UNIVERSITY OF GHANA DEPARTMENT OF MEDICAL MICROBIOLOGY UNIVERSITY OF GHANA MEDICAL SCHOOL IN-VITRO EVALUATION OF ANTIBACTERIAL PROPERTIES OF EUPHORBIA HIRTA AGAINST SELECTED MULTIDRUG-RESISTANT BACTERIA IN GHANA BY JONES GYABENG (10806233) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL IN MEDICAL MICROBIOLOGY DEGREE JUNE, 2022 University of Ghana http://ugspace.ug.edu.gh IN-VITRO EVALUATION OF ANTIBACTERIAL PROPERTIES OF EUPHORBIA HIRTA AGAINST SELECTED MULTIDRUG-RESISTANT BACTERIA IN GHANA University of Ghana http://ugspace.ug.edu.gh i DECLARATION I, Jones Gyabeng, declare that the work presented in this thesis is the result of my own research work carried out in the Department of Medical Microbiology - University of Ghana, The Centre for Plant Medicinal Research (CPMR) Akuapem-Mampong - Eastern region. The Central Laboratory of Kwame Nkrumah University of Science and Technology under the supervision of Nicholas T.K.D. Dayie (PhD) and Simon K. Attah (PhD)- Department of Medical Microbiology and that all references cited in this work have been duly acknowledged. 20 /06 / 2022 Signature Date Jones Gyabeng (10806233) ____________________ 20/06/ 2021 Signature (Supervisor) Date Nicholas T.K.D. Dayie (PhD) Department of Medical Microbiology, University of Ghana Medical School 20 /06/2021 Date Signature (Co-supervisor) Simon K. Attah (PhD) Department of Medical Microbiology, University of Ghana Medical School University of Ghana http://ugspace.ug.edu.gh ii DEDICATION I dedicate this work to my loving and supportive family, friends, participants, supervisors, collaborators and the Medical Microbiology Department of University of Ghana Medical School. This momentous milestone would not have been achieved without your commitment, support and love. University of Ghana http://ugspace.ug.edu.gh iii ACKNOWLEDGEMENT I give thanks to the Almighty God for his mercy, guidance, and favour. I would like to convey my sincere appreciation to Dr. Nicholas T.K.D. Dayie and Dr. Simon K. Attah of the Medical Microbiology Department, University of Ghana Medical School. They have been fantastic mentors to me due to their tireless efforts, which included script editing and proofreading, constructive criticism, regular supervision and training. I am much thankful to Mr. Christopher Mfum Owusu-Asenso, Mr. Fleischer C. K. Kotey and Miss Mary–Magdalene Osei, Department of Medical microbiology, UGMS for their special assistance and proofreading of this write-up. I wish to express my gratitude to Dr. Daniel Boamah, Mr. Borge – Leth Frimpong, Mr. Sylvester Kaminta, Mr. Charles Aidoo, Miss. Susan Oteng and Miss. Lydia Annoh of the Microbiology Department, Dr. Maxwell M. Sakyiamah, Mr. Frederick Ayertey, Mr. Henry Brew- Daniel, Mr. Ebenezer Ehun, Mr. peter Bolah, Mr. Prince Kyei Baffour and Miss. Christian Opare also at the Photochemistry Department - Centre for Plant Medicinal Research (CPMR) at Akuapem- Mampong. I really appreciate their tireless effort for assisting in the laboratory work. I owe my parents a debt of gratitude for their love, prayers, care, and sacrifices in teaching and preparing me for the future. Finally, I want to express my gratitude to everyone who has helped me accomplish the research work, whether directly or indirectly. University of Ghana http://ugspace.ug.edu.gh iv TABLE OF CONTENTS DECLARATION ............................................................................................................................. i DEDICATION ................................................................................................................................ ii ACKNOWLEDGEMENT ............................................................................................................. iii LIST OF TABLES .......................................................................................................................... x LIST OF FIGURES ....................................................................................................................... xi LIST OF PLATES ........................................................................................................................ xii LIST OF ABBREVIATIONS ...................................................................................................... xiii ABSTRACT ................................................................................................................................. xv CHAPTER ONE ............................................................................................................................. 1 1.0 INTRODUCTION .................................................................................................................... 1 1.1 Background ............................................................................................................................... 1 1.2 Problem Statement .................................................................................................................... 3 1.3 Justification ............................................................................................................................... 4 1.4 Aim ........................................................................................................................................... 6 1.5 Specific Objectives ................................................................................................................... 6 CHAPTER TWO ............................................................................................................................ 7 2.0 Literature Review...................................................................................................................... 7 2.1 Antimicrobials........................................................................................................................... 7 2.2 Antibiotics ................................................................................................................................. 7 University of Ghana http://ugspace.ug.edu.gh v 2.2.1 Classification and Mechanism of Action of Antibiotics ........................................................ 8 2.2.1.1 Cell Wall Synthesis Inhibitors ............................................................................................ 8 2.2.1.2 Denaturing / Cell Membrane Synthesis Inhibitors.............................................................. 9 2.2.1.3 Protein Synthesis Inhibitors ................................................................................................ 9 2.2.1.4 Folic Acid Synthesis Inhibitors ......................................................................................... 11 2.2.2.1 Mechanism of Resistance ................................................................................................. 11 2.3 The Problem of Antibiotic Resistance .................................................................................... 13 2.4.1 Medicinal Plants................................................................................................................... 14 2.4.2 Uses of Medicinal Plants ..................................................................................................... 14 2.4.3 Plants as Antimicrobials ...................................................................................................... 15 2.4.4 The Present Use of Plants as Antimicrobials ....................................................................... 15 2.4.5 Euphorbia hirta..................................................................................................................... 16 2.4.6 Pharmacological Importance of Euphorbia hirta ................................................................ 18 2.5.1 Extended Spectrum Beta-Lactamase Producers (ESBLs) ................................................... 19 2.5.2 Extended Spectrum Beta-Lactamase Escherichia coli Producers ....................................... 20 2.5.3 ESBL Klebsiella pneumoniae Producers ............................................................................. 20 2.5.4 Methicillin-Resistant Staphylococcus aureus (MRSA) ....................................................... 22 2.5.5 Salmonella typhi .................................................................................................................. 23 2.6 Antimicrobial Susceptibility Testing (AST) ........................................................................... 25 2.6.1 Principles of Antimicrobial Susceptibility Testing .............................................................. 26 University of Ghana http://ugspace.ug.edu.gh vi 2.6.1.1 The Diffusion Method....................................................................................................... 