BIOLOGY INVESTIGATORY PROJECT

TOPIC: STUDY OF DRUG RESISTANCE IN

BACTERIA USING ANTIBIOTICS
NAME:
______________________________________
__________
SCHOOL:
______________________________________
________
ROLL NO:
YEAR: 2014-15
INDEX
CONTENTS PAGE NO.
1. Certificate of Authenticity
2. Acknowledgement
3. Aim of the project
4. Introduction
5. Need of the Experiment
6. Material Required for the experiment
7. Experimental Procedure
8. Observation and Conclusions
9. References
CERTIFICATE OF AUTHENTICITY

This is to certify that, __________________________
student of class XII has successfully completed the
research project on the topic STUDY OF DRUG RESISTANCE
IN BACTERIA USING ANTIBIOTICS under the guidance of
_____________________________ (Biology
Teacher).
This project is absolutely genuine and does not indulge
in plagiarism of any kind.
The references taken in making this project have been
declared at the end of this Report.


Signature (Subject Teacher) Signature (Examiner)





I would like to express my special thanks of
gratitude to our Biology teacher Mr.
_________________________ as well as our
Principal Mr. ___________________________
who gave me the golden opportunity to do
this wonderful project on the topic STUDY OF
DRUG RESISTANCE IN BACTERIA USING ANTIBIOTICS
which also helped me in doing a lot of
Research and I came to know about so
many new things I am really thankful to
them.
Secondly I would also like to thank my
parents and friends who helped me a lot in
finalizing this project within the limited
time frame.










To study the Drug
resistance in bacteria
using Antibiotics.



























Introduction

What is Antibiotic?
An antibiotic is an agent that either kills or inhibits the
growth of a microorganism.
The term antibiotic was first used in 1942 by Selman
Waksman and his collaborators in journal articles to
describe any substance produced by a microorganism that
is antagonistic to the growth of other microorganisms in
high dilution.
[3]
This definition excluded substances that kill
bacteria but that are not produced by microorganisms (such
as gastric juices and hydrogen peroxide). It also
excluded synthetic antibacterial compounds such as the
sulfonamides. Many antibacterial compounds are
relatively small molecules with a molecular weight of less
than 2000 atomic mass units.
With advances in medicinal chemistry, most modern
antibacterial are semi synthetic modifications of various
natural compounds.
[4]
These include, for example, the beta-
lactam antibiotics, which include the penicillin (produced by
fungi in the genus Penicillium), the cephalosporin, and
the carbapenems. Compounds that are still isolated from
living organisms are the amino glycosides, whereas other
antibacterialfor example, the sulfonamides, the
quinolones, and the oxazolidinonesare produced solely by
chemical synthesis. In accordance with this, many
antibacterial compounds are classified on the basis of
chemical/biosynthetic origin into natural, semi synthetic,
and synthetic. Another classification system is based on
biological activity; in this classification, antibacterial are
divided into two broad groups according to their biological
effect on microorganisms: Bactericidal agents kill bacteria,
and bacteriostatic agents slow down or stall bacterial
growth.
What is Antibiotic Resistance?
Antibiotic resistance is a form of drug resistance whereby
some (or, less commonly, all) sub-populations of
a microorganism, usually a bacterial species, are able to
survive after exposure to one or more antibiotics; pathogens
resistant to multiple antibiotics are considered multidrug
resistant(MDR) or, more colloquially, superbugs.
Antibiotic resistance is a serious and growing phenomenon
in contemporary medicine and has emerged as one of the
pre-eminent public health concerns of the 21st century, in
particular as it pertains to pathogenic organisms (the term
is especially relevant to organisms that cause disease in
humans). A World Health Organization report released April
30, 2014 states, "this serious threat is no longer a prediction
for the future, it is happening right now in every region of the
world and has the potential to affect anyone, of any age, in
any country. Antibiotic resistancewhen bacteria change so
antibiotics no longer work in people who need them to treat
infectionsis now a major threat to public health."
In the simplest cases, drug-resistant organisms may have
acquired resistance to first-line antibiotics, thereby
necessitating the use of second-line agents. Typically, a
first-line agent is selected on the basis of several factors
including safety, availability, and cost; a second-line agent
is usually broader in spectrum, has a less favorable risk-
benefit profile, and is more expensive or, in dire
circumstances, may be locally unavailable. In the case of
some MDR pathogens, resistance to second- and even third-
line antibiotics is, thus, sequentially acquired, a case
quintessentially illustrated byStaphylococcus aureus in
some nosocomial settings. Some pathogens, such
as Pseudomonas aeruginosa, also possess a high level of
intrinsic resistance.
It may take the form of a spontaneous or induced
genetic mutation, or the acquisition of
resistance genes from other bacterial species by horizontal
gene transfer via conjugation, transduction, or transformation. Many
antibiotic resistance genes reside on
transmissible plasmids, facilitating their transfer. Exposure
to an antibiotic naturally selects for the survival of the
organisms with the genes for resistance. In this way, a gene
for antibiotic resistance may readily spread through an
ecosystem of bacteria. Antibiotic-resistance plasmids
frequently contain genes conferring resistance to several
different antibiotics. This is not the case for Mycobacterium
tuberculosis, the bacteria that causes Tuberculosis, since
evidence is lacking for whether these bacteria have
plasmids. Also M. tuberculosis lack the opportunity to
interact with other bacteria in order to share plasmids.
Genes for resistance to antibiotics, like the antibiotics
themselves, are ancient. However, the increasing
prevalence of antibiotic-resistant bacterial infections seen
in clinical practice stems from antibiotic use both within
human medicine and veterinary 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.
The growing prevalence and incidence of infections due to
MDR pathogens is epitomized by the increasing number of
familiar acronyms used to describe the causative agent and
sometimes the infection; of these, MRSA is probably the
most well-known, but others including VISA (vancomycin-
intermediate S. aureus), VRSA (vancomycin-resistant S.
aureus), ESBL (Extended spectrum beta-lactamase), VRE
(Vancomycin-resistant Enterococcus) and MRAB (Multidrug-
resistant A. baumannii) are prominent
examples. Nosocomial infections overwhelmingly dominate
cases where MDR pathogens are implicated, but multidrug-
resistant infections are also becoming increasingly common
in the community.
Although there were low levels of preexisting antibiotic-
resistant bacteria before the widespread use of
antibiotics,
[7]

