DEPARTMENT OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY.

DETERMINING THE ANTIBACTERIAL ACTIVIY OF MEDICINAL PLANT EXTRACTS:

NEEM (AZADIRACHTA INDICA) AND ROSEMARY (SALVIA ROSMARINUS) AGAINST

ESCHERICHIA COLI, STAPHYLOCOCCUS AUREUS AND PROTEUS MIRABILIS.

BY
HONU GIDEON
(10864690)

SUPERVISOR:
DR. GLORIA AMEGATCHER

A PROJECT SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY, CELL AND

MOLECULAR BIOLOGY, UNIVERSITY OF GHANA, IN PARTIAL FULFILLMENT OF

THE REQUIREMENT FOR THE AWARD OF A BACHELOR OF SCIENCE DEGREE IN

BIOCHEMISTRY CELL AND MOLECULAR BIOLOGY.

20th September 2024.

TOPIC: Determining the antibacterial activity of medicinal plant extracts: Neem (Azadirachta

indica) and Rosemary (Salvia rosmarinus) against Escherichia coli, Staphylococcus aureus and

Proteus mirabilis.

BY

HONU GIDEON
(10864690)

SUPERVISOR:

DR. GLORIA AMEGATCHER

ii
 DECLARATION

I, Gideon Honu do hereby declare that with the exclusive references made in relation to work

done by other scientists for which has been acknowledged, the experimental outline described in

this project was performed by me, at the department of Biochemistry, Cell and Molecular

Biology under the supervision of Dr. Gloria Amegatcher.

…………………………… ………………………………
HONU GIDEON (STUDENT) DATE

…………………………………. …… …10-09-2024………………….........
DR. GLORIA AMEGATCHER (SUPERVISOR) DATE

SEPTEMBER 2024.

iii
 DEDICATION

This thesis is dedicated to God Almighty for His never- ending grace and guidance throughout

my undergraduate program. I also dedicate this project work to my family and friends especially

Mr. Ekpe Godwin Honu Sallah, Torgbui Shitsi III, Honu Emmanuella and Honu Augusta who

were there for me throughout these few years of my undergraduate studies, God bless you all

most especially the lecturers of the Biochemistry department including my mentor and

supervisor.

iv
 ACKNOWLEDGEMENT

I would like to offer my gratitude to my supervisor, Dr. Gloria Amegatcher for the privilege to

work under her and her supervision and mentorship. Also, my gratitude goes out to Mr. Godwin

Batsa Kpodjei for providing all the reagents needed for this project. Finally, I thank the

Department of Biochemistry Cell and Molecular Biology for providing me with the laboratory

equipment and the research-friendly environment, and the University of Ghana for the

opportunity.

v
Table of Contents
CHAPTER ONE........................................................................................................................................1
1.0 INTRODUCTION...........................................................................................................................1
1.1 Background....................................................................................................................................1
1.2 Hypothesis.....................................................................................................................................2
1.3 Aim................................................................................................................................................3
1.4 Specific Objectives.........................................................................................................................3
CHAPTER TWO.......................................................................................................................................4
2.0 LITERATURE REVIEW...............................................................................................................4
2.1 Introduction to Antibacterial Resistance and Medicinal Plants.....................................................4
2.2 Historical and Ethnobotanical Context of Medicinal Plants...........................................................5
2.3 Ancient Civilizations and Medicinal Plants.....................................................................................5
2.4 Medicinal Plants in Modern Context.............................................................................................6
2.5 Phytochemicals and Mechanisms of Antibacterial Action.............................................................7
2.6 Phytochemicals present in Neem (Azadirachta indica)..................................................................9
2.7 Phytochemicals present in Rosemary (Salvia rosmarinus).............................................................9
2.8 Pathogenic bacteria and their impact on human health..............................................................10
2.9 Methodologies for Assessing Antibacterial Activity.....................................................................11
2.9.1 Synergistic effects in antibacterial therapy...............................................................................12
2.9.2 multi-drug resistance (MDR) of pathogenic bacteria................................................................12
CHAPTER THREE.................................................................................................................................13
3.0 MATERIALS AND METHODS..................................................................................................13
3.1 Collection of the plant sample.....................................................................................................13
3.2 Preparation of the plant extracts.................................................................................................13
3.3 Subculturing of bacterial strains..................................................................................................13
3.5 Preparation of Mueller Hinton Agar............................................................................................14
3.6 Antibiotic Susceptibility Test (AST)..............................................................................................14
3.6.1 Minimum Inhibitory Concentration Determination..................................................................14
CHAPTER FOUR...................................................................................................................................16
4.0 RESULT.........................................................................................................................................16

vi
 4.1 Minimum inhibitory concentration (MIC)....................................................................................22
CHAPTER FIVE.....................................................................................................................................27
5.0 DISCUSSION.................................................................................................................................27
CONCLUSION....................................................................................................................................30
REFERENCES....................................................................................................................................31
APPENDIX..........................................................................................................................................36

vii
viii
 ABSTRACT

This study explores the antibacterial activity of Neem (Azadirachta indica) and Rosemary

(Salvia rosmarinus) against resistant strains of Escherichia coli, Staphylococcus aureus and

Proteus mirabilis, aiming to address the growing challenge of antibiotic resistance. Extracts of