26 2.6.1.2 The Dilution Method......................................................................................................... 27 2.7 Role of Chromatography in Science ....................................................................................... 27 2.8.1 Thin Layer Chromatography ................................................................................................ 29 2.8.2 Column Chromatography..................................................................................................... 30 2.8.3 Principles of Column Chromatography ............................................................................... 32 2.9 Gas Chromatography Mass Spectrometer .............................................................................. 32 2.9.1 General Principles for Chromatographic Techniques. ......................................................... 34 CHAPTER THREE ...................................................................................................................... 35 3.0 Materials and Methods ............................................................................................................ 35 3.1 Study Design .......................................................................................................................... 35 3.2 Study Area ............................................................................................................................. 35 3.3 Media and Reagents ................................................................................................................ 37 3.4 Preparation of Plant Extracts .................................................................................................. 37 3.4.1 Collection and Identification of plant material .................................................................... 37 3.4.2 Preparation of Plant Material ............................................................................................... 37 3.4.3 Extraction of Euphorbia hirta using the Cold Maceration Process ..................................... 37 3.4.4 Extraction of Euphorbia hirta using the Soxhlet Maceration Process ................................ 38 3.4.5 Preparation of Stock Solution for Antimicrobial Assay ...................................................... 38 3.5.1 Test Organisms .................................................................................................................... 38 University of Ghana http://ugspace.ug.edu.gh vii 3.5.2 Confirmation of Bacteria Isolates Identity ........................................................................... 39 3.5.2.1 Gram Stain ................................................................................................................. 39 3.5.2.2 Biochemical Test ....................................................................................................... 39 3.5.2.3 Phenotypic Screening Test for ESBL Producing Escherichia coli and Klebsiella pneumoniae ............................................................................................................................ 40 3.5.3 Standardization of Inoculums .............................................................................................. 41 3.6 Evaluation of the Antibacterial Activity of Extracts............................................................... 41 3.6.1 Qualitative Phytochemical Analysis .................................................................................... 41 3.7 Collection of Active Fractions ................................................................................................ 44 3.7.1 Determination of Solvent System ........................................................................................ 44 3.7.2 Collection of Active Fractions Using Column Chromatography ........................................ 44 3.7.3 Evaluation of the Antibacterial Activity of Ethyl-acetate Extract Fractions ....................... 45 3.7.4 Determination of Minimum Inhibitory Concentration (MIC). ............................................ 46 3.7.5 Determination of Minimum Bactericidal Concentration (MBC)......................................... 46 3.7.6 Gas Chromatography Mass Spectrometer Analysis ............................................................ 47 3.8 Ethical Clearance .................................................................................................................... 47 3.9 Statistical Analysis .................................................................................................................. 48 CHAPTER FOUR ......................................................................................................................... 49 4.0 Results ..................................................................................................................................... 49 4.1 Determination of Yield Using Both Soxhlet and Cold Extraction Method ............................ 49 4.2 Antimicrobial Activity of the Extracts .................................................................................... 50 4.3 Phytochemical Components of E. hirta Crude Extracts. ........................................................ 57 University of Ghana http://ugspace.ug.edu.gh viii 4.4 Determination of Fractions Using Column Chromatography ................................................. 59 4.5 Antimicrobial Activity of Ethyl-Acetate Active Fractions ..................................................... 60 4.6 Determination of Possible Compounds Present in the Active Fraction. ................................. 62 4.7 The Minimum Inhibition Concentration (MIC) of the Extracts ............................................. 63 4.8 The Minimum Bactericidal Concentration (MBC) of the Extracts ........................................ 64 CHAPTER FIVE .......................................................................................................................... 68 5.0 Discussion ............................................................................................................................... 68 5.1 Limitations .............................................................................................................................. 71 CHAPTER SIX ............................................................................................................................. 72 CONCLUSION AND RECOMMENDATIONS ......................................................................... 72 6.0 Conclusion .............................................................................................................................. 72 6.1 Recommendations ................................................................................................................... 73 REFERENCES ............................................................................................................................. 74 APPENDICES .............................................................................................................................. 90 Appendix 1: Column Chromatography Work Sheet ..................................................................... 90 Appendix II: Ethical Clearance Form ........................................................................................... 96 Appendix III: MIC Work Sheet for Crude Ethyl Acetate Extracts ............................................... 97 Appendix IV: MIC Work Sheet for Crude Ethyl Acetate Extract ................................................ 98 Appendix V: Data Showing Antimicrobial Activity of Methanol Cold Extracts ....................... 100 Appendix VI: Data Showing Antimicrobial Activity of Methanol Soxhlet Extracts ................. 101 University of Ghana http://ugspace.ug.edu.gh ix Appendix VII: Data Showing Antimicrobial Activity of Petroleum Ether Soxhlet Extracts ..... 102 Appendix XIII: Data Showing Antimicrobial Activity of Fractions Obtained from Column Chromatography ......................................................................................................................... 108 Appendix XIV: Data Analysis of the Yield of E. Hirta Crude Extracts using Soxhlet and Cold Extraction Methods ..................................................................................................................... 110 Appendix XV: Analysis of Ethyl-Acetate Active Fractions ....................................................... 111 Appendix XVI: Data Analysis on the MIC Values for Crude Ethyl Acetate Extracts and Active Fractions ...................................................................................................................................... 