Experiment To Observe Antibiotic Resistant Bacteria Biology Essay

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An experiment was conducted to observe the differences in amounts of antibiotic resistant bacteria obtained from 3 East Lansing locations, Burger King, Meijer, and Déjà Vu. A hypothesis was developed which stated that the bathrooms would have more bacteria which is resistant to antibiotics, meaning there would be difference between the two areas, with a null of there being no significant difference between the two. This was due to the idea that bathrooms are constantly exposed to antibiotics, whereas the trafficked areas are rarely exposed to antibiotics in comparison. A sample was taken from each bathroom, as well as a sample taken from a highly trafficked area in that location. These locations were swabbed onto growth plates to grow a colony and then transferred onto plates with Ampicillin, Tetracycline, and Kanamycin to test for resistance to antibiotics. A gram stain, KOH test, MacConkey, and EMB plate tested gram-positivity or negativity, gel electrophoresis tested for plasmids and cut DNA length, PCR tested for what bacteria was present, and restriction digestion tested for addition of antibiotic resistant bacteria. Conclusions show that there was no significant difference in the amount of antibiotic resistant bacteria grown in bathrooms versus highly trafficked areas, and the types of bacteria have not gotten results at this time.

Introduction

With antibiotic resistant bacteria becoming increasingly hazardous, it is important to understand where the bacteria tend to conjugate. The purpose of this experiment is to answer this question by determining whether areas of heavy human traffic at popular locals or the bathrooms at those locals house more antibiotic resistant bacteria.

Independent variables of the experiment include anything that the researchers have control over. Independent variables of this experiment are the locations chosen, the temperature the samples were incubated at (37 degrees Celsius), number of plates made, whether the bacteria is gram positive or gram negative, the antibiotic that the bacteria were exposed to, and the amount of time they were incubated for. Dependent variables are variables that change depending on the independent variables. For this experiment that would include amount of bacteria grown, the antibiotic resistance, and how the bacteria separates in the gel electrophoresis.

In modern society, antibiotics are a common commodity and are often given out without just cause. They were often thought of as a cure all drug and given out to anyone who it could help, as well as those who could just not accept being sick without a cure (Campbell et al. 2008). Moreover, when given these antibiotics for true medical reasons, people may not take the full dose of the prescription and stop treatment at a stage where the bacteria are not completely killed. The bacteria that are still alive are more resistant to the antibiotics that they have been exposed to. Moreover, they can spread their resistance easily through a process called conjugation. The bacteria that already house the antibiotic resistant DNA can use a structure called the sex pilus to form a bridge with bacteria that does not have the resistance (Campbell et al. 2008). The donor bacteria can give the non-resistant bacteria a resistance plasmid, which binds to its DNA, making it more resistant to antibiotic threats. This increases the amount of antibiotic resistant bacteria in the world. Locations constantly exposed to antibiotics are finding that the antibiotics are no longer working. The constant exposure to antibiotics has lead to resistant forms of the bacteria living on and reproducing (Campbell et al. 2008). Due to this issue, it has come crucial to understand antibiotic resistant bacteria and where they are commonly located.

Bathrooms have a better resistance to antibiotics than the highly trafficked areas due to continual exposure to antibiotic cleaning products and human wastes that are laced with antibiotics due to their availability. Since humans are continually taking in antibiotics, their waste is filled with antibiotics. The antibiotic waste is a constant source of natural selection for bacteria in restrooms, causing the weak to die out and the strong to live on. Therefore, it can be inferred that bathrooms would have more exposure to antibiotics than heavily trafficked areas, leading to increased levels antibiotic resistant bacteria.

Finally, aims of his experiment are to determine the magnitude of bacteria grown in each environment, and to determine which types of bacteria are present.

This experiment is being held to see between areas of heavy human traffic and contact, such as a bar, a shelf, or a play place for children, and bathrooms constantly exposed to cleaning and therefore antibiotics, which area had more antibiotic resistance.