[8]
evolutionary pressure from their use has
played a role in the development of multidrug-resistant
varieties and the spread of resistance between bacterial
species.
[9]
In medicine, the major problem of the emergence
of resistant bacteria is due to misuse and overuse of
antibiotics.
[10]
In some countries, antibiotics are sold over
the counter without a prescription, which also leads to the
creation of resistant strains. Other practices contributing to
resistance include antibiotic use in livestock feed to
promote faster growth.
[11][12]
Household use of antibacterial
in soaps and other products, although not clearly
contributing to resistance, is also discouraged (as not being
effective at infection control).
[13]
Unsound practices in the
pharmaceutical manufacturing industry can also contribute
towards the likelihood of creating antibiotic-resistant
strains.
[14]
The procedures and clinical practice during the
period of drug treatment are frequently flawed usually no
steps are taken to isolate the patient to prevent re-infection
or infection by a new pathogen, negating the goal of
complete destruction by the end of the
course
[15]
(see Healthcare-associated
infections and Infection control).
Certain antibiotic classes are highly associated with
colonization with "superbugs" compared to other antibiotic
classes. A superbug, also called multiresistant, is a
bacterium that carries several resistance genes.
[16]
The risk
for colonization increases if there is a lack of susceptibility
(resistance) of the superbugs to the antibiotic used and high
tissue penetration, as well as broad-spectrum activity
against "good bacteria". In the case of MRSA, increased
rates of MRSA infections are seen
with glycopeptides, cephalosporins, and
especially quinolones.
[17][18]
In the case of colonization
with Clostridium difficile, the high-risk antibiotics include
cephalosporins and in particular quinolones
and clindamycin.
[19][20]

Of antibiotics used in the United States in 1997, half were
used in humans and half in animals; in 2013, 80% were used
in animals.




Need of this Experiment

Antibiotic resistance is becoming more and more common.
Antibiotics and antimicrobial agents are drugs or chemicals
that are used to kill or hinder the growth
of bacteria, viruses, and other microbes. Due to the
prevalent use of antibiotics, resistant strains of bacteria are
becoming much more difficult to treat. These "super bugs"
represent a threat to public health since they are resistant
to most commonly used antibiotics.

Current antibiotics work by disrupting so-called cell viability
processes. Disruption of cell membrane assembly or DNA
translation are common modes of operation for current
generation antibiotics. Bacteria are adapting to these
antibiotics making them ineffective means for treating these
types of infection. For example, Staphylococcus aureus have
developed a single DNA mutation that alters the organism's
cell wall. This gives them the ability to withstand antibiotic
cell disruption processes. Antibiotic resistant Streptococcus
pneumoniae produce a protein called MurM, which
counteracts the effects of antibiotics by helping to rebuild
the bacterial cell wall.
Fighting Antibiotic Resistance
Researchers are attempting to develop new types of
antibiotics that will be effective against resistant strains.
These new antibiotics would target the bacteria's ability to
become virulent and infect the host cell. Researchers at
Brandeis University have discovered that bacteria have
protein "switches" that when activated, turn "ordinary"
bacteria into pathogenic organisms. These switches are
unique in bacteria and are not present in humans. Since the
switch is a short-lived protein, elucidating its structure and
function was particularly difficult. Using nuclear magnetic
resonance (NMR) spectroscopy, the researchers were able
to regenerate the protein for one and one half days. By
extending the time frame that the protein was in its "active
state," the researchers were able to map out its structure.
The discovery of these "switches" has provided a new target
for the development of antibiotics which focus on disrupting
the activation of the protein switches.

