Download Comparing the Prevalence of Verotoxin

Comparing the prevalence of verotoxin-producing
Escherichia coli between conventional and organic
ground beef samples collected from local grocery stores located in
Thurston County, Washington
Shannon Davis
Senior Seminar
May 6, 2008
Final Draft
Table of Contents
2
Abstract
3
Introduction
4-9
Materials and Method
9-14

Sample collection

Pouring agar plates

Detection and isolation of E. coli

Isolation of verotoxin producing E. coli

Polymerase chain reaction and electrophoresis

PCR primer selection

Isolation of DNA template

Preparation of master mix

DNA amplification

Agarose gel electrophoresis

Loading Samples Into Gel Walls

Positive and negative controls
 Data analysis
Results
14-19
Discussion
19-22
Acknowledgements
23
Literature Cited
24-25
2
Abstract:
Due to the dangers of Escherichia coli contaminated ground beef, the prevalence of
verotoxin producing E. coli was investigated in conventional and organic samples collected in
Thurston County, Washington. E. coli was isolated using two E. coli specific media. DNA
extracted from E. coli isolates were tested for the verotoxin producing genes, VT1 and VT2,
using Polymerase Chain Reaction (PCR). Results indicate the presence of VT1 and VT2, but
show no differences between conventional and organic ground beef samples. Detecting the
verotoxin genes in the ground beef samples reinforces the need for consumers to educate
themselves on food borne illnesses as well as how to safely prepare and cook food.
3
Introduction:
On October 2, 2007, the Centers for Disease Control (CDC) reported an outbreak of E.
coli O157:H7 that caused a recall of 21.7 million pounds of ground beef (CDC, 2007). Over a
four month period, July thru September, the CDC identified 40 cases that spanned over 8 East
Coast states. In 1999, the CDC released a report that estimated 73,000 cases of E. coli O157:H7
occur in the United States per year (CDC, 2006). Of those estimated 73, 000, cases 61 resulted in
fatalities. The report stated that while there were other sources of E. coli O157:H7, most of these
cases were associated with contaminated ground beef.
The CDC’s web site provides current information regarding E. coli O157:H7 and describes E.
coli O157:H7 as an opportunistic pathogen that is classified as a gram negative rod (CDC, 2006).
E. coli is an enteric bacterium, meaning it is found within the intestines of animals. This
bacterium can reside in healthy cattle, sheep, deer, and goat intestines. E. coli O157:H7 is a
serotype of E. coli that produces toxins known as Shiga toxins or verotoxins. Verotoxins are
produced by a multisubunit protein. This protein consists of 1 molecule of an A-subunit and 5
molecules of a B-subunit. The A-subunit molecule enables the toxogenic proteins to bind to the
target cells that line the intestinal wall. Once the protein has entered the cell the B-subunit
molecules deactivate the ribosome of the cell. Without working ribosomes, protein synthesis
ceases and the cell dies. If these toxin producing bacteria are ingested by humans, they can cause
severe bloody diarrhea, which is also known as hemolytic colitis. Secondary to the bloody
diarrhea, other more serious illnesses can occur, such as renal failure and destruction of red
blood cells known as hemolytic uremic syndrome (HUS). Because children and the elderly are
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more likely to have a compromised or undeveloped immune system, they are at a higher risk for
developing HUS upon infection with verotoxin producing E. coli.
Ground beef becomes contaminated with pathogenic E. coli during the time the animal is
eviscerated (J. Bolker, Washington State DOH Officer, personal communication, Sept. 2007). If
proper procedures are not followed during evisceration of the animal, the intestines can be
perforated, releasing the intestinal contents onto and into the meat supply. Once the infected
meat is ground into ground beef, the bacteria are spread throughout the ground beef.
In light of the recent E. coli O157:H7 outbreaks, questions are being raised by consumers
about the quality and safety of ground beef. Is organic ground beef safer than conventional
ground beef? The USDA (2007) qualifies organic ground beef as ground beef that has come
from cattle that have not been treated with antibiotics or given any growth hormones. The
question remains whether there is a higher prevalence of verotoxin producing E. coli in ground
beef from cattle that has not been treated with antibiotics than ground beef that has.
Tuttle et al. (1999) analyzed a 1992 outbreak of E. coli 0157:H7 in the western United States.
To determine the source of the outbreak, suspect lots from a meat processing plant were located
