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shamebagΒιοτεχνολογία

22 Φεβ 2013 (πριν από 4 χρόνια και 7 μήνες)

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Lisa Michelle Hathaway


Ampicillin
vs. E.Coli:



Who’s the Real Winner?


Purpose


The purpose of this experiment was to get a
better understanding of how the bacteria,
Escherichia Coli reacts to the frequent use of
antibiotics and how it affects the bacteria’s long
term resistance. I became interested in this
experiment after reading about the new super
bugs that are antibiotic resistance and how they
are gaining force. The information gained in this
experiment will help others understand how the
overuse of antibiotics may lead to the growth and
multiplication of a new population of resistant
bacteria.


P
roblem


The excessive use of antibiotics has resulted in
bacteria developing a resistance and then
immunity to the antibiotics rendering the
antibiotics no longer effective. This is leading to
the growth and multiplication of a new
population of resistant bacteria also known as
“Super Bugs”. In this project I set out to test how
the bacteria Escherichia Coli (E. coli) is affected by
frequent and repeated use to the antibiotic
ampicillin and what resistance it develops.


Hypothesis


The higher the concentration of the antibiotic
Ampicillin used against the bacteria
Escherichia Coli (E.Coli) the faster it will
develop a resistance to it when exposed to
repeated doses of
the
Ampicillin.

Variables


Independent variable: The concentration of the antibiotic
Ampicillin used.




Dependent variable: The size of the inhibition zone which is
free from bacteria.




Constants:
The same Agar solution, same Ampicillin, same
P
etri dishes same room and environmental controls.




Control:
Experiment was preformed with one Petri dish of
E. Coli in the Agar Solution with no added Ampicillin
Solution.


Research

: E.Coli


Escherichia coli commonly abbreviated as E. coli is a Gram
-
negative
or rod
-
shaped bacterium that is commonly found in the lower
intestine. Most E. coli strains are harmless, but some can cause
serious food poisoning in humans. The harmless strains are part of
the normal flora of the gut, and can benefit their hosts by
producing vitamin K, and preventing the establishment of
pathogenic bacteria within the intestine. E. coli. related bacteria
constitute about 0.1% of gut flora, and fecal
-
oral transmission is the
major route through which pathogenic strains of the bacterium
cause disease. Cells are able to survive outside the body for a
limited amount of time. The bacterium can also be grown easily and
inexpensively in a laboratory setting, and has been intensively
investigated for over 60 years. E. coli is the most widely studied
prokaryotic model organism, and an important species in the fields
of biotechnology and microbiology, where it has served as the host

organism for the majority of work with recombinant DNA.


Research: Ampicillin


Ampicillin is a
beta
-
lactam antibiotic that
has been used
extensively to
treat baterial infections since
1961.
Ampicillin demonstrated activity against
Gram
-
negative
organisms such as
influenza,
E.Coli,
coliforms
and
Proteus.
Ampicillin was the first of a number of broad spectrum
penicillin subsequently introduced by Beecham. Ampicillin
is part of
the amino penicillin
family and is roughly
equivalent to its successor
, amoxicillin
in terms of spectrum
and level of activity. It can sometimes result in reactions
that range in severity from a
rash
and many people had
allergic reactions such
as

anaphylaxis.
However, as with
other penicillin drugs, it is relatively non
-
toxic and adverse
effects of a serious nature are encountered only rarely.


Research: Super Bugs


A superbug is a term used to describe a strain of bacteria that is resistant to most prescribed
antibiotics. These emerging strains of bacteria can be pretty frightening, since treatments for
infection are usually very limited. The following are examples of several super bugs already know to
exist. Staphylococcus aureus ( “Staph”) manifests itself in many ways, but is probably most
famously known as “flesh
-
eating bacteria”. For many years, penicillin and methicillin were
considered excellent treatments for Staph infections. Strains of Staph that are resistant to
methicillin were first observed in hospitals and other healthcare facilities. Tuberculosis, also known
as “consumption”, is a disease that is acquired by inhalation into the lungs, where it can cause
disease and can spread to other organs in the body. Prior to the discovery of antibiotics,
tuberculosis was untreatable. However, even with the widespread use of antibiotics that began in
the 1940s, multidrug
-
resistant tuberculosis (MDR
-
TB) has emerged and is a leading cause of death,
particularly among HIV
-
infected individuals. MDR
-
TB is caused by strains of Mycobacterium
tuberculosis that are resistant to at least the antibiotics isoniazid and rifampicin Enterococcus
faecalis and Enterococcus faecium are found in the bowel and female genital tract and can cause
urinary tract infections, blood infections, and meningitis. Enterococci can cause fatal infections in
individuals with compromised health, such as infants and the elderly. Several strains of drug
-
resistant enterococci have emerged in the last 30 years, including those that are resistant to
penicillin, vancomycin, and linezolid. Streptococcus pneumoniae is a common cause of ear
infections in children, meningitis, systemic infection, and pneumonia. Strains that are resistant to
penicillin and other penicillin
-
like antibiotics have increased over the last 30 years.