Independent variables of the experiment include anything that the researchers have control over. Independent variables of this experiment are the locations chosen, the temperature the samples were incubated at (37 degrees Celsius), number of plates made, the antibiotic that the bacteria were exposed to, and the amount of time they were incubated for. Dependent variables are variables that change depending on the independent variables. For this experiment that would include amount of bacteria grown, the antibiotic resistance, whether the bacteria is gram positive or gram negative, and how the bacteria separates in the gel electrophoresis.

In modern society, antibiotics are a common commodity and are often given out without just cause. They were often thought of as a cure all drug and given out to anyone who it could help, as well as those who could just not accept being sick without a cure. Moreover, when given these antibiotics for true medical reasons, people may not take the full dose of the prescription and stop treatment at a stage where the bacteria are not completely killed. The bacteria that are still alive are more resistant to the antibiotics that they have been exposed to. They can spread their resistance easily, and if the same medical issue that they caused arises again, it may be much harder to cure.

This increases the amount of antibiotic resistant bacteria in the world. Locations constantly exposed to antibiotics are finding themselves facing the problem of the antibiotics no longer working. Due to this issue, it has come crucial to understand antibiotic resistant bacteria and where they are commonly located.

Bathrooms have a better resistance to antibiotics than the highly trafficked areas due to continual exposure to antibiotic cleaning products. Also, since humans are continually taking in antibiotics and selecting for antibiotic resistance in their immune systems, the bathrooms would have more exposure to antibiotic resistant bacteria excreted in human waste. Therefore, this would produce a null of no difference between levels of bacteria.

Finally, aims of his experiment are to determine the magnitude of bacteria grown in each environment, and to determine which types of bacteria are present.

Methods

First, environmental samples of bacteria were obtained. This was carried out by taking a sterile swab with a sterile solution of phosphate buffered saline (PBS), and passing it over the surface where the bacteria were hoping to be obtained. The swabs were then taken into lab and swabbed onto agar only plates. Lawns of bacteria were grown after being incubated for 24 hours, and from these plates, more agar only plates were made from these and used as master plates. Once these were grown, colonies were taken and put onto patch plates, each containing an antibiotic including Kanamycin, Ampicillin and Tetracycline, and also a plate containing only LB base, which was used for a control. Individual colonies were grown in each of the 16 sections that the plates had been divided into, and these colonies were resistant to the antibiotic that was in the plate that they were grown on. To ensure that these plates stayed healthy, they were redone every 2 weeks. These were incubated for 24 hours, and then counted to analyze the antibiotic resistance of the bacteria from each environment.

Next, Gram stains were preformed. A colony of bacteria was diluted in water and placed onto a slide. To adhere the bacteria onto the slide, it was passed over a slide 2 to 3 times. The slide was then flooded with crystal violet for 60 seconds and rinsed with water. It was then flooded with iodine for 60 seconds and rinsed with ethanol. After, it was flooded with safranin as a counter stain and rinsed with water. The slide was air-dried. Immersion oil was placed on the slide and the stain was viewed at 100x magnification.

The KOH test was also conducted to determine if the bacteria was Gram positive or Gram negative. Bacteria was placed on a microscope slide and exposed to KOH. A metal hoop was touched to the mixture of bacteria and KOH. If the mixture stuck to the hoop, then it is Gram negative, if it did not stick to the hoop, it is gram positive.

Gel electrophoresis was used to separate DNA nucleotides into bands. The gel was made out MacConkey Agar, which when cooled, forms pores in the matrix to let the DNA run through when exposed to an electric current. To make the gel, either 40mL or 60mL of the TBE is mixed with either .4g or .6g of agarose respectively. These were heated, mixed together, and left to cool. Following, 2l of ethidium bromide was added. The DNA ran from the negative end of the cell to the positive end and formed columns of bands with specific lengths.

Plasmids were isolated from the environmental samples using a miniprep protocol. After antibiotic resistance was developed, the colonies were converted into liquid cultures. These cultures were incubated for 24 hours. After incubation, 10 ml of the sample were placed in a centrifuge, spun, and the bacteria were separated from the solution. It was then re-suspended and lysed to remove genetic material and proteins. The sample was spun again, and the plasmids were drained and put into a spin column. Using nuclease free water, plasmids were removed from the spin column into a centrifuge tube. The isolated plasmids are then run through a gel electrophoresis, and using the NEB cutter V2.0 program from New England Biolabs, a plasmid map can be produced. After the plasmid was produced, it was run through gel electrophoresis and shown under UV light to confirm the procedure worked.