and identified as coming from the implicated plant on November 19-20, 1992. Once the source
had been determined, analysis was conducted to further understand the effects of the outbreak.
Included in the study were 76 ground beef patty samples collected from 16 of the 17 lots that
were identified to have been contaminated. Various testing methods were used to isolate and
identify E. coli 0157:H7. Data on the bacterial content of the infected patties were analyzed, and
from that data, they estimated the number of bacteria required to cause illness in humans.
5
Tuttle et al. (1999) isolated E. coli 0157:H7 from 7 of the 21 lots tested. Although they
determined the lot numbers of the contamination source, it is noteworthy that the beef samples
that were ground to make the beef patties were combined from several different suppliers,
foreign and domestic. Upon inspection of the various meat processing plants, numerous
procedures were identified as possible mechanisms that could have lead to infected ground beef.
The most identified error in procedure was allowing parts of the carcass to touch the floor or
allowing items that had touched the floor to touch the carcass. Based upon the analysis of the
samples and the people infected, they estimated that fewer than 700 bacteria can cause infection.
Tuttle et al. (1999) showed how a minute amount of contamination is enough to cause
infections in human. They also identified problems in the process of meat handling that could
possibly lead to future outbreaks. The employees did not wash their hands in between handling
the outside of the carcass and handling the inside of the carcass These results reinforce
consumers concerns regarding contaminated ground beef and the need for further investigation
for ground beef contamination.
For preventative measures and during an outbreak, E. coli 0157:H7 and other pathogens
need to be detected expeditiously. Using artificially contaminated samples, Wang et al. (2006)
developed a detection method by using Multiplex Real-Time Polymerase Chain Reaction (PCR).
Their objective was to develop a way to simultaneously detect multiple pathogens using
Multiplex Real- Time PCR.
Multiplex PCR uses probes and molecular beacons to select specific DNA regions from
specific pathogens and amplify them simultaneously. As amplification increases, so does the
fluorescence of the probes, allowing for the quantitative measurement of multiple pathogens.
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This detection method simultaneously detected E. coli, Salmonella, and Shigella. Not only did
this method identify multiple pathogens at once, but it also decreased the amount of time and
money spent.
Multiplex PCR is beneficial in research that has time and budget constraints. By using this
method of detection, I can decrease the amount of money and time spent on detection during my
investigation.
In Argentina, Chinen et al. (2001) studied the beef supply in retail outlets. Because HUS is
endemic in Argentina, they wanted to determine the prevalence of E. coli 0157:H7 in the meat
supply and the antibiotic sensitivities of E. coli 0157:H7.
During the study, Chinen et al. (2001) sampled 279 various types of beef products collected
through the months of February and May. They isolated the bacterium using MacConkey II agar
with sorbitol and PCR. Of the 279 samples taken, 4% tested positive for E. coli 0157:H7. All but
one of the verotoxin producing bacteria isolated was susceptible to all the antibiotics used. Out of
the samples collected 10 of the 11 positives came from different retail meat outlets.
In the Chinen et al. (2001) study of the isolated E. coli 0157:H7 strains, the strain showed
susceptibility to all the antibiotics. Since the verotoxin producing E. coli was susceptible to the
antibiotics, cattle that are treated with antibiotics should have a lower presence of this bacteria
residing in their intestinal tract. If this is the case than it would give support to the theory that
conventional ground beef that has been treated with antibiotics may have a lower prevalence of
verotoxin producing E. coli.
Bohaychuk et al. (2006) investigated the prevalence of E. coli 0157:H7 and other pathogens
in the meat supply of Edmonton, Alberta, in Canada. Four retail chains were selected from
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telephone listings, and samples were collected every Sunday and Monday of the month during a
four month period. An unspecified rapid detection method was used to determine the presence of
a particular pathogen. Once the pathogens had been detected, enrichment media and PCR were
used to isolate and identify the pathogens. Out of the 100 samples tested for E. coli 0157:H7,
only 1% was positive for E. coli, which was a different serotype than E. coli 0157:H7.