Research: Antibiotic Resistance


Antibiotic resistance is a type of
drug resistance where
a
micro
-
organism
is
able to survive exposure to
an antibiotic.
While a spontaneous or induced
genetic mutation in bacteria may confer resistance to antimicrobial drugs,
genes
that confer resistance can be by
conjugation, transduction,
or
transformation.
Therefore a gene for antibiotic resistance which had
evolved via
natural selection
may be shared. Evolutionary stress such as
exposure to antibiotics then selects for the antibiotic resistant trait. Many
antibiotic resistance genes reside on
plasmids,
facilitating their transfer. If
a bacterium carries several resistance genes, it is called a superbug or
super bacterium. The increasing prevalence of antibiotic
-
resistant
bacterial infections seen in clinical practice stems from antibiotic use both
within human medicine and veterinary medicine. Any use of antibiotics
can increase selective pressure in a population of bacteria to allow the
resistant bacteria to thrive and the susceptible bacteria to die off. As
resistance towards antibiotics becomes more common, a greater need for
alternative treatments arises. Antibiotic resistance therefore poses a
significant problem.


Research: Inhibition Zone


The inhibition zone is an area around a paper disk or colony of
bacteria where no other organisms are growing. For example, when
the growth of bacteria susceptible to the antibiotic is inhibited.
Typically several million bacterial cells are spread on the agar plate,
and if their growth is inhibited, a clear "zone of inhibition" is
observed around the antibiotic impregnated disc. If the bacteria are
resistant to the antibiotic, a confluent "lawn" of growth is observed.
To tell if it is an inhibition zone, it starts with the microbe
distributed across the entire plate. If nothing is added to the plate,
each microbe will grow, and produce a new microbe by cell division.
After this happens for a while (1
-
3 days depending on the
temperature), the agar will get a cloudy appearance because of all
of the microbial growth and cell divisions. If no growth occurred in a
small area of agar, this area would remain clear.


Materials


15 Petri dishes with an agar solution in them


15 disinfected swabs


1 bottle of disinfected water


1 hole puncher


4 Test tubes


1measuring cylinder


1 digital weighing scale


1 beaker of water


1 forceps


1 marker


1 stirring rod


100mg of Ampicillin


E Coil Bacteria


Filter Paper


1 Inoculating needle, looped end


Access to a room and ability to control the temperature at a constant 78 degrees.


Procedures


Prepare

18 Petri dishes using an agar solution and store


in a refrigerator.


Bring 3

of the Petri dishes

to room temperature before the start of experiment.


Label the

Petri dishes

1mg, 3mg and 5mg.


Prepare and label the antibiotic mixture.


Labeled one 1mg
-


1mg of ampicillin is mixed with 10ml water


Labeled one 3mg
-

3mg of ampicillin is mixed with 10ml water


Labeled one 5mg
-


5mg of ampicillin is mixed with 10ml water




Mix 10ml of E Coli bacterium culture

with 40ml water in the 4th test tube.


Rinse the inoculation needle using the disinfected water.


Dip it into

the test tube containing the bacteria culture and swipe it

gently

over the agar surface of the 3 Petri
dishes.


Punch out circular plates of filter paper

using a hole puncher.


Label the circular plates of filter paper

1mg, 3mg and 5mg.


Pick up the filter paper plates using the forceps and dip them

into the ampicillin solution according to the labeled
concentrations.


Place the filter paper plates at the center of the Petri dish according to their labels and cover them.


Keep the dishes in a cool dry place for the bacteria to incubate for 4 days.


After 4 days, measure the diameter of this area around the filter paper in the inhibition zone.


Collect the surviving bacteria


along the border of
the inhibition
zone by using a swab and use it to prepare the
bacteria culture needed for subsequent experiment.


Repeat 5 more times.


Measure the diameter of the inhibition zone for the repeated labs and record the results
.

Photos

1st Generation

2nd Generation

3rd Generation

4th Generation

5th Generation

6th Generation

Live
E.Coli

Bacteria

Agar Solution

Data



Ampicillin Concentration

1st


generation

2nd
generation

3rd


generation

4th


generation

5th


generation

6th


generation

1mg

24


22.5


19


15.5


11.5


7.5

3mg

22.5


21.5


20.5


18


16.5


12.5

5mg

21.5


21


20


19


18


17

Graph

0
5
10
15
20
25
30
1 mg
3 mg
5 mg
1st Generation
2nd Generation
3rd Generation
4th Generation
5th Generation
6th Generation
Ampicillin Concentration and Bacterial Resistance

Results


In the first generation set of the experiment the Perti dish with 1mg of Ampicillin had an
inhibition zone of 24mg. The Perti dish with 3mg of Ampicillin had an inhibition zone of
22.5mg. The Perti dish with 5mg of Ampicillin had an inhibition zone of 21.5mg.