113 Appendix XVII: Data Analysis on the MBC Value for Ethyl Acetate Crude Extracts and Active Fractions Method ........................................................................................................................ 114 University of Ghana http://ugspace.ug.edu.gh x LIST OF TABLES Table3.1: Qualitative Phytochemical Analysis ............................................................................. 42 Table 4.1: Yield of E. Hirta Crude Extracts Following the Use of Methanol, Dichloromethane, Methanol for Soxhlet and Cold Extraction Methods…………………………………………….49 Table 4.2: Presence of Phytoconstituents of Euphorbia Hirta in the Crude Extracts of Various Solvents. ........................................................................................................................................ 58 Table 4.3 Fractions Obtained From Crude Ethyl Acetate Extracts of Euphorbia Hirta Using Column Chromatography.............................................................................................................. 60 Table 4.4 Summary of Gas Chromatography Mass Spectrometer Results ................................... 63 University of Ghana http://ugspace.ug.edu.gh xi LIST OF FIGURES Figure 2.1: Image of Euphorbia Hirta……………...…………………………………………….17 Figure 3.1: Map of Ghana showing the study site …………….……….…….……………….…36 Figure 4.1: Antimicrobial Activity of Petroleum Ether Extracts Against Test Organism.….…...51 Figure 4.2: Antimicrobial Activity of Dichloromethane Extracts Against Test Organism……...52 Figure 4.3: Antimicrobial Activity of Methanol Extracts Against Test Organisms.……….…....53 Figure 4.4: Antimicrobial Activity of Aqueous Extracts Against Test Organisms …….........….54 Figure 4.5: Antimicrobial Activity of Ethyl Acetate Ether Extracts Against Test Organisms......55 Figure 4.6: Image of TLC Plates Showing Bands Formed from Ethyl Acetate Extracts Fractions………………………………………………………………………………………….59 Figure 4.7: Antimicrobial Activity of Active Fraction (CF5A) Against Test Organisms.…....…61 Figure 4.8: Graph Showing Retention Time of Compound Against Test Organisms ….….……62 Figure 4.9: Graph Showing Molecular Weight of Compound Against Test Organisms….…….62 Figure 4.10: Minimum Inhibitory Concentration of Crude Ethyl Acetate and Active Fraction (CF5A) Of E. Hirta Against Test Organisms …………………………… ……………….….….64 Figure 4.11: Minimum Bactericidal Concentration of Crude Ethyl Acetate and Active Fraction (CF5A) of E. Hirta On Test Organisms…………………………….………….………...………65 University of Ghana http://ugspace.ug.edu.gh xii LIST OF PLATES Plate 1: A, B, C, and D Showing the Antimicrobial Susceptibility Test of E. Hirta Against Some Test Organisms…………………………………………………...................……………………57 Plate 2: A, B, and C Showing the MBC of Crude Ethyl Acetate and Fraction (CF5A) Against Some Test Organisms....................................................................................................................67 University of Ghana http://ugspace.ug.edu.gh xiii LIST OF ABBREVIATIONS ANOVA Analysis of Variance AST Antimicrobial Susceptibility Testing ATCC American Type Culture Collection CF Color Form CPMR Centre for Plant Medicinal Research DDST Double-Disc Synergy Test DMSO Dimethyl Sulphoxide DNA Deoxyribonucleic Acid ESBLs Extended Spectrum Β-Lactamases EUCAST European Committee on Antimicrobial Susceptibility Testing GCMS Gas Chromatography Mass Spectrometer GCMS Gas Chromatography Mass Spectrometer H2SO4 Sulfuric Acid HCl Hydrochloric Acid IBC Inflammatory Breast Cancer ICU Intensive Care Units INT P-Iodonitrotetrazoliumviolet L Litre M/Z Mass Charge Ratio MBC Minimum bactericidal concentration MDR Multidrug-resistance Mg Milligram MHA Mueller Hinton Agar MIC Minimum Inhibitory Concentration University of Ghana http://ugspace.ug.edu.gh xiv Ml Milliliter Mm Millimeters mRNA Messenger Ribonucleic Acid MRSA Methicillin Resistant Staphylococcus Aureus NCCLS National Committee for Clinical Laboratory Standards NCTC National Collection of Type Cultures NIST National Institute of Standard and Technology PBP Penicillin- Binding Proteins PCR Polymerase Chain Reaction PMT Proton Motive Force RNA Ribonucleic Acid rRNA Ribosomal Ribonucleic acid TLC Thin layer chromatography UGMS University of Ghana Medical School UTI Urinary Tract Infection UV Ultraviolet WHO World Health Organization µg Microgram µl Microliter University of Ghana http://ugspace.ug.edu.gh xv ABSTRACT Background: Treatment of infections is an important area of public health concern as the prevalence of multidrug-resistant (MDR) bacteria is on the rise. MDR bacteria are associated with high morbidity and mortality worldwide. Medicinal plants including Euphorbia hirta have shown effectiveness in the treatment of infections and have been one area of interest worldwide for the treatment of diseases due to their high antimicrobial properties against MDR bacteria. In Ghana, the continuous spread of MDR bacteria has resulted in prolonged illness, increased healthcare costs and heightened fatalities which can suddenly cripple the country’s economy. One way to reduce the burden of MDR bacteria is to screen for new classes of antimicrobials from natural products and medicinal plants. Thus, this research aimed to evaluate the antimicrobial properties of E. hirta against selected MDR bacteria in Ghana. Methodology: Five solvents systems (methanol, distilled water, ethyl acetate petroleum ether and dichloromethane) with varying polarities were used to extract E. hirta via cold and Soxhlet extraction methods. The agar-well diffusion method was used to determine the antimicrobial activity of the various extracts against some selected MDR bacteria. Column chromatographic technique was used to the separate most potent crude extract into fractions and their antimicrobial activity were determined. Fractions that showed antimicrobial activity were further purified using column chromatography. Purified fractions were analyzed for the functional groups of compounds present using gas chromatography mass spectrometer (GCMS). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of crude ethyl acetate extracts and active fraction was determined. Results: Results from this study showed that, soxhlet maceration process had higher yield than cold maceration but the antimicrobial activity of extracts from both methods were the same. The University of Ghana http://ugspace.ug.edu.gh xvi Antimicrobial susceptibility test (AST) results revealed that K. pneumoniae isolates. recruited in the study were resistant to all extracts used. Furthermore, all test organisms were resistant to dichloromethane and petroleum ether extracts. Out of the 15 test organisms used, methanol and aqueous extracts were potent against 5 test organisms. Phytochemical analysis revealed the presence of phytoconstituents such as reducing sugars, phenolic compounds, saponins, flavonoids, anthracenosides and phytosterols. GC-MS analysis shows that 1,2,3-Benzenetriol is the probable sugars present in the active fraction. MIC and MBC results indicated that ethyl acetate extracts and the active fraction had the same MBC values with 3.13 mg/ml as their lowest MBC concentration. The MIC value recorded for crude ethyl acetate was between 50- 6.25 mg/ml whiles that of the active fraction (CF5) was between 50 -12.5 mg/ml. Conclusion; Polar extracts of whole E. hirta plant have antimicrobial activity with ethyl acetate extracts being the highest. The plant has the potential to be used as an antimicrobial agent. Therefore, in-vivo studies should be conducted using different polar solvents to extract the whole plant of E. hirta to exploit its antimicrobial activity in living things. University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 INTRODUCTION 1.1 Background Plants have been the main source of food, shelter and clothing as they usually form the base of the food chain and food web in an ecosystem. Ethnobotanical studies have provided reliable information on the usefulness of traditional plants worldwide (Hassan, 2012). Currently, research into plant medicine, identifying the active ingredient that plays a role in disease treatment has been an area of much interest (WHO, 2019). In the traditional system of disease treatment, medicinal plants have been documented to have pharmacological properties (Larsen et al., 2015). A considerable amount of research into plants to determine their antimicrobial properties to develop new drugs are currently ongoing (WHO, 2019). It is particularly important to produce drugs that will be effective against multidrug-resistant microbes to solve the problems posed by these multidrug-resistant microbes (Saravanan et al., 2012). According to Abah & Egwari (2011), the bioactive compounds of plants can be described on a larger scale as lipids, phytochemicals, pharmaceutics, pigments, flavors, and fragrances. The