By using this rapid detection method, Bohaychuk et al. (2006) decreased the cost of having
to use other more expensive resources to determine the presence of the pathogen as well as
determining the contaminated beef products expeditiously.
Samadpour et al. (2001) presented a study that determined the threat to Seattle consumers by
testing for the prevalence of Shiga toxin-producing E. coli in the ground beef of local retail
stores and the prevalence of these pathogens in cattle feces. The study compared the prevalence
of Shiga toxin-producing E. coli among retail stores, different fat contents of ground beef, and
the presence of Shiga toxin-producing E. coli in feces samples collected from local cattle. The
study was conducted by obtaining samples from various retail outlets located in King County,
Washington. There were 296 ground beef samples selected from the display cases of the retail
stores, and 165 samples were separated into groups based on their fat content to allow for
possible differences between grades.
The process by which Samadpour et al. (2001) used to isolate the pathogen was modified to
allow for further testing. Isolation was accomplished by using modified Trypticase soy broth.
Samples from the plates that showed the most isolated colonies were transferred to filter paper.
Bacteria colonies on the filter paper were processed and the bacterial DNA was extracted for use
8
as the template in the PCR process to identify the pathogen. The plates with remaining colonies
were returned to continue incubation for future testing.
Samadpour et al. (2001) results showed that 17% out of the 296 samples tested positive for
Shiga toxin-producing E. coli. These were not the E. coli serotype O157:H7, although the
serotypes isolated were determined to cause bloody diarrhea in humans. Out of the 52 fecal
samples tested, 15% of the samples tested positive for Shiga toxin-producing bacteria.
The results from the Samadpour et al. (2001) study reinforce the need to further investigate
the prevalence of verotoxin producing E. coli in the meat supply of Seattle and its surrounding
area. The positive results show the presence of verotoxin producing bacteria in the Seattle
ground beef supply. If verotoxin producing bacteria was shown to be present in the Seattle
ground beef supply, it can be present in the Thurston County ground beef supply and therefore
should be investigated.
In this study, I asked the following question: Does organic ground beef have a higher
prevalence of verotoxin producing E. coli than conventional ground beef? The objective of my
research was to determine the prevalence of verotoxin producing E. coli in the ground beef
supply in Thurston County, Washington. The prevalence of verotoxin producing E. coli was
compared between organic ground beef samples and conventional ground beef samples. Since
conventional ground beef is treated with antibiotics; I hypothesized that conventional ground
beef samples would show a lower prevalence of verotoxin producing E. coli.
Materials and Methods:
Sample collection. Eight samples were collected from four different grocery stores
located in Thurston County. From each store, 1 organic sample and 1 conventional sample were
9
collected. To keep the samples consistent; there was no more than 10% fat content in each
sample, the date stamps of samples collected were within no more than 4 days from expiration,
and the temperature of display cases were recorded. Samples were transported on ice to the lab
where they were refrigerated until processed.
Pouring agar plates. I measured out 12.5 grams of MacConkey II agar containing 4methyllumbelliferryl-β-D-glucuronide (MUG) on a balance. MacConkey II agar containing
MUG was chosen for the initial media because it was selective and differential to enteric bacteria.
The agar was then placed into a beaker with 250 mL of deionized water. The agar was then
heated to a boil and stirred on a hot plate for five minutes to insure it was fully dissolved. I
measured the pH of the agar to insure that it was within the tolerated pH of 6.5-7.5. The agar was
then autoclaved at 21 psi for 15 minutes at 121°C. Once the beaker was ≈ 50°C I poured the agar
into sterile Petri dishes. I then waited for the agar to solidify and placed them in the refrigerator
upside down for storage until use. The same procedures and concentrations were used for the
MacConkey II agar with sorbitol. Verotoxin producing E. coli can be detected on MacConkey II
agar with sorbitol because most of the flora that live in the intestines ferment sorbitol and
therefore appear as pink colonies on the agar. Because verotoxin producing E. coli do not
ferment sorbitol they appeared as colorless colonies on the agar.
Detection and isolation of E. coli. I used the following procedures suggested by Mr.