Neem and Rosemary was extracted from their leaves dried under shade for one week and

subjected to comprehensive antibacterial assays using agar disc diffusion and well diffusion

methods. Contrary to my initial hypothesis, extract of Neem did not show any significant

antimicrobial activity against Escherichia coli. The Neem extract shows antibacterial activity

against strains of Staphylococcus aureus and Proteus mirabilis. The negative outcome of Neem

extract against Escherichia coli may be due to part of the plant used since different plant parts

have different levels of antibacterial compounds and concentration of the extract used against the

bacterial strain. This research was carried out with careful consideration of various factors

including plant extracts concentration, environmental conditions, bacterial strain resistance

profiles and potential interactions with other compounds.

ix
 CHAPTER ONE
1.0 INTRODUCTION

1.1 Background

The increasing incidence of antibiotic-resistant bacteria is a global public health concern,

necessitating the exploration of alternative antimicrobial agents. Plants are potential sources of

antibacterial agents in many countries and a high percentage of populations in developing

countries use medicine derived from plants. Crude plant extracts are traditionally used as herbal

medicine for the treatment of many human infectious diseases (Macharia, 2023). Medicinal

plants contain several phytochemicals including tannins, terpenoids, alkaloids, and flavonoids

which have been found in vitro to have antimicrobial properties. Bacteria species such as

Staphylococcus aureus, Escherichia coli are the most common pathogens causing serious

infections (Adnan et al., 2020). Understanding the mechanisms by which medicinal plants exert

antibacterial effects and evaluating their efficacy against clinically relevant pathogens is crucial

for expanding inventory of modern medicine. Crude extracts from medicinal plants can display a

range of biological activities such as anti-microbial, anti-inflammatory and antioxidant activities

(Sarker & Nahar, 2024). The antimicrobial compounds from medicinal plants may inhibit the

growth of bacteria, fungi, viruses, and protozoa by different mechanisms than those of presently

used antimicrobials and may have a significant clinical value in the treatment of resistant

microbial strains (Mishra et al., 2016). Some of those active compounds show both intrinsic

antibacterial activity and antibiotic resistance-modifying activities and some of them, while not

effective as antibiotics by themselves, when combined with antibiotics, can help overcome

antibiotic resistance in bacteria (Mehraj & Parry, 2023). Chemically complex compounds have

great therapeutic potential as they have fewer side effects compared to synthetic drugs and low

1
chances of developing resistance (Arruebo et al., 2011). Bacteria can develop resistance to

medicinal plants treatment through several mechanisms such as genetic mutation where the

bacteria can undergo genetic mutations that alter the target site of the medicinal plant compound

making it less effective, enzymatic degradation where the bacteria can produce enzymes that

break down the medicinal plant compound, rendering it inactive (Elfadadny et al., 2024). Also,

efflux pumps where the bacteria can develop efflux pumps that actively transport the medicinal

plant compound out of the cell reducing its effectiveness, biofilm formation where the bacteria

can form biofilms, which provide a protective environment that reduces the penetration of

medicinal plant compounds and also cell membrane modification where the bacteria can modify

their cell membrane to reduce the uptake of the medicinal plant compound (Veeresham, 2010).

The effectiveness of medicinal plant extracts to inhibit bacteria growth is related to the

synergistic effect between the active compounds of the extracts. The synergism action come from

different effects, such as the emergence of multi-target mechanisms, the existence of compounds

capable of suppressing bacterial resistance mechanisms, pharmacokinetic or physicochemical

effects resulting in enhanced bioavailability, solubility and resorption rate, neutralization of

adverse effects and reduction of toxicity (Lin et al., 2015).

The aim of this study focused on seeing the antibacterial property of medicinal plants such as

Neem (Azadirachta indica), Rosemary (Salvia rosmarinus) against human pathogenic bacteria

including Escherichia coli, Staphylococcus aureus and Proteus mirabilis.

1.2 Hypothesis

Extracts of Neem and Rosemary will inhibit the growth of Escherichia coli, Staphylococcus

aureus and Proteus mirabilis.

2
1.3 Aim

To determine the antibacterial properties of Neem and Rosemary against Escherichia coli,

Staphylococcus aureus and Proteus mirabilis using the agar diffusion assay.

1.4 Specific Objectives

a. To isolate the active compounds in the plant parts.

b. To test the activity of the plant extracts and their active compounds against human pathogenic

bacteria.

c. To evaluate the potential synergistic effects of neem and rosemary extracts in combination

with conventional antibiotics(cefuroxime) by conducting the agar diffusion assay using both the

plant extract and antibiotics to determine enhanced antibacterial activity.

d. To determine the minimum inhibitory concentration (MIC) of the most active plant extracts

and compounds.

3
 CHAPTER TWO
2.0 LITERATURE REVIEW

2.1 Introduction to Antibacterial Resistance and Medicinal Plants

Antibacterial resistance represents one of the most pressing health challenges of the 21st century.

