Drug Resistance in Bacteria

A Project Report
Submitted by
Aashray Kotha
CBSE GRADE XII
BIOLOGY

Nehru Smarka Vidyalaya Kendra 244C,

32 Cross,7th block, 2nd Main Rd

Jayanagar, Bengaluru, Karnataka 560082

2016-17

CERTIFICATE

This is to certify that Aashray Kotha of Grade XII in Nehru Smarka

Vidyalaya Kendra, Bangalore, with registration number
has successfully completed the project in Biology on the topic
Drug Resistance in Bacteria, in partial fulfilment of the requirements stated
by CBSE in the year 2016-17.

Signature of the Candidate

Signature of Teacher In Charge

Signature of Principal

Signature of External Examiner

ACKNOWLEDGEMENT

I warmly acknowledge the continuous encouragement and timely

suggestions offered by our dear Chairman Mr. D Muniraju, and Mr.
Ashish. I extend my hearty thanks also to our principal, Mr. Arokia Raj,
for giving me the opportunity to make use of the facilities available in
the campus to carry out the project successfully.

I am highly indebted to Mrs. Deepthi Kiran for the constant supervision,

providing necessary information and support in completing the project. I
would like to express my gratitude towards her for her kind co-operation
and encouragement.

Finally, I extend my gratitude to one and all who are directly or indirectly
involved in the successful completion of this project work.

Aashray Kotha

INDEX
1. Introduction
2. Apparatus
3. Procedure
4. Observation
5. Conclusion
6. References

Aim of the Project

To study the Drug
resistance in bacteria
using Antibiotics.

Introduction
What is Antibiotic?
Anantibioticis an agent that either kills or inhibits the
growth of amicroorganism.
The termantibioticwas first used in 1942 bySelman
Waksmanand his collaborators in journal articles to
describe any substance produced by a microorganism that
isantagonisticto the growth of other microorganisms in
high dilution.[3]This definition excluded substances that kill
bacteria but that are not produced by microorganisms (such
asgastric juicesandhydrogen peroxide). It also
excludedsyntheticantibacterial compounds such as the
sulphonamides. Many antibacterial compounds are
relativelysmall moleculeswith amolecular weightof less
than 2000atomic mass units.
With advances inmedicinal chemistry, most modern
antibacterial aresemi syntheticmodifications of various
natural compounds.[4]These include, for example, thebetalactam antibiotics, which include thepenicillin(produced by
fungi in the genusPenicillium), thecephalosporin, and
thecarbapenems. Compounds that are still isolated from
living organisms are theamino glycosides, whereas other
antibacterialfor example, thesulfonamides, the
quinolones, and theoxazolidinonesare produced solely by
chemical synthesis. In accordance with this, many
antibacterial compounds are classified on the basis of
chemical/biosyntheticorigin 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:Bactericidalagents kill bacteria,
and bacteriostatic agentsslow down or stall bacterial
growth.

What is Antibiotic Resistance?

Antibiotic resistanceis a form ofdrug resistancewhereby
some (or, less commonly, all) sub-populations of
amicroorganism, usually a bacterial species, are able to
survive after exposure to one or moreantibiotics; pathogens
resistant to multiple antibiotics are consideredmultidrug
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). AWorld Health Organizationreport 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 riskbenefit 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 thirdline antibiotics is, thus, sequentially acquired, a case
quintessentially illustrated byStaphylococcus aureusin
somenosocomialsettings. Some pathogens, such
asPseudomonas aeruginosa, also possess a high level of
intrinsic resistance.
It may take the form of a spontaneous or induced
geneticmutation, or the acquisition of
resistancegenesfrom other bacterial species byhorizontal
gene transferviaconjugation, transduction,
ortransformation. Many antibiotic resistance genes reside
on transmissibleplasmids, facilitating their transfer.
Exposure to an antibioticnaturally selectsfor 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 forMycobacterium
tuberculosis, the bacteria that causesTuberculosis, since
evidence is lacking for whether these bacteria have
plasmids.AlsoM. tuberculosislack 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 andveterinary medicine. Any use of antibiotics
can increaseselective pressurein 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,MRSAis probably the
most well-known, but others including VISA (vancomycinintermediateS. aureus), VRSA (vancomycin-resistantS.
aureus), ESBL (Extended spectrum beta-lactamase), VRE
(Vancomycin-resistantEnterococcus) and MRAB (MultidrugresistantA. baumannii) are prominent
examples.Nosocomial infectionsoverwhelmingly dominate
cases where MDR pathogens are implicated, but multidrugresistant infections are also becoming increasingly common
in the community.
Although there were low levels of preexisting antibioticresistant bacteria before the widespread use of
antibiotics,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
includeantibiotic use in livestockfeed to promote faster
growth. 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). 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 (seeHealthcareassociated infectionsandInfection control).
Certain antibiotic classes are highly associated with
colonisation with "superbugs" compared to other antibiotic
classes. A superbug, also called multiresistant, is a
bacterium that carries several resistance genes.The risk for
colonisation 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 ofMRSA, increased
rates of MRSA infections are seen
withglycopeptides,cephalosporins, and
especiallyquinolones. In the case of colonisation
withClostridium difficile, the high-risk antibiotics include
cephalosporins and in particular quinolone andclindamycin.
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

ofbacteria,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 ofcell membraneassembly orDNA
translationare 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 aureushave
developed a singleDNAmutation that alters the organism's
cell wall. This gives them the ability to withstand antibiotic
cell disruption processes. Antibiotic resistantStreptococcus
pneumoniaeproduce a protein called MurM, which
counteracts the effects of antibiotics by helping to rebuild
thebacterial 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 hostcell. 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 aproteincomplex 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 defences could lead to the reversal of
antibiotic resistance in pathogens such
asStaphylococcusandE.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. Drugresistant microbes in medical facilities lead to patient
infections that are more costly and difficult to treat.
Resistantbacteria in turkeyand 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 Petri
dishes will be clear. In other areas, colonies of
bacteria will be observed. Then I examined the clear
area in each Petri dishes 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