extracts obtained from plants are extensively used in production industries including pharmaceuticals, cosmetics, food production and processing industries. In October 2017, the World Health Organization stated in their Fact Sheet number 134 that, about 80% of people in rural areas in developing African countries relies on traditional medicines as their first point of contact for their basic health care services (WHO, 2017). University of Ghana http://ugspace.ug.edu.gh 2 In developing countries like Ghana, the traditional method of disease treatment is high since the ratio of physician to patient is as low as 1:1000. In reality, the low level of physician to patient ratio gives credence to the relevance of traditional methods of disease treatment (Appiah et al., 2019). Based on the high level of benefit of the traditional method of disease treatment, the government of Ghana set up the Centre for Plant Medicine Research (CPMR) at Akuapem- Mampong in the Eastern region of Ghana to coordinate and promote various scientific activities that would improve herbal medicines as well as to carry out studies to confirm therapeutic evidence of herbal remedies (Mensah et al., 2019). Owing to this, the usage of herbal medicines and the traditional health care system have improved in Ghana (Joshi et al., 2020). Euphorbia hirta, popularly known as the asthma plant, belongs to the family Euphorbiacae. It is an official plant included in the African pharmacopoeia since 1985 (Kumar & Kumar, 2010). It is a small, pantropical plant located along roadsides, pathways, and found abundantly on refuse dumpsites. It is popularly noted for its several medical importance including wound healing, asthma, diarrhoea, cough, athlete's foot, bronchial infection and stomach upset (Kuta et al., 2013). According to Tuhin et al. (2017), E. hirta possesses several antimicrobial properties including septic, inflammatory, diabetic, plasmodium, bacterial, viral, fungal, convulsion, fertility, aphrodisiac and other characteristics which have been documented previously. Several studies point to the emergence and wide-spread of multidrug-resistant bacteria (Dayie et al., 2015, Opintan et al., 2015; Donkor et al., 2018). As a result, there is high mortality, morbidity and prolonged duration of infection treatment in hospitals despite a high level of development in healthcare service world-wide (Borquaye et al., 2019). Evidence shows that continual use of a particular antimicrobial agent has a direct effect on the rate of resistance against that antimicrobial agent (Saravanan et al., 2012). Studies by Donkor et al. (2012) and Borquaye et al. (2019) stated University of Ghana http://ugspace.ug.edu.gh 3 that, the rise in antimicrobial resistance is influenced by two main factors; the misuse of the antimicrobial compounds and the evolution of newly modified resistance genes. The inappropriate use of antimicrobials put microorganisms under selective pressure. Resistance strains, on the other hand exploit their resistant genes to evade the antimicrobial agent's effects (Donkor et al., 2012; Borquaye et al., 2019). 1.2 Problem Statement As explained by Richardson (2017), the widespread of antimicrobial agents and antimicrobial resistance were discovered right after the introduction of the first antibiotics. A greater number of pathogenic microorganisms are becoming increasingly resistant to common, potent, and commonly accessible antibiotics. This accounts for the high level of infection-related morbidities and mortalities (Nweneka et al., 2009). A six-month nationwide surveillance study by Opintan et al. (2015) also reported that bacterial resistance to antimicrobials has reached alarming rates. Other studies conducted by Newman & Opintan (2015), Agyepong et al. (2018), and Borquaye et al. (2019) also proved the existence of multidrug-resistant strains in Ghana and many African countries. The widespread of multidrug-resistant microbes is attributed to the inappropriate use of antimicrobials and the evolution of antibiotic resistance genes (Newman et al., 2015; Borquaye et al., 2019). Other elements contributing to the widespread of antimicrobial resistance are; insufficient infrastructure and resources to carry out surveillance systems in deprived areas, poor infection prevention and control (Iwu-Jaja et al., 2021). The widespread of multidrug-resistant microbes has resulted in crippling economies of developing countries like Ghana's. (Borquaye et University of Ghana http://ugspace.ug.edu.gh 4 al., 2019). In the treatment of infections like gonorrhea, antimicrobial resistance is the main problem (Klausner et al., 2021). Over the years, a number of plant materials have been used in the preparation of drugs; these includes Cinchona sp. and Artemesia annua which have been used in the preparation of quinine and artemisinin respectively for the treatment of malaria. The brain behind the success in the preparation process of these drugs was based on the laid down information obtained through experimental research studies undertaken to investigate phytochemicals like phenolic acids, polyphenols, phenanthrenes, flavonoids, and terpenoids. None of these components have been accepted as the main agent in the manufacturing process for antimicrobial agents due to the fact that, there is luck of data on the mode of action for the phytoconstituents after purification (Mohammadi et al., 2020). Furthermore, there is paucity of data in relation to the biological activities of E. hirta against multidrug resistant (MDR) bacteria. Also, active fractions and their phytochemical compounds responsible for the antimicrobial activity of E. hirta extracts have not been discovered. 1.3 Justification In 1998, the World Health Organization stated that the main challenge associated with traditional health care is the safe use of medicinal plants. However, according to Magiorakos et al. (2011), plant extract medications have reduced toxicity and lower adverse effect than other convention method of disease treatment. Although studies have documented the phytochemical constituents of E. hirta, there is paucity of data on the active fractions of crude extracts that show activity against various microorganisms studied (Kuta et al., 2015). Although plant extracts have shown effectiveness against all classes of bacteria, research have shown that the difference in cell wall University of Ghana http://ugspace.ug.edu.gh 5 arrangement in these two categories of bacteria render plant manufactured products more potent against Gram positive bacteria. That is why Gram negatives are the most common plant pathogens. This suggest that, a mixture of different plant material in manufacturing biological antimicrobial products against gram negatives will yield higher antimicrobial activity than single plant products (Zheng et al., 2013). In-depth knowledge into plants products and their mode of action after conducting a series of experiments will provide knowledge based evidence on the bioactive components of plant extracts that will yield equal benefits in its therapeutics applications (González-Lamothe et al., 2009). Tracing from ancient times, nature has been the main source of antimicrobial agent in the treatment of various ailments which cannot be over emphasized (Saravanan et al., 2012). Over the last few years, natural products are used as the main component in preparing nearly half of all newly manufactured drugs either directly or indirectly. These natural products are usually plant products (Newman & Cragg, 2016). Apart from the great pharmacological properties of plants, they have high dependent ratio for the manufacturing of drugs due to their potency, availability, and lesser side effects compared to other materials (Kunwar & Bussmann, 2008). Despite their extensive traditional history, medicinal plants have had a short-lived research and ethno-pharmacology history over the last 50 years (Yeung et al., 2019). The testing of plant extracts against a wide range of diseases to uncover new bioactive components in plants is a remedy for the short history of the ethno-pharmacological capabilities of therapeutic plants (Joshi et al., 2020). It is therefore imperative to establish scientific evidence that E. hirta has antimicrobial properties against multidrug-resistant bacteria. This study will also seek to screen for the active fractions and the phytoconstituents of E. hirta that show activity against an array of multidrug-resistant bacteria University of Ghana http://ugspace.ug.edu.gh 6 in Ghana. This will represent a new dimension in dealing with the menace of antibiotic resistance in Ghana. 1.4 Aim To determine the antibacterial activity of E. hirta against some selected multidrug-resistant bacteria. 