This phenomenon occurs when bacteria evolve mechanisms to withstand the drugs that once

killed them or inhibited their growth (Lin et al., 2015b). The overuse and misuse of antibiotics in

medicine and agriculture have accelerated this process, leading to a situation where common

infections and minor injuries, once easily treatable, can become life-threatening. As traditional

antibiotics become less effective, there is an urgent need for new strategies to combat bacterial

infections. One promising avenue of research involves the use of medicinal plants, which have

been employed in traditional medicine for centuries and offer a diverse array of bioactive

compounds with potential antibacterial properties (Engineeri et al., 2022). Antibacterial

resistance arises through several mechanisms. Bacteria can mutate, altering their genetic material

in ways that reduce the effectiveness of antibiotics. They can also acquire resistance genes from

other bacteria through horizontal gene transfer. Bacteria can also produce enzymes that

inactivate antibiotics, such as beta-lactamases, which degrade beta-lactam antibiotics (Tan,

2020). Bacteria can also use efflux pumps where they can expel antibiotics from the cell using

efflux pumps, reducing the intracellular concentration of the drug. Once these resistance traits are

established, they can spread rapidly within bacterial populations, especially in environments

where antibiotics are frequently used (Nguyen et al., 2024). The misuse of antibiotics, such as

taking them for viral infections or not completing a prescribed course, further exacerbates this

issue by creating selective pressure that favors resistant strains. The implications of antibacterial

4
resistance are profound. It leads to longer hospital stays, higher medical costs, and increased

mortality (De J Sosa et al., 2009). Resistant infections require more expensive and toxic

treatments, and in some cases, there may be no effective treatments available. This situation has

prompted global health organizations to call for better stewardship of existing antibiotics,

increased surveillance of resistant infections, and the development of new antimicrobial agents.

2.2 Historical and Ethnobotanical Context of Medicinal Plants

The use of medicinal plants spans across cultures and epochs, deeply rooted in the history of

human civilization. Long before the advent of modern medicine, ancient societies relied on the

flora around them to treat illnesses, heal wounds, and maintain health (Rosen, 2015). The

knowledge of these plants and their medicinal properties was accumulated through trial and

error, passed down through generations, and often intertwined with cultural and spiritual

practices (“Body Matters: Exploring the Materiality of the Human Body,” 2019).

2.3 Ancient Civilizations and Medicinal Plants

In ancient Egypt, as early as 1500 BCE, the Ebers Papyrus documented over 800 medicinal

recipes, including the use of aloe vera for skin conditions and garlic for cardiovascular ailments.

The Egyptians' sophisticated understanding of medicinal plants influenced later Greek and

Roman medicine. Hippocrates, often called the "Father of Medicine," and Dioscorides, a Greek

physician and botanist, documented numerous herbal remedies that formed the basis of Western

herbal medicine (Hanif et al., 2019). The ancient Indian system of Ayurveda, which dates back

over 3,000 years, extensively uses medicinal plants. Texts like the Charaka Samhita and Sushruta

Samhita describe hundreds of plant species and their applications in treating various diseases

(Premila, 2012). Similarly, Traditional Chinese Medicine (TCM), with its roots in texts like the

5
Shennong Ben Cao Jing, has utilized plants such as ginseng, ephedra, and licorice for their

therapeutic properties for thousands of years. Indigenous cultures across the Americas also have

rich traditions of plant-based medicine. Native American tribes used plants like echinacea for

infections and willow bark, which contains salicin (a precursor to aspirin), for pain relief. In the

Amazon rainforest, indigenous peoples have a profound knowledge of the local flora, using it to

treat a wide array of ailments (Gupta et al., 2016). Ethnobotany, the scientific study of the

relationships between people and plants, plays a crucial role in understanding how different

cultures use medicinal plants. This field combines anthropology and botany to document

traditional knowledge and practices, providing insights into how societies have utilized plant

resources for medicinal purposes (“A Reader in Ethnobotany and Phytotherapy,” 2014).

Ethnobotanists often work closely with indigenous and local communities, learning from their

knowledge systems to identify plants with potential medicinal properties. This collaboration has

led to the discovery of several important pharmaceuticals. For instance, the rosy periwinkle

(Catharanthus roseus), used in traditional medicine in Madagascar, led to the development of

cancer-fighting drugs like vincristine and vinblastine. Ethnobotanical research emphasizes the

importance of preserving traditional knowledge, which is often at risk due to cultural

assimilation and loss of biodiversity (Vallisuta & Olimat, 2015). Many plants used in traditional

medicine are endemic to specific regions, and the loss of these habitats threatens both the plants,

and the knowledge associated with them.

2.4 Medicinal Plants in Modern Context

Despite the advances in synthetic pharmaceuticals, medicinal plants remain vital in

contemporary medicine. The World Health Organization (WHO) estimates that up to 80% of the

world's population relies on traditional medicine for primary healthcare, particularly in

6
developing countries. Even in developed nations, there is a growing interest in herbal remedies

and natural health products (Vallisuta & Olimat, 2015). Modern science continues to explore and

validate the therapeutic potential of medicinal plants. Techniques such as bioassay-guided

fractionation, phytochemistry, and molecular biology are used to isolate active compounds and

understand their mechanisms of action. This scientific validation is essential for integrating

traditional herbal medicine into evidence-based medical practice. Furthermore, the concept of

ethnopharmacology has emerged, focusing on the pharmacological properties of traditional

medicines (Veeresham, 2010b). This interdisciplinary approach combines ethnobotany,

pharmacology, and clinical research to develop new drugs and therapies. It respects and

acknowledges the value of traditional knowledge while applying rigorous scientific methods to

ensure safety and efficacy.

