ANTIBIOTIC

RESISTANCE
HOW BACTERIA DEVELOP
RESISTANCE AGAINST
ANTIBIOTICS?
CERTIFICATE
This is to certify that this biology project report entitled
‘ANTIBIOTIC RESISTANCE (HOW BACTERIA DEVELOP
RESISTANCE AGAINST ANTIBIOTICS)' by GAURI SINGH
student of class XII-A4, submitted in fulfilment of credit for
biology practical evaluation of CBSE, during the academic
year 2024-2025, is a bonafide record of work carried out
under the guidance and supervision of my Biology teacher
'MRS. BHAVANA JOSHI MA'AM' and has been completed
successfully.

Teacher’s signature:- ………………………………………

Examiner’s signature:- ………………….

Rani laxmi bai memorial school
Session: 2024-2025
Subject: biology

Investigatory project on
Antibiotic resistance (how bacteria
develop resistance against antibiotics)

Submitted to: Mrs. Bhavana Joshi Ma’am

Submitted by: Gauri Singh
Class: XII A4
 ACKNOWLEDGEMENT

I would like to convey my sincere gratitude to my

biology teacher MRS. BHAVANA SRIVASTAVA, for her
support and guidance which directed me to
successfully complete this project and to my principal
MRS. UMA PANDEY who gave me this golden
opportunity to do this interesting project.

I would also like to present heartfelt thanks my

parents and friends who helped me a lot across this
journey of completing the project.
 INDEX:
 INTRODUCTION
 MECHANISMS OF ANTIBIOTIC

RESISTANCE
 FACTORS CONTRIBUTING TO

ANTIBIOTIC RESISTANCE
 Detection of antibiotic resistance
 PUBLIC HEALTH IMPLICATIONS OF

ANTIBIOTIC RESISTANCE
 CURRENT STRATEGIES TO COMBAT

ANTIBIOTIC RESISTANCE
 EXPERIMENT

 CONCLUSION

 BIBLIOGRAPHY
 INTRODUCTION
Antibiotics can be defined as any of a large group of chemical substances, as
penicillin or streptomycin, produced by various microorganisms and fungi,
having the capacity in dilute solutions to inhibit the growth of or to destroy
bacteria and other microorganisms, used chiefly in the treatment of infectious
diseases. In other words, it is a drug used to treat infections caused by bacteria
and other microorganisms. Antibiotics targets and impair several essential
mechanisms involved in bacterial metabolism, growth or multiplication. It also
causes the bacterial lyses by distortion and damage to the cell membrane that
cause leakage of vital cell materials and death. The discovery of antibiotics, such
as penicillin by Alexander Fleming in 1928, marked a milestone in medical
history, saving countless lives worldwide.

Antibiotics work by targeting specific aspects of bacterial biology that human

cells do not possess, such as cell wall synthesis, protein production, or DNA
replication. This targeted action helps eliminate bacteria while minimizing harm
to human cells. There are several classes of antibiotics, each with its own
mechanism of action and spectrum of activity against different types of bacteria.
These include broad-spectrum antibiotics effective against a wide range of
bacteria and narrow-spectrum antibiotics that target specific types of bacteria.
For e.g. Penicillin G and V are used to treat infections caused by Grampositive
Staphylococcus pyogenes (strep throat), and Streptococcus pneumoniae
(respiratory tract infections). Erythromycin (Macrolide group) is a very safe
antibiotic, it is effective orally, bacteriostatic, and active against Gram-positive
infections, especially those of the respiratory tract caused by streptococci.

While antibiotics are invaluable in treating bacterial infections, their overuse and
misuse have led to the emergence of antibiotic-resistant bacteria. Antibiotic
resistance is a significant global health concern characterized by bacteria
evolving to withstand the effects of antibiotics designed to kill them. This
phenomenon arises when bacteria mutate or acquire genes that allow them to
survive exposure to antibiotics, rendering these medications less effective.
 MECHANISMS OF ANTIBIOTIC
RESISTANCE:-

Different mechanisms are known to enhance the antimicrobial resistance.

