Plasmid Isolation and Attempted Identification of Kanamycin Resistant Bacteria

By Jessica Levely

Cell and Molecular Biology – LBS 145

Dr. Luckie

4/29/2005

Abstract

The discovery of antibiotics has been very beneficial to medicine and the sciences (Upshall, 1998). However, many strains of bacteria are becoming resistant to certain antibiotics (Anonymous 1, unknown). Our experiment involved finding a strain of bacteria resistant to the ampicillin, tetracycline or kanamycin. It was predicted that resistant bacteria would be found. Since antibiotics are used many places around campus, the only bacteria still growing will be antibiotic resistant. It was also predicted that the plasmid of the resistant bacteria would be able to be identified by gel electrophoresis. Kanamycin and ampicillin resistant bacteria was found in all locations. The kanamycin resistant bacteria from the bathroom sink was used for the experiment. The resistant bacteria was the isolated by growing it on an antibiotic agar plate. The colonies resistant to the antibiotic thrived while those not resistant to the bacteria died out. One type of bacteria was then isolated by taking one colony growing on the antibiotic and streaking it onto another plate. The bacteria was then lysed to extract the DNA. Lysis was run three times and was positive for DNA the first and third time. Restriction digest was then used to generate smaller fragments of DNA. This was done three times with restriction enzymes Pst I and Hind III, Eco RI and Hind III and Bam HI and Hind III. Then the unknown plasmid was run through gel electrophoresis. None of the restriction digests were successful. The plasmid was not identified contrary to the prediction.

Introduction

When antibiotics such as penicillin were discovered they were an answer to many commonly occurring illnesses that otherwise would be life threatening, such as strep throat (Upshall, 1998). A growing problem in the medical field is antibiotic resistant bacteria. Due to natural selection and mutations, bacteria are beginning to become antibiotic resistant, thus rendering them useless to human infections (Upshall, 1998).

Bacteria can be antibiotic resistant because of extra-chromosomal DNA called a plasmid. These plasmids can protect the cell from extreme environments, such as antibiotic, by either attacking the formation of the bacterial cell wall or attacking mechanism involved in the reproduction of the cell. Or the plasmid can affect the uptake of the bacteria into the cell (Anonymous -1, 2005). Bacteria can double in population approximately every 20 minutes (Upshall, 1998). When antibacterial is used, resistant bacteria become more prevalent in the population. The nonresistant bacteria are eradicated from the environment and the resistant bacteria can thrive, as it no longer has competition. In addition resistance can spread when plasmids are passed from one bacterium to another (Upshall, 1998). A recent discovery determined that plasmids can be passed to bacteria of different species, thus spreading antibiotic resistance at a faster rate than previously understood (Bal, M. and Bhakta, M., 2002).

Many studies have been conducted to test the antibiotic resistance of bacteria plasmids. For example Gill et al, showed the plasmid of lactic acid bacteria is transmissible to other bacteria; it was previously thought otherwise. This discovery was important due to the fact that this type of gene transfer is very detrimental to the human gut (Gill, 2004). Another study showed that the plasmids of Enterococcus spp. can be resistant to many antibiotics including penicillin, which is commonly prescribed for infection. The multiple resistant strain caused a problem in Turkish hospitals by causing infections in patients (Coleri, A. et al, 2004).

Places that frequently use antibacterial, such as hospitals, are increasing the risk of antibacterial resistance. Using antibiotic on bacteria that is resistant to multiple antibiotics is ineffective in killing it and isolates the resistant bacteria. This is an especially dangerous situation in hospitals, where many people are already dealing with weak immune systems and are more susceptible to infection. Many people believe that new treatments and new drugs are needed to combat the problem of antibiotic resistant bacteria (Upshall, 1998).

In this experiment, bacteria samples were collected from various locations in Holmes Residence Hall at Michigan State University. These locations included a bathroom sink in room 261 on the east wing of Holmes Hall, a toilet in the same room, the door handle at the front entrance, the handrail on the stairs on the 5th floor landing, the 5th floor trash chute, a students athletic bag and the inside of a student’s shoe and a student’s computer keyboard. It was hypothesized that antibiotic resistant bacteria would be found in these places, because they are high traffic areas that many people frequently come in contact with. It was also hypothesized that the plasmids contained in these resistant bacteria would be identifiable, by implementing restriction digest and gel electrophoresis generating a banding pattern and characterizing it by comparing the pattern to those of known plasmids. This is because plasmids resistant should be cut the same way by restriction enzymes (Anonymous-3, 2002).

