TO STUDY DRUG RESISTANCE IN BACTERIA USING

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
Candidate

Signature of the
Principal

Signature of the
Teacher In-Charge

Signature of the
External Examiner

Table of Contents

INTRODUCTION........................................................................................................................ 1
HISTORY.................................................................................................................................... 3
OBJECTIVE................................................................................................................................ 9
SCOPE AND LIMITATIONS.....................................................................................................10
THEORY................................................................................................................................... 11
EXPERIMENT.......................................................................13
PROCEDURE...........................................................................18

OBSERVATION......................................................................................................................... 22
RESULT.................................................................................................................................... 23
BIBLIOGRAPHY...................................................................................................................... 24

ACKNOWLEDGEMENT
I would like to thank my teachers, Mrs. Neelam and Mrs. Nikhila for
guiding me through this project and for their valuable inputs which provided
me with a constant nudge for improvement.
It is imperative to thank our Principal, Mrs. Seema Goel for providing me
the opportunity to work on this project.
It goes without saying that my classmates, especially Madhumathi Mandal,
Arathi Nair and Sushrutha Sricharan for their help in due course of this
project. My parents have also played a part in helping me in this project. My
thanks goes out to them also.
This project and reading-up on the same has provided me with an in depth
understanding of the topic. It has nurtured my scientific temperament and
curiosity.

Signature of the
Candidate

ABBREVIATION
Sl.No
1
2
3
4
5
6
7
8

Abbreviation
MDR
MRSA
HAQs
SR
MDRTB
ml
mg
g

Expansion
Multidrug Resistance
Methicillin Resistant Staphylococcus Aureus
4-hydroxy-2-alkylquinolines
Stringent response
Multidrug Resistant TB
Milliliter
Milligram
Gram

INTRODUCTION
In this project we will study and attempt to cultivate bacteria that are
resistance to drugs.
According to external sources, Antibiotic resistance is a type of drug
resistance where microorganism 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 that
transferred between bacteria in a horizontal fashion by conjugation,
transduction, or transformation. Thus, a gene for antibiotic resistance that
evolves via natural selection may be shared. Evolutionary stress such as
exposure to antibiotics then selects for the antibiotic resistance trait. Many
antibiotic resistance genes reside on plasmids, facilitating their transfer. If a
bacterium carries several resistance genes, it is called multidrug resistance
(MDR) or, informally, a super bug or super bacterium.
What this tells us is that drug resistance bacteria can be a product of natural
selection.
For example, bacteria are cultivated in the lab and the drug that can kill these
bacteria is added to the culture. Most of the bacteria will di, but the ones that
survive have genetic mutation that enables them to survive in the presence of
the antibiotic. The bacteria are drug resistance.

Schematic representation on how antibiotic resistance develops under

natural condition.
As we can see from the above explanation, drug resistance bacteria are
caused due to the overuse of antibiotics. In some countries like India
antibiotics are sold over the counter. This results in misuse or overuse of
antibiotics which cause drug resistance bacteria to proliferate. Once a
bacterium becomes drug resistance it is very hard to get rid of it as it is a
new drug and it has not been exposed to work against it.

HISTORY
Before the early 20th century, treatments for infections were based primarily on
medicinal folklore. Mixtures with antimicrobial properties that were used in
treatment of infections were described over 2000years ago. Many ancients
cultures, including the ancient Egyptians and ancient Greeks, used specially
selected mold and plant materials and extracts to treat infections. More recent
observation made in the laboratory of antibiotics between microorganisms led
to the discovery of antibacterial produced by microorganisms. Louis Pasteur
observed, If we could intervene between antagonism observed between some
bacteria it would offer perhaps the greatest hopes of therapeutics. The terms,
antibiosis, meaning, Against life, was introduced by French bacteriologist
Vuillemin as descriptive name of the phenomenon by these early antibacterial
drugs. Antibiosis was first described in 1877 in bacteria when Louis Pasteur
and Robert Koch observed that an airborne bacillus could inhibit the growth of
Bacillus anthracis.

Bacillus anthracis.
Synthetic antibiosis chemotherapy as a science of antibacterial began in
Germany by Paul Ehrlich in 1880s. Ehrlich observed that certain dyes could
color humans, animal, or bacterial cells, while other did not. He then proposed
the idea that it would be possible to create chemicals that would act as a
selective drug that would bind and would kill the bacteria without harming the
human host. After screening hundreds to dyes against various organisms, he
3

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 antibacterial 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

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.

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.
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 efflux
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.

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 New
York, Arizona, Florida and Massachusetts between 1989 and 1992.
[87] Geographically dispersed 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.

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
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.

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.

OBJECTIVE

Our objective is to identify these drug resistant bacteria by cultivating the

bacteria and placing then in an environment of bacteria.

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.

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:
a.

Apparatus Requirement

Petridish

12

 Sterilized culture tubes

Forceps

Flasks

13

 Beakers

Burner

b.

Chemical Requirements

Penicillin and other antibiotics

14

 Agar

Starch

15

 Distilled water

16

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

1.

Levoflaxacin

Number of bacterial
colonies
5

2.

Amoxicillin

3.

Cefixime

16

4.

Roxithromycin

29

5.

Ciprofloxacin

6.

Cefpodoxime

31

7.
8.
9.

Ofloxacin
Azithromycin
Control

34
14
0

Description
Few medium colonies near
the edge of the Petri dish
Few medium sized colonies
around the filter paper
Small to medium size
colonies
Dense growth of small
colonies of bacteria.
Few medium to large sized
colonies
Large number of small to
medium
sized
colonies
around the edge.
Small sized colonies
Medium sized colonies
N/A

21

B. After two weeks

Sl.no.

Antibiotic used

1.

Levoflaxacin

Number of bacterial
colonies
3

2.

Amoxicillin

3.

Cefixime

4.
5.

Roxithromycin
Ciprofloxacin

15
3

6.

Cefpodoxime

23

7.
8.
9.

Ofloxacin
Azithromycin
Control

21
11
0

C. After three weeks

Sl.no.
Antibiotic used
1.

Levoflaxacin

Number of bacterial
colonies
2

2.

Amoxicillin

3.

Cefixime

4.
5.

Roxithromycin
Ciprofloxacin

12
3

6.

Cefpodoxime

19

Description
Medium colonies near the
edge of the petri dish
Medium
sized colonies
around the filter paper
Small to medium size
colonies
Small sized colonies
Medium to large sized
colonies
Large number of small to
medium
sized
colonies
around the edge.
Small sized colonies
Medium sized colonies
N/A

Description
Medium colonies near the
edge of the petri dish
Medium
sized colonies
around the filter paper
Small to medium size
colonies
Small sized colonies
Medium to large sized
colonies
Large number of small to
medium
sized
colonies
22

7.
8.
9.

Ofloxacin
Azithromycin
Control

13
9
0

around the edge.

Small sized colonies
Medium sized colonies
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.

23

BIBLIOGRAPHY
Wikipedia - The free encyclopedia - (http://en.wikipedia.org)
Comprehensive Practical Chemistry

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