Microbes could be intrinsically resistant and may lack a target for the
antibiotics. Some common mechanisms are:-

1. Intrinsic resistance: Some microbes may lack a target for antibiotics,

making them intrinsically resistant. Chlamydiae do not have
peptidoglycan and are not susceptible to the action of penicillins

2. Inaccessibility of antibiotic targets: Changes in the membrane can

block antibiotic entrance and penetration into the cell. Peptidoglycan
in Gram-negative bacteria is inaccessible to penicillin that cannot
penetrate the Gram-negative outer membrane.

3. Efflux pumps: These pumps actively remove antibiotics from cells,

contributing to resistance. Efflux systems function via an energy
dependent mechanism (Active transport) pump out unwanted toxic
substances through specific efflux pumps. Some efflux systems are
drug-specific, whereas others may accommodate multiple drugs.

4. Modification of antibiotic targets: Bacteria can modify ribosomes

or enzymes to prevent antibiotic action.
 5. Chemical modification or destruction: Enzymes can degrade or
inactivate antibiotics through reactions like phosphorylation,
adenylation, or acetylation. Chloramphenicol resistance is due to the
presence of an intracellular enzyme called chloramphenicol
transacetylate. This enzyme acetylates hydroxyl groups on the
chloramphenicol structure which causes decreased binding

6. Elaboration of alternative pathways: Bacteria can develop new

enzymes or pathways that are not affected by antibiotics.

7. Mutation and selection: Bacteria can rapidly mutate, leading to

changes in proteins or DNA that confer resistance. Bacteria grow and
mutate rapidly at a rate of 1 in every 100,000 to 1 in every million

8. Transfer of antibiotic resistance: DNA and resistance traits can be

transferred between bacteria through processes like transformation
(DNA escapes from damaged or dying cells, and live bacterial cells uptake
one strand of the genes and incorporate those into the full DNA gene
package), transduction (Transduction occurs when chromosomal or
plasmid DNA is transferred from one bacterium to another by
bacteriophages [58]. Bacteriophages are viruses that attack bacteria), and
conjugation (One bacterium attaches to another bacterium via a pilus
(protein transfer tube) that transfers a portion of its genes to a receiving
bacterium).

These mechanisms contribute to the ability of bacteria to survive and

reproduce in the presence of antibiotics that were previously effective
against them.
 FACTORS CONTRIBUTING TO
ANTIBIOTIC RESISTANCE:-
 Over prescription and Misuse: Inappropriate use of antibiotics, such as
prescribing them for viral infections (where they are ineffective), not completing
prescribed courses, or sharing antibiotics, accelerates the development of
resistance. This misuse creates selective pressure favoring resistant bacteria.

 Incomplete Treatment: Not completing a full course of antibiotics allows

surviving bacteria with resistance traits to proliferate, as they are not completely
eradicated. This can lead to the emergence of resistant strains.

 Use in Agriculture and Livestock: Antibiotics are extensively used in

agriculture for disease prevention and growth promotion in livestock. This
widespread use can contribute to the development of resistant bacteria, which
may spread to humans through food consumption or environmental
contamination.

 Poor Infection Control in Healthcare Settings: Inadequate infection

prevention and control measures in hospitals and healthcare facilities can lead to
the transmission of resistant bacteria among patients, healthcare workers, and
visitors.

 Global Travel and Trade: Resistant bacteria can spread internationally

through travel, trade, and migration. Infected individuals or contaminated goods
can introduce resistant strains to new geographic regions.

 Lack of New Antibiotics: There has been a decline in the development of

new antibiotics, partly due to scientific challenges and economic disincentives.
Without new antibiotics, treatment options become limited, and existing
antibiotics may become less effective over time.