Monash University researchers have demonstrated that
bacteria contain a protein complex called Translocation and
Assembly Module (TAM). TAM is responsible for exporting
disease causing molecules from the inside of the bacterial
cell to the outer cell membrane surface. TAM has been
discovered in several antibiotic resistant bacteria. The
development of new drugs to target the protein would inhibit
infection without killing the bacteria. The researchers
contend that keeping the bacteria alive, but harmless, would
prevent the development of antibiotic resistance to the new
drugs.

Researchers from the NYU School of Medicine are seeking
to combat antibiotic resistance by making resistant bacteria
more vulnerable to current antibiotics. They discovered that
bacteria produce hydrogen sulfide as a means to counter the
effects of antibiotics. Antibiotics cause bacteria to undergo
oxidative stress, which has toxic effects on the microbes.
The study revealed that bacteria produce hydrogen sulfide
as a way to protect themselves against oxidative stress and
antibiotics. The development of new drugs to target
bacterial gas defenses could lead to the reversal of
antibiotic resistance in pathogens such
asStaphylococcus and E.coli.

These studies indicate how highly adaptable bacteria are in
relation to the application of antimicrobial treatments.
Antibiotic-resistant bacteria have become a problem not
only in hospitals, but in the food industry as well. Drug-
resistant microbes in medical facilities lead to patient
infections that are more costly and difficult to treat.
Resistant bacteria in turkey and other meat products have
caused serious public health safety issues. Some bacteria
may develop resistance to a single antibiotic agent or even
multiple antibiotic agents. Some have even become so
resistant that they are immune to all current antibiotics.
Understanding how bacteria gain this resistance is key to
the development of improved methods for treating antibiotic
resistance.





Material Required for the experiment

1. Sterilized Petri dishes
2. Sterilized culture tubes with media
3. Transfer loops
4. Forceps
5. Flask
6. Beaker
7. Burner
8. Penicillin
9. Aureomycin
10. Hay
11. Alcohol
12. Agar
13. Starch
14. Distilled water









EXPERIMENTAL PROCEDURE

1. To 200ml of distilled water in a flask, I added 8
grams of agar powder and 2 grams of starch. Then
putting a few pieces of dry hay into the medium I
covered the flask with an Inverted beaker. Boiling
the medium for 5 minutes and then cooling the
medium to room temperature. After that placing
the flask in a warm place. Within 2-3 days,
formation of scum of cloudy suspension appeared
on the medium indicating the growth of Bacillus
subtilis.

2. Taking culture tubes with agar medium and
heating the test tubes in warm water to melt agar.
Cooling each test tube so that I can hold it in my
hand and the agar remains liquid. After that
removing the cotton plug and I passed the mouth
of the test tube through the burner flame twice.
Flaming the transfer loop after dipping it in alcohol
and I let it cooled. After that picking up a loop full
of bacterial culture from flask and then I
transferred it to the warm agar in the culture tube.
Flaming the loop and the mouth of the culture tube
and then I replaced the cotton plug. Rolling the
culture tube of warm agar between palms to I
mixed the bacteria well with agar.

*Transferring the bacteria should be done as quickly as possible.




3. After that I took sterilized petridishes. Removing
the cotton plug and flamed the mouth of the
culture tube. Then I lifted the cover of the Petridish
at an angle 45 Degree and then quickly pouring
the medium of the culture tube into the bottom
half the dish. Removing the culture tube and
replacing the cover tube into the bottom half of the
dish. Removing the culture tube, and replace the
cover of the Petridish. Moving the covered Petridish
along the table top to distribute the medium
evenly. Then I allowed the agar to cool. After that I
prepared two petridishes and marked them A & B.

4. I prepared Penicillin and Aureomycin solution by
dissolving the powdered drugs in distilled water.
Then I cut down a few discs of filter paper of 1 cm
diameter. Then I soaked a disc in each of the
penicillin and Aureomycin solutions. Dipping the
forceps in alcohol and the I passed the forceps tip
quickly over the burner flame. Using the sterilized
forceps I put Penicillin and Aureomycin soaked
discs at two distant sites of Petridish A. Considering
Petridish B as control. Then I kept both the
Petridishes undistributed in warm place to allow
the bacteria to grow. Then I observed the
Petridishes for several days.


OBSERVATION:

The area around the antibiotic discs in the Petridishes will
be clear. In other areas, colonies of bacteria will be
observed. Then I examined the clear area in each
Petridishes for few more days. A few very colonies may
appear in the clear areas. These are the colonies of
resistant strains of the bacteria.

CONCLUSION:

Antibiotic drugs killed most of the bacterial strain, hence
the areas appeared clear. However, a few strains which
were resistant in the bacterial population survived and
produced colonies later. This proves the resistant strain to
antibiotics were present in the bacterial population.

REFERENCES:

1. Comprehensive Laboratory Manual In Biology-XII
2. Biology Text For Class XII NCERT

SITES USED

1. http://www.wikipedia.org/
2. http://www.sciencedaily.com/articles/a/antibiotic_resistance.htm
3. http://www.betterhealth.vic.gov.au/bhcv2/bhcarticles.nsf/pages/Anti
biotic_resistant_bacteria
4. http://www.rxlist.com/antibiotic_resistance-page3/drugs-
condition.htm