1.5 Specific Objectives 1. To determine and compare the effectiveness of the crude extracts of E. hirta against multidrug- resistant bacteria using different extractants. 2. To determine the fraction of the crude extracts that show antimicrobial activity using chromatographic techniques such as thin layer chromatography (TLC) and column chromatography. 3. To determine the phytochemical constituents of the active fractions of E. hirta that show activity. 4. To investigate the functional groups of compounds present in the active fraction. 5. To determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the phytoconstituents identified University of Ghana http://ugspace.ug.edu.gh 7 CHAPTER TWO 2.0 Literature Review 2.1 Antimicrobials There are both beneficial and harmful microbes to plants and animals which humans are not exception. The harmful microbes have great negative effect on health and economy. As a remedy to the negative effect imposed by the harmful microbes, a variety of substances that inhibit or kill microbes have emerged (Begum et al., 2021). Compounds or substance that kills, slows or retards the growth of microorganisms is termed as antimicrobials. In the application of antimicrobial agents, antimicrobial chemotherapy is the usage of antimicrobial agents in managing microbial infections whilst antimicrobial prophylaxis is the use of antimicrobial agents in preventing microbial infections. In relation to this, the branch of medicine that studies antimicrobial agents have categorized antimicrobial agents based on the type of microorganisms it work against as; antibiotics, antifungal, antiseptic etc. (Hassan et al., 2021). 2.2 Antibiotics When we talk of drugs, antibiotics first comes in mind. Antibiotics have been used for decades. The name antibiotics was coined from the Greek word antibiosis, which literally means against life. As one’s survival may either have positive or negative impact on the lives of others, antibiotics were first considered as substances (chemical compounds) obtained from microbial source that have the ability to kills or prevent other microorganisms from multiplying. These Chemical compounds were of higher concentrations thereby acting as toxic substances to those of lower concentrations (Sengupta et al., 2013) Antibiotics have been classified into two broad categories University of Ghana http://ugspace.ug.edu.gh 8 as bacteriostatic and bactericidal based on their ability to kill or inhibit bacterial (microbial) growth. Whilst bacteriostatic antibiotics inhibit bacterial (microbes) growth, bactericidal antibiotics kills bacterial (microbes) completely (Hassan et al., 2021). 2.2.1 Classification and Mechanism of Action of Antibiotics There are quite a number of antimicrobial agents that are readily available for use worldwide. It is known that all antibiotics have their effect through one of the following; cell-wall synthesis inhibition, inhibition of protein synthesis, destruction of bacterial RNA or DNA (Cunha et al., 2021). This implies that all antibiotics target a specific part of the bacterial cell in other to kill or inhibit its growth. Antibiotics are divided into classes based on how they work as; cell wall synthesis inhibitors, denaturing or cell membrane synthesis inhibitors, protein synthesis inhibitors and folic acid synthesis inhibitors. 2.2.1.1 Cell Wall Synthesis Inhibitors This group of antibiotics target specific part of the cell walls in other to manifest its complete activity in inhibiting the cell wall synthesis. Antibiotics like the beta-lactams inhibit cell wall synthesis by inhibit peptidoglycan cross linkage. Although Gram positive bacteria have thick (higher) cell wall made of peptidoglycan, the synthesis of this structure is a necessary requirement for the survival of all bacterial cells (Beak et al., 2021). The peptidoglycan is made of a cross linking proteins in the form of peptides bonds called the ß-(1-4)-n-acetyl hexamine. Before bacteria can synthesize peptidoglycan, it must activate the penicillin binding proteins (PBP) called trans-peptidase and trans-glycosylase. These PBP are the main target of antibiotics that inhibit cell wall synthesis. Since beta-lactam share similar chemical structure with the D-alanyl D-alanine portion of the cross linked that binds with the penicillin binding proteins (PBPs). The beta lactams University of Ghana http://ugspace.ug.edu.gh 9 mimic and binds at the PPBs instead. After binding, the PBPs becomes unavailable for bacterial cells to synthesize new peptidoglycan for the cell which eventually leads to cell lysis (Wirtz et al., 2021). In the case of penicillin, cephalosporin and carbapenems, they inhibit cell wall synthesis by inhibiting the peptide bond formation process which subsequently block the cross-linking unit of peptidoglycan present in the cell wall. Although all antibiotics in this group inhibit cell wall synthesis, they sometimes have different mode of action in performing their activity. For example, the binding of beta lactams and vancomycin to PBPs (Bæk et al., 2021). Examples of antibiotics in this group includes penicillin’s, cephalosporin’s, vancomycin, beta- lactamase inhibitor, carbapenems, azeteronams, polymycin and bacitracin (Wirtz et al., 2021). 2.2.1.2 Denaturing / Cell Membrane Synthesis Inhibitors. Antibiotics in this class inhibit or kills bacteria by denaturing the bacterial cell membrane. The polar nature of the cell membrane makes it easier for the synthesis of macromolecules in the cell membrane. Antibiotics in this class like the daptomycin depolarize cell membranes that depends on calcium. This depolarization prevents the synthesis of macromolecules in the bacterial cell membrane, causing the cell membrane to rupture (Epand et al., 2016). 2.2.1.3 Protein Synthesis Inhibitors Antibiotics in this group includes aminoglycosides (gentamicin) which inhibit the 30s subunits, macrolides, chloramphenicol, clindamycin, linezolid and streptogramins which also inhibit the 50s subunit. Through transcription, DNA is transcribed into RNA and RNA into proteins through translation. As genetic materials transcribe from DNA to messenger RNA (mRNA), the ribosome synthesizes the proteins content of the mRNA in the translation process. The bacterial 70s University of Ghana http://ugspace.ug.edu.gh 10 ribosome is made up of two smaller subunits, i.e. the 30s subunit and the 50s subunit, which are the main target site for antibiotics that inhibit or kill bacterial cell through the inhibition of protein synthesis (Mccoy et al., 2011). Aminoglycosides also inhibit protein synthesis by targeting the 30s subunits of the ribosomes. Aminoglycosides are positively charged molecules that attract and attach negatively charged particles of the cell membrane. The attached particles in the cell membrane creates larger pores for the entry of the antibiotics. After the entry of the antibiotics, energy in the form of oxygen and active proton motive force (PMT) is required to pass through the cytoplasmic membrane before getting into the bacterial ribosomes. This energy requirement account for the reason why these antibiotics works poorly against anaerobic bacteria’s but effective against aerobic organisms. This antibiotic has synergistic effect with other antibiotics that inhibit cell wall synthesis (beta lactams) as it allows penetration for lower dosage of these antibiotics. Aminoglycosides interact with the 16s ribosomal RNA (rRNA) of the 30s subunit closer to the cell membrane through hydrogen bonds formation which eventually leads to the termination of mRNA in the translation process (Arenz & Wilson, 2016). Tetracycline, another 30s ribosomal subunit inhibitor also prevent the binding of 30s ribosomal subunit thereby interfering with the 16s rRNA to disrupt the binding activity of its transfers RNA. Others like chloramphenicol and macrolides are 50s subunit inhibitors that interfere with peptidyl transferase activity of its 23s ribosomal (rRNA). This action prevents protein synthesis as binding of the transfer (tRNA) to the ribosomal site is ceased. This occurs in chloramphenicol but in macrolides, the action results in detachment of peptide chains at their immature state (Wirtz et al., 2021). University of Ghana http://ugspace.ug.edu.gh 11 2.2.1.4 Folic Acid Synthesis Inhibitors Folic acids are needed to enhance metabolism of nucleic acid and amino acids in the bacterial cells. Antibiotics that inhibit folic acid synthesis do so by imitating the bacterial substrate called the tetrahydrofolate which is obligatory in the synthesis of folic acid in the bacterial cell. Sulfonamides also inhibits folic acid synthesis in the bacterial cell or termination the production of nucleic acids (DNA and RNA). This group of antibiotics have synergic activity with trimethoprim since but both drugs have dissimilar step in their biosynthetic pathways of folic acid. Other classification of antibiotic based on their chemical structure includes; aminoglycoside, monobactams, carbapenems, etc. (Fernández-Villa et al., 2019). 