ANTIBACTERIAL ACTIVITY OF ESSENTIAL OILS FROM EUCALYPTUS GLOBULUS, ROSMARINUS OFFICINALIS AND CITRUS LIMON AGAINST SALMONELLA TYPHI, ESCHERICHIA COLI, STAPHYLOCOCCUS AUREUS, ENTEROCOCCUS FAECALIS AND PSEUDOMONAS AERUGINOSA.

ABSTRACT

Essential oil is an aromatic volatile substance that has antibacterial, antifungal, antiviral, insecticidal, preservative and aromatic properties. They are widely used in pharmaceutical, food, cosmetic and agricultural industries. The antibacterial activity of essential oils of Eucalyptus globulus, Rosmarinus officanalis and Citrus limon was tested against Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and Stapylococcus aureus using the agar well diffusion method, a range of 7-32mm zone of inhibition was obtained. Gram positive organisms were more sensitive to the action of the oil than Gram negative organisms in which Stapylococcus aureus was the most sensitive of the bacteria having 32mm zone of inhibition while Pseudomonas aeruginosa was the least sensitive bacteria having 7mm zone of inhibition. The minimum inhibitory concentration and minimum bactericidal concentrations was evaluated using the micro dilution broth method and the result showed that essential oil of Eucalyptus (1.11µl/ml) and Rosemary (0.11µl/ml) had greater antibacterial activity than lemon (visible growth). Also a time-kill analysis was done to access the antibacterial effect of the essential oil over time at 1, 5, 10 and 20 minutes. The antibacterial effect of essential oils against some of the most important pathogens provide an exciting potential for the future especially as there is an increased interest towards the use of natural products as alternatives for synthetic antibacterial agents.

TABLE OF CONTENTS

Title page i

Certification ii

Dedication iii

Acknowledgements iv

Table of contents v

List of Tables vii

List of figures viii

Abstract ix

CHAPTER ONE

1.1 Introduction 1

1.2 Statement of problem 3

1.3 Aim and objectives 4

CHAPTER TWO

2.0 Literature review 5

2.1 Extraction of Essential oils 6

2.2 Composition of essential oils 8

2.3 Interactions between components of essential oils 10

2.4 Mechanism of action 11

2.5 Bacterial cell targets 12

2.6 Susceptibility of Gram positive and Gram negative bacteria 15

2.7 Application of essential oils 15

CHAPTER THREE

3.0 Materials and methods 17

3.1 Screening for antibacterial activity 17

3.2 Determination of minimum inhibitory concentrations and minimum bactericidal

      Concentrations 18

3.3 Time-kill analysis 19

CHAPTER FOUR

4.0 Results 20

CHAPTER FIVE

5.0 Discussion 29

5.1 Conclusion 30

5.2 Recommendation 31

References 32

Appendices 39

LIST OF TABLES

Table                                                           Title                                                                        Page

1.1 Major raw materials used for the extraction of essential oil 7

4.1 Antimicrobial properties of plant essential oil 21

4.2 Minimal Inhibition Concentration values of the essential oils .22

4.3 Minimal Bactericidal Concentration values of the essential oils on the organisms 23

LIST OF FIGURES

Figure                                                           Title                                                                       Page

2:1 Components of essential oil 9

4:1 Survivor curves for Enterococcus faecalis 24

4:2 Survivor curves for Salmonella typhi 25

4:3 Survivor curves for Escherichia coli 26

4:4 Survivor curves for Staphylococcus aureus 27

4:5 Survivor curves for Pseudomonas aeruginosa 28

CHAPTER ONE

INTRODUCTION

Essential oils are volatile liquids obtained from herbs, spices and different plants mainly by steam distillation. They can have more than 50 components in different ratios. Some of the essential oils and their constituents are known to possess antibacterial, antifungal, antiparasitic, antiviral, antioxidant and insecticidal properties (Tserennadmid, 2010). These characteristics are possibly related to the function of these compounds in plants (Mahmoud and Croteau, 2002). They are widely used in medicine, cosmetic, agricultural and the food industry for these purposes.

