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Samantha Arnold, Sec. 07
Antibiotic Resistance
Introduction:
Bacteria are prokaryotic single celled organisms. They are classified according to their
appearance and structure and by specific characteristics of their metabolism and growth. Most
bacteria are either gram-positive or gram-negative. Specific antibiotics are more effective against
gram-positive than gram-negative bacteria. A coliform bacterium is defined as aerobic or
faculatively anaerobic, gram-negative rods that ferment lactose with the production of acid and
gas (Cornell University: Department of Food Science).
During week two of this experiment, we chose to streak an E.coli colony onto our LB
plates. An E. coli colony was either dark blue or purple in color. My plate from which I
attempted to collect bacterial colonies during week one did not have E. coli colonies (or any
bacterial colonies for that matter) so I scraped some from one of my lab partner’s plates. I chose
to scrape from the largest E. coli colony because then I would be able to transfer more cells onto
my LB plate and therefore be able to observe a larger colony.
Antibiotics are medicines that damage or kill bacteria and can be used to treat bacterial
diseases (National Institute of Allergy and Infectious Diseases, 2008). Antibiotics are able to
distinguish prokaryotic bacteria cells from eukaryotic cells because antibiotics are created to
target and destroy certain structures that are only found in bacteria such as their peptidoglycan
cell walls. Peptidoglycan is a polymer that makes up the cell walls of bacteria. Antibiotics also
target certain proteins in bacteria that develop differently than proteins found in eukaryotic cells
(St. Edwards University: Department of Chemistry and Biochemistry).
Antibiotic resistance occurs when an antibiotic has lost its ability to attack bacterial
growth. Bacteria can become resistant to antibiotics either by genetic mutation (rare, spontaneous
Samantha Arnold, Sec. 07
changes of the bacteria’s genetic material) or by acquiring resistance by another bacterium. This
can happen through conjugation or viruses. Conjugation is the transfer of genetic material that
contains genes encoding resistance to antibiotics. Bacteria can obtain antibiotic resistance from
viruses when resistance traits from one bacterium are packaged into the head of the virus and the
virus then injects those traits into another bacterium. (Alliance for the Prudent Use of
Antibiotics). The plasmids (small, circular DNA strands) in bacteria contain genes that code for
antibiotic resistance (Bennet, 2008).
Humans contribute towards antibiotic resistance in a number of ways. For example, the
use of antibiotics in agriculture, particularly for growth enhancement, has been shown to
contribute to the increased prevalence of antibiotic-resistant bacteria. An incredibly large amount
of antibiotics are used for agriculture and that leads to a higher chance that bacteria will acquire
traits to protect themselves against the antibiotics. The increase has been large enough to be of
significance to humans (AG, Matthew, 2007). The use of antibiotics in agriculture is not the only
way bacteria become antibiotic resistant. Antibiotics are becoming more commonly overused by
people because they are easier to obtain. In some countries, and over the Internet, antibiotics can
be purchases without a prescription. People also are taking antibiotics uncessisarily, such as for
viral infections like the common cold (Alliance for the Prudent Use of Antibiotics).
The objectives of this experiment was to collect and identify different kinds of bacteria,
observe the effectiveness of different antibiotics on bacteria, observe the variable levels of
antibiotic resistance on bacteria, and generate informative and appropriate figures and tables
from data. I collected different types of bacteria by sampling water from the N. River and
filtering it with a Coliscan filter. I observed the different levels of antibiotic resistance on
bacteria by growing E. coli on a TSA plate and then placing different antibiotic discs (Cipro,
Samantha Arnold, Sec. 07
Tetracycline, and Penicilin) and a water disk to be the control group, onto the colony. I then
measured the effectiveness of the antibiotics by measuring the diameter of the “zone of
inhibition” on the TSA plate. This zone indicates where bacteria did not grow. The data was then
shown in graphs, tables, and images that are provided in this lab report. My hypothesis is that the
bacteria isolated from the N. River are resistant to antibiotics.
Results:
Table 1. Number of Different Bacteria Colonies Collected
E. Coli (dark
Coliforms (pink
blue or purple
or teal)
colonies)
# of Colonies
0
0
Total %
0
0
Non-coliforms
(pale green or
colorless)
0
0
Total # of
colonies
0
100%
40
35
% E. Coli
30
25
20
15
10
5
0
1-Oct
2-Oct
3-Oct
4-Oct
Day
Figure 1. Average Percentage E. Coli in Bacteria Sample Collected on various days. Water was
collected and filtered through a coliscan filter and then left to colonize. The percentage of E. coli
in each sample was then measured using an EMB plate.
Samantha Arnold, Sec. 07
Figure 2. EMB Plate Image. The bacteria sample extracted from the N. River was smeared onto
an EMB plate. A purple color indicated that E. coli was not present in the bacteria sample. Purple
with a bright green sheen indicated the presence of E. coli. Our sample did not have E. coli.
Figure 3. Bacterial Plate with Antibiotic Discs Image. Part of the bacteria sample was smeared
onto the bacterial plate and then different antibiotic disks (Cipro, Tetracyline, and Penicillin)
along with a water disk to be the control group were placed onto the disk and then the “zone of
clearing” was measured to measure the antibiotic resistance of the bacteria had to each antibiotic.