2.5 Phytochemicals and Mechanisms of Antibacterial Action

Phytochemicals are naturally occurring compounds found in plants, playing crucial roles in their

growth, reproduction, and defense against pathogens. Over centuries, humans have harnessed

these compounds for medicinal purposes, particularly for their antibacterial properties. With the

rise of antibiotic resistance, there is renewed interest in phytochemicals as potential alternatives

or supplements to conventional antibiotics (Alamgir, 2019). Medicinal plants are rich in

phytochemicals, including alkaloids compounds such as berberine and quinine have shown

antibacterial activity against a broad spectrum of bacteria. Phenolics and Polyphenols such as

flavonoids, tannins, and phenolic acids, terpenoids and essential oils and Plant glycosides, such

as saponins, have demonstrated activity against bacterial pathogens (Ferdes, 2018). These

phytochemicals combat bacterial infections through various mechanisms. One of the primary

mechanisms by which they combat bacterial infections is through the disruption of bacterial cell

7
membranes. The bacterial cell membrane is a critical structure composed of a phospholipid

bilayer embedded with proteins, which is essential for maintaining cell integrity and homeostasis

(Naidu et al., 2024). Phytochemicals such as essential oils, alkaloids, and phenolic compounds

can integrate into this membrane due to their lipophilic nature. For example, essential oils, rich in

terpenoids and phenylpropanoids, have been shown to insert themselves into the lipid bilayer,

causing increased membrane fluidity and permeability. This disruption results in the leakage of

vital intracellular contents, such as ions, ATP, and metabolites, leading to cellular dysfunction

and death. Another crucial mechanism through which phytochemicals exert their antibacterial

effects is the inhibition of cell wall synthesis (Arnason et al., 2013). The bacterial cell wall is a

rigid structure that provides shape and protection to the cell, composed of peptidoglycan in most

bacteria. Phytochemicals can interfere with the biosynthesis of peptidoglycan, leading to

weakened cell walls and bacterial lysis. One of the ways phytochemicals inhibit cell wall

synthesis is by targeting the enzymes involved in peptidoglycan assembly. For example, certain

alkaloids and flavonoids have been found to inhibit transglycosylase and transpeptidase

enzymes, which are crucial for the polymerization and cross-linking of peptidoglycan strands

(Abdulra’Uf, 2017). By inhibiting these enzymes, phytochemicals prevent the formation of a

robust peptidoglycan network, making the bacterial cell wall prone to osmotic pressure and

mechanical stress, leading to cell rupture and death. Additionally, phytochemicals like tannins

and terpenoids can interfere with the precursor molecules required for peptidoglycan synthesis.

For example, tannins can bind to and inactivate bactoprenol, a lipid carrier that transports

peptidoglycan precursors across the cell membrane. Without functional bactoprenol, the delivery

of necessary building blocks for cell wall construction is halted, resulting in incomplete and

defective cell walls (Patra, 2012). Furthermore, some phytochemicals can target specific

8
membrane proteins, impairing their function. For example, flavonoids and saponins can form

complexes with membrane proteins, disrupting their normal activity. This interaction can hinder

essential processes like nutrient transport and signal transduction, further compromising bacterial

viability (Damyanova et al., 2024). The disruption of membrane potential and the collapse of

proton gradients are additional effects that impair bacterial energy production and metabolic

function. This multifaceted approach can reduce the likelihood of bacteria developing resistance.

2.6 Phytochemicals present in Neem (Azadirachta indica)

Neem (Azadirachta indica) are known for their extensive medicinal properties, which are

attributed to their rich phytochemical compositions. Neem is renowned for compounds such as

azadirachtin, which serves as a potent insecticidal agent. Other notable phytochemicals in neem

include nimbin and nimbidin, both recognized for their anti-inflammatory, antifungal, and

antibacterial properties. Additionally, neem contains quercetin, a flavonoid with strong

antioxidant capabilities, and nimbolide, a compound with significant anticancer and

antimicrobial activities. Gedunin and salannin further enhance neem’s medicinal profile with

their antimalarial, antifungal, and insect repellent properties.

2.7 Phytochemicals present in Rosemary (Salvia rosmarinus)

Rosemary contains an array of phytochemicals that provide numerous health benefits, from

antioxidant and anti-inflammatory effects to antimicrobial and anti-cancer activities. One of the

key phytochemicals in rosemary is rosmarinic acid, a potent antioxidant that protects cells from

damage caused by free radicals. Rosmarinic acid also has anti-inflammatory properties, making

it useful in treating conditions like arthritis. Rosemary also contains flavonoids such as apigenin,

luteolin, and diosmin, which support cardiovascular health by improving blood circulation and

9
reducing the risk of heart disease. Diterpenes like carnosic acid and carnosol are another

important group of phytochemicals in rosemary. These compounds have strong antioxidant

properties and have been studied for their potential in cancer prevention. Carnosic acid helps

protect the skin from UV damage and may inhibit the growth of certain cancer cells. Rosemary's

essential oils, including 1,8-cineole, camphor, and alpha-pinene, are known for their

antimicrobial effects. These oils can help fight bacterial and fungal infections and are also used

in aromatherapy to enhance cognitive function and reduce stress.