To inoculate liquid media, the following procedure was followed. 5mL tubes with liquid medium were obtained. Next, 5 L of appropriate antibiotic were added. With a sterilized loop, a single colony from an antibiotic streak plate was taken and added to the mixture. Finally, the tube was capped and placed in the shaker for 24 hours.

After plasmids were isolated, in this case only the blue control, the control plasmids and plasmids from competent E. coli cells were taken and a heat shock procedure was used to force the bacteria to obtain the plasmid. Three samples, two containing plasmids from blue control and from E. coli, and one with P. Litmus and LB only plates as controls, were heat shocked for ninety seconds, then mixed with warm LB broth and incubated. The samples were then spread onto an LB and antibiotic plate to determine resistance transferred from one bacterium to another.

A restriction digest was completed as follows. 10 l of the restriction enzyme, in this case Ava 1 was chosen, and 20l of the DNA from the miniprep were mixed. Next, 3l of the buffer number 4 were added to the mixture. 100l of BSA was added last. This concentration was then incubated for an hour. 3.3l of gel loading buffer was added after the incubation right into the reaction. The reaction was then stored in the freezer until we were ready to run the gel. A gel electrophoresis was run to determine the results of the digest.

Results

Written by Stacy Tipton

The experiment began with gathering two bacteria samples from three different locations. All bacteria were then grown on LB only plates, which served as master plates for the bacteria in the experiment (Figure 1). All bacteria for the experiment were taken ultimately from these plates. Bacteria from these plates were then grown onto patch plates that contained Ampicillin, Tetracycline, and Kanamycin in the LB, so that only bacteria resistant to that antibiotic would grow (Figure 2).

Gram Negative bacterium grows lawns of bacteria on MacConkey plates. Using this test, the Déjà vu bathroom AMP resistant bacteria, and Burger King Play area TET resistant bacteria came out to be Gram negative. The Meijer bathroom AMP resistant bacteria and the Déjà vu bar AMP resistant bacteria came out Gram positive (Figure 3). Gram stain tests for both gram negativity and positivity. Gram-positive stains pink during a Gram stain, while Gram negative stains purple (Figure 4 ). The Déjà vu bathroom came up gram negative, while Meijer bathroom, Déjà vu bar, and Burger King play structure came up gram-positive (Table 1). The KOH test is sticky if it is gram-negative and not sticky for gram-positive bacteria. Déjà vu bathroom came up gram-negative, while the Meijer bathroom, Déjà vu bar, and Burger King play structure came back gram-positive (Table 2).

Mini-preps on cultured bacteria were preformed. The mini-prep isolates plasmids from a bacterium. The mini-preps were then run through a gel electrophoresis. This was to test for plasmids in the bacteria. A ladder was run with the bacteria to identify plasmids, and a control was run to ensure the test worked properly. No plasmids were found in the cultured bacteria (Table 3), and a second gel was run to ensure the result was correct (Figure 9).

EMB plates were run. Gram-negative bacterium grows on EMB agar. A bacterial colony from each cultured antibiotic resistant plate was taken and exposed to the EMB agar. The results were that all colonies exposed to the EMB were gram positive.

A chi square test was conducted. It included 1 degree of freedom, and had a significance value of 0.05. This meant that the Chi square distribution was at 3.84 (Table 4). Results said there was no definite difference in the amount of antibiotic resistant bacteria found in bathrooms than in highly trafficked areas.

A PCR was run to identify the bacteria by reproducing the DNA inside of it and comparing known samples of DNA to the ones being tested. These results have not been completed yet.

A restriction digest was also run, but correct results have not been yielded from this experiment yet. Bacteria has not yet been properly cut and observed on a gel electrophoresis experiment.