 Environmental Contamination: Antibiotics and resistant bacteria can enter

the environment through wastewater from healthcare facilities, pharmaceutical
manufacturing, and agricultural runoff. Environmental reservoirs can promote
the survival and spread of resistance genes.
 Detection of antibiotic
resistance:
Some common methods used for detecting antibiotic
resistance:

1. Disk Diffusion (Kirby-Bauer) Test: This is a simple and widely

used method where paper disks containing specific antibiotics are
placed on an agar plate inoculated with the bacteria in question.
The zone of inhibition around each disk indicates the
susceptibility of the bacteria to the antibiotic. A smaller zone or
no zone indicates resistance.
2. Minimum Inhibitory Concentration (MIC) Test: This method
determines the lowest concentration of an antibiotic that inhibits
the growth of the bacteria. It provides a quantitative measure of
antibiotic susceptibility and is usually performed using broth
dilution or agar dilution methods.
3. Agar Dilution Method: In this method, antibiotics are
incorporated into agar plates at different concentrations. Bacteria
are then streaked onto the plates, and the minimum inhibitory
concentration is determined by observing the growth or lack
thereof.
4. Molecular Methods (PCR, Sequencing): Molecular techniques
can detect specific resistance genes or mutations associated with
resistance. Polymerase chain reaction (PCR) is used to amplify
and detect resistance genes directly from bacterial DNA.
Sequencing can provide detailed information about genetic
mutations conferring resistance.
5. Phenotypic Tests for Specific Resistance Mechanisms: Some
tests are designed to detect specific mechanisms of resistance,
such as beta-lactamase production (e.g., the Modified Hodge
Test) or carbapenemase production (e.g., Carba NP test).
 PUBLIC HEALTH IMPLICATIONS OF
ANTIBIOTIC RESISTANCE:-
Antibiotic resistance poses significant public health implications globally,
affecting treatment outcomes, healthcare costs, and patient mortality rates.
Here are some key statistics and implications related to antibiotic
resistance:

1. Increased Mortality: Antibiotic-resistant infections are associated

with higher mortality rates compared to infections caused by
susceptible bacteria. According to the Centers for Disease Control and
Prevention (CDC), in the United States alone, at least 35,000 deaths
per year are attributed to antibiotic-resistant infections.
2. Healthcare Costs: Treating antibiotic-resistant infections is more
expensive due to prolonged hospital stays, additional diagnostic tests,
and the need for more expensive antibiotics. In the European Union,
the economic burden of antibiotic resistance is estimated to exceed
€1.5 billion annually in healthcare costs and productivity losses.
3. Limited Treatment Options: As antibiotic resistance spreads, fewer
effective treatment options are available for common infections. This
can lead to treatment failures, persistent infections, and the need for
more aggressive therapies.
4. Impact on Vulnerable Populations: Vulnerable populations, such as
elderly individuals, children, and immunocompromised patients, are
particularly at risk from antibiotic-resistant infections due to weaker
immune systems and frequent healthcare exposure.
5. Global Health Security: Antibiotic resistance undermines global
health security by reducing the effectiveness of antibiotics used to
treat infectious diseases, including potential pandemics caused by
resistant pathogens.
6. Challenges in Healthcare Settings: Hospitals and healthcare
facilities face challenges in infection prevention and control, as
resistant bacteria can spread easily among patients.
CURRENT STRATEGIES TO COMBAT
ANTIBIOTIC RESISTANCE:
Current strategies to tackle antibiotic resistance include:
1. Optimizing Antibiotic Use: Sensible use of antibiotics is crucial to
minimize the development of resistance. This involves prescribing
antibiotics only when necessary, completing the full course of treatment,
and avoiding the unnecessary use of broad-spectrum antibiotics.

2. Research on Alternative Measures: Exploring and developing

alternative treatments such as probiotics, vaccines, bacteriophages,
antimicrobial peptides, and bioactive phytochemicals to reduce reliance
on antibiotics.

3. Surveillance and Monitoring: Regular surveillance and monitoring of

antibiotic use in both human and animal settings to track resistance
patterns and identify emerging resistance mechanisms.

4. Education and Awareness: Educating healthcare professionals, patients,

and the public about the importance of responsible antibiotic use, the
consequences of resistance, and the need for adherence to treatment
protocols.

5. Regulation and Policy: Implementing regulations and policies to

control the use of antibiotics in healthcare settings, agriculture, and
veterinary medicine to reduce the selective pressure that drives
resistance.

6. Infection Prevention and Control: Emphasizing infection prevention

measures in healthcare facilities to reduce the spread of resistant
bacteria and infections.