Garman, Microbiology Professor at St. Martin’s University. To attempt to isolate E. coli
organisms, I used sterile procedures and collected samples from each ground beef sample using a
metal spatula. Each sample was transferred to a test tube containing 3mL of 1% CaCl2. The
CaCl2 provided an ideal osmotic environment for the bacterial cells. I then used a sterile glass
stirring rod to mix and macerate the ground beef in the test tube. MacConkey II agar with MUG
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plates were incubated by pouring the liquid sample directly onto the agar. This was done because
the fat in the sample prevented using a pipette. To spread the liquid sample on the agar I swirled
the plate in a circular motion. E. coli produces β-D-glucuronidase, an enzyme that when in
contact with MUG in the agar will hydrolyze and produce 4-methyllumbellife. Following the
media’s package insert, I incubated the agar plates at 37°C for 48 hrs under aerobic dark
conditions to provide the optimum environment for E. coli growth. The compound 4methyllumbellife fluoresced under UV light, and allowed for E. coli colonies to be identified.
After incubation, I used the long wave fluoresce UV light in St. Martin’s lab to identify
fluorescing colonies.
Isolation of verotoxin producing E. coli. Using sterile procedures I inoculated
MacConkey II agar containing sorbitol with the fluorescing E. coli colonies from the
MacConkey II agar containing MUG. I then incubated the agar plates at 37°C for 48 hours under
aerobic dark conditions. Because verotoxin producing E. coli do not ferment sorbitol they will
appear as colorless colonies on the agar. I used these colorless colonies for Polymerase Chain
Reaction (PCR).
Polymerase chain reaction and electrophoresis. I used PCR and electrophoresis to
determine the presence of verotoxin 1 and 2 (VT1/VT2) genes in the colorless colonies from the
MacConkey II agar with sorbitol. For PCR and electrophoresis, I followed procedures from the
Bio-Rad PCR kit manual (2006).
PCR primer selection. E. coli may have genes that code for the production of
verotoxins 1 and 2 as described in a study by Blanco et al. (1997). Primers for verotoxins 1 and 2
shown in Table 1 were selected from a previously published study by Pass et al. (1999). These
11
primers were shown to be specific to VT1 and VT2; therefore I used them to determine the
presence of VT1 and VT2 in my samples.
Table 1. Olyigonucleotide Primers used for PCR.
Primer Pairs
Gene
Amplified
VT1
Estimated
Length of PCR
Products (bp)
Nucleotide Sequence
121
fp: 5'-ACGTTACAGCGTGTTGCTGGGATC-3'
rp: 5'-TTGCCACAGACTGCGTCAGTRAGG-3'
VT2
102
fp: 5'TGTGGCTGGGTTCGTTAATACGGC-3'
rp: 5'-TCCGTTGTCATGGAAACCGTTGTC-3
Isolation of DNA template. I followed the procedures listed on the educational Access
Excellence website and collected bacterial cells from identified colonies for use as the DNA
template for PCR. I used the wood end of a sterile cotton swab to I transfer bacterial cells from
the cultures to a microtest tube containing 500 μL of sterile water. I then froze and thawed the
test tubes three times to lyse the bacterial cells, which released the bacterial DNA. The samples
were then boiled at 95°C in a hot water bath for 5 minutes to deactivate the DNAase. DNAase is
a DNA enzyme that catalyzes the bonds in DNA, without deactivating this enzyme the PCR
process will not work. I then mixed the samples for 10 seconds using a vortex. I centrifuged the
test tubes for 2 minutes at 13,000 x g to allow the cell debris and the suspended genetic material
to separate. Taking care not to re-suspend the pellet at the bottom of the microtest tube, I pipetted
12
≈ 500 μL of the supernatant into a new microtest tube. I then refrigerated the samples until they
were used for PCR.
Preparation of master mix. To run PCR I prepared a master mix containing 5.0μL of
TE buffer, 6.0μL of 25 mM MgCl2, 1.0μL of dNTP’s, 1.0μL for each forward and reverse
primers of VT1 and VT2 (total of 4.0μL of primers), 28.5μL of sterile water, 0.5μL of Taq DNA
polymerase, and 5.0μL of the DNA template for a total of a 50μL reaction.
DNA amplification. The complete reaction mixture (master mix) was run through the
thermal cycle shown in Table 1.2 used by Pass et al. (1999).
Table 2. Thermal Cycle for Multiplex PCR
Phase
Temperature C°
Time
Initial
Denaturation
95°
5 minutes
Denaturation
95°
30 seconds
72°
1 minutes
Primer Annealing
95°
30 seconds
Extension
63°
30 seconds
Extension
72°
30 seconds
Final Extension
72°
5 minutes
Number of Cycles
1
X5
X 20
1
Agarose gel electrophoresis. I prepared the gel beds using a casting tray and a mixture
of 1X TAE buffer and 1% agarose powder. I combined this mixture in a 500ml flask. The flask
was then heated in a microwave for 2 minutes. The mixture was completely dissolved until the
13