% Resistant to Antibiotic
120.0
100.0
80.0
60.0
40.0
E. coli
20.0
Other Antibiotics
0.0
Antibiotic
Samantha Arnold, Sec. 07
Average Diameter of zone of clearing
(mm)
Figure 4. Percent of E. coli vs other bacteria species resistant to antibiotics. A portion of the
bacteria sample collected was smeared onto a bacterial plate and 4 antibiotic disks were placed
on it. After a week, the zone of clearing was measured to measure the antibiotic resistance of the
bacteria to each antibiotic. The percent of bacteria sample resistant was then graphed.
25.0
20.0
15.0
E. coli
10.0
Other Bacteria
5.0
0.0
Water
Cipro
Tetracycline Penicillin
Antibiotic
Figure 5. Avg zone of clearing in E. coli versus other species of bacteria. A portion of the
bacteria sample collected was smeared onto a bacterial plate and 4 antibiotic disks were placed
on it. After a week, the zone of clearing was measured to measure the antibiotic resistance of the
bacteria to each antibiotic.
Discussion:
The main objective of this experiment was to measure the antibiotic resistance of
different kinds of bacteria. My hypothesis was the bacteria isolated from the N. River are
resistant to antibiotics. The data did not entirely support my hypothesis because not all the
antibiotic discs were successful in prohibiting the growth of the bacteria.
I think there was a difference in the percent of E. coli collected at the river during
different days because the weather was different on each of the days. Water tends to be more
contaminated during rainy weather rather than when the weather is calm. This variability affects
the accuracy of the results of water sampling. To account for this variability, researchers should
test river water for many consecutive days in order to get an accurate reading.
Samantha Arnold, Sec. 07
The EMB plate helps to determine whether the sample of bacteria I used contained E. coli
or not. If E. coli was present in the sample, then the smear would have been purple with a bright
green sheen. The sample I used was purple in color, indicating that it did not contain E. coli.
If a large amount of bacteria become resistant to antibiotics, it can become more difficult
to treat human bacterial infections. When antibiotics do not work, it can result in hospitalization,
a need for more expensive antibiotics to replace the ineffective ones, and in some cases death
(Alliance for the Prudent Use of Antibiotics). The overuse of antibiotics in agriculture also leads
to antibiotic resistance which can affect the health of animals. Some experts have urged the ban
of growth-enhancement antibiotics in animals, which has been shown to decrease the prevalence
of antibiotic resistance in animals. However, the ban of antibiotics in agriculture all together
could also increase animal morbidly and mortality (AG, Matthew, 2007).
Both E. coli and the other gram negative bacteria were virtually resistant to Penicillin.
The average zone of clearing around Penicillin was equal to or close to zero indicating that it did
not stop the growth of the bacteria. Both E. coli and the other strains of bacteria did not appear to
be very resistant to Cipro with an average zone of clearing of 23.5 for E. coli and 22.1 for the
other bacteria. These averages are close to each other which indicates that E. coli and other gram
negative bacteria are close in levels of resistance against Cipro. The average zone of clearing
around Tetracycline was 9.7 for E. coli and 7.8 for the other gram negative bacteria. Both E. coli
and the other strains of bacteria were close in levels of resistance against all antibiotics used. E.
coli showed to be slightly less resistant to the antibiotics because it had a larger zone of clearing.
To continue research on this experiment, I would expand it by testing water from
different water sources. Even though the N. River is a good place to test antibiotic resistance due
to the large amount of agriculture in the area, this experiment could be improved upon by testing
Samantha Arnold, Sec. 07
other bodies on water. I would even test bodies of water where agriculture is not prevalent to
show the difference agriculture makes on antibiotic resistance. If more bacteria was collected
from more than just one body of water, the results of the experiment would be more accurate is
portraying the problem of antibiotic resistance.
Samantha Arnold, Sec. 07
Works Cited
AG, Matthew, 2007. Antibiotic resistance in bacteria associated with food animals: a United
States perspective of livestock production. http://www.ncbi.nlm.nih.gov/pubmed/17600481
Alliance for the Prudent Use of Antibiotics, General background: about antibiotic resistance.
http://www.tufts.edu/med/apua/about_issue/about_antibioticres.shtml.
Bennet, Peter M. 2008. Plasmid encoded antibiotic resistance: acquisition and transfer of
antibiotic resistance genes in bacteria. http://www.ncbi.nlm.nih.gov/pubmed/18193080.
Cornell University: Department of Food Science. Basic dairy bacteriology.
http://foodscience.cornell.edu/cals/foodsci/extension/upload/CU-DFScience-Notes-BacteriaGeneral-Dairy-Micro-06-10.pdf
National Institute of Allergy and Infectious Diseases, 2008. Microbes.
http://www.niaid.nih.gov/topics/microbes/Pages/glossary.aspx
St. Edwards University: Department of Chemistry and Biochemistry. Antibiotics.
http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/Antibiotics/Antibiotics.HTM
L#general