EHSC 3910 ReportInternship in EHSC with Dr. Lipp

This report outlines the course EHSC 3910 under Dr. Erin Lipp and led by Keri Lydon in the analysis of Vibrio vulnificus resistance to triclosan. This report includes an overview of the course as well as background on the bacterium of focus in reference to potential public health risks associated with climate change.

AJ Shockley5/1/2015

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

bacterium can also enter the body through open wounds and infect the bloodstream resulting in the 1 http://textbookofbacteriology.net/V.vulnificus_2.html2 http://www.cdc.gov/vibrio/vibriov.html

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% fatal to 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

major public health concerns with the growth of vibrio concentrations especially in coastal sub-tidal

regions of the country. 3 http://www.cdc.gov/features/necrotizingfasciitis/4 http://emergency.cdc.gov/disasters/vibriovulnificus.asp

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 coastal and 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 well as 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%2Fs00248-012-0163-27 http://pubs.acs.org/doi/ipdf/10.1021/es401919k

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 plate incubation and stabs and broth for

overnight cultures. Vibrio grows very well in this media at 30-35⁰C.

Meuller Hinton (MH) broth was the other 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

positive bacterial growth. I made this media for Jason Westrich who took a trip to the Keys to

perform field work with Dr. Lipp.

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 45μL of master mix. This combined mixture is

called prepared master mix and can now be transferred to the DNA hood. 2μL of each sample along with

a positive and a negative control are aliquoted into 23μL 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 2μL 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

vibrio to triclosan but have yet to perform the tests and dilutions on my own. I plan on continuing and

logging my work with Keri over the summer and into late Fall. We plan on being done by the end of this

coming fall semester where Keri will 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 mediaNov Write up of results/experiences