ANTIBIOTICS

A BIOLOGY PROJECT REPORT

SUBMITTED BY ANUSHA PRASAD

IN PARTIAL FULFILMENT OF THE

CBSE GRADE XII

IN

BIOLOGY

AT

AECS MAGNOLIA MAARUTI PUBLIC

SCHOOL
#36/909, ARAKERE, BANNERGHATTA

ROAD,

BANGALORE- 560076.
2013
2014
CERTIFICATE

This is to certify that ANUSHA PRASAD of Grade XII, AECS MAGNOLIA

MAARUTI PUBLIC SCHOOL, BANGALORE with register number
____________________ has satisfactorily completed the project in Biology on
TO STUDY DRUG RESISTANCE IN BACTERIA USING ANTIBIOTICS in
partial fulfillment of the requirements of All India Secondary School Certificate
Examination (AISSCE) as prescribed by CBSE in the year 2013-2014.

Signature of the Signature of the

Candidate Teacher In-Charge

Signature of the Signature of the

Principal External Examiner
discovered medicinally useful drug, the synthetically antibacterial
Salvarsan now called arspheamine.

In 1895, Vincenzo Tiberio, of university of Naples discovered that a mold in

water has antibacterial action. After this initial chemotherapeutic compound
proved effective other perused similar lines of inquiry, but it was not until 1928
that Alexander Fleming observed antibiosis against bacteria by a fungus genus
of the Penicillium. Fleming postulated that the effect was mediated by an anti-
bacterial compound named penicillin. He initially characterized some of its
properties, but he did not pursue its further development.

The first sulfonamide and first commercially available antibacterial

antibiotic, Pronstosil, was developed by a research team led by
Gerhard Domagk in 1932 in Bayers laboratory Germany. Domagk
received the 1939 Noble Prize for Medicine for his efforts.
Penicillin, the first natural antibiotic discovered by Alexander Fleming

4
SOME RESISTANT PATHOGENS

Staphylococcus aureus

Staphylococcus aureus is one of the major resistant pathogens. Found on

the mucous membranes and the human skin of around a third of the
population, it is extremely adaptable to antibiotic pressure. It was one of
the earlier bacteria in which penicillin resistance was foundin 1947, just
four years after the drug started being mass-produced. Methicillin was
then the antibiotic of choice, but has since been replaced by oxacillin due
to significant kidney toxicity. Methicillin resistant Staphylococcus aureus
(MRSA) was first detected in Britain in 1961, and is now "quite common" in
hospitals. MRSA was responsible for 37% of fatal cases of sepsis in the
UK in 1999, up from 4% in 1991. Half of all S. aureus infections in the US
are resistant to penicillin, methicillin, tetracycline and erythromycin.X

Streptococcus and Enterococcus

Streptococcus and Enterococcus infections can usually be treated with many

different antibiotics. Early treatment may reduce the risk of death from
invasive group A streptococcal disease. However, even the best medical care
does not prevent death in every case. For those with very severe illness,
supportive care in an intensive care unit may be needed. For persons with
necrotizing fasciitis, surgery often is needed to remove damaged tissue.
Strains of S. pyogenes resistant to macrolide antibiotics have emerged;
however, all strains remain uniformly sensitive to penicillin.X

Pseudomonas aeruginosa

Pseudomonas aeruginosa is a highly prevalent opportunistic pathogen. One

of the most worrisome characteristics of P. aeruginosa is its low antibiotic
susceptibility, which is attributable to a concerted action of multidrug effluxX
5
 pumps with chromosomally encoded antibiotic resistance genes and the low
permeability of the bacterial cellular envelopes. Pseudomonas aeruginosa
has the ability to produce HAQs and it has been found that HAQs have
prooxidant effects, and over expressing modestly increased susceptibility to
antibiotics. The study experimented with the Pseudomonas aeruginosa
biofilms and found that a disruption of relA and spot genes produced an
inactivation of the Stringent response (SR) in cells who were with nutrient
limitation which provides cells be more susceptible to antibiotics.X

Clostridium difficile

Clostridium difficile is a nosocomial pathogen that causes diarrheal disease in

hospitals worldwide. Clindamycin-resistant C. difficile was reported as the
causative agent of large outbreaks of diarrheal disease in hospitals in NewX

York, Arizona, Florida and

Massachusetts between 1989 and 1992.
[87] Geographically dispersedX
outbreaks of C. difficile strains resistant

to fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin,

were also reported in North America in 2005.