liquid was clear. I then added 6μL of ethidium bromide to allow for ultraviolet transillumination
viewing of the DNA bands. Next, I poured the agarose mixture into a casting tray and allowed it
to solidify, which took about 20 minutes. I placed the gel into the electrophoresis chamber, and
added buffer to the chamber until the gel was completely submerged.
Loading samples into gel wells. The first well was loaded with a benchtop 100 base pair
DNA ladder. The DNA ladder allowed for measurement of the molecular weight of the samples.
The remaining wells were loaded with subsequent samples and the positive and negative controls.
Each well was loaded with 15μL of the sample using a calibrated micro pipette Electrophoresis
was run at 120V for 20 minutes to allow migration of the DNA bands. A photograph of final
electrophoresis results was taken.
Positive and negative controls. Shigella flexneri was used for the positive control. Shigella
flexneri is a bacterium that produces VT1 and VT2 and therefore displayed bands for my primers.
For my negative control I used a sterile cotton tip and ran it through the same methods as the
bacterial samples. Using a sterile sample allowed me to determine the accuracy of my procedures.
The negative control for PCR was sterile water because it left no bands.
Data analysis. Data was analyzed qualitatively by determining the presence of vertoxin
producing E. coli in the ground beef samples tested. The verotoxin genes were determined to be
present in a given sample if the PCR product for that sample amplified the targeted nucleotide
sequence for VT1 and or VT2 after gel electrophoresis.
Results:
Experiments were conducted to detect verotoxin producing bacteria, E. coli, on samples
of conventional and organic ground beef collected from four different stores. Growth after
14
inoculation and 48 hour incubation of possible E. coli colonies on the MacConkey II agar with
MUG resulted in pink to red colored colonies. The red colonies were counted and the average for
the three replicate plates was calculated and shown in Figure 1.
Conventional Replicates 1-3
Organic Replicates 1-3
Figure1. Average number of red colony growth per three replicate plates of conventional and organic samples from each store on the MacConkey
agar with MUG after 48 hours of incubation at 37˚C. Red colonies indicate possible E. coli colonies. Error bars represent one standard error for
eight samples.
Figure 2 is a picture showing an example of the red colored colonies that grew on the
MacConkey II agar with MUG.
15
Figure 2. Picture of MacConkey II agar with MUG with rose colored colony growth. Arrows point to red colony growth.
The red colonies were observed under 365nm long wave UV light to determine if the E. coli
colonies produce β-D-glucuronidase, an enzyme that when in contact with the MUG in the agar
hydrolyzes and produces 4-methyllumbellife, which fluoresce blue green under the UV light. As
shown in Figure 3, every replicate plate showed fluorescing colonies.
Store A
Store B
Store C
Store D
Figure 3. Number of blue/green fluorescing colonies per replicate plate on MacConkey II agar with MUG under 365nm long wave UV light.
Error bars represent one standard error for eight samples.
16
Figure 4 is a picture of blue-green fluorescing colonies under UV light.
Figure 4. Picture of 4 fluorescing colonies on MacConkey II agar with MUG under a 365nm long wave UV light. Arrow points to a region of
fluorescence.
The MacConkey II agar with sorbitol plates are selective for vertoxoin producing E. coli which
results in clear to colorless colony growth. Table 3 shows 20 out of the 24 plates that were
inoculated with the fluorescing colonies grew clear to colorless colonies.
Table 3: Number of plates with positive colorless growth for verotoxin producing E. coli on MacConkey agar with sorbitol after incubation for 48
hours at 37 ˚C.
Store/ Replicate#:
A-1
A-2
A-3
F-1
F-2
F-3
S-1
S-2
S-3
T-1
T-2
T-3
Total Positive:
Conventional:
Positive growth
+
+
+
+
+
+
+
+
+
+
+
11
Organic:
Positive growth
+
+
+
+
+
+
+
+
+
9
17
Figure 5 is a picture showing the clear to colorless growth on the MacConkey II agar with
sorbitol indicating the presence of verotoxin producing E. coli.
Figure 5. Example of clear to colorless colony growth on MacConkey II agar with sorbitol. Arrow points to region of colorless growth.
Polymerase chain reaction and electrophoresis was run on each sample using primers
VT1 and VT2. Figure 6A shows PCR amplifications for conventional and organic samples from
store A. Conventional and organic VT1 samples amplified the targeted 121 base pair product.
Conventional and organic VT2 samples amplified the targeted 102 base pair product. Figure 6B
shows PCR amplifications for conventional and organic samples from store B. Conventional and