Use of synthetic antimicrobial is posing a lot of threat to human environment which may lead to negative health consequences.Besides, the use of synthetic compounds have significant drawbacks, such as increasing cost, handling hazards, concerns about residues on food and threat to human environment. In spite of modern improvements in slaughter hygiene and food production techniques, food safety is an increasingly important public health issue (Burt, 2004). It has been estimated that as many as 30% of people in industrialized countries suffer from a food borne disease each year and in 2000 at least two million people died from diarrheal disease worldwide (WHO, 2002a). Preservatives in cosmetics, though usually used in small concentrations, are considered as one of the main factors causing allergies to users. Therefore growing demands for more natural and preservative-free cosmetics promoted an idea of the replacement of synthetic preservatives with essential oils of antimicrobial properties (Dreger and Wielgus, 2013). Control of insect populations is primarily dependent upon continued applications of liquid and gaseous insecticides. Although effective, their repeated use for several decades has disrupted biological control system by natural enemies and led to outbreaks of insect pests, widespread development of resistance, undesirable effects on non-target organisms, and environmental and human health concerns. As an alternative strategy to prevent the spread of diseases, natural components of plants are being tested for their antimicrobial activity (Vasinauskiene et al., 2006). Also, use of antibiotics may lead to antimicrobial resistance resulting in treatment failure, increased treatment costs as well as the rate of fatalities and create even broader infection control problems, spreading resistance bacteria from hospital, to community and even the environment (Yap et al., 2014). The persistence of antibiotic resistance urges the need of finding new therapies against the multi-drug resistant bacteria. Therefore, there has been increasing interest to replace synthetic antimicrobials with natural, effective and non-toxic compounds.

The increased interest in alternative natural substances is driving the research community to find new uses and applications of these substances (Bassole et al., 2012).Some natural substances of plant origin have good antimicrobial properties and have been used as seasonings for centuries. One such possibility is the use of essential oils as antibacterial agents. In the first place, extracts and essential oils of spices and herbs were used as antimicrobials. As natural foodstuffs, plant appeal to all who question safety of synthetic food additives and demand high-quality products that at the same time are safe and stable.

Plants produce a high diversity of secondary metabolites with a prominent function of protecting plants against predators and microbial pathogens due to their biocidal properties against microbes or repellence to herbivores. There are three major groups of secondary metabolites, including terpenes, phenylpropenoids and N- and S-containing compounds (Wink, 1999). Among these secondary metabolites, it is estimated that over 3,000 essential oils are known, of which about 300 are commercially important and used by the flavor and fragrance industries (Van de Braak and Leijten, 1999). Dorman and Deans (2000) demonstrated that the individual oil components (mainly with phenolic structures) were able to exhibit a wide spectrum of antibacterial activity and that the chemical structures greatly affect the components effectiveness and their mode of antibacterial action.

The antimicrobial effects of three essential oils from Eucalyptus globulus, Citrus limon and Rosmarinus officinalis were tested against five pathogenic microorganisms. Three main factors can influence the results of test of the antimicrobial activity of essential oil namely:

· The composition and solubility of the oil,

· The microorganisms and

· The method of growing and enumerating the surviving bacteria.

A unit commonly used in the measurement of antimicrobial activity is the diameter of the zone of inhibition of bacterial growth on solid medium. For essential oil samples, the zone of inhibition will depend on the ability of oil to diffuse uniformly through an agar medium and the effect on bacteria of oil vapors that may be released (Friedman et al., 2002).

1.1 Statement of problem

It is well known that bacteria and other microorganisms are causative agents for several infectious disease posing a great threat to human, animal and plant health. The indiscriminate use of synthetic antimicrobial medicines, preservatives, biocides and commercially available antibiotics are leading to hypersensitivity reactions in sensitive persons. In addition,these microorganisms have developed resistance to many of the currently used antimicrobials thereby posing a great challenge to human environment and its control. It is therefore desired and necessary to develop effective, safe, natural methods for the treatment of infectious diseases. In recent years, essential oils which are extracted from wild or cultivated aromatic plants have received attention of researches as potent bioactive compounds to combat several species of microorganisms.

1.2 Aim and objectives

The aim of the study is to investigate the antibacterial activities of some essential oil against some Gram positive and Gram negative microorganisms which include Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus.  

The objectives of this work are:

1. To investigate the effects of the selected essential oils – Eucalyptus (Eucalyptus globulus), Rosemary (Rosmarinus officinalis) and lemon (Citrus limon) on the growth parameters of bacteria in agar. This work shall ascertain whether this essential oil could affect growth inhibition on the test organisms.

2. To determine the minimal inhibitory concentrations (MICs) of the selected essential oils.

3. To determine the minimal bactericidal concentrations (MBCs) of the essential oils that showed high bactericidal activity.

4. To access the bactericidal effect of the essential oil with the lowest bactericidal and bacteriostatic concentrations over time (survival curve- time-kill analysis).

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