Salmonella and E. coli

Escherichia coli and Salmonella come directly from contaminated food. When
both bacteria are spread, serious health conditions arise. Many people are
hospitalized each year after becoming infected, with some dying as a result. By
1993, E. coli resistant to multiple fluoroquinolone variants was documented. X

Acinetobacter baumannii

On November 5, 2004, the Centers for Disease Control and Prevention (CDC)
reported an increasing number of Acinetobacter baumannii bloodstream
infections in patients at military medical facilities in which service members X
6
injured in the Iraq/ Kuwait region during Operation Iraqi Freedom and
in Afghanistan during Operation Enduring Freedom were treated. Most
of these showed multidrug resistance (MRAB), with a few isolates
resistant to all drugs tested.X

Mycobacterium tuberculosis

Tuberculosis is increasing across the globe, especially in developing

countries, over the past few years. TB resistant to antibiotics is called
MDR TB (Multidrug Resistant TB). The rise of the HIV/AIDS epidemic
has contributed to this.X

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OBJECTIVE

Our objective is to identify these drug resistant bacteria by cultivating

the bacteria and placing then in an environment of bacteria.

8
SCOPE AND LIMITATIONS

In our experiment we plan to take antibiotics in a dish and selectively grow

bacteria that are resistant to drugs. The main limitations are the equipments.
For us to correctly identify the drug resistant bacteria out of the mass of the
normal bacteria, we need powerful microscopes that are not easily available.

After culturing the bacteria, we also need to be able to verify these bacteria
are truly drug resistant. For this, we need to be able to count the number of
bacteria in the sample oe at least know that their density in a given area.
Once again this will be difficult without the required equipment.

This field of bacteria has immense scope and can provide more
efficient healthcare system. Today a lot of people are suffering from
drug resistant bacteria because none of the drugs are working to cure
them. With more research in this field, we provide more targeted
approach to kill these harmful bacteria.

Existing Scientific Literature on new medication for drug resistant bacteria:

Until recently, research and development (R&D) has provided new drugs in
time to treat bacteria that became resistant to older antibiotics. That is no
longer the case. The potential crisis at hand is the marked decrease in
industry R&D, the increasing prevalence of resistant bacteria. Infectious
diseases physicians are alarmed by the prospect that effective antibiotics
may not be available to treat seriously ill patients in the near future.

As bacterial antibiotic resistance continues to exhaust the supply of

effective antibiotics, a global public health disaster appears likely.
Poor financial investment in antibiotic research has exacerbated the
situation. A call to arms raised by several prestigious scientific
organizations a few years ago rallied the scientific community, and
now the scope of antibacterial research has broadened considerably.

9
THEORY
Antibiotics are the chemical substances produces by microorganisms
to kill other organisms or retard their growth. Tetracycline,
Streptomycin, Penicillin are a few examples of the antibiotics which
have been useful in treating various bacterial diseases.

Continuous use of particular antibiotic against any microorganism reduces

its effect because of a few bacterial cells develop resistance to antibiotic,
may be due to mutation and thus such resistant stains keep on growing
even in the presence of antibiotic and do not respond to treatment.

Genes for resistance to antibiotics, like the antibiotics themselves, are

ancient. However, the increasing prevalence of antibiotic-resistant bacterial
infections stem from antibiotic in 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.
However, despite a push for new antibiotic therapies there has been a
continued decline in the number of newly approved drugs. Antibiotic
resistance therefore poses a significant problem.