2.8 Pathogenic bacteria and their impact on human health

Bacteria are microorganisms that are a few micrometers in length and take the shapes of spheres,

rods, or spirals. Bacteria sense and respond to temperature and pH changes, nutritional

starvation, stresses to the outer and inner membranes, new food sources, toxins, and quorum

sensing signals. Pathogenic bacteria are harmful species that cause bacterial infections and

contagious diseases that result in many serious complications. There are two types of pathogenic

bacteria which includes gram- negative bacteria such as Escherichia coli and Proteus mirabilis

and gram - positive bacteria such as Staphylococcus aureus, Streptococcus spp (Dyakov et al.,

2007). Common pathogenic bacteria and their effects include Escherichia coli (food poisoning).

Staphylococcus aureus (boils, cellulitis, abscesses, wound infections, toxic shock syndrome,

pneumonia, and food poisoning), and Streptococcus spp. (pneumonia, meningitis, ear infections,

and pharyngitis). Epidemic diseases caused by pathogenic bacteria are major health issues, which

necessitate an immediate detection strategy. Sensing strategies have been developed for early

screening and proper enumeration of the identified pathogenic strains (Taussig & Landau, 2008).

The rapid and accurate detection of pathogenic bacteria is vital for the administrations of

appropriate antibiotic treatment to control the spread of the disease and to assess the drug

10
resistance information. Detection of pathogenic agents is also a vital step for the identification of

infection source anywhere from home, hospital to outdoor settings (“Topic Sessions,” 2019). The

fundamental identification of bacteria relies on the morphological features of the cells, which can

be visualized via microscopic observations. Other common methods include Gram staining,

culturing, biochemical assays and sequence-based detection. In addition, probes (e.g. antibodies)

that are specific to surface/flagellin proteins of the bacterial or the bacterial whole cell, are also

used to detect the presence of bacteria. These probes are often applied in biosensors for the

detection of specific types of bacteria.

2.9 Methodologies for Assessing Antibacterial Activity

Various methodologies exist to assess antibacterial activity, each with specific applications,

strengths, and limitations. A widely used method for assessing antibacterial activity is the agar

diffusion test, which includes both disk diffusion (Kirby-Bauer test) and well diffusion

techniques. In the disk diffusion method, paper disks soaked in the test substance are placed on

an agar plate that has been inoculated with the target bacteria (Cavalieri, 2009). After incubation,

the zones of inhibition around the disks are measured indicating where bacterial growth has been

prevented, providing a straightforward indication of antibacterial activity. The well diffusion

method is similar but involves placing the test substance in wells cut into the agar. Both methods

are valued for their simplicity and cost-effectiveness, making them suitable for initial screenings

however, they provide only qualitative results and can be influenced by the diffusion properties

of the substance being tested, limiting their utility for precise quantitative analysis. Another

method for assessing antibacterial activity include the broth dilution tests, which come in

microbroth and microbroth formats (Atta-Ur-Rahman et al., 2001). In macrobroth dilution, serial

dilutions of the test substance are prepared in a liquid medium, to which a standard bacterial

11
inoculum is added. The minimum inhibitory concentration (MIC) is then determined as the

lowest concentration that inhibits visible bacterial growth. Beyond these traditional methods,

modern techniques like time-kill assays, flow cytometry, and molecular methods offer more

detailed insights into antibacterial efficacy.

2.9.1 Synergistic effects in antibacterial therapy

Synergistic effects occur when the combined action of two or more antimicrobial agents results

in enhanced bacterial killing compared to the sum of their individual effects. Synergy can be

particularly beneficial in treating infections caused by multi-drug-resistant bacteria, where

single-agent therapy may be insufficient. Synergistic effects enhance antibacterial efficacy,

reduce the likelihood of resistance development, and potentially lower the required doses of

individual drugs, thereby minimizing toxicity.

2.9.2 multi-drug resistance (MDR) of pathogenic bacteria

MDR occurs when bacteria develop resistance to multiple antibiotics, rendering many standard

treatments ineffective. This resistance arises through several mechanisms: mutation, horizontal

gene transfer, and selective pressure from antibiotic overuse and misuse (Kardos, 2017).

Mutations in bacterial DNA can lead to changes in the structure of antibiotic targets, reducing

drug binding and efficacy. Horizontal gene transfer, where bacteria acquire resistance genes from

other bacteria via plasmids, transposons, or bacteriophages, accelerates the spread of resistance

traits. The overuse of antibiotics in medicine and agriculture further compounds the problem,

creating environments where only resistant bacteria survive and proliferate (Harper et al., 2021).

Infections caused by multi-drug-resistant bacteria are harder to treat, often requiring more

expensive alternatives, leading to longer hospital stays, higher medical costs, and increased

12
mortality. Common diseases such as pneumonia, tuberculosis, and urinary tract infections are

becoming increasingly difficult to manage due to the ineffectiveness of antibiotics.

CHAPTER THREE
3.0 MATERIALS AND METHODS

3.1 Collection of the plant sample.

Fresh leaves of Neem (Azadirachta indica) and Rosemary (Salvia rosmarinus) were collected

from their natural habitat, washed thoroughly with distilled water and air dried under shade for

one week. The leaves of each plant were grinded using mortar and pestle and their powered

forms collected in an Eppendorf tube.

