plates I labeled and streaked each plate with its corresponding .5mL
tube. After incubating the sample over a 24 hour period at 30℃ I took the plates and made stabs of each
that needed to be re-isolated (some had been done by the previous intern). After making stabs I took a
small amount of agar in each stab to make an overnight culture in 4mL of Marine Broth (24 hours at
30℃). This process took a couple of months to process about 250 tubes. After the overnight cultures
reached log phase,a period marked by cell doubling, I aliqoted 100𝜇𝐿 of the isolates into a strip tubes (8
Marine broth and agar were the predominate
media used in my research under Keri. I spent
the first month of this internship making agar
for plateincubation and stabs and broth for
overnight cultures. Vibrio grows very well in
this media at 30-35⁰C.
Meuller Hinton (MH) broth was theother
media used in this research. MH is a
microbiological growth media used to analyze
antibiotic susceptibility to tricolsan by finding
the minimum inhibitory concentration.
One of the most used media was mTec used to
make small 5 mL plates. This media is most
commonly used for detecting E. coli levels and
proved useful to our research at Lake Herrick
and Parvo Pond in analyzing recreational risk.
This semester the agar was used for Keri’s
Environmental Microbiology lab.
Thiosulfate-citrate-bile salts-sucrose(TCBS)
agar was an agar I made in high volume near
the end of the semester. TCBS is used to
isolate vibrio in salt water and inhibits gram
positivebacterial growth. I made this media for
Jason Westrich who took a trip to the Keys to
perform field work with Dr. Lipp.

6. isolate tubes per strip). After thermocycling the tubes for 10 minutes on BOIL the isolates were then
placed in the refrigerator in the teaching lab.
The stored strip tubes were then prepped for PCR in order to amplify the DNA within the isolate
samples so that they can be sent to the EPA for sequencing. In the first month I was taught to prep the
isolates by performing the overnight cultures and prepping for PCR as I made the strip tubes, but as I got
the knack of the process I was able to prep a few strips at a time. This maximized efficiency as the strips
must be cycled for 4 hours before PCR can be performed. This way I was able to cycle 16 or even 24
isolates at a time instead of 8. In order to prep the tubes for PCR before thermocycling them a few very
precise steps must be followed. First of all, this preparation process takes place in the clean room on the
2nd
floor of EHS. The clean room is heavily sterilized to prevent contamination of samples being
prepared. For this reason it is imperative that anyone using the clean room take precautions by showering
and changing clothes before entering the room if they have come in contact with PCR product or any
other DNA samples. In the clean room there is an automated hood for reagents and another for DNA.
Products made under the reagent hood can be transferred to the DNA hood, but in order to prevent
contamination of the reagents, products made under the DNA hood must never then be transferred to the
reagent hood. The reagent is first prepared under the reagent hood by mixing 126𝜇𝐿 of molecular grade
water,18𝜇𝐿 of both the forward and reverse reagents,and 45 𝜇𝐿 of master mix. This combined mixture is
called prepared master mix and can now be transferred to the DNA hood. 2 𝜇𝐿 of each sample along with
a positive and a negative control are aliquoted into 23 𝜇𝐿 of the prepared master mix. These strip tubes
are then placed in the thermocylcer for 4 hours to complete prep for PCR. In the meantime the PCR gel
can be prepped using 50mL of tris buffer, .75g of agar, and 2 𝜇𝐿 of ethidium bromide. The PCR process
takes 30 minutes to set the gel and an hour and a half to amplify the samples. After that a photo is made of
the bands which can then be sent off for sequencing.
Though my work with Keri is not yet complete this is as far into the process as we got for the
internship. I have gotten some preliminary training on finding the maximum inhibitory concentration of

7. vibrio to triclosan but have yet to perform the tests and dilutions on my own. I plan on continuing and
logging my work with Keriover the summer and into late Fall. We plan on being done by the end of this
coming fall semester where Keriwill be able to submit her work and findings to her doctoral board. Here
is an outline of the work we have completed and plan to complete:
 .Jan-Feb Training in lab: Media Prep,Sterile Technique
 Mar PCR,DNA Extraction, Gel Electrophoresis
Apr Minimum Inhibitory Concentration Assays for triclosan &
Vibrio/ Gel/ PCR, prepare media
May-Jul Blind study; testing virbio isolates exposed to triclosan for
MIC, identify if isolates are vibrios w. pcr, prepare media
(1trip to the field in July)
Aug Process summer samples for DNA extraction for future 16S
sequencing on Illumina platform
Sep-Oct Analyze samples; prepare media
Nov Write up of results/experiences