Study of Drug Resistance in Bacteria Using Antibioti

A Study of the Presence of Antibiotic Resistant Bacteria in Fitness
Centers and Their Susceptibility to a Variety of Antibiotics

Jill Schroeder
Latta High School
Abstract:
The purpose of this project was to see if there was a presence of antibiotic
resistant bacteria in fitness centers and also to determine their susceptibility to a
variety of antibiotics. The hypothesis was that there would be a significant amount of
the bacteria encountered in fitness centers and they would exhibit a degree of
resistance. Also, it was further hypothesized that greater numbers of bacteria would
be resistant to penicillin, since it is the oldest known antibiotic.
Ten machines were swabbed from the fitness center and then the bacteria
were swabbed onto LB plates. Then five colonies were chosen from each plate and
cultured. The cultures were then put onto LB plates and antibiotic disks from six
different antibiotics were placed onto the plates.
After 24 hours the zones of inhibition from the antibiotic disks were measured
and classified as resistant, intermediate, and susceptible according to the zone radii.
The results showed that there were more colonies resistant to the penicillin and
ampicillin as was hypothesized.
Introduction:
Imagine contracting something and doctors are frantically trying to figure out
what the source of it is. Eventually, they determine it is an infection caused by
antibiotic resistant bacteria. Now what? Where does one go from there? It is not as if
the doctors can pick from an array of antibiotics for a cure. These questions among
others go through a persons head when diagnosed with an infection which is
antibiotic resistant disease. The most powerful antibiotic used as a last resort is
Vancomycin. What happens, however, if the bacteria become resistant to
Vancomycin? Current research is being done on these problems. Scientists question
where people are getting these antibiotic resistant diseases. The scary part is that a
lot of it can be contracted at any given public facility, even upper class facilities are
at risk.
Penicillin was the first antibiotic that was developed. In 1928, Sir Alexander
Fleming isolated it from the mold found growing in his lab experiments (2). This
revolutionized medical practice and improved the health of people all over the world.
Since that time numerous antibiotics have been developed, but with each bacteria
have mutated and developed resistance to many of them.
Vancomycin was introduced into the medical community about forty years
ago, and it has played a very important role since. It is often been referred to as the
antibiotic of last resort (1). Doctors turn to Vancomycin when no other antibiotics
will work. An increasing number of diseases however are beginning to become
resistant to even Vancomycin. Doctors wonder what to do? If an antibiotic is
introduced into a hospital, it can be as little as a few months before resistant strains
of bacteria can appear (1). When Vancomycin was first introduced doctors thought
of it as a cure-all antibiotic. This thought ended in 1987 when the first strains of
Vancomycin resistant bacteria appeared in hospitals.
Now cases of Vancomycin resistant bacteria are as frequent as ever, and
serious research is being done to determine how to handle this growing problem.
Scientists are a closer to understanding how antibiotic resistance spreads because
the genome of the two main antibiotic resistant bacteria have been sequenced. Dr.
Keiichi Hiramatsu and other colleagues at Juntendo University sequenced the
genome of Staphlococcus aureus, one of the two main antibiotic resistant bacteria
(2). Dr. Hiramatsu said To avoid emergence of resistance, we should restrict use of
antibiotics (2). Scientists say that part of the problem is that bacteria are very
resilient and can develop ways to survive drugs that should kill them. Studies show
that about 70% of bacteria are resistant to at least one of the drugs most commonly
used to treat infections (3). To try to help this problem doctors should avoid giving
antibiotics if possible. If doctors continue to prescribe so many antibiotics then the
problem of antibiotic resistant bacteria will continue to rise steadily.
To fully understand antibiotic resistant bacteria, scientists must question
where does it come from? Fitness centers provide an excellent site for bacterial
transmission and growth. They provide warm, moist environments for bacteria to
grow. To prevent contracting bacteria while using a fitness center, one should
always use a barrier between them and the equipment.
The purpose of this project was to determine whether patrons of fitness
centers are being exposed to antibiotic resistant bacteria during their routine
workouts. How many antibiotic resistant bacteria are present in fitness centers? If
there are any antibiotic resistant bacteria, to which antibiotic are they most resistant?
The hypothesis for this project was that a significant amount of the bacteria
encountered in fitness centers will exhibit some degree of resistance. It was further
hypothesized that greater numbers of bacteria will be resistant to penicillin, since it is
the oldest known antibiotic.
Methods and Materials:
Materials:
10 LB Agar Plates
Sterile swabs
Antibiotic disks (ampicillin, tetracycline, streptomycin, penicillin, kanamycin, and
chloramphenicol)
LB Broth
Sterile Water
Inoculating loops
Bacteria spreader
Disinfectant used at fitness center (100 mL)
10 Ziploc bags
50 Falcon Tubes (15 mL)
Antimicrobic susceptibility test guide
Procedure:
1. After choosing a fitness center location, choose 10 machines to test.
2. Moisten sterile swab with sterile water and then thoroughly swab the handle
area of the machine. (repeat with each machine)
3. Streak an LB plate for each machine. Let grow for 24 hours at 37 degrees
Celsius.
4. Using an inoculating loop, select 5 colonies from each plate and start different
cultures of each in 15 mL Falcon Tubes of LB broth. Let grow for 24 hours at
37 degrees Celsius in a shaking water bath.
5. Prepare pour plates by adding 150 uL of each bacteria culture into 3 mL
tubes of melted R-top soft agar and vortex.
6. Pour onto LB agar plate and swirl to cover entire plate.
7. Place each of the 6 different antibiotic disks onto the agar plate in a circular
pattern, separated equally from each other.
8. Seal with para-film and incubate.
9. After 24 hours, measure the zones of inhibition in millimeters for each