2.2.2.1 Mechanism of Resistance Bacteria use one or combination of the processes bellow in other to form resistance against antibiotics; Reduction in Antibiotic Uptake: Gram positive bacteria have thick peptidoglycan which prevents the entry of antibiotic due to its rigidity. In Gram negative bacterial, the capsule is made of lipopolysaccharides molecules that confer protection in the cell wall and decreases antibiotics uptake. Although bacteria cells are made of small pores that allow the entry of very minute molecules. Reduction in pore sizes provide resistance by decreasing antibiotics uptake (Uruén et al., 2021). Development of Enzymes that Inactivate Antibiotics: Another mode of antibiotic inactivation within bacteria is the production of enzymes that inactivate antibiotics. These enzymes have the ability to inactivate or render antibiotic inactive. Example is the penicillinase, an enzyme produced by bacteria that inactivate penicillin (Uruén et al., 2021). University of Ghana http://ugspace.ug.edu.gh 12 Efflux Pumps: Efflux pump is a resistant mechanism structure that pump to expel (elute) substances out of the organism’s body. This structure is present in organisms like pseudomonas which expel toxic substance out of the organism’s body. This structure is sometimes considered as reduction of antibiotic uptake mechanism (Uruén et al., 2021). Mutation: Antibiotics affect a cells by targeting a specific part of the cell which can either be the cell wall, cell membrane, ribosomes, cell proteins etc. Through genetic mutations, microorganisms confer resistance by altering the target sites of the antibiotics. The reduction or alternation of the binding site renders the antibiotics inactive. Mutation in microorganisms sometimes occurs at their receptors where antibiotics have specific ligands for. The alternation in binding sites through genetic mutation marks the binding inability of antibiotics to the bacterial cell. Most resistant bacteria undergo spontaneous mutation or point mutation. Methicillin resistant staphylococcus aureus (MRSA) is one of the most common bacteria to exhibit this form of resistance. Through mutation, bacteria develop structural mechanisms thereby providing resistance against antibiotics (Read & Woods, 2014). Biofilms: Biofilms are nonliving tissues serving as a protective shelf for microorganisms. When number of microorganisms affect a tissue, they secrete substances that crumps and lay over the surface of the fresh. This are usually seen in wounds that do not respond treatment therapies. The nonliving substances serve as community and harbor number of microorganisms and confer resistance for microbes present by preventing the antibiotics from coming in contact with the microorganism. Not all microorganisms secrete substances in the formation of biofilms, but the most interesting thing is microbial biofilms produced by one microorganisms serve as resistant mechanism for other microorganisms in the dead tissues (Wu et al., 2021). University of Ghana http://ugspace.ug.edu.gh 13 2.3 The Problem of Antibiotic Resistance Antibiotic resistance has been a major health problem worldwide ( Newman et al., 2011; Li et al., 2017; Borquaye et al., 2019; García-Vello1 et al., 2020). The rise in antibiotic-resistant levels has been the cause of threat to the health of both humans and animals. It has affected both economic and social development (Li et al., 2017). Newman et al., (2015) explained that the main cause of resistance to antimicrobial agents is the misuse of antimicrobials substances which gives a selective advantage for resistant strains over non-resistant strains. As an initiative to control the problem imposed by antimicrobial resistance, the WHO implemented the creation of a taskforce and development of indicators to monitor and evaluate the impact of antimicrobial resistance (WHO, 2019). Globally, B-lactamase antibiotics are the widely consumed antibiotic. In Ghana , amoxicillin, penicillin, and metronidazole accounts for about 75% of all B-lactamase antibiotics used (Borquaye et al., 2019; Ngumba et al., 2020). The B-lactamase; amoxicillin and penicillin work against microorganisms by suppressing the production peptidoglycan in the cell wall resulting in cell growth inhibition leading to cell death. Metronidazole, an antibiotic from the nitroimidazole interferes with DNA synthesis, causing cell death (Rice, 2012). Review of several studies by Newman et al. (2014) revealed the development of resistant strains of bacteria in many African countries like Nigeria, Uganda, Zimbabwe, and Ghana. The bacteria have developed resistance to antibiotics like ampicillin, tetracycline, and cotrimoxazole, which have highly been used for decades due to their relatively cheaper prices. In Ghana, antimicrobial resistance has been reported in two (2) teaching hospitals suited at Accra and Kumasi (Newman et al., 2014). Ghana has also recorded the highest resistance rate in microbial isolates from humans, animals, food, and environmental samples ( Newman et al., 2014; Li et al., 2017). University of Ghana http://ugspace.ug.edu.gh 14 2.4.1 Medicinal Plants According to Mack et al. (2019), Medicinal plants known variously as herbal medicines, botanical medicines, phototherapy or phytomedicines. These plants are used in whole or their parts are used in making products like medicines, flavors in food, soap and perfumes. He also described medicinal plants as plants with one or more organs constituents of which are useful for therapeutic purposes. 2.4.2 Uses of Medicinal Plants History of medicinal plants and their antimicrobial purposes can be traced from ancient time. Medicinal plants have served different purposes ranging from traditional to industrial uses. Plants have been the main source for the preparation of medicines before the introduction of Western medicine (Pan et al., 2014). According to the WHO on traditional medicines, medicinal plants such as E. hirta are relatively freely available, resulting in an increasing demand for their utilization to provide primary health care for about 80% of the rural dwellers who depend on traditional medicines for their primary healthcare (WHO, 2019).In the 1990s, it was documented that there are about 250,000 to 500,000 plant species. With this, 10% serve as food, and over 80,000 species are used for therapeutic purposes (Razzaghi-abyaneh et al., 2012). This substantiate that Plants used in the preparation of traditional medicines have the potential to be used in developing new conventional potent drugs. In biological classification, the kingdom plantae are made up of inexhaustible materials acting as ingredients for the treatment of number of diseases (Abah & Egwari, 2011). University of Ghana http://ugspace.ug.edu.gh 15 2.4.3 Plants as Antimicrobials The emergence of multidrug resistant (MDR) bacteria over the past years have call for number of researches aimed at investigating plants to discover their antimicrobial properties against these microorganisms. The rise in research into plants derivatives is attributed to the high emergence of multidrug resistance (MDR) bacteria, rendering the currently available drugs ineffective in the treatment of various ailment. In addition to the reasons for the rise in plant research is that, number of plant extracts possess high antimicrobial agents which works against wide range of microorganisms with additional benefits (García-Vello and González-Zorn, 2020). VanEtten (1991) explained that plants produce phyto-anticipins that undergoes constant synthetic process by plants in forming a barrier against microorganisms. In other to respond to external changes, plants produce a substance called phytoalexin which also perform other function in impeding microbial attacks ( González-Lamothe et al., 2009; García-Vello and González-Zorn, 2020) 2.4.4 The Present Use of Plants as Antimicrobials Currently, the raw form of plant materials used in the traditional method of treating various ailments have been modified for the preparation of commercial drugs. It is documented that plants serve as the main source for the production of about 50% of western drugs. The use of plants in the production of commercial drugs provide additional advantage in terms of cost, availability and safety with greater therapeutic value as compared to drugs prepared from synthetic sources (Namsa et al., 2011). University of Ghana http://ugspace.ug.edu.gh 16 2.4.5 Euphorbia hirta In the tropics, Euphorbia hirta also known as the asthma-plant, is a weed and a native of India. This hairy weeds grow profusely