3.2 Preparation of the plant extracts.

A mass of 0.6 g of each plant powder was dissolve in 5.5 ml of 96 % pure ethanol and then kept

on shaking incubator overnight at 37 ◦C. The extracts were filtered using Whatman No.1 filter.

The supernatant was collected and centrifuged at 4400rpm for 7 minutes. The supernatant and

pellet were then collected in separate falcon tubes and kept in the refrigerator at 4 ◦C.

3.3 Subculturing of bacterial strains.

A mass of 1.5 g of nutrient broth powder was weighed and transferred into a media bottle. A

volume of 100 ml of distilled water was added, shaken thoroughly. The mixture was autoclaved

for one hour, allowed to cool and poured into four different falcon tubes. A volume of 500 ul

13
liters of each bacterial strains was pipetted into each falcon tubes containing the nutrient broth

and the mixture placed on the shaking incubator overnight.

3.5 Preparation of Mueller Hinton Agar

A mass of 26.6 g of Mueller Hinton agar powder was weighed and transferred into a media

bottle. A volume of 700 ml of distilled water was added, shaken thoroughly. The mixture was

autoclaved for one hour, allowed to cool, and poured into sixteen agar plates.

3.6 Antibiotic Susceptibility Test (AST)

The Antibiotic Susceptibility Test (AST) was performed using the Disc diffusion assay and Agar

well diffusion assay. A nutrient broth was added to each bacterial suspension in a capped test

tube. The suspension was vortex-mixed, and the cell density was adjusted to that of McFarland

0.5 standard. A sterile swab dipped into the bacterial suspension and evenly spread over the

entire surface of a Mueller-Hinton agar plate, ensuring complete coverage. About 20 µl of each

plant extract (pellet) impregnated disks were placed on the surface of the inoculated agar plates

using sterile forceps. The disks were evenly spaced and adequately pressed to ensure contact

with the agar. Antibiotic (cefuroxime)-impregnated disks were also placed on the surface of the

inoculated agar plates using sterile forceps as positive control. The plates were incubated for 24

hours at 37 ◦C.

14
The same procedure was repeated with the supernatant of each plant extracts to check activity of

the supernatant and the antibiotic against the bacterial strains.

3.6.1 Minimum Inhibitory Concentration Determination

A series of dilution of the crude extracts was prepared (70 %, 50 %, 30 % and 10 % dilution) in

four different falcon tubes. A sterile swab dipped into the bacterial suspension and evenly spread

over the entire surface of a Mueller-Hinton agar plate, ensuring complete coverage. Each plant

extract impregnated disks were placed on the surface of the inoculated agar plates using sterile

forceps. The disks were evenly spaced and adequately pressed to ensure contact with the agar.

Antibiotic (cefuroxime)-impregnated disks were also placed on the surface of the inoculated agar

plates using sterile forceps as positive control. The plates were incubated for 24 hours at 37 ◦C

and observe for visible bacterial growth. The MIC is the lowest concentration of the

antimicrobial that prevents visible growth.

15
 CHAPTER FOUR
4.0 RESULT
Neem extracts(crude) inhibition zone against Staphylococcus aureus on the agar

plates after 24 hrs.

16
Figure 1.0 shows the growth and zone of inhibition of Neem extracts against

resistant strains of Staphylococcus aureus with cefuroxime as the positive control.

Neem extracts (crude) inhibition zone against Proteus mirabilis on the agar plates

after 24 hrs.

17
Figure 1.1 shows the growth and zone of inhibition of Neem extracts against

resistant strains of Proteus mirabilis with cefuroxime as the positive control.

Rosemary extracts(crude) inhibition zone against Staphylococcus aureus on the agar

plates after 24 hrs.

18
Fig 2.0 Rosemary extracts(crude) inhibition zone against Staphylococcus aureus

on the agar plates after 24hrs.

Figure 2.0 shows the growth and zone of inhibition of Rosemary extracts

against resistant strains of Staphylococcus aureus with cefuroxime as the positive

control.

Figure 2.0 shows the growth and zone of inhibition of Rosemary extracts

against resistant strains of Staphylococcus aureus with cefuroxime as the positive control.

19
Rosemary extracts (crude) inhibition zone against Proteus mirabilis on the agar

plates after 24 hrs.

20
Figure 2.1 shows the growth and zone of inhibition of Rosemary extracts

against resistant strains of Proteus mirabilis with cefuroxime as the positive control.

The table below represents the diameter of the zones of inhibition measured and recorded for

each plant extract and antibiotics (cefuroxime) against each bacterial strain.

Plant extracts Bacterial strains Zone of inhibition Control (cefuroxime)

(mm)

Rosemary(supernatant) Proteus 11 17

Rosemary (debris) Proteus 11 2

Neem (supernatant) Proteus 8 12

Neem (debris) Proteus 6 15

Rosemary(supernatant) E. coli _ 5

Rosemary(debris) E. coli 14 8

Neem (supernatant) E. coli _ 7

Neem (debris) E. coli _ 5

Neem (supernatant) Staphylococcus 10 4

Neem (debris) Staphylococcus 7 6

Rosemary(supernatant) Staphylococcus 13 4

21
 Rosemary(debris) Staphylococcus 5 7

Table 1.0 zone of inhibition of each plant extract (crude) and antibiotics against each bacterial

strain.

