Identification of the Sources of Kanamycin Resistant Bacteria in Chicken Farms Melissa Luyster Bio 110 Section 034 Introduction Antibiotics have been used in poultry farming since the 1940s They are added to chicken feed to produce healthier chickens and prevent the invasion of harmful bacteria into the products However it was not long before the overuse of certain antibiotics caused the populations of chickens to become resistant to these antibiotics One of the antibiotics most commonly used is kanamycin Kanamycin kills bacteria as a result of interference with protein synthesis during bacterial growth An outbreak of kanamycin resistance has occurred in the three farms Tucker s Cluckers Lucky s Eggs and Mia s Egg cellent eggs Scientists know that it are three genes responsible for the kanamycin resistance Each different gene codes for a different enzyme This enzyme chemically alters the kanamycin molecule in different ways The farms are geographically separate from each other thus it is important to know whether they share a common source for the contamination or whether each occurrence of resistant bacteria is unique to each farm Bacterial plasmids are small circular extra chromosomal elements of DNA molecules found in many bacteria They can replicate separate from the host They also may carry genes that provide resistance to certain antibiotics The antibiotic resistance genes are found on these plasmids The research questions for the following investigation are as follows Is the bacterial contamination at these three farms due to the same gene or different genes What is the frequency of kanamycin resistant bacteria in the chicken farm cultures These questions will be answered through the procedure of the experiment To identify the strain of resistant bacteria samples from each of the three farms were collected Petri dishes were coated with the bacteria to create a lawn and allow the colonies to develop Once they were developed they needed to be serially diluted to make them manageable and countable After they were serially diluted and counted another process was performed to compare the DNA strains of the farms PCR was performed to obtain a large amount of DNA for gel electrophoresis Gel electrophoresis uses an agarose gel and an electric field to separate DNA fragments by size A DNA ladder was used as a molecular size scale to compare the fragments to known lengths The larger fragments moved slower than the smaller fragments which migrated toward the positive electrode in the apparatus Gel electrophoresis was run and the gels were observed and photographed under a UV light The purpose of this experiment was to determine the source of an outbreak of gastroenteritis linked to consumption of raw or partially cooked eggs that were linked to the three chicken farms we investigated Tucker s Cluckers Lucky s Eggs and Mia s Egg cellent Eggs It was determined whether the kanamycin resistant bacteria in the outbreaks came from the same farm or source or if they come from the same gene that causes resistance In addition the kanamycin resistance frequency needed to be found to prevent future contamination or outbreaks Methods And Materials In week 1 of the investigation a sample of bacteria from one of the farms was serially diluted to a lower concentration to make a more manageable observation The three 100 fold serial dilutions 10 2 10 4 and 10 6 were labeled with their dilution factor on separate plates Using a sterile technique 990 l of water was mixed with 10 l of the sample in a microtube to create a 10 2 dilution This process was repeated for the 10 4 and 10 6 dilutions to create three dilutions of the chicken farm sample Sterile glass beads were used to spread the bacteria evenly over the agar surface on the plates The dishes were then inverted stacked and incubated for 24 hours for the colonies to develop After the colonies were incubated the plates were examined for bacterial colonies The plates that contained individual bacterial colonies and could be countable were recorded In week 2 of the investigation a Polymerase Chain Reaction was performed to determine whether the three farms share a common form of resistant bacteria or whether each farm has a unique strain of resistant bacteria Three tubes containing control plasmids and DNA primers as well as three tubes containing bacterial samples from three different colonies and DNA primers were put through PCR PCR amplifies DNA and produces millions of copies of a DNA sequence Thus after PCR enough DNA is produced to run agarose gel electrophoresis Table 1 The following table summarizes what is in each of the tubes While the PCR reaction was running the numbers of bacteria colonies growing on plates with and without kanamycin were counted and recorded Also while the PCR reaction was running the gels for agarose gel electrophoresis were prepared To prepare the gel 300 mg of agarose were mixed with 30 mls of 1X TAE buffer in a 125 ml Erlenmeyer flask The flask was heated for 35 seconds cooled and 1 l of ethidium bromide dye was added The molten agarose was then poured into the gel apparatus to harden Electrode buffer was poured into the unit as well As a molecular size standard 5 microl of PCR DNA ladder was loaded into the first well Next 2 l of 6x loading dye was added to each of the six tubes containing the samples 15 l of each of the colored tubes was loaded into the next six wells of the gel The apparatus was then turned on allowing the electric field to separate the DNA fragments by size Larger fragments have slower migration than smaller fragments After the gel was electrophoresed the gel was observed under a UV light and photographed to observe Table 2 The table below gives a summary of what was loaded into each of the wells of the the migrated DNA strands agarose gel Results Table 3 Numbers of bacterial colonies on kanamycin dilution plates Treatment Volume plated Kanamycin No Kanamycin 10 2 100 l 26 Lawn Dilutions 10 4 100 l 308 720 10 6 100 l 13 21 Formula to calculate the original number of colonies B n D B original number of colonies per 100 l n number of counted colonies at a certain dilution D Dilution factor 10 4 dilution with kanamycin B 308 10 4 x 10 1 308 10 5 308 x 105 10 4 without kanamycin B 720 10 4 x 10 1 720 10 5 720 x 105 Formula for calculating Antibiotic Resistance Frequency of Resistant Bacteria Colonies x 100 of Non Resistant Bacteria colonies 5 419 x 100 41 9 308 x 10 720 x 105 Table 4 Antibiotic