Discussion

Written and Revised by Stacy Tipton

There is little difference in the amount of antibiotic resistant bacteria found in bathrooms versus found in highly trafficked areas. It was hypothesized that when the experiment was done to see which environment, the bathrooms or the highly trafficked areas, would have the greatest antibiotic resistance, that the bathroom would have greater resistance. This was due to the theory that the bathrooms are constantly exposed to antibiotics, where as locations such as a store shelf, stripper bar, or play area are rarely if ever cleaned with antibiotics. It was thought that the more the bacteria were exposed to antibiotic resistant bacteria, the more likely it would be that antibiotic resistance was naturally selected for. The results contradict the hypothesis. There was found to be little difference in the amount of antibiotic resistant bacteria in each location. According to the Chi square test, no significant difference between the two locations.

As we knew from previous research, exposure to antibiotics selects for antibiotic resistance. If the antibiotics that the bacteria is exposed to does not kill all bacteria, the strongest bacteria that are still alive reproduce and pass on their resistance both through reproduction and the passing of plasmids. Our findings suggest that when exposed to the same bacteria, it doesn't matter how many times the bacteria are exposed to the antibiotics, once it is selected for, antibiotic resistance stays in the population.

There were specific problems in the research that could have affected the results. First, no plasmids were found in the bacteria. This means that the passing of the resistance via plasmid spreading could not be tested. Also, several experiments had to be done repeatedly to get results. This means that the exact same samples were not used, but reproduced as exactly as possible. However, since there is variation in which colonies were tested in each experiment, it is possible that the results are skewed somewhat.

There are several options for further studies in this experiment. Further experiments could be used to find plasmids in the bacteria. One could go farther to see if the antibiotics that were used were supposed to kill the bacteria found. Also, one could experiment to find out if the bacteria were resistant to other antibiotics that use mechanisms similar to the ones tested.

References

Written and Revised by Stacy Tipton

Campbell, N.A., J.B. Reece, L.A.Urry, M.L. Cain, S.A. Wasserman, P.V. Minorsky, and R.B. Jackson. 2008. Biology 8th ed. Pearson Education, Upper Saddle River, NJ

Cognato, A. 2010. Recitatation_031710 PowerPoint.  Pages 1-10.

Lu, H., X. Wang , X. Lang, Y. Wang, Y. Dang, F. Zhang, J. Tang, X. Li, X. Feng. 2009. Preparation and application of microarrays for the detection of antibiotic resistance genes in samples isolated from Changchun, China. Molecular Biology Reports. Volume 37. 1857-1865.

Rusin, P., P. Orosz-Coughlin, C. Gerba. 1998. Reduction of faecal coliform, coliform and heterotrophic plate count bacteria in the household kitchen and bathroom by disinfection with hypochlorite cleaners. Journal of Applied Microbiology. Volume 85. 819-828.

Figures

Written and Revised by Stacy Tipton

Figure 1: Swab Plates. Each different environment was swabbed and two different locations were taken from each. The bacteria were grown on an LB only master plate before exposed to any antibiotics. Each small dot on the plate represents a different colony of bacteria. These plates served as the master plate for all bacteria in the experiment and all tested bacteria ultimately came from this plate. The small dots throughout the plate in the figure each represent a different colony of bacteria. These could be the same species of bacteria, or there could be several species on one plate due to the fact that each environment the samples were taken from was exposed to all kinds of bacteria. Afterward, these bacteria will be taken and exposed to antibiotics to test for resistance. These bacteria were swabbed from an environment using a swab coated with PBS. A shows Burger King play structure, with individual colonies of bacteria. B shows Burger King bathroom with individual colonies of bacteria. C shows Déjà vu bar with a lawn of bacteria. D shows Déjà vu bathroom with a lawn of bacteria. E shows Meijer bathroom with a lawn of bacteria. F shows Meijer shelf with a lawn of bacteria.