It is important that the scientific community studies and understands antibiotic resistant bacteria, as it could be a danger to health. In addition it is important that the general community be educated to understand the consequences antibacterial in everyday use. It is also important that antibiotics taken as medication for various infection be prescribed in moderation by physicians, in order to allow antibiotics to continue to be a possible solution to health problems (Upshall, 1998).

Methods and Materials

Isolation of resistant bacteria

The materials used for this experiment included cotton swabs, LB, agar, ampicillin, kanamycin, tetracycline, GTE, Lysis buffer, KOAc, Ethanol, Tris/EDTA buffer and RNAase. Also restriction enzymes Hind III, Pst I, Eco RI, Bam HI and KGB buffer were used. To begin the experiment cotton swabs were used to obtain bacteria samples from areas around Holmes Hall (Michigan State University, East Lansing MI) including; a bathroom sink and toilet in a dorm room (261 East Holmes), the door handle at the front entrance, the handrail on the stairs on the 5th floor landing, the 5th floor trash chute, a students athletic bag, a student’s computer keyboard and the inside of a student’s shoe. The bacteria swabs were placed in test tubes with 5mL of LB broth and was agitated and allowed to incubate at 33.4° C for 24 hours. The LB was prepared with 20g of LB dissolved in one liter of water and autoclaved at 248°F at 15 psi for 15 minutes. Then a sample of each bacteria culture was transferred to test tubes containing 6 μL ampicillin or kanamycin and 3mL LB. These cultures were agitated and incubated at 33.4° C for 24 hours. The bacteria samples were then transferred to agar plates. The plates were prepared with 1.15g agar base and 32mL of distilled water per plate. The solution was autoclaved at 248°F at 15 psi for 15 minutes. Then 64μL of ampicillin or kanamycin was added, and control plates without antibiotic were made as well. The plates were poured and allowed to cool in the refrigerator over night. Then 100μL of bacteria was added to each plate and spread across the surface with a sterilized bent glass rod. Each sample was put on a control plate, a plate containing ampicillin and a plate containing kanamycin. The plates were incubated for 24 hours at 33.4°C. The plates were then inverted, Para filmed and stored in the refrigerator. The bacteria taken from the sink, and resistant to the kanamycin is the only sample used from now on. Three single bacteria colonies were taken from the plate with a sterilized metal loop and streaked onto fresh plates containing the kanamycin. The plates were prepared as stated above. The plates were incubated for 24 hours at 33.4° C. The plates were then inverted, Para filmed and stored in the refrigerator.

Plasmid Extraction:

Three single colonies from the streaked plates were put into three separate test tubes containing 3mL LB broth and 6 μL kanamycin and was agitated and allowed to incubate at 33.4° C for 24 hours. The bacteria were then lysed following the procedures on page 211-215 of the LBS 145 lab manual. Gel electrophoresis was then run to ensure the presence of DNA. The Gel was prepared according to the methods on page 231-235 of the LBS 145 lab manual. Rubber stoppers were used in lieu of autoclave tape. The gel was run for 20 minutes at 130 volts and then placed on a UV light. This was repeated 3 times.

Plasmid Identification:

