The Washington 1 Strain of Escherichia coli does not grow when treated with Kanamycin

Abstract:

A new strain of Escherichia coli bacteria was discovered in a hospital in the Washington area. This strain of the bacteria was resistant to some of the antibiotics commonly used to treat the infection. A plasmid was isolated from the strain. The hypothesis was that the plasmid did not confer resistance to the antibiotics, but this was shown wrong. The plasmid was incorporated into laboratory E. coli cells by transforming them and they also showed resistance to the antibiotics used in the hospital. However, one of the antibiotics tested, kanamycin, was effective in preventing bacteria growth. It is recommended that this antibiotic be used in treatment of further cases.

Introduction:

Escherichia coli is a kind of bacteria that lives in the human intestine. They help to digest food. However, there are many different strains of E.coli. Some of the strains can be harmful. A new strain was discovered in hospitals near Washington that causes severe diarrhea. This strain, Washington 1, is resistant to several of the common antibiotics used to treat the infection.

Testing Washington 1 E. coli directly to find what it is resistant to is not possible. It was determined that the Washington 1 strain of bacteria does not grow well in the lab. Therefore, a different process will take place instead. Before this process is detailed, a little more background information is needed.

There are small circular pieces of DNA called plasmids that contain genes for different proteins. Some plasmids have genes that make enzymes. Some of these enzymes can destroy antibiotic molecules of a specific type near the cell. A cell containing a plasmid can therefore show a resistance to antibiotics, and colonies can grow even when the antibiotics are present.

In this case, a plasmid was isolated from the Washington 1 E. coli. We tested wether the Does the _In this study, we tested to see if the plasmid contained genes for enzymes that digest the antibiotics used in the hospital.? The initial hypothesis was that the new strain of E. coli (Washington 1) has a plasmid in it that confers resistance to some antibiotics. The null hypothesis was “Plasmids isolated from Washington 1 E. coli do not confer immunity to the antibiotics.”

To test the hypothesis, advantage can be_was( always write in passive voice) taken of a special property bacteria have. When heat shocked, they can take up free plasmids. The bacteria then become transformed. After transformation, the cell can use the plasmid DNA to manufacture proteins. If these proteins are enzymes that digest antibiotics, then the transformed cell will show immunity to the antibiotics in the same way as the Washington 1 E. coli.

TO DO: ADD THE REFERENCES TO THIS AND OTHER SECTIONS. ALSO FIND OUT WHY E.COLI IS IMPORTANT IN THE HUMAN BODY. WHAT IS IT GOOD FOR? WHY IS IT IMPORTANT? ALSO CHECK THROUGH THE LAB BOOK TO PUT IN THE REFERENCES FOR THAT TOO.

Materials and Methods:

Exercise 10:

A plasmid was isolated from the Washington 1 strain of E. coli responsible for the outbreak. Two 100-microliter samples of complement E. coli cells were prepared. These samples were thawed and stored on ice to prevent the cells from multiplying. One sample (labeled “-”) was kept as a control group, while the other sample (“+”) had 5-microliters of plasmid solution added to it. The tubes were then allowed to rest for 30 minutes to ensure that the plasmids would mix with the cells in the experiment sample. The cells were then heated to 42°C to heat-shock them and create transient pores on their surfaces. The plasmids that are in the solution with the cells can then enter the bacteria. .This enables the plasmids to gain entry..and force their pores open_. After spending 90 seconds in the high temperature environment, the samples were then placed on ice to force the pores in the cells to close, trapping the plasmids inside.

At this point, 400-microliters of growth media were added to each sample, and the cells were incubated for 20 minutes in a 37°C warm water bath to allow them to multiply. After this incubation period was over, the cells were then placed _plated on petri plates.

100-microliters of each sample containing both transformed cells and normal cells were placed on three different kinds of petri plate containing ampicillin, tetracycline, and kanamycin. These plates were labeled with the name of the antibiotic and the type of E. coli (Transformed or Normal). The petri plates were then placed in a 37°C incubator for 24 hours to allow the E. coli to grow.

Exercises 11 and 12:

To evaluate the quality of the experiment the plasmid DNA was then purified from the cells used in the 10^th exercise. A gel electrophoresis was run on the purified DNA to verify that the plasmid-treated E. coli was in fact transformed.

TO DO: WRITE A BIT MORE ABOUT EXERCISES 11 and 12-ENOUGH SO THAT THERE ARE SOME MECHS DISCUSSED HERE...

Results:

After a 24-hour incubation period elapsed, the petri dishes were examined for growth. The growths are summarized in TABLE 1. When the experiment was repeated several times, the results differed from the initial attempt. The results from these experiments were in TABLE 2.

The electrophoresis gel made in Exercises 11 and 12 is shown in FIGURE 1.