in refuse dumpsites, grasslands, roadside and walking paths with open spaces (Kumar & Kumar, 2010; Tuhin et al., 2017). The plant prefers to grow on acidic, neutral or alkaline soils but shady or dry moist soil does not favor its growth. This is an annual herb that prostrates and grows up to 60 cm long and produces white latex in abundance. The stem bears two simple leaves arranged in opposite pairs. The simple, elliptical, hairy leaves have fine dentate edges and stipules. Each leaf node has axillary cymes with unisexual flowers on stalks without petals. The capsulated fruits have three valves that produces small rectangular red seeds. It releases its ripe seeds through an explosion from the seed capsule. The plant have taproot system for better anchorage and transport substances ( Kuta et al., 2013; Tuhin et al., 2017). University of Ghana http://ugspace.ug.edu.gh 17 Figure 2.1: Image of Euphorbia hirta University of Ghana http://ugspace.ug.edu.gh 18 2.4.6 Pharmacological Importance of Euphorbia hirta According to Tuhin et al. (2017), the leaves of E. hirta has been used in the Indian traditional system of disease treatment for decades. It has shown high level of effectiveness against numerous infections caused by pathogenic parasites, bacteria, fungus, viruses and other ailments. Whole plant exhibits high anti-inflammatory, anxiolytic, analgesic, and antipyretic activities from its aqueous extracts. Also, when used in combination with plants that show anti-asthma properties, it helps in controlling respiratory problems for asthma patients. Preparations of the whole plant of E. hirta is used in the management of bacterial and fungal infections including tinea pedis commonly called the athlete’s foot, dysentery, stomach upsets, warts, scabies, thrush and aphthae (Shih & Cherng, 2012). Titilope et al. (2012), in Nigeria, stated that E. hirta crude extracts are used in the management of Cellulitis and ear infections. Triterpenes, βamyrin, 24-methylenecycloartenol, and β-Sitosterol are phytoconstituents obtained from the aerial part of E. hirta using n-hexane extract. These phytoconstituents showed a higher level of dose-dependent effectiveness for controlling inflammation in mice and rats. In 1995, Mathur and colleagues undertook a study to determine toxic substances in different cell lines. It was revealed after their study that whole plant extract of E. hirta showed non-cytotoxic effect with high level of effectiveness as anti-bacterial agents. According to Ubaid et al. ( 2018), Studies conducted by Tona and colleagues to determine the traditional uses plant extracts in the treatment of diarrheal showed that, whole-plant extract of E. hirta and seven other extracts have anti-diarrheal activity at an average of 17.39%. In-vitro studies carried out using a whole plant extract of E. hirta showed a 60% zone of inhibition of Plasmodium parasite growth. Oral administration of E. hirta extracts showed significant level of effectiveness against the parasitemia. University of Ghana http://ugspace.ug.edu.gh 19 Attah et al. (2013) also conducted an in-vitro study to determine the anti-filarial activity of E. hirta and concluded that E. hirta possesses anti-filarial properties with ethyl acetate fraction being the most effective. They also concluded that E hirta extract possess low toxicity against monkey kidney cell lines. Tuhin et al. (2017) also explained after his study on the in-vivo application of E. hirta in wound treatment that oral application of ethanolic extract promoted wound healing. 2.5.1 Extended Spectrum Beta-Lactamase Producers (ESBLs) The extended spectrum beta-lactamase (ESBLs) are mostly produced by Gram-negative Enterobacteriacea like E. coli, Klebsiella Pneumoniae, Proteus sp. Pseudomonas Aureginosa and Salmonella sp. (Riccio et al., 2021; Matloko et al., 2021). The extensive use of broad-spectrum antibacterial agents is mostly cited to be the cause of the acquisition of resistant mechanism to beta-lactamases (Al-Hammadi et al., 2018). ESBLs are classified into A and B lactamases based on their ability to hydrolyses antibiotics like penicillin, oxyimino-cephalosporins, and monobactams as they produce ESBLs by using genes that were earlier used in beta-lactam production. ESBLs that do not affect cephamycins or carbapenems (Matloko et al., 2021). Bacteria that produce ESBLs have genes that arise as a result of point mutations at sites of their previous B-lactamase like TEM-1, SHV-1, and CTX-M being the most common among the genotype. Genes like VEB, PER, BEL-1, BES-1, SFO-1, TLA, and IBC are other genotypes with great clinical importance among ESBLs. These are enzymes that are usually mediated by plasmids within their cells (Jamborova et al., 2017; Riccio et al., 2021; Matloko et al., 2021). Detailed studies into the genetic makeup of ESBLs shows that these bacteria have genes that confer resistance against some groups of antibiotics. Treatment of infection has been a serious battle between humans and microorganisms that produces extended-spectrum beta-lactamase (Shakya et al., 2017; Riccio et al., 2021). ESBLs were first isolated in Germany in 1983 with increasing University of Ghana http://ugspace.ug.edu.gh 20 reports to date. The prevalence of ESBLs depends on factors like the species themselves, geographic area, health service settings, group of patients infected, and variation among strains. Research into multidrug resistant Gram-negative bacteria that produce ESBLs is an area with much concern (Matloko et al., 2020; Riccio et al., 2021). 2.5.2 Extended Spectrum Beta-Lactamase Escherichia coli Producers Escherichia coli is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium. A normal flora in the lower intestine (Mohammadi et al., 2020) but the most prevalent causative agents of UTI with more than 80% of all UTI cases (Abernethy et al., 2015; Fitzpatrick et al., 2016; Widodo et al., 2020). Lipworth et al., (2021) described E. coli as the deadliest Gram- negative pathogen which accounts for 18% of all mortality cases among gram negative pathogen. Another condition associated with both ESBL E. coli and ESBL Klebsiella infection is sepsis. A condition resulting in multiple damage to organ systems, leading to organ failure or death (Al- Hammadi et al., 2018). The widespread of ESBL E. coli has complicated treatment of infections caused by E. coli. This is due to the high adaptability of resistance mechanisms against routinely used antibiotics for E. coli bacteremia infections such as the broad-spectrum cephalosporin. This has led to the widespread and onset of many urinary tract infections (Abernethy et al., 2015; Widodo et al., 2020; Ochien & Atieno, 2021). One advantage possessed by ESBL E. coli is the production of extended-spectrum beta-lactamase which serves as resistant mechanisms against beta-lactam antibiotics (Ochien & Atieno, 2021). 2.5.3 ESBL Klebsiella pneumoniae Producers The ESBL K. pneumoniae are strain of K. pneumoniae that have develop resistance against extended-spectrum beta-lactam antibiotics (Sarojamma & Ramakrishna, 2011; Riwu et al., 2020). University of Ghana http://ugspace.ug.edu.gh 21 They show resistance to antibiotics like aminoglycoside through their enzymatic activities. This enzymatic activity is peculiar as it is the only mechanism of resistance to aminoglycosides derivatives. This activity occurs in the 16s rRNA in the ribosome leading to its high resistance. The identified 16s methylase genes are armA, rmtB, rmtB, rmtC, rmtD, rmtA and npmA ( Daehre et al., 2018; Riwu et al., 2020). ESBL K. pneumoniae are Gram-negative pathogenic bacterium, facultative, lactose fermenters that causes most nosocomial and community-acquired infections. It can be identified on an agar media due to the mucoid nature of its outer-membrane. A leading causative agent for nosocomial infections in the United States (Pfaller et al., 2018). Carl Friedlander first described K. pneumoniae as bacteremia after its isolation from dead pneumococcal patients in 1882 (Ashurst & Dawson, 2018). In recent years, the emergence and spread of K. pneumoniae resistant strains have increasingly been reported in many countries as it has been reported to be one of the most important causative agents for hospital-acquired infections. According to Magill et al. (2014) and Kalanuria et al. (2014), Klebsiella spp. are the major causative agent of pneumonia that arise from poor ventilation in all of the United States, the second causative agent for all Gram-negative Enterobacteriaceae bacteremia infections and the third leading cause of all hospital-acquired pneumonia (Martin & Bachman, 2018). Sarojamma and Ramakrishna explained that the prevalence of ESBL-producing Klebsiella is 17% out all cases recorded on ESBL producing organisms in India ( Sarojamma & Ramakrishna, 2011; Riwu et al., 2020). Recent studies attest to the fact that ESBL rate is increasing in all parts of the world with K. pneumoniae and E. coli being the major ESBL-producing bacteria. This opportunistic pathogen account for about 33.3% of all Enterobacteriaceae infection and the third most common cause of nosocomial infections (Magill et al., 2014). University of Ghana http://ugspace.ug.edu.gh 22 K. pneumoniae cause infections such as urinary tract infections, cystitis, pneumonia, surgical wound infections, endocarditis, and septicemia. They are resistant to the entire beta-lactam class of antibiotics due to the presence of the blaKPC gene that arises as a result point mutation (Martin & Bachman, 2018). The ESBL producing organisms are noted to affect a variety of organisms ranging from humans to plants. In 2017 and 2018, it was reported to affect healthy broilers (Yossapol et al., 2017). Hartmann et al. (2012) reported its infection in the dairy cow. Germany also recorded the infection of ESBL-producing K. Pneumoniae hatcheries in connection to boilers in a farm as of 2017 (Daehre et al., 2018). The zoonotic aspect of this pathogen is documented to arise from contact or consumption of contaminated meat (Smet et al., 2010; Riwu et al., 2020). 