organic VT1samples did not amplify. Conventional and organic VT2 samples amplified the
targeted 102 base pair product.
18
100 bp
120 bp
120 bp
100 bp
110 bp
110 bp
110 bp
110 bp
1 2
3
4
5
6
7
1
8
6
7
3
4
5
6
7
8
Figure 6B. Electrophoresis result of PCR amplification,
using VT1 and VT2 primers, to detect verotxoin 1and 2 in
ground beef samples from store B. Well 1: 100 base pair
bench top marker. Well 2: Positive VT1 control, which
showed amplification at ≈120 base pairs. Well 3:
Conventional VT1 sample. Well 4: Organic VT1 sample.
Well 5: Negative control of sterile water. Well 6: Positive
VT2 control, which showed amplification at ≈100 base
pairs. Well 7: Conventional VT2 sample. Well 8: Organic
VT2 sample.
Figure 6A. Electrophoresis result of PCR amplification,
using VT1 and VT2 primers, to detect verotxoin 1and 2 in
ground beef samples from store A. Well 1: 100 base pair
bench top marker. Well 2 : Positive VT1 control, which
showed amplification at ≈120 base pairs. Well 3 :
Conventional VT1 sample. Well 4: Organic VT1 sample.
Well 5: Negative control of sterile water. Well 6: Positive
VT2 control, which showed amplification at ≈100 base
pairs. Well 7: Conventional VT2 sample. Well 8: Organic
VT2 sample.
4
2
8
Figure 7A shows PCR amplifications for conventional and organic samples from store C. All
ground beef samples from store C did not amplify the targeted 102 or121 base pair product.
110 bp
Figure 7B shows PCR amplifications for conventional and organic samples from store D.
110 bp
Conventional and organic VT1 samples amplified the targeted 121 base pair product. The
conventional and organic VT2 samples did not amplify.
3
19
110-120 bp
110-120 bp
100 bp
1
2
3
4
5
6
7
100 bp
110 bp
110 bp
110 bp
110 bp
8
1
Figure 7A. Electrophoresis result of PCR
amplification ,using VT1 and VT2 primers, to detect
verotxoin 1and 2 in ground beef samples from store
C. Well 1: 100 base pair bench top marker. Well 2:
Positive VT1 control, which showed amplification at
≈120 base pairs. Well 3: Conventional VT1 sample.
Well 4: Organic VT1 sample. Well 5: Negative
control of sterile water. Well 6: Positive VT2
control, which showed amplification at ≈100 base
pairs. Well 7: Conventional VT2 sample. Well 8:
Organic VT2 sample.
2
3
4
5
6
7
8
Figure 7B. Electrophoresis result of PCR
amplification ,using VT1 and VT2 primers, to detect
verotxoin 1and 2 in ground beef samples from store
D. Well 1: 100 base pair bench top marker. Well 2:
Positive VT1 control, which showed amplification at
≈120 base pairs. Well 3: Conventional VT1 sample.
Well 4: Organic VT1 sample. Well 5: Negative
control of sterile water. Well 6: Positive VT2
control, which showed amplification at ≈100 base
pairs. Well 7: Conventional VT2 sample. Well 8:
Organic VT2 sample.
Discussion:
My results confirmed the presence of verotxoin producing E. coli in the Thurston County
ground beef supply. Out of the 8 ground beef samples collected, 50% of the conventional
samples and 50% of the organic samples were positive for at least 1 of the 2 verotoxins tested for.
My findings suggest that there is no difference between the prevalence of verotxoin producing E.
coli in conventional ground beef when compared to organic ground beef. Based on my results
my experiment failed to support my hypothesis that conventional ground beef would have a
lower prevalence of verotxoin producing E. coli.
I expected the conventional ground beef to have a lower prevalence of verotoxin
producing E. coli because conventional cattle are treated with antibiotics. According to a study
20
conducted by Elder, R.O. et al. (2002), E. coli 0157:H7 levels in cattle’s fecal samples were
reduced to non-detectable levels 24 hours after treating the cattle with the oral antibiotic
neomycin sulfate. Non-pathogenic E. coli levels were also reduced considerably after treatment
with the antibiotic.
A possible explanation for why the conventional ground beef did not show a higher
prevalence of vertoxin producing E. coli could be that the vertoxin producing strain of E. coli
that was isolated was not susceptible to the antibiotics that the cattle were treated with.
Antibiotics are selective in which bacteria they are able to kill and if the cattle were not treated
with an antibiotic that was selective for the vertoxin producing E. coli, the conventional cattle
could still be reservoirs for the pathogen. The package does not list the names or types of
antibiotics that the cattle were treated with. If the strain of E. coli was not susceptible to the
antibiotics the cattle were treated with then there would have been no difference between the
conventional and organic samples. Of the possible explanations for why conventional ground
beef did not show a lower prevalence of verotxin producing E. coli, sample size probably