Figure 2: Patch Plates. In the above figure A represents Meijer Bathroom Ampicillin, B represents Meijer Shelf LB only patch plate, C represents Burger King Play Structure, D represents Tetracycline Déjà Vu Bathroom, and E represents Déjà Vu Bar Ampicillin patch plates. Each cultured bacteria was taken from the original master plate and exposed to a different antibiotic plate. Each plate was divided into sixteen sections, and a different colony of bacteria from the same location were placed on each section. This means several different species of bacteria could be on one plate, but did not necessarily have to be. Bacteria grown on these plates are resistant to the antibiotic that is in the LB that they grew on. A, B, C, and F all had 16 colonies of growth, D had 8 colonies of growth. These are important in determining the amount of bacteria that was resistant in each environment so that statistical tests could be run to determine if there was significant difference in the amount of antibiotic resistance in each area. Ampicillin plates contained 100g of antibiotic permL, and Tetracycline and Kanamycin both contained 50g of antibiotic per mL.

Figure 3: MacConkey Plates. MacConkey plates test for gram-positive or gram-negative bacteria. Gram-negative bacteria grow on MacConkey plates, gram-positive bacteria does not. Clockwise from top left: Déjà Vu bathroom, Burger King Play Structure, Meijer Bathroom, and Déjà Vu bar. Only one colony of bacteria was selected to grow onto each plate, and therefore it is only one species of bacteria represented per plate. B and C have growth on the plates, and are therefore gram-negative. The A and D show no growth and are therefore gram-positive.

Figure 4: Gram Stain. B: Déjà Vu Bar AMP Gram Stain, B and C: Burger King Play Structure TET Gram Stain D: Déjà Vu Bathroom AMP Gram Stain, E: Meijer Bathroom AMP Gram Stain. The dark color purple color shows gram-negativity. Gram-negative bacteria lack a thicker cell wall, and have their layer of peptidoglycan more exposed than gram-positive bacteria. Gram stain goes about identifying gram negativity or positivity by coloring the layer under the wall. Since the wall isn't as thick, the dye is able to better stain that layer of peptidoglycan. These are rod-shaped bacteria, otherwise known as bacillus shaped.The pink color illustrated gram positivity. Gram-positive bacteria have a thicker cell wall, which means their layer of peptidoglycan is less exposed to the environment. A gram stain goes about identifying gram negativity or positivity by coloring the layer under the wall. Since the wall is thicker, the dye is not able to stain that layer of peptidoglycan as well, and it shows up as a pink color. This is a lawn of bacteria, mostly coccus (round) shaped, but some are bacilli shaped as well.

Figure 5: Gel Electrophoresis Plasmid Isolation. The gel separates nucleic acids and proteins. Lane 1 shows the 1 KB ladder, which is used to help identify unknown plasmids by showing lanes of known base pairs. Lane 2 illustrates the blue control plasmid. This Plasmid ensures that the gel worked properly. A control of E. Coli bacteria was used. Lanes 3 through 6 show where a gel was run with lanes 1 through 6 loaded using a micropipetter, and where no plasmids were found. These lanes were samples form each of the environmental plates that had already been exposed to antibiotics. Meijer bathroom, Déjà vu bar, and Déjà vu bathroom in lanes 3 through 5 were all from Ampicillin plates. Lane 6 represents the sample from the Burger King play structure that had been exposed to tetracycline. It is obvious no plasmids were found, because there are no bright bands that appear in the gel in lanes 3 through 6. None of the environments had plasmids isolated from them. This was significant because resistance is transferred through plasmids. To make this gel, .4 g of TBE were mixed with 40 mL of agarose. After these cooled, .2L of ethidium bromide was added.

Tables

Written and Revised by Stacy Tipton

Table 1: Gram Positive/Negative Results

 

Gram Stain

KOH Test

Miejer Bathroom

Pos

Pos

déjà vu bathroom

Neg

Neg

Déjà vu bar

Pos

Neg

BK play structure

Neg

Pos

Table 3: Gel Electrophoresis Results

 

Positive*

Negative*

Meijer bathroom

 

X

Déjà Vu bathroom

 

X

Déjà Vu bar

 

X

BK play structure

 

X

*Indicates positive or negative for plasmids

Table 4: Chi Square Test

 

Antibiotic resist. colonies

Non-antibiotic resist. colonies

Total

Bathroom

167

25

192

Highly Trafficked Area

153

39

192

Total

320

64

384

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