Bar chart visually representing the zones of inhibition for the plant extracts (Rosemary and

Neem) and the control antibiotic (cefuroxime) against various bacterial strains ( Proteus

mirabilis, Staphylococcus aureus and Escherichia coli)

22
The bar chart compares the effectiveness of the different treatments, providing a clear

visualization of the differences in inhibition zones across each bacterial strain and

treatment method.

4.1 Minimum inhibitory concentration (MIC).

23
Below are pictures showing serial dilution (70 %, 50 %, 3 % and 10 % dilution) of each crude

plant extract against each resistant bacterial strain to determine the least concentration of each

plant extract that can inhibit the growth of each bacterial strain.

Rosemary 70 % + proteus Rosemary 50 % + proteus

Rosemary 30 % + proteus Rosemary 10 % + proteus

Rosemary 70 % + staphylococcus Rosemary 50 % + staphylococcus

24
Rosemary 30 % + staphylococcus Rosemary 10 % + staphylococcus

Rosemary 70 % + E. coli Rosemary 50 % + E. coli

Rosemary 30%+ E. coli Rosemary 10%+ E. coli

25
 Neem 50 % + proteus Neem 10 % + proteus

Neem 50 %+ E. coli Neem 30 %+ E. coli

The table below shows the minimum concentration of each plant extract that can inhibit the

growth of bacteria.

26
 Extracts concentration Bacteria Zone of inhibition (mm)

Rosemary 10 % Staphylococcus 6

Rosemary (70 %, 50 %, 30 E. coli _

%, 10 %)

Rosemary 50 % Proteus 6

Neem 10 % Proteus 3

Neem 30 % Staphylococcus 4

Neem (70 %, 50 %,30 %,10 E. coli _

%)

Table 1.1 above shows the minimum concentration of each plant extract that can inhibit the

growth of each bacterial strain. 10 %, the least concentration of Rosemary extract to inhibit the

growth of Staphylococcus aureus with zone of inhibition being 6 mm. 50 % concentration of

Rosemary extract inhibited the growth of Proteus mirabilis with zone of inhibition also being

6mm but against Escherichia coli, there was no inhibition. For Neem extract, 10 % is the least

concentration to inhibit the growth of Proteus mirabilis with zone of inhibition of 3 mm and 30

% being the least concentration to inhibit the growth of Staphylococcus aureus with zone of

inhibition of 4 mm but against Escherichia coli, none of the concentration inhibited the growth

of Escherichia coli and this was confirmed as the crude extract of Neem did not show any

inhibition against Escherichia coli

27
The bar chart represents the minimum concentration of each plant extract that can inhibit the

growth of bacteria, along with their corresponding zones of inhibition in millimeters.

Each bar above shows the inhibition effect of specific extracts like Rosemary and Neem on

different bacteria such as Staphylococcus aureus and Proteus mirabilis.

28
 CHAPTER FIVE
5.0 DISCUSSION
There are many works done on investigating the antibacterial activity of

medicinal plants against pathogenic bacterial strains in recent decades due

to the rising incidence of antibiotic-resistant bacteria which causes global

health challenges over the years leading to increased mortality rates in the

world. My study aims to investigate the potential antibacterial properties of

Neem and Rosemary against clinically stored bacterial strains and how

effective would these plants be in treating bacterial infections caused by

these resistant or multi- drug-resistant bacterial strains (Mares et al.,

2021). To do this pure extract of Neem and Rosemary was extracted from the

leaves of their plant and then tested against the various strains of bacteria of

interest. The investigation into the antibacterial activity of Neem

(Azadirachta indica) and Rosemary (Salvia Rosmarinus) extracts against

Escherichia coli, Staphylococcus aureus and Proteus mirabilis revealed

significant insights into the potential of these medicinal plants as alternative

antimicrobial agents. My study's findings demonstrate that both Neem and

Rosemary possess varying degrees of antibacterial activity against the

tested bacterial strains, which supports their traditional use in herbal

medicine for treating infections (Patra, 2012b). Neem extract exhibited

substantial antibacterial activity, particularly against Staphylococcus aureus

which is a gram-positive bacterium. The zones of inhibition observed for

these strains were larger compared to those for Escherichia coli and Proteus

mirabilis, which are gram-negative bacteria which means that the gram-

29
positive bacteria are more susceptible to the bioactive compounds in the

Neem (Méndez-Vilas, 2011). The effectiveness of Neem extract against gram

positive bacteria might be attributed to the structural characteristics of the

cell wall which are made of a thick peptidoglycan layer and the bioactive

compounds in Neem such as nimbin, azadirachitin and quercetin may disrupt

this thick peptidoglycan layer which might lead to cell lysis and death of

bacteria. The antibacterial activity of Neem against gram negative bacteria

specifically Escherichia coli and Proteus mirabilis was less pronounced ass

seen in the zones of inhibition (U-Islam & Butola, 2018). These gram-

negative bacteria possess an outer membrane called lipopolysaccharides

and this can act as a barrier to many antimicrobial agents and thus

explaining the reduced susceptibility of these gram-negative bacteria to the

Neem extract however, the existence of some inhibitory activity of Neem

extracts against these bacterial strains shows that Neem may still inhibit the

growth of these bacterial strains but not as effective as against gram positive

bacteria (Fuertes & Corral, 2023). Rosemary extracts exhibited high

antibacterial activity against all the tested bacterial strains with the largest

zone of inhibition observed in Proteus mirabilis and Staphylococcus aureus.