Restriction digest was done using three treatments. The first was 16μL of DNA, 2μL of KGB buffer, 1μL of restriction enzyme (PST I) and 1μL of double distilled water, the second treatment was 16μL of DNA, 2μL of KGB buffer, 2μL of restriction enzyme (1 of PST I and one of Hind III), and the third was 16(L of DNA, 2μL of KGB buffer, 1μL of restriction enzyme (Hind III) and 1μL of double distilled water (Table 1). The samples were incubated at 37° C for 2 hours. The samples were then run through gel electrophoresis, with a 1Kb ladder on the same gel, for 30 minutes at 84 volts and 15 minutes at 94 volts. The gel was prepared with the same methods used previously. Restriction digest was done a second time using three different treatments. The first was 16μL of DNA, 2μL of KGB buffer, 1μL of restriction enzyme (Eco RI) and 1μL of double distilled water, the second treatment was 16μL of DNA, 2μL of KGB buffer, 2μL of restriction enzyme (1 of Eco RI and one of Hind III) and the third was 16μL of DNA, 2μL of KGB buffer, 1μL of restriction enzyme (Hind III) and 1μL of double distilled water (Table 1). The samples were incubated at 37° C for 2 hours. The samples were then run through gel electrophoresis, with a Hind III ladder on the same gel, for 30 minutes at 84 volts and 15 minutes at 94 volts. Restriction digest was done a third time using three different treatments. The first was 16μL of DNA, 2μL of KGB buffer, 1μL of restriction enzyme (Bam HI) and 1μL of double distilled water, the second treatment was 16μL of DNA, 2μL of KGB buffer, 2μL of restriction enzyme (1 of Bam HI and one of Hind III) and the third was 16μL of DNA, 2μL of KGB buffer, 1μL of restriction enzyme (Hind III) and 1μL of double distilled water (Table 1). The samples were incubated at 37° C for 2 hours. The samples were then run through gel electrophoresis, with a Hind III ladder on the same gel, for 30 minutes at 84 volts and 15 minutes at 94 volts.

Results

Isolation of Resistant Bacteria:

There was bacteria growth in each place tested; a bathroom sink and toilet in a dorm room, the door handle at the front entrance, the handrail on the stairs on the 5th floor landing, the 5th floor trash chute, a students athletic bag, a student’s computer keyboard and the inside of a student’s shoe. The sink bacteria sample was selected for further research (Figure 1). A sample of the bacteria was put in test tubes containing kanamycin and LB, positive control (bacteria and LB) and a negative control (LB). Bacteria grew in all but the negative control (Figure 2). These bacteria were allowed to grow on an agar plate containing kanamycin and a control plate containing only agar. Bacteria grew in both (Figure 3). Another plate containing kanamycin was streaked with one colony from the original kanamycin plate and colonies grew (Figure 3). One colony was taken from the plate and grown in LB and kanamycin, ampicillin or tetracycline. Positive and negative controls were also used (bacteria and LB and only LB respectively). Bacteria grew in only the positive control and kanamycin (Figure 4).

Identification of Resistant Bacteria Plasmids:

Lysis was performed run through gel electrophorsis. There were thick, pink fluorescent bands in two of the lanes when put under a UV light (Figure 5). Restriction digest was done with Pst I and Hind III. There were individual fluorescent bands in the wells where there was KB ladder and one thick band from each sample well (Figure 6). Lysis was performed again and there were no fluorescent bands when put under UV light (Figure 5). Lysis was performed a third time and there were fluorescent bands in each well (Figure 5). Restriction digest was performed with Eco RI and Hind III and no bands appeared in the lanes from the sample wells. There were many bands in the lanes of the ladder (Figure 6). A digest was done, using DNA from a different well from the same lysis, with Hind III and Bam HI and there were no bands from the sample wells (Figure 6). There were bands from the ladder (Figure 6).

Discussion

Isolation of Antibiotic Resistant Bacteria:

Antibacterial resistance is a growing problem in the health care profession. The use of antibiotics in public places contributes to this problem. The antibiotic kills all bacteria not resistant to the antibiotic and allows the bacterium that are resistant to thrive (Upshall, 1998). These resistant bacteria can then reproduce or pass their resistant plasmid on to other species of bacteria thus increasing the resistant population (Anonymous 1, 2004). In this experiment it was predicted that antibiotic resistant bacteria would be found in high traffic areas in Holmes Residence Hall at Michigan State University. The areas tested included a bathroom sink and toilet in a dorm room, the door handle at the front entrance, the handrail on the stairs on the 5th floor landing, the 5th floor trash chute, a students athletic bag and the inside of a student’s shoe and a students computer keyboard. As a lab group we collected and isolated the bacteria. The steps that follow this section, beginning with lysis, were completed independently. The swabs of bacteria were allowed to grow in LB containing kanamycin, ampicillin and just broth. A tube of only the LB was also incubated at the same time, to ensure that the LB had been properly autoclaved and was sterile. Bacteria grew in all but the broth alone. Indicating that there were resistant bacteria in all of these locations and the broth was sterile. The sink bacteria seemed to be the best sample in terms of healthy bacteria (Figure 2), so this sample was used for the rest of the experiment.