In the second generation set of the experiment the Perti dish with 1mg of Ampicillin
had an inhibition zone of 22.5mg. The Perti dish with 3mg of Ampicillin had an inhibition
zone of 21.5mg. The Perti dish with 5mg of Ampicillin had an inhibition zone of 21mg.



In the third generation set of the experiment the Perti dish with 1mg of Ampicillin had
an inhibition zone of 19mg. The Perti dish with 3mg of Ampicillin had an inhibition zone of
20.5mg. The Perti dish with 5mg of Ampicillin had an inhibition zone of 20mg.



In the fourth generation set of the experiment the Perti dish with 1mg of Ampicillin
had an inhibition zone of 15.5mg. The Perti dish with 3mg of Ampicillin had an inhibition
zone of 18mg. The Perti dish with 5mg of Ampicillin had an inhibition zone of 19mg.



In the fifth generation set of the experiment the Perti dish with 1mg of Ampicillin had
an inhibition zone of 11.5mg. The Perti dish with 3mg of Ampicillin had an inhibition zone of
16.5mg. The Perti dish with 5mg of Ampicillin had an inhibition zone of 18mg.



In the sixth generation set of the experiment the Perti dish with 1mg of Ampicillin had
an inhibition zone of 7.5mg. The Perti dish with 3mg of Ampicillin had an inhibition zone of
12.5mg. The Perti dish with 5mg of Ampicillin had an inhibition zone of 17mg.

Conclusion


I was able to conclude from the results that my hypothesis was incorrect.
The higher dose of Ampicillin killed off the E.Coli faster but the E.Coli did
not develop a resistance to the stronger dose quicker. The experiment
proved that the smaller dose allowed for the E.Coli to become resistant to
the antibiotic quicker than the higher level doses did. The bacteria was
able, at the lower dose after repeated use of the antibiotic to develop a
resistance and in turn caused the antibiotic to become less effective in
eliminating the bacteria faster than at the higher dose. However the high
dose did mimic the same results of resistance just at lower numbers. In
conclusion of these results antibiotics should be avoided unless absolutely
necessary. The experiment proves that overuse of antibiotics causes
resistance in turn causing the antibiotic to become ineffective
. Newer and
stronger antibiotics need to be developed to combat the bacteria that have
developed a resistance but these antibiotics will be more expensive and
with unknown side effects.

Abstract


Antibiotics are among the most frequently prescribed medications in today’s world
of medicine. Antibiotics cure disease by killing or injuring the bacteria. I set out to
get a better understanding of how the bacteria, Escherichia Coli reacts to the
frequent use of the antibiotic Ampicillin. I became interested in this topic after
researching the new “Super Bugs”, (bacteria that has become resistant to
antibiotics) and how they are currently gaining force. My experiment centered
around how quickly the E.Coli would develop a resistance to the antibiotic. I
hypothesized that the higher the concentration of the antibiotic Ampicillin used
against the bacteria
Escherichia
Coil the faster it would develop a resistance to it
when exposed to repeated doses of the Ampicillin. I preformed six generations of
experiments with three concentration levels of the antibiotic to determine the rate
of resistance it would develop. The data I collect from the results of six generations
of experiments showed that the bacteria died quicker the higher the dose of
antibiotic but also that the higher the dose of antibiotic the slower the bacteria
was in developing a resistance. My hypothesis was incorrect in the end as the
lower the dose of Ampicillin given the quicker the E.Coli was able to develop a
resistance to it. However, now people can know these results and avoid the
overuse of antibiotics to avoid more strains of bacteria becoming resistant to
antibiotics and the need for stronger and more harmful antibiotic to be developed
to combat them.


Bibliography & Acknowledgements


Rosenberg, S, “Inhibition of Mutation and Combating the Evolution of
Antibiotic Resistance”, Research Article. Published May 10, 2005.
http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.00
30176



WebMD, “What is an E.Coil Infection”, E.Coil Infections. Created
September 2005, Updated June 14, 2010.


http://
www.webmd.com/a
-
to
-
z
-
guides/e
-
coli
-
infection
-
topic
-
overview?page=2




World Health Organization, “Antimicrobial Resistance", Key Facts on
Antimicrobial Resistance. Created and Copy
-
written 2011.


http://www.who.int/mediacentre/factsheets/fs194/en/



Wikipedia, “Ampicillin,” Mechanism of Action. Created 1998, Updated
September 25, 2011
.


http://en.wikipedia.org/wiki/Ampicillin



Centers for Disease Control and Prevention, “Antibiotic Resistance”,
Questions and Answers. Created June 30, 2009, Updated September
10, 2010.



Http://
www.cdc.gov/getsmart/antibiotic
-
use/antibiotic
-
resistance
-
faqs.html









I would like to
acknowledge the
following people
for their help and
support in the
completion of this
science project:
Mrs. Garciga, Mr.
Brezina, Jeannette
Hathaway and Ann
House.