The primary bioactive compound in Rosemary is rosmarinic acid and this

compound is known for its broad-spectrum antimicrobial properties including

disrupting bacteria membrane integrity, generating reactive oxygen species

(ROS) and inhibiting cell signaling pathways (Ivanišová, 2023). The overall

effectiveness of Rosemary extracts was higher than that of Neem extracts

30
and this could be due to several factors with one of them being rich variety

of potent phytochemicals, such as rosmarinic acid, carnosic acid, and

essential oils like 1,8-cineole, which exhibit strong antimicrobial, antioxidant,

and anti-inflammatory properties (Cavalieri, 2009). These compounds work

synergistically to enhance the extract's overall potency against pathogens

and other harmful agents. Neem extract (supernatant and debris) did not

show any zone of inhibition against Escherichia coli which implies that

Escherichia coli is not susceptible to the bioactive compounds in neem plant.

Rosemary extract (supernatant) did not show any zone of inhibition against

Escherichia coli, and this may be attributed to rosemary lower concentration

of bioactive compounds in the supernatant than the debris after the

centrifugation which enables the debris to inhibit Escherichia coli growth but

not the supernatant (Adnan et al., 2020). The dilution of crude Rosemary extract

(debris) reduces its concentration making the reduced concentration not

effective to inhibit Escherichia coli growth. The comparison between the

plant extracts (both rosemary and neem) and the control antibiotic

(cefuroxime) offers valuable insight into alternative treatments. Rosemary

supernatant against Proteus mirabilis showed a zone of inhibition of 11 mm,

which is lower than cefuroxime (17 mm). While this result confirms some

level of antibacterial activity, the lesser inhibition compared to cefuroxime

suggests that rosemary extract might not be as potent as a first-line

treatment. However, its natural origin and reduced resistance profile in

bacteria could make it a useful alternative or adjunct therapy in cases of mild

31
infections. The rosemary debris also had an inhibition zone of 11 mm, identical to the

supernatant. Interestingly, both forms of rosemary produced consistent results against Proteus

mirabilis. The debris showed higher efficacy than the cefuroxime control in this specific

context, as the control produced only a 2 mm zone of inhibition indicating higher concentration

of antibacterial compounds that may work better against Proteus mirabilis.

Staphylococcus aureus a gram-positive bacterium responsible for various

infections, including skin infections and more serious conditions like

pneumonia (Patra, 2012b). The plant extracts and cefuroxime showed

notable differences in their inhibition zones. The rosemary supernatant

demonstrated significant antibacterial activity against Staphylococcus

aureus with a 13 mm inhibition zone, far surpassing the 4 mm zone produced

by cefuroxime. This indicates that rosemary in its supernatant form could be

highly effective against Staphylococcus aureus (Taussig & Landau, 2008).

Conversely, rosemary debris displayed only a 5 mm zone of inhibition,

suggesting that its antibacterial compounds are less concentrated or less

effective in this form against Staphylococcus aureus. This study highlights

the importance of natural alternatives in the ongoing battle against bacterial

infections. Both rosemary and neem demonstrated effective antibacterial

properties, and their potential as alternative treatments deserves further

investigation.

CONCLUSION
This study has provided valuable insights into the antibacterial properties of

Neem and Rosemary and its efficacy as an antibiotic against resistant strains

32
of Escherichia coli, Staphylococcus aureus and Proteus mirabilis. Experimental results confirms

that Neem and Rosemary extract exhibit significant antibacterial activity as seen in their zones

of inhibition against the bacterial strains with Rosemary showing stronger effects particularly

against gram positive bacteria. These findings support the traditional use of these medicinal

plants in treating bacterial infections and enable their potential as alternative or complementary

therapies in fighting antibiotic resistant pathogens.

33
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APPENDIX
Concentration of plant extracts

Mass of powder = 0.6g

Volume of ethanol = 5.5ml
Concentration of crude extract = mass/ volume
= 0.6g/ 5.5ml

39
 = 0.11g/ml
1g = 1000mg
The concentration = 110mg/ml
Nutrient broth concentration
Mass = 1.5 g
Volume of distilled water = 100 ml
Concentration = mass/ volume
= 1.5 g/ 100 ml
= 0.015 g/ ml
MHA concentration
Mass (MHA powder) = 26.6 g
Volume (distilled water) = 700 ml
Concentration = mass/ volume
= 26.6 g/ 700 ml
= 0.038 g/ ml
Serial dilution
For 70% dilution,
C1V1= C2V2
100 *V1 = 70*1
V1 =70/100
= 0.7 ml
= 700 µl
Pick 700 µl of crude extract and add 300 µl of distilled water.
For 50% dilution,
C1V1= C2V2
100 *V1 = 50*1
V1 =50/100
= 0.5 ml
= 500 µl

40
For 30% dilution,
C1V1= C2V2
100 *V1 = 30*1
V1 =30/100
= 0.3 ml
= 300 µl
For 10% dilution,
C1V1= C2V2
100 *V1 = 10*1
V1 =10/100
= 0.1 ml
= 100 µl

41
42