The bacteria were allowed to grow on agar plates and colonies formed. One plate had kanamycin in it and the other had only agar and was a control. Bacterial colonies grew on both (Figure 3). It is possible that multiple types of bacteria that are resistant to kanamycin were growing together on the plate. For this reason one bacterial colony was taken from the kanamycin plate and streaked onto another plate with kanamycin in it (Figure 3). This process spread out the bacteria so that each colony of bacteria that grew on the streaked plate contained only one type of bacteria and thus only one type of plasmid.

Plasmid Extraction and Identification:

One colony from the streaked plate was taken and placed in broth and antibiotic. Three antibiotics, kanamycin, ampicillin and tetracycline, were used to determine if there was dual resistance. The bacteria were only resistant to the kanamycin. Bacteria grew also in the control broth containing no antibiotic and did not grow in the tube containing only broth (Figure 4). This indicates that the broth was sterile. This information was helpful in predicting what restriction enzymes would be the best to use later in the experiment. There were three tubes of kanamycin and broth incubated each time so that there were enough resistant bacteria to work with. The bacteria were then lysed to isolate the plasmid DNA. After lysis, a small amount of the DNA was run on a gel to determine if the DNA in fact was present. The first time this was done it appeared that there was possible RNA present. There were fluorescent bands in two of the wells (Figure 5). However there were not individual bands, indicative of DNA, just one large band in each well. DNA would appear in smaller bands that are longer than they are tall. The DNA travels at a rate that is inversely proportional to the log₁₀ of the number of base pairs (Anonymous 2, 2000). The gel must be made accordingly; as the more agarose used the harder it is for large fragments to travel through. The less agarose used the easier it is for smaller fragments to travel quickly and possibly run off the gel (Anonymous 2, 2000) Also circular pieces of DNA travel more rapidly than linear fragments of the same molecular weight (Anonymous 2). All gels in this experiment contained 0.9% agarose and it is possible that a different amount would have resulted in more accurate results. A restriction digest was performed on the sample to determine if it was DNA or RNA. The enzymes Pst I and Hind III were randomly chosen and used for the digest. They were used individually and together (Table 1). This digest did not work (Figure 6). It appeared that there was in fact RNA not DNA present, as there was one band instead of multiple individual bands. It is also most likely that the enzymes were not effective in cutting the strands, as they were chosen randomly.

Lysis was performed again using the same methods since it was difficult to determine where the last process failed. This time there were no fluorescent bands indicating that there was no DNA at all (Anonymous 2, 2000). This is most likely due to human error at some step in the lysis process. Lysis was performed again. This time the methods were slightly changed and twice as much bacteria were used for the lysis. This time the lysis was successful (Figure 5) and DNA appeared in all wells. Wells 2 and 5 (from left to right) appeared to have the strongest bands so they were used in the next digest. This time Eco RI and Hind III were randomly chosen. They were used together and alone (Table 1). This digest was also unsuccessful (Figure 6). The ladder in the last well on the right and in the well next to it there appears to be some bands, however they are identical to the ladder so it is most likely that something went wrong in the preparation of the gel or the loading of the gel so that the ladder some how moved into the adjoining well. Since the ladder worked, the concentration of agarose in the gel was not the problem (Figure 6). The enzymes were probably not effective because they were chosen randomly.

Since this digest did not work, the DNA in well one, from the third lysis, was used for a digest (Figure 5). As shown earlier the plasmid was only resistant to kanamycin, not ampicillin or tetracycline. This plasmid can then be compared to pKAN, which is known to be resistant to kanamycin and not ampicillin or tetracycline (Anonymous - 3, 2002). The enzymes were chosen by analyzing the plasmid map of pKAN (Figure 7). This map showed that Hind III and Bam HI cut the pKAN (total of 4194 base pairs) at 234 and 2095 respectively. This would result in fragment sizes of approximately 1,861 and 2,333 base pairs. The enzymes were used together and alone (Table 1). The digest was unsuccessful and there was no fluorescence on the gel (Figure 6). The ladder appeared as it should, indicating that there was no problem with the gel itself.

