INDEX

S.no
CONTENT
:
1. Certificate

2. Acknowledgements

3. Objective

4. Introduction

5. What is antibiotic resistance?

6. Need of this Experiment

7. Fighting antibiotic resistance

8. Materials required

9. Experimental procedure

10. Observation

11. Conclusions

12. Bibliography

13. End of project

 OBJECTIVE

To study of drugs
resistance in bacteria
using antibiotics.

introduction
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. 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
sulphonamides . 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. 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
antibacterial—for example, the sulphonamides, the quinolones, and
the oxazolidinones—are 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 resistance–when bacteria change so antibiotics no
longer work in people who need them to treat infections–is 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 by Staphylococcus 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 pre-existing antibiotic-resistant
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. In medicine, the major problem of the emergence of
resistant bacteria is due to misuse and overuse of antibiotics. 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.] 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. 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 (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 multi-resistant, is a bacterium that carries several resistance
genes. 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. In the case of colonization with Clostridium difficile, the
high-risk antibiotics include cephalosporins and in particular
quinolones and clindamycin.
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
sulphide 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
sulphide 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 as Staphylococcus 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

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 petri dishes. Removing the cotton plug
and flamed the mouth of the culture tube. Then I lifted the cover of
the Petri dish 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 Petri
dish. Moving the covered Petri dish along the table top to distribute
the medium evenly. Then I allowed the agar to cool. After that I
prepared two petri dishes 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 Petri dish A. Considering Petri dish B as control.
Then I kept both the Petri dishes undistributed in warm place to allow
the bacteria to grow. Then I observed the Petri dishes 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.

CONCLUSIONS

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.
Reference

1. Comprehensive Laboratory Manual in Biology-XII 2. Biology Text

for Class XII – NCERT

2. http://www.wikipedia.org/

3. http://www.sciencedaily.com/articles/a/antibiotic_resistance.htm

4.
http://www.betterhealth.vic.gov.au/bhcv2/bhcarticles.nsf/pages/Antibi
otic_resistant_bacteria

5. http://www.rxlist.com/antibiotic_resistance-page3/drugs-
condition.htm