10
11
EXPERIMENT

Aim:

To study the drug resistance in bacteria using antibiotics.

Requirement:

Apparatus Requirement

Petridish

12
 Sterilized culture tubes

Forceps

Flasks
13
 Beakers

Burner
Chemical Requirements

Penicillin and other antibiotics

14
 Agar
 Starch

1
5
 Distilled water

1
6
PROCEDURE

Prepare pure culture of bacteria:

100ml distilled water; 4gms of agar and 1g of starch was added to a test
tube and shaken well.

The test-tube was placed over a burner ad boiled for 5minutes.

While the solution was boiled, 10pieces of dry hay were added.

The resulting mixture was then kept in a warm place for 5days for
bacterial growth.
17
Culture medium:

Beaker was taken and molten agar was prepared in it.

 Samples from the bacterial culture were taken in the petridish.

Transfer bacteria from agar test tube into petri dish containing antibiotics:

Petridish were taken.

18
The warm culture medium prepared in the previuos step was poured into
each petridish. Care was taken to sterilize the petridish so as to avoid any
unnecessary growth.

The culture was spread evenly.

Antibiotics:

A solution of antibiotics was made by dissolving different antibiotic tablets

in distilled water. Eg: penicillin solution, tetracycline solution.

19
Filter paper cut in circles were soaked in antibiotic solution ad placed
in one part of the petriplate with culture.

Another petridish with agar was taken and labeled Control. No

antibiotic was added to this.

20
OBSERVATION

A. After one week

Sl.no.
Antibiotic used
Number of bacterial

Description

colonies

1.
Levoflaxacin
5
Few medium
colonies
near
2.

the edge of the Petri dish

Amoxicillin
8
Few medium sized colonies

3.

around the filter paper

Cefixime
16
Small
to
medium
size

4.

colonies

Roxithromycin
29
Dense
growth
of
small

5.

colonies of bacteria.
Ciprofloxacin
4
Few medium to large sized

6.

colonies

Cefpodoxime
31
Large number of small to

medium
sized
colonies

7.

around the edge.

Ofloxacin
34
Small sized colonies

8.
Azithromycin
14
Medium sized colonies

9.
Control
0
N/A

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B. After two weeks

Sl.no.
Antibiotic used
Number of bacterial

Description

colonies

1.
Levoflaxacin
3
Medium
colonies
near the

2.
edge of the petri dish

Amoxicillin
4
Medium
sized
colonies

3.

around the filter paper

Cefixime
9
Small
to
medium
size

4.

colonies

Roxithromycin
15
Small sized colonies

5.
Ciprofloxacin
3
Medium
to
large
sized

6.

colonies

Cefpodoxime
23
Large number of small to

medium
sized
colonies

7.

around the edge.

Ofloxacin
21
Small sized colonies

8.
Azithromycin
11
Medium sized colonies

9.
Control
0
N/A
C. After three weeks

Sl.no.
Antibiotic used
Number of bacterial

Description

colonies

1.
Levoflaxacin
2
Medium
colonies
near the

2.

edge of the petri dish

Amoxicillin
3
Medium
sized
colonies

3.

around the filter paper

Cefixime
6
Small
to
medium
size

4.

colonies

Roxithromycin
12
Small sized colonies

5.
Ciprofloxacin
3
Medium
to
large
sized

6.

colonies
Cefpodoxime
19
Large number of small to

medium
sized
colonies

22
7.

around the edge.

Ofloxacin
13
Small sized colonies

8.
Azithromycin
9
Medium sized colonies

9.
Control
0
N/A

RESULT

The observed trend was that the number of bacterial colonies decreased with time
and family remained constant. We can infer that the decrease was because
bacteria with the mutation survived. Over time as only the mutated bacteria
remained, and the population stabilized. We can predict that after a few more
weeks, the population may show an upward trend with mutations produce progeny
with mutations.
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BIBLIOGRAPHY

Wikipedia - The free encyclopedia - (http://en.wikipedia.org)

Comprehensive Practical Chemistry

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