One thing that is most likely a major problem in the design of this experiment is the purity of the DNA. One way to purify plasmid DNA is equilibrium centrifugation in CsCl – ethidium bromide (Krha et al, 2005). The fact that the DNA was acquired from a swab of unknown bacteria and the purification step was skipped is very possible explanation for the failure of the identification portion of this experiment.

References

1) Anonymous – 1. Unknown. Antibiotic Resistance. http://www.biochem.ucl.ac.uk/bsm/PLASMID/AntibioticR.htm/ Accessed 4/19/2005.

2) Anonymous – 2. 2000. Gel Electrophoresis of DNA and RNA. http://arbl.cvmbx.colostate.edu/hbooks/genetics/biotech/gels/

3) Anonymous – 3. 2002. Recombinant DNA: Dual Antibiotic Resistant Genes. http://www.biotech.iastate.edu/lab_protocols/DNA_Transformation_recom.html/

4) Bal M, and Bhakta M. 2002. Identification and Characterization of a Shuttle Plasmid with Antibiotic Resistance Gene from Staphylococcus aureus. Current Microbiology 46: 413-417

5) Coleri A, et al. 2004. Determination of Antibiotic Resistance and Resistance of plasmids of Clinical Enterococcus species. Journal of General and Applied Microbiology 50 (4): 213-219

6) Gill et al. 2004. Antibiotic Susceptibiolity Profiles of New Probiotic Lactobacillus and Bifidobacterium strains. International Journal of Food Micorbiology 98(2): 211-217

7) Krha, et al. 2005. LBS-145 Cell and Molecular Biology; Lecture/Lab Spring 2005 Course Packet

6) Upshall, R. 1998. Antibiotic Resistance. http://www.whingiel.co.uk/MW2/Page1.html/ Accessed 4/19/2005

Figure 1. The above gels are from restriction digest. (A) Well 1 (from left to right) contains the digest with Pst I, 2 contains the digest with Pst I and Hind III and 3 is only Hind III. Well 4 contains a Kb ladder. Wells 5-7 contain a second trial of the same treatments and well 8 contains Kb ladder. The digest was not successful as there should be individual spread out bands for the digest. (B) Well 1 (from left to right) contains the digest with Eco RI, 2 contains the digest with Eco RI and Hind III and 3 is only Hind III. Well 4 contains a Kb ladder. Wells 5-7 contain a second trial of the same treatments and well 8 contains Kb ladder. This digest was unsuccessful as there was no movement of DNA at all through the gel. (C) Well 1 (from left to right) contains the digest with Bam HI, 2 contains the digest with Bam HI and Hind III and 3 is only Hind III. Well 4 contains a Kb ladder. Wells 5-7 contain another students experiment. This digest was unsuccessful as there was no movement of DNA at all through the gel.

Figure 7. The plasmid map pKAN. The total number of base pairs is 4194. This map only shows where Hind II an Bam HI would cut, as these were the enzymes used together in a digest. However there are many more enzymes that will cleave this plasmid. The cutting results in fragments of 1,861 and 2,333 base pairs. Each section around the circumference represents a fragment of DNA and the length is representative of the number of base pairs in each fragment. This map was used to help pick the enzymes used in the digest with the unknown plasmid, since it is also resistant to kanmycin.

Table 1. The Amounts of enzymes, buffer, DNA and water used in restriction digest. Different enzymes were used for each trial of restriction digest. The total volume of the digest was 20μL. Water was used as filler when the rest of the ingredients to not equal this volume. Each enzyme was used alone and with another enzyme. This was done in order to determine exactly where each enzyme cleaved the plasmid. None of the above digest were successful in this experiment.

Trial	Enzyme (μL)	DNA (μL)	KB Buffer (μL)	Water (μL)
1	1 Pst I	16	2	1
1	1 Pst I and 1 Hind III	16	2	0
1	1 Hind III	16	2	1
2	1 Eco RI	16	2	1
2	1 Eco RI and 1 Hind III	16	2	0
2	1 Hind III	16	2	1
3	1 Hind III	16	2	1
3	1 Hind III and 1 Bam HI	16	2	0
3	1 Ban HI	16	2	1