TABLE 1: Initial Petri Plate Media Growth Results

Plate: Color and Label	Antibiotic	Plasmid	Result
yellow: “Media/no drug/P”	none	yes	growth
yellow: “Media/no drug/NP”	none	no	growth
red: “Media/ampicillin/P”	ampicillin	yes	no growth
red: “Media/ampicillin/NP”	ampicillin	no	growth
green: “Media/tetracycline/P”	tetracycline	yes	no growth
green: “Media/tetracycline/NP”	tetracycline	no	no growth
blue: “Media/kanamycin/P”	kanamycin	yes	no growth
blue: “Media/kanamycin/NP”	kanamycin	no	no growth

Each petri plate was prepared with a matrix of Luria Bertani agar agar9this is called luria bertani media…just for your information. treatedmedia treated with the antibiotic indicated in the Antibiotic column of the table. 100 microliters of the E. coli bacteria (either treated with Plasmid (transformed) or not (control)) was added to each plate and carefully spread over the surface with a sterile rod. The plates were then incubated for 24 hours at 37°C, then removed and examined. The Result indicates whether growth was observed or not.

TABLE 2: Final Petri Plate Media Growth Results

Plate: Color and Label	Antibiotic	Plasmid	Result
yellow: “Media/no drug/P”	none	yes	growth
yellow: “Media/no drug/NP”	none	no	growth
red: “Media/ampicillin/P”	ampicillin	yes	growth
red: “Media/ampicillin/NP”	ampicillin	no	no growth
green: “Media/tetracycline/P”	tetracycline	yes	growth
green: “Media/tetracycline/NP”	tetracycline	no	no growth
blue: “Media/kanamycin/P”	kanamycin	yes	no growth
blue: “Media/kanamycin/NP”	kanamycin	no	no growth

Each petri plate was prepared with a matrix of agar treated with the antibiotic indicated in the Antibiotic column of the table. 100 microliters of the E. coli bacteria (either treated with Plasmid or not) was added to each plate and carefully spread over the surface with a sterile rod. The plates were then incubated for 24 hours at 37°C, then removed and examined. The Result indicates whether growth was observed or not.

Figure 1: The Electrophoresis Gel

This matrix of agar gel was prepared for experiment 12.

The wells were initially prepared with samples set up as follows:

K: a 5-microliter sample of the plasmid taken directly from Washington 1 E. coli.

The other four wells contained the purified plasmid DNA extracted from the E. coli colonies grown during experiment 10.

C_52: 52 microliters of the control sample.

C_155: 15 microliters of the control sample.

T2change this too5: 52 microliters of the transformed sample.

T15: 15 microliters of the transformed sample.

The gel was then placed in the electrophoresis chamber for 30 minutes to allow the DNA to migrate through the matrix. When the time was up, the gel was removed from the chamber and placed in a visualizing chamber. It showed the sets of three bands (about 2/3 of the way down the gel).

Discussion:

The initial results from the E. coli’s treatment with different antibiotics (see Table 1) differed from the results obtained when the experiment was repeated. This could have been caused by several different things. Possibilities include cross-contamination of the bacteria used in the experiment, or possibly the petri plates were mislabeled. Alternatively, the spreader used to form an even layer of cells on the agar matrix might have been too hot, sterilizing the plates. A fourth possibility is that the concentrations of antibiotics were too high for the transformed bacteria to digest.

The results obtained from repeated experimentation (see Table 2) show clearly that the transformed bacteria are growing in environments that the control strain of E. coli cannot survive in. The transformed bacteria showed growth on both the ampicillin petri plate and the tetracycline plate, while the control bacteria did not grow on either one. This means that the transformed E. coli is resistant to the two antibiotics because of the proteins represented on the plasmid.

This fact was further verified by purifying the plasma from the transformed bacteria. Although some of the plasmids were recovered from the transformed bacteria, the control sample did not contain any. This reinforces the hypothesis that the plasmid is indeed the cause of the drug resistance.

The technique that we used to recover the plasmids from the bacteria was called Alkalilysis. It takes advantage of the difference in structure between the plasmids and the bacteria’s chromosomal DNA. While plasmids are usually in a supercoiled closed covalent state, chromosomal DNA is in a linear structure. This makes the chromosomal DNA much more sensitive to changes in pH than the plasmids. In Alkalilysis advantage is taken of this property.

After separation, the plasmid DNA was run in a Gel Electrophoresis chamber for forty-five minutes with a sample of the plasmid taken from Washington 1 E. coli cells. The results (see Figure 1) show that the plasmid collected from the lab bacteria is the same as the one found in Washington 1.

Because of these results the null hypothesis, “Plasmids isolated from Washington 1 E. coli do not confer resistance to the antibiotics,” was shown to be false. The plasmids isolated from the Washington 1 E. coli are the reason that the cells are resistant to the action of ampicillin and tetracycline.

Looking at the results of the bacteria growth part of the experiment (Table 2), neither the transformed nor the control E. coli showed any growth in the media treated with the antibiotic kanamycin. This shows that the plasmid does not confer resistance to kanamycin, so this drug is the one to give to the patients in the hospital. As long as the Washington 1 strain of E. coli behaves in the same manner (i.e. dies) as the strain used in the lab, a course of kanamycin should be effective in treating the infection.

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