influenced the results the most. The small sample size and the number of stores sampled could
have affected the results. Although each organic sample that was tested was from a different
supplier, most stores only had one brand of organic ground beef to choose from. This limited the
organic sample size. For further studies, the sample size for conventional and organic ground
beef could be expanded by including samples for the different fat contents. Also, all the samples
were collected on the same day. Expanding the collection date, increasing the number of samples
and increasing the number of stores that samples were collected from would give a better
qualitative idea on the prevalence of vertoxin producing E. coli in the ground beef supply of
Thurston County. Increasing the sample size would also decrease the sampling error.
21
To better understand the prevalence of verotxoin producing E. coli in Thurston County,
an additional study should be conducted using the above suggestions. Chalmers et al. (1999)
conducted a four year study on a population based surveillance of the vero cytoxin producing E.
coli 0157; and suggested that cases that involved E. coli serotypes with VT1 and VT2 genes
were more often associated with hemolytic colitis and cases that involved E. coli serotypes with
only VT2 were more often associated with HUS. Because the E. coli serotype that contains the
VT1 and VT2 genes was isolated from store A and the E. coli serotype that contains the VT2
gene was isolated from the samples collected from store F, these factors should also be
investigated.
Of the 4 stores sampled, 3 stores had both positive conventional and positive organic
ground beef samples. I discovered a correlation between the conventional and organic samples.
For each store that showed a positive conventional sample for VT1, VT2, or both, the results for
the organic sample paralleled that of the conventional samples. A possible explanation for this
correlation between the conventional and the organic samples from the same store could be
contributed to in-store contamination or cross contamination during processing and or packaging
at the stores.
During the collection of store F’s ground beef samples, I asked the meat counter
attendant for the temperature of the meat cooler. The attendant informed me that the
thermometer was broken and had been broken for some time. Perhaps an additional study should
be conducted on grocery stores located in Thurston County, to investigate the possible dangers of
in-store contamination and or cross contamination of ground beef.
22
As the number of food borne illnesses in the United States climbs to more than 76 million
per year, strategies for combating pathogens in the food supply is continually being developed
(Mead et al., 1999). The use of antibiotics to treat cattle as a preventive for food born illness was
one of these strategies developed to help fight food borne illness (World Health Organization,
1997). Initially treating cattle with antibiotics was thought to be an efficient method for
combating pathogens that cause food borne illness but now the World Health Organization (1997)
warns of the unknown impact that the use of antibiotic in food animal production will have on
public health. It is now known that the increased use of antibiotics in food animal production
leads to antibiotic resistant bacteria. This poses an increased threat to humans by creating food
borne illnesses that are more difficult to treat due to the resistance to the antibiotics that would
normally treat the illness.
Although my hypothesis was not supported, detecting E. coli with the VT1 and VT2
genes in the ground beef samples collected shows the importance of this study. Not only do the
positive results reinforce the need for consumers to practice good food and safety practices but
also show the threat of being infected with a pathogen that is resistant to most antibiotics is an
actual possibility. Consumers need to seriously consider this threat when consuming under
cooked ground beef. Cooking ground beef to a temperature of at least 160°F/70˚C should kill
any pathogenic bacteria that the ground beef could be potentially contaminated with (CDC,
2006).
Acknowledgments:
I would like to extend my thanks and appreciation to Dr. Coby and Dr. Hartman for their
assistance and suggestions through out my project. I would like to thank Cheryl Guglielmo for
23
her assistance with the location and operation of lab equipment and supplies in the lab. I would
also like to extend a special thanks to Dr. Olney for the late night and weekend hours she stayed
to help and guide me through my project. Finally, I would like to thank Enjoli Washington for all
her help and for all the hours she accompanied me in the lab.
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