Identification of Pathogenic Agent Isolated from

1
Identification of Pathogenic Agent Isolated
from Hamburger Sample in Virulent, PA
Aaron Weston, Joe Julian, Jess Savino, Steve
Berman
Abstract
Results
Fifty residents of Virulent, PA became ill
after consuming beef patties from Buffs Big Bad
Burgers. Samples of the hamburger were collected
and DNA samples were isolated from the burger.
The DNA was replicated via the PCR process with
16S rRNA primers. The samples were then run
through a 1 agarose gel with EtBr. Although the
control wells were contaminated and did not
conclusively show the presence of bacteria in the
hamburger, the experimental well results matched
what would be expected from a bacterial presence.
However, when the replicated DNA was isolated,
sequenced, and run through the BLAST database,
the DNA sample positively matched the 16S region
of E. coli, the bacterial bandit.
Introduction
Fifty occupants of Virulent, PA, became
violently ill in a 48-hour span after consuming a
Bad-Boy Burger at Buffs Big Bad Burgers.
Symptoms included nausea, severe abdominal pain,
cramping, explosive diarrhea, and projectile
vomiting. Predictably, Buff denied claims that
his hamburgers were contaminated and has provided
hamburger samples for analysis. The Virulent
Department of Health demanded to know if the
hamburgers were indeed contaminated and if so, by
which bacterial species. The list of suspected
bacteria was narrowed down to the following
Salmonella enterica, Campylobacter jejuni,
Escherichia coli, and Listeria monocytogenes.
Salmonella enterica can cause disease in humans
with the presence of at least 1 million infecting
organisms (Washington, 2004). Onset of symptoms
occurs within 12-14 hours after ingestion and
last for 2-3 days (2004). These symptoms include
diarrhea, which can progress to containing blood,
abdominal cramps, nausea, vomiting, headache,
prostration, muscle weakness, and chills (2004).
Campylobacter jejuni can cause disease in
humans with a minimum number 400-500 infecting
organisms (USFDA, 2004). Onset of symptoms
occurs 2-5 days after ingestion and can last for
7-10 days (2004). These symptoms include
diarrhea, which can contain blood and become
watery (2004). Complications can possibly result
in hemolytic uremic syndrome (HUS), which can
lead to renal failure (2004). Escherichia
coli can cause disease in humans with as a few as
10 organisms (Washington, 2004). Onset of
symptoms occurs 3-8 days after ingestion (2004).
These symptoms include nausea, vomiting,
diarrhea, and severe abdominal cramps (2004).
Like C.jejuni, E.coli can lead to the
complication HUS (2004). However, this
complication only occurs in 2-7 of the people
the bacteria infects (2004). Listeria
monocytogenes is rarer than the aforementioned
bacteria. Onset of symptoms occurs 1-2 days
after ingestion and can last for 1-3 days
(Weinstein, 2001). These symptoms include fever,
muscle aches, nausea, and diarrhea (CDCP, 2003).
Symptoms such as headache, stiff neck, confusion,
loss of balance, and convulsions can occur if the
bacteria infect the nervous system (2003).
Infection of the nervous system can result in
death due to brain abscesses in about 10 of all
CNS infections (Weinstein, 2001).
Discussion Conclusions
The DNA sample obtained from the hamburger
(Figure 1.) was replicated via the polymerase
chain reaction using primers designed to target
the 16S rRNA gene promoter region of bacteria.
An agarose gel was then run with the replicated
sample (Figure 2.). Although the control wells
were contaminated, the experimental wells
results indicated that bacteria were present in
the hamburger. The bacterial DNA was sequenced
at the Seq-a-Lot company and the sequence
obtained from the company was run through the
BLAST nucleotide database. The BLAST analysis of
the 16S rRNA segment taken from the hamburger was
a direct match to the 16S rRNA segment of the
bacterium Escherichia coli. Not only did this
corresponding sequence have the highest amount of
matching bits, but it also had an e value of 0.0.
This showed that the infectious bacteria was,
without a doubt, E. coli. E. coli infections
are usually left untreated. Treatment with
antibiotics could lead to serious kidney
complications. Antidiarrheal medication should
be avoided because the E. coli bacteria are
expelled from the body in the waste. Generally,
the patient should drink large amounts of water
to avoid dehydration from the diarrhea and get
lots of bed rest. Infections usually last from 5
to 10 days (AAFP, 2000). Though E. coli
infections are not treated proactively, hemolytic
uremic syndrome (HUS), a serious complication of
E. coli infections, must be treated aggressively
because this condition can be fatal (2000).
Between 5 and 15 of all HUS cases are lethal
(Shapiro, 2004). Symptoms of HUS are hemolytic
anemia, thrombocytopenia, and renal failure
(2000). If the patient develops HUS, fluid
intake is monitored closely (Shapiro, 2004). If
the HUS is associated with diarrhea, a precise
fluid balance must be achieved (2004). Treatment
of hyperkalemia and renal failure is done through
blood transfusions and/or dialysis (2004).
Methods
Polymerase Chain Reaction Into a
0.2mL micro centrifuge tube, 15µL Ready Mix PCR
Reaction Mix, 2µL forward primer, 2µL reverse
primer, 6µL hamburger DNA, and 20µL sterile water
were added. A separate 0.2µL was prepared in the
same fashion, but with 6µL sterile water in place
of the hamburger DNA. The second tube was to
function as the control. The tubes were placed
in the thermal cycler and run for 95C, 4
minutes 94C, 1 minute 55 C, 1 minute 72 C,
2 minutes repeated 30 times and 72 C for 5
minutes. Analysis of Products A
1 agarose gel with EtBr in 1 X TAE was prepared.
Five microliters of the DNA obtained from the
PCR was placed into 4 wells, 5µL of the Hin
dIII-digested Lambda DNA standard was placed into
one well, and 5µL of the sterile water control
were placed in 4 wells. The gel was run at 100
volts for 45 minutes until the dye migrated
halfway down the gel. The gel was then
photographed on a UV transilluminator. A PCR DNA
sample was also sent to the Seq-a-Lot Company
where it was sequenced. The sequence was then
searched on the nucleotide database located on
the BLAST website.
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