1. Growth Media Directed Selection for Clinical Strains of Vibrio vulnificus
by
Nhat Ho
REU Final Report
Presented to the Faculty of the
NSF-REU Program at
Texas A&M University at Galveston
APPROVED BY:
________________________________________
Dr. Robin Brinkmeyer, Ph.D., REU Advisor

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ACKNOWLEDGEMENTS
We thank Jamie Steichen for collecting the Galveston Bay water samples and the
personnel in the Coastal Health and Estuarine Microbiology Lab for assistance and guidance in
laboratory procedures.
This study was funded in part by a National Science Foundation ‘Research Experience
for Undergraduates’ grant awarded to TAMUG.

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ABSTRACT
Vibrio vulnificus is a closely related cousin of the more familiar Vibrio cholerae, the
agent responsible for Cholera worldwide. Vibrio vulnificus is highly virulent to humans and can
be lethal in immuno-compromised individuals. There is much to learn about the ecology of V.
vulnificus including why clinical strains, i.e. those isolated from human infections, are more
virulent than those detectable in the environment. Lack of detection is likely limited by the
methodology. Currently, water is added to selective growth media that enriches for Vibrio
bacteria. Then the Vibrios are detected either by gene probing or polymerase chain reaction).
Galveston Bay water samples and ballast water were enriched with Luria-Bertani broth (LB),
alkaline peptone water (APW) and brain heart infusion broth (BHIB) at varied concentrations.
LB is a nutrient rich medium that stimulates heterotrophic bacterial growth. APW is a medium
that selects for all strains of Vibrios, while BHIB contains additional proteins found in humans
and other mammals that may select for the elusive clinical strains. Inoculated media was
incubated overnight at 35°C, harvested by centrifugation, DNA was extracted, and then
quantitative PCR assays were conducted to detect and traditional PCR with primers targeting
virulence enhancing genes found only in clinical strains were used to enumerate V. vulnificus and
differentiate environmental from clinical strains, respectively. Sample salinity will also be
measured as to decipher its’ significant role in Vibrio growth efficiency in parts per thousand
(ppt).

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INTRODUCTION
Vibrio vulnificus, ubiquitous in the estuarine environment, is a highly lethal opportunistic
human pathogen that is responsible for much of the cause of seafood related deaths in the United
States (Felhousen, 2000). Symptoms of this disease includes fevers, chills, nausea, septic shock,
lesions across the patient’s body, bullae and necrotizing faciitis (B,C; Jones and Oliver 2009).
Clinical strains, i.e. from infected humans, of V. vulnificus are typically more virulent than strains
isolated from seawater, oysters, and surfaces such as shells, plants, sediments, or rocks in the
marine environment. However, the more virulent strains originate from the marine environment
and so detection must be limited by methodology. Recently, it was determined that clinical strains
contain several housekeeping genes, gyrB, mdh, rpoD, and groEL1, encoded on chromosome I and
two housekeeping genes, pyrC and groEL2,encoded on chromosome II, that are lacking in most
environmental strains (Cohen et al., 2007). These genes appear to enable clinical strains to better
attach to and infect the human host and therefore facilitate a selective process of these strains
versus ‘environmental’ strains. We used three types of growth media for selection for clinical
strains of V. vulnificus from Galveston Bay water to test our hypotheses:
Clinical strains of Vibrio have thus far been undetectable in the Earth’s ecosystem, but that
does not mean they’re not there. Open wounds exposed to marine waters could lead to infections
followed by symptoms of septicemia and, or bullae formations. These symptoms often lead to
death. When examining the patients of the immune-compromised bodies, unique strains are found
here that exists nowhere else in the world or as far as we know.
Persistent studies of the disease determined the need for virulence to fall under four
categories: Locomotion, attachment, protein and toxins. The bacterium uses flagella to propel itself
to make contact with a human cell. The pili structure then attaches itself to the cell. The protein

