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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 4 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. 6 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 7 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 10 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. Literature Cited: Access Excellence [Internet]. Access Excellence: the national health museum: 2007 [cited 2007 Dec 03]. Available from http://www.accessexcellence.org. 24 Blanco, J.E., Blanco, M., Mora, M., Blanco, J., 1997, Production of toxins (enterotxoins, verotoxins, and necrotoxins) and colicins by Echerichia coli strains isolated from septicemic and healthy chickens: relationship with in vivo pathogenicity. Journal of Clinical Microbiology. 35: 2953-2957. Bohaychuk, V.M., Gensler, G.E., King, R.D., Manninen, K.I., Sorensen, O., Wu, J.T., Stiles, M.E., McMullen, L.M. 2006. Occurrence of pathogens in raw and ready-to-eat meant and poultry products collected from the retail marketplace in Edmonton, Alberta, Canada. Journal of Food Protection. 69: 2176-2182. Chalmers, R. M., Parry, S.M, Salmon, R.L., Smith, R.M.M., Willshaw, G.A., Cheasty, T. 1999. The surveillance of Vero Cytotoxin-producing Escherichia coli O157 inWales, 1990 to 1998.Emerging Infectious Diseases. 5: 566-569. Chinen, I., Tanaro, J.D., Miliwebsky, E., Lound, L.H., Chillemi, G., Ledri, S., Baschkier, A., Scarpin, M., Manfredi, E., Rivas, M. 2001. Isolation and characterization of Escherichia coli 0157:H7 from retail meats in Argentina. Journal of Food Protection. 64: 1346-1351. Division of Bacterial and Mycotic Diseases. Echerichia coli O157:H7 [Internet]. Atlanta, (GA): Center for Disease Control; 2006 Dec [cited 2007 Oct 03] 3 p. Available from http://www.cdc.gov/nczved/dfbmd/disease_listing/stec_gi.html. Division of Foodborne, Bacterial and Mycotic Diseases. Echerichia coli O157:H7 [Internet]. Atlanta, (GA): Center for Disease Control; 2006 Dec [cited 2007 Oct 03] 3 p. Available from http://www.cdc.gov/ecoli/2007/october/100207.html. Elder, R. O., Keen, J. E., Wittum, T. E., Callaway, T. R., Edrington, T. S. , Anderson, R. C., and Nisbet, D. J. 2002. Intervention to reduce fecal shedding of enterohemorrhagic Escherichia coli O157:H7 in naturally infected cattle using neomycin sulfate. J. Anim. Sci. 80(Suppl. 1):15. Fey, P.D., R.S. Wickert, M.E. Rupp, T.J. Safranek, and S.H. Hinrichs. 2002. Prevalence of NonO157:H7 Shiga Toxin-Producing Escherichia coli in Diarrheal Stool Samples from Nebraska. Emerging Infectious Diseases. 6: 530-533. Food safety and education [Internet]. United States Department of Agriculture, Food Safety and Inspection Service; 2007 Oct [cited 2007 Oct 03] p1. Available from http://www/fsis.usda.gov/food_safety_education/ask_karen/index. Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee, J.S., Shapiro, C., Griffin, P.M., Tauxe, R.V. 1999. Food-related illness and death in the United States. 5: 840-842. Pass, M.A., Odedra, R., Batt, R.A. 1999. Multiplex pcrs for identification of Escherichia coli virulance genes. Journal of Clinical Microbiology. 2001-2004. 25 Samadpour, M., Kubler, M., Buck, F.C., Depavia, G.A., Mazengia, E., Stewart, J., Yang, P., Alfi, D. 2001. Prevalence of shiga toxin-producing Escherichia coli in ground beef and cattle feces from King County, Washington. Journal of Food Protection. 65: 1322-1325. The medical impact of antimicrobial use in food animals[Internet]. World Health Organization: 1997 Oct. [cited 2008 April 20] pl. Available form http://whqlibdoc.who.int/hq/1997/WHO_EMC_ZOO_97.4.pdf. Tuttle, J., Gomez, T., Doyle, M.P., Wells, J.G., Zhao, T., Tauxe, V., Griffin, P.M. 1999. Lessons from a large outbreak of Escherichia coli O157:H7 infections: Insights into the infectious dose and methods of widespread contamination of hamburger patties. Epidemiology of Infection.122: 185-192. Wang,L. Li, Y., Mustapha, A. 2007. Rapid and simultaneous quantitation of Escherichia coli 0157:H7, Salmonella, and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation. Journal of Food Protection. 70: 1366-1372. 26