To Study Drug Resistance in Bacteria Using Antibiotics

A Project Report
Submitted by

Arathi Nair
In partial fulfillment of the

CBSE GRADE XII

IN
Biology
AT

AECS MAGNOLIA MAARUTI PUBLIC SCHOOL

Arakere, Off Bannerghatta Road, Bangalore-560076

2013-2014

CERTIFICATE

This is to certify that Arathi Nair of Grade XII, AECS MAGNOLIA

MAARUTI PUBLIC SCHOOL, BANGALORE with Register Number
<bold,underline>has satisfactorily completed the project in Biology on To study
drug resistances in bacteria using antibiotics in partial fulfillment of the
requirements of All Inida Senior Secondary Certificate Examination(AISSCE)
as prescribed by CBSE in the year 2013-14.

Signature of the
Candidate

Signature of the
Principal

Signature of the
Teacher In-Charge

Signature of the
External Examiner

ACKNOWLEDGEMENT

I warmly acknowledge the continuous encouragement and timely suggestions offered by

our dear Principal Dr.Seema Goel. I extend my hearty thanks for giving me the
opportunity to make use of the facilities available in the campus to carry out the project
successfully.
I would like to thank CBSE for including project work in syllabus as it has taken my
learning outside classroom.
I am highly indebted to Mrs.Neelam Ahlawatfor for the constant supervision, providing
necessary information and supporting in completing the project. I would like to express
my gratitude towards them for their kind co-operation and encouragement.
Finally I extend my gratefulness to one and all who are directly or indirectly involved in
the successful completion of this project work.

Signature of the
Candidate

Table of Contents

Sl.no

Topic

Page no.

1.

Introduction

1-2

2.

History

2-4

3.

Some resistance pathogens

5-6

4.

Objective

5.

Scope and limitations

6.

Theory

7.

Experiment

10-15

8.

Procedure

16-18

9.

Observation

19

10.

Result

20

11.

Bibliography

21

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.
3

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 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

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. Methicillinresistant 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 pyogenes 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 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 overexpressing modestly increased susceptibility to
antibiotics. The study experimented with thePseudomonas 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.
5

Clostridium difficile
Clostridium difficile is a nosocomial pathogen that causes diarrheal disease in hospitals
world wide. 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 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 limitation 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.

EXPERIMENT
Aim: To study the drug resistance in bacteria using antibiotics.
Apparatus Requirements:
Petridish

Sterilized culture tubes

10

Forceps

Flasks

11

Beakers

Burner

12

Chemical Requirements
Penicillin and other antibiotics

Agar

13

Tetracycline

Starch

14

Distilled water

15

PROCEDURE

Prepare pure culture of bacteria:

1. 100ml distilled water; 4gms of agar and 1g of starch was added to a test tube and
shaken well.
2. The test-tube was placed over a burner ad boiled for 5minutes.
3. While the solution was boiled, 10pieces of dry hay were added.
4. The resulting mixture was then kept in a warm place for 5days for bacterial
growth.

16

Culture medium:
1. Beaker was taken and molten agar was prepared in it.

2. Samples from the bacterial culture were taken in the petridish.

Transfer bacteria from agar test tube into petri dish containing antibiotics:
1. Petridish were taken.
2. 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.
3. The culture was spread evenly.

17

Antibiotics:

1. A solution of antibiotics was made by dissolving different antibiotic tablets in

distilled water. Eg: penicillin solution, tetracycline solution.
2. Filter paper cut in circles were soaked in antibiotic solution ad placed in one part
of the petriplate with culture.
3. Another petriplate with agar wa taken and labeled Control. No antibiotic was
added to this.

18

OBSERVATION

After one week

Antibiotic used

Number of bacterial
colonies

19

RESULT

Description

The observes 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.

20