antibiotic disk.
Results:
Bacteria were isolated from 10 different fitness machines. Five morphologically
different colonies from each machines plates were cultured in tubes of LB broth for
a total of 50 cultures. After plating the bacteria onto LB plates, the six antibiotic disks
were placed on each of them. Examination after 24 hours of incubation revealed
considerable differences between the antibiotics and also differences in the degree
of susceptibility to the antibiotic. The greatest number of plates were most resistant
to ampicillin. 34% of all the cultured bacteria plates were highly resistant to it. There
was also a high degree of resistance, 32%, to penicillin. On the remaining plates,
less than 10% were highly resistant to streptomycin, kanamycin, chloramphenicol,
and tetracycline.
Many of the bacteria cultures exhibited high susceptibilityto the antibiotics.

Interestingly enough, 56% and 44% of the plates showed high sensitivity to
ampicillin and penicillin respectively. Tetracycline, streptomycin, and kanamycin all
ranged from 26% up to 58% that were susceptible.
The graphs below show the degree of susceptibility that the bacteria cultures
had to all six antibiotics.
Analysis and Conclusion:

The results of this experiment provide evidence that people are being
routinely exposed to antibiotic resistant bacteria in public facilities. It is concluded
that there are considerable differences among the resistance of the bacteria to the
different antibiotics.
It was found that the greatest amount of bacterial resistance was to ampicillin
and penicillin. This is likely due to the fact that penicillin is the oldest and most widely
used antibiotic, and ampicillin is a derivative of penicillin. However, penicillin and
ampicillin were quite effective in destroying many of the cultured bacteria, thus
implying that they do continue to be an effective antibiotic. The high resistance
percentages for penicillin and ampicillin were 32% and 34% respectively. The low
susceptibility percentages were 44% and 56% respectively. This shows that the
antibiotic is at one extreme or the other. This is somewhat of a double-edged sword.
The results of this project do support the hypothesis that there are a
significant numbers of bacteria that are highly resistant to typical antibiotics. Also,
greater numbers of bacteria were resistant to both ampicillin and penicillin, further
supporting the hypothesis.
One possible source of error is the likelihood that the same bacteria were
present on the different machines and may have been used as a sample more than
once. Without further identification of these bacteria, this would be difficult to
overcome this problem.
The results also reaffirm the concern for overuse of antibiotics. The more that
antibiotics are used the greater risk there will be for resistance. This project taught
me the significance of antibiotics and also the dangers they can posses. I learned
that they are not to be used generously so to keep resistance levels low. If I could
have done this project differently, I would have tested more of a variety of machines.
Possible future studies for this project might include testing other fitness
centers and other public facilities to compare percentages of antibiotic resistant
bacteria. Also, it would be extremely interesting to run other tests like Gram
staining to determine more specifically what these bacteria are.
Acknowledgements:
A special thank you goes out to my teacher, Mrs. Stevens, who encouraged
me all throughout my project and spent many hours helping me to complete this
project. Also, I would like to thank my parents who love and support me in all my
efforts. Finally, a big thank you goes to my step brother Bodie who was strong
enough to fight off even the toughest bacteria.
References:
1. Bells, M. 1995. The History of Penicillin. Retrieved from the World Wide Web at:
http://inventors.about.com/library/inventors/blpenicillin.htm, February 17, 2005.
2. 2005. The Microbial World: Penicillin and other antibiotics. Retrieved from the
World Wide Web at: http://helios.bto.ed.ac.uk/bto/microbes/penicill.htm, February
17, 2005.
3. FDA. 2005. Antibiotic Resistance. Retrieved from the World Wide Web at:
http://www.fda.gov/oc/opacom/hottopics/anti_resist.html, January 5, 2005.
4. Kalke, K. 2003. Mobile DNA: Genomic Studies Illuminate Antibiotic Resistance.
Retrieved from the World Wide Web at:
http://www.genomenewsnetwork.org/articles/04_03/mobile.shtml, January 5, 2005.
5. April 2004. The Problem of Antibiotic Resistance. Retrieved from the World Wide
Web at: http://www.niaid.nih.gov/factsheets/antimicro.htm, January 5, 2005.
Data Table:
Number of Plates
Per Amount of Resistance
Type of
Antibiotic
Resistant Intermediate Susceptible
Penicillin
16
12
22
Ampicillin
17
28
Streptomycin
30
13
Kanamycin
24
24
Chloramphenicol
17
29
Tetracycline
14
27

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A Study of the Presence of Antibiotic Resistant Bacteria in Fitness

Centers and Their Susceptibility to a Variety of Antibiotics

9. After 24 hours, measure the zones of inhibition in millimeters for each

Many of the bacteria cultures exhibited high susceptibilityto the antibiotics.

Analysis and Conclusion:

Resistant Intermediate Susceptible

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