7. Research and Development: Continued research into new antibiotics,

diagnostic tools, and treatment strategies to combat resistant strains of
bacteria.
 EXPERIMENT
AIM: To study the drug resistance in bacteria using antibiotics.

Materials Needed:
 Sterilized Petri dishes
 Sterilized culture tubes with media
 Transfer loops4. Forceps
 Flask
 Beaker
 Burner
 Penicillin
 Aureomycin
 Hay
 Alcohol
 Agar
 Starch
 Distilled water

Procedure:
1. I put 200ml of distilled water in a flask, then 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. Boiled
the medium for 5 minutes and then allowed the medium to cool down
to reach room temperature. After that, I placed 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. I took the tubes including the agar medium and heated them in warm
water to cause the agar to melt. Allowed each test tube to cool so I
can hold it in my hand and the agar remains liquid. After removing
the cotton plug, I passed the mouth of the test tube through the
3. Bunsen burner twice. I dipped the transfer loop in alcohol before
flaming it and allowed it to cool. Picking up a loop full of bacterial
culture from the flask, I transferred it quickly to the warm agar in the
culture tube. I flamed the loop and the mouth of the culture tube
before putting back the cotton plug. Rolling the culture tube of warm
agar in between the palms of my hands, I mixed the bacteria with the
agar well.

4. I prepared my sterilized Petri dishes before removing the cotton plug

and flaming the mouth of the tube another time. I then lifted the cover
of the Petri dish at an angle of 45 degrees and quickly poured the
medium of the culture tube into the bottom half of the Petri dish. And
then, moved the covered Petri dish around the table top to distribute
the medium evenly, I allowed the agar to cool. After that I prepared 2
Petri dishes and marked them as A & B.

5. I prepared Penicillin and Aureomycin by dissolving both powder

drugs in distilled water. Then I cut out a few discs of filter paper 1cm
in diameter. Then I soaked a disc in each of the Penicillin and
Aureomycin solutions. I dipped the forceps in alcohol before quickly
passing its tip over the Bunsen flame. Using the sterilized forceps, I
put the 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, undisturbed, in a warm place to allow the bacteria to
grow. I then observed the Petri dishes for several days.
OBSERVATIONS:-
Bacteria that are susceptible to the antibiotic will not grow in the
section where the antibiotic disk is placed, creating a clear zone
(called the zone of inhibition), while resistant bacteria will
continue to grow.

 Sensitive Strains: On plates with antibiotics to which the

bacteria are sensitive, you will observe clear zones of inhibition
around the antibiotic discs. The diameter of these zones can vary
depending on the susceptibility of the bacteria.
 Resistant Strains: On plates with antibiotics to which the
bacteria are resistant, there will be no or very small zones of
inhibition. Bacterial growth will be observed up to the edge of the
antibiotic disc.

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

PRECAUTIONS:-
1. Handle all materials in a sterile manner to prevent
contamination.
2. Use appropriate safety measures when working with bacteria
and antibiotics.
 CONCLUSION:-

Antibiotic resistance remains an internationally

worrisome problem that requires urgent intervention.
The idea of antibiotic potentiation by molecules or
approaches that block key metabolic pathways is a
viable alternative to the “one compound, one target”
model that has dominated antibiotic drug development.
Although employing this combinatorial technique to
build and improve antibiotics present obstacles in
terms of clinical trials and regulatory hurdles. The
development of agents with the dual activity of
inhibiting bacteria while also enhancing the immune
system appears to be a promising strategy.
Furthermore, antibiotic-induced suppression of
bacteria's downstream repair processes could be the
ultimate haymaker for tackling bacterial pathogens.
 BIBLIOGRAPHY

 Antibiotic_resistance_The_challenges_and_some_
emer.pdf
 CLASS XII BIOLOGY NCERT
 WHO, Antibiotics resistance, WHO; 2022. Accessed
April 21, 2022; https://www.who.int/news-
room/fact-sheets/detail/antibiotic –resistance.
 NRayamajhiChaSBYooHS2010.Antimicrobialresista
ncepastpresentandfuture.Reviewpaper.JournalofB
iomedicalResearch.pdf