Abstract

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 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. 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 sulfonamides, 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 preexisting 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 multiresistant, 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 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
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.

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.

Antibiotic resistance tests: Bacteria are streaked on dishes with white disks, each
impregnated with a different antibiotic. Clear rings, such as those on the left, show
that bacteria have not grown—indicating that these bacteria are not resistant. The
bacteria on the right are fully susceptible to only three of the seven antibiotics tested.
[1]

Antimicrobial resistance (AMR) is the ability of a microbe to resist the effects of

medication previously used to treat them.[2][3][4] The term includes the more
specific "antibiotic resistance", which applies only to bacteria becoming resistant to
antibiotics.[3] Resistant microbes are more difficult to treat, requiring alternative
medications or higher doses, both of which may be more expensive or more toxic.
Microbes resistant to multiple antimicrobials are called multidrug resistant (MDR); or
sometimes superbugs.[5]

Resistance arises through one of three mechanisms: natural resistance in certain types
of bacteria, genetic mutation, or by one species acquiring resistance from another.[6]
All classes of microbes can develop resistance: fungi develop antifungal resistance,
viruses develop antiviral resistance, protozoa develop antiprotozoalresistance, and
bacteria develop antibiotic resistance. Resistance can appear spontaneously because of
random mutations.

Preventive measures include only using antibiotics when needed, thereby stopping
misuse of antibiotics or antimicrobials.[7][8] Narrow-spectrum antibiotics are
preferred over broad-spectrum antibiotics when possible, as effectively and accurately
targeting specific organisms is less likely to cause resistance.[9] For people who take
these medications at home, education about proper use is essential. Health care
providers can minimize spread of resistant infections by use of proper sanitation and
hygiene, including handwashing and disinfecting between patients, and should
encourage the same of the patient, visitors, and family members.[10]

Rising drug resistance is caused mainly by use of antimicrobials in humans and other
animals, and spread of resistant strains between the two.[7] Antibiotics increase
selective pressure in bacterial populations, causing vulnerable bacteria to die; this
increases the percentage of resistant bacteria which continue growing. With resistance
to antibiotics becoming more common there is greater need for alternative treatments.
Calls for new antibiotic therapies have been issued, but new drug development is
becoming rarer.[11]
Antimicrobial resistance is on the rise. Estimates are that 700,000 to several million
deaths result per year.[12][13]Each year in the United States, at least 2 million people
become infected with bacteria that are resistant to antibiotics and at least 23,000
people die as a result.[14] There are public calls for global collective action to address
the threat include proposals for international treaties on antimicrobial
resistance.Worldwide antibiotic resistance is not fully mapped, but poorer countries
with weak healthcare systems are more affected