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HlyU was first found within clinical patients and is said to be responsible for regulation and
production of the cytotoxin of the bacterium. The rtxA1 gene in V. vulnificus encodes for the
primary toxin used to attack its hosts causing tissue necrosis (Jones and Oliver 2009).
There is still much to learn about the ecology of V. vulnificus including why clinical
strains, i.e. those isolated from human infections, are virulent than those detectable in the
environmental. This lack of detection is likely limited by the methodology since the clinical strains
must have been present in seawater or oysters etc. in order for the host to become infected. Current
methods involve addition of samples to a selective growth media that enriches for Vibrio bacteria.
Then the Vibrios are detected either by gene probing or PCR.
Hypothesis:
H1: APW selects for non-clinical i.e. environmental strains of V. vulnificus.
H2: Growth media such as BHIB and LB that have proteins similar to those in humans will
preferentially select for clinical strains of V. vulnificus.
Objective:
Use selective media such as APW + BHIB to enrich for the clinical strains and then
enumerate them with QPCR followed by screening with PCR with primers specific for ‘clinical’
toxicity genes.
METHODS AND MATERIAL
Sample Collection—Samples used in this study was collected from several sites off
Galveston such as Galveston Bay, Trinity Bay, where the Gulf meets the Galveston Bay estuaries
and etc. APW, BHIB and LB media (7ml each) were added to 15ml of the sample, incubated

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overnight at 35°C, harvested by centrifugation, and then frozen at -20°C until later DNA
extraction.
DNA will be extracted with a modified CTAB method. Frozen samples will be re-
suspending in 500ul of 3% CTAB, incubated for 40 minutes in a water bath at 70°C, and purified
with addition of 500ul of chloroform-isoamyl alcohol and centrifugation at 13,000 RPM. The
supernatant will be collected and 300ul of isopropanol is added and left overnight to precipitate the
DNA. The precipitated DNA is harvested with centrifugation at 13,000 RPM, and the supernatant
is removed. Ethonol is added to remove co-precipitated salts. After leaving it to dry, the last step is
to add 200ul of LB buffer, amount is variable to the amount of DNA in this tube. A Nanodrop
Spectrophometer will be used to determine its DNA concentration and purity. Quantitative PCR
using assays specific for the vvhA gene in V. vulnificus (Panicker and Bej, 2005). Samples testing
positive for V. vulnificus will be further screened with primers specific for the additional toxicity
genes found in clinical strains (Table 1). Amplicons of toxicity genes will be viewed with gel
electrophoresis.
Results & Discussion
Quantitative PCR of our initial (Fig. 3) enrichment experiment, using seawater collected
on 5/20/10, found that APW consistently selected for more V. vulnificus (expressed as colony
forming units; CFU) at all stations than APW + BHIB and LB. No consistent trend of
presence/absence of virulence enhancing genes was observed for the different media in this
experiment (Table 2), however VvGroEL1b and VvGro2 were the most prevalent among all
treatments and sampling stations. Enrichments using seawater collected at station 6 located in the
middle of Galveston Bay tested positive for most of the genes (LB 5/5; APW + LB 4/5; and APW

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5/5). Salinity at this station is 12 ppt on average and falls within the optimal range (6-17 ppt) for V.
vulnificus growth and survival (Jones and Oliver, 2009). Vibrio vulnificus CFUs were low at
(<1000 CFU) at Station 12, located near the mouth of the Trinity River. Average salinity at this
station is 1 ppt and below the optimal range. CFUs at station 29 were also < 1000, however at this
station salinity is typically 25 ppt and above the optimal range for V. vulnificus. A follow up
enrichment experiment (Fig. 4), using seawater collected on 7/5/10, tested different proportions of
APW + BHIB, APW, and LB. The overall CFUs for all media tested were more than two fold
greater than for the previous experiment and the combination of of ¼ BHIB and ¾ APW was the
most effective enriching V. vulnificus even at stations 12 and 29. We are currently screening all
treatments from this experiment for presence/absence of virulence genes.

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Table 1. Primers specific to toxicity genes found only in clinical strains of V. vunificus.

9. Figure 1. An overview of the experiment in a flow chart.
6
Experimental Design
Figure 1. An overview of the experiment in a flow chart.

10. Figure 2. The timeline for the study.
7
Timeline
Figure 2. The timeline for the study.

11. Figure 3. Comparison of V. vulnificus
BHIB (1:1) enrichment media for samples collected 5/20/10.
8
V. vulnificus colony forming units (CFU) in LB, APW, and APW+
BHIB (1:1) enrichment media for samples collected 5/20/10.
colony forming units (CFU) in LB, APW, and APW+
BHIB (1:1) enrichment media for samples collected 5/20/10.

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Table 2. Presence (+)/absence (-) of V. vulnificus virulence enhancing genes found in clinical
strains.

13. Figure 4. Comparison of V. vulnificus
(varied proportions) enrichment media for samples collected 7/5/10.
10
vulnificus colony forming units (CFU) in LB, APW, and APW+ BHIB
(varied proportions) enrichment media for samples collected 7/5/10.
colony forming units (CFU) in LB, APW, and APW+ BHIB

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