Session:-2018-2019

Biology Investigatory Project File

Submitted by:- Supervised by:-

Ma.Gautam Bhawsar Mrs.mamta Patidar

XII “A” PGT , Biology
 Certificate
This is to certify that Gautam Bhawsar of Grade
XII “A” KAKKA INTERNATIONAL SCHOOL
NALKHEDA satisfactorily completed the
project in Biology under my guidance 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 2018-
2019………

Signature of Signature of the

Candidate teacher In-Charge

Signature of Signature of the

Principal External Examiner
 Acknowledgement

I would like to thank my teacher, Mrs.Mamta Patidar 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.Kiran Mundra

ma’am for providing me the opportunity to work on this project.

It goes without saying that especially my classmates,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.
Gautam Bhawsar
XII “A”
Kakka International School,
Nalkheda
Table of Contents………!
 Introduction
 History
 Objective
 Scope & Limitations
 Theory
 Experiment
 Procedure
 Observation
 Result
 Bibliography
 And of the Project
 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 1880’s. 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 genusof 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 Bayer’s laboratory Germany.
Domagk received the 1939 Noble Prize forMedicine for his efforts.

The first true antibiotic, was 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 found—in 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 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 injured in the
Iraq/Kuwait region during Operation Iraqi Freedom and in
Afghanistan during Operation Enduring Freedom were treated.
Most ofthese 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.
 EXPERIMENT
Aim:
To study the drug resistance in bacteria using antibiotics.

Requirement:
a. Apparatus Requirement

 Petridish

 Sterilized culture tubes

 Forceps

 Flasks
 Beakers

 Burner
b. Chemical Requirements

 Penicillin and other antibiotics

 Agar
 Starch

 Distilled water
 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.
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.
 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.

 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.
 OBSERVATION

 After one week

Sl Antibiotic used No. of bacterial Description

no. colonies found
1 Levoflaxacin 5 Few medium colonies near
the edge of the Petri dish
2 Amoxicillin 8 Few medium sized colonies
around the filter paper
3 Cefixime 16 Small to medium size
colonies
4 Roxithromycin 29 Dense growth of small
colonies of bacteria
5 Ciprofloxacin 4 Few medium to large sized
colonies
6 Cefpodoxime 31 Large number of small to
medium sized colonies
around the edge.
7 Ofloxacin 34 Small sized colonies
8 Azithromycin 14 Medium sized colonies
9 Control 0 N/A
 After two weeks
Sl Antibiotic used No. of bacterial Description
no. colonies found
1 Levoflaxacin 3 Medium colonies near
the edge of the petri
dish
2 Amoxicillin 4 Medium sized colonies
around the filter paper
3 Cefixime 9 Small to medium size
colonies
4 Roxithromycin 15 Small sized colonies
5 Ciprofloxacin 3 Medium to large sized
colonies
6 Cefpodoxime 23 Large number of small
to medium sized colonies
around the edge
7 Ofloxacin 21 Small sized colonies
8 Azithromycin 11 Medium sized colonies
9 Control 0 N/A
 After three weeks

Sl Antibiotic used No.of bacterial Description

No. colonies found
1 Levoflaxacin 2 Medium colonies near the
edge of the petri dish
2 Amoxicillin 3 Medium sized colonies
around the filter paper
3 Cefixime 6 Small to medium size
colonies
4 Roxithromycin 12 Small sized colonies
5 Ciprofloxacin 3 Medium to large sized
colonies
6 Cefpodoxime 19 Large number of small to
medium sized colonies
around the edge.
7 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.
 BIBLIOGRAPHY
 Wikipedia - The free encyclopedia -
(http://en.wikipedia.org)
 Comprehensive Practical Chemistry
 NCERT BIOLOGY CLASS XII
 Xam idea Class XII
 https://www.google.com/search?q=Penicillin%2C+the+firs
t+natural+antibiotic+discovered+by+Alexander+Fleming
&ie=utf-8&oe=utf-8&client=firefox-b-ab
 https://www.google.com/search?client=firefox-b-
ab&ei=VeL8W6rUOIum9QO2xLPIDA&q=penicillin+ant
ibiotic&oq=Penicillin+&gs_l=psy-
ab.1.0.0i67l7j0l3.4360501.4366725..4368900...0.0..
0.349.3424.0j6j7j2......0....1..gws-
wiz.......33i10._bdqu_C5Z1A
Thanking You ………………….