2.5.4 Methicillin-Resistant Staphylococcus aureus (MRSA) Staphylococcus aureus is a Gram-positive, facultative anaerobe that colonizes epithelial cells causes infections to organs like the skin, bone, lung, heart, brain, and the entire circulatory system (Msed et al., 2012; Sharaf et al., 2021). The continual use of unprescribed and overuse of antibiotics triggers the emergence of MRSA. MRSA is a specie of Staphylococcus aureus that are resistant to methicillin. The gene mecA has a low-affinity to penicillin-binding protein PBP 2 and the newly discovered, mecC providing resistance to methicillin. In classification of infections, MRSA infection was previously considered as hospital-associated as it is usually acquired in hospitals. It was classified as a community-associated MRSA infection as a result of its communicability among individuals in the community. In veterinary medicine, it was also classified as livestock-associated MRSA (Grema et al., 2015; Sharaf et al., 2021). The World Health Organization categorized the burden of MRSA in antibiotic resistance reports as a rapidly increasing resistant strain, highly bacteremia, and the leading cause of mortality in hospitals (Wangai et al., 2019). In disease epidemiology, MRSA is noted for the high rate of septic University of Ghana http://ugspace.ug.edu.gh 23 shocks and prolong treatment compared to methicillin-susceptible strains (Otto, 2012; Sharaf et al., 2021). This effect on health is seen as high economic burden associated with increased duration in hospitals. In the United State, it is reported that S. aureus isolates account for more than 60% of all hospital- acquired infections at Intensive Care Units (ICU) (Wangai et al., 2019). In 2014, the World Health Organization following a surveillance study, reported that MRSA infection exceeded 20% in all WHO selected countries put together but more than 80% in some isolated regions. The prevalence of MRSA between and within African countries is believed to be derived from different species (Garoy et al., 2019). Surveillance on MRSA prevalence in 9 African countries shows that the rate of spread is between 12% to 80%, with some individual countries leading with more than 82 % (Falagas et al., 2013). The prevalence between the health worker and patients in Uganda is between 31.5 and 42% (Wangai et al., 2019), 31 and 82% in Ruanda (Ntirenganya et al., 2015; Seni et al., 2016), and 50% in Tanzania but with a grate reduction rate of 34 to 24% in southern African between 2011 and 2014 (Dsani et al., 2020; Sharaf et al., 2021). In 2012, Bagbin undertook a study to determine the angiogram of identified agents of bacterial infections in Ghana and concluded that MRSA was found to be 42.3% and recorded an outbreak of the bacteria infection in the Children’s ward of the Korle Bu Teaching Hospital, which led to the temporal closure of the Children’s Emergency Ward (Bagbin, 2012). 2.5.5 Salmonella typhi Salmonella typhi is a gram-negative, bacillus shaped bacterium with flagella associated with blood stream infection. It is the causative agent for typhoid fever. Typhoid fever was first coined by Pierre Louis in 1829 but the causative organism was not known until its discovery in 1880 by Karl University of Ghana http://ugspace.ug.edu.gh 24 Eberth and later cultured in 1884 by George Gaffky ( Mills-robertson et al., 2002; Griffith et al.,2019) In other to prevent infections caused by S. typhi, scientist started producing drugs and vaccines against this pathogen. Among them is Almroth Wright who developed a vaccine for the diseases. This has been a public health problem ranging from developing to developed countries. In the early 2000s, it was estimated that typhoid fever is associated with 21.7 million illness with 216,000 deaths worldwide. In 2010, the international vaccine institute explained that typhoid fever is accountable for 119 million cases and 12900 deaths in developing countries with low level of sanitation. In relation to this, it was further estimated that S. typhi associated infections ranges between 200 to 300 million each year (Joshi et al., 2020). Typhoid fever has been reported in both south and east Asia, west and central Africa with high emergence rate in areas with deprived portable water and lower standard of sanitations. Resistant strains of non-typhoidal salmonella infections have arose in several African countries with increasing frequency over the last generation. Before the introduction of antibiotics, the mortality rate associated with S. typhi infection was on a rise with estimated value of about 15% or more, but reduced drastically to 1% after the introduction of antibiotics. Although chloramphenicol has been the main antibiotics of choice when it comes to the treatment of salmonella infections (Griffith et al., 2019). Studies conducted by Mills-Robertson et al., (2002) and Arshad et al., (2021) point to the rise of resistant strains of salmonella typhi against ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole, a common potent antibiotics used in managing typhoid fever. They further explained that the presence of multidrug resistant in S. typhi to first line antibiotic may be on a continual rise. It is noted from epidemiological studies that; Salmonella infections are common in Ghana during the rainy season. All this resistance observed in S. typhi is associated with plasmid encoded resistance to antibiotics (Arshad et al., 2021). University of Ghana http://ugspace.ug.edu.gh 25 Patience with plasmid encoded resistant strains are susceptible to fluoroquinolones but other studies prove the resistance against fluoroquinolones. In the early 2000s, S. typhi was noted as the most prevalent pathogen in hospitals despite its high susceptibility to ciprofloxacin. In diagnosis of this infection, there is positive serological test for the O9 and O12 antigens present in the lipopolysaccharide components of their cell wall (2n 3). The commonly used serological test in most low-income countries is the widal test, which measures antibody titters forming agglutination against the lipopolysaccharide O components of their cell wall and antigen H present in their flagella. Other tests for diagnosis can be done by culturing blood or stool samples. Bone marrow aspirates are samples with high sensitivity when it comes to diagnosis of S. typhi (Joshi et al., 2020). 2.6 Antimicrobial Susceptibility Testing (AST) Techniques for detecting the susceptibility and resistance levels of pathogens to antimicrobial agent are collectively called antimicrobial susceptibility testing. The most commonly used method in checking antimicrobial AST are; disk diffusion method, agar well diffusion, the broth or the agar dilution and the E-test. Some AST methods such as the disk diffusion was developed right after the discovery of the first antibiotics and still in use in laboratories (Pfaller et al., 2010; Doerna, 2018). These methods of ASTs detect the susceptibility level of microorganisms phenotypically. The phenotypic method exhibits some advantages compared to other newly developed methods that deals with genotypic constituents of the organism. This advantage includes the opportunity to predict the resistance levels of microbes as well as their susceptibility level. In addition to its advantages, it has the ability to quantitatively measure the level of susceptibility of a pathogenic microbe against the antimicrobial. Meanwhile, these phenotypic method is time consuming (Hsueh et al., 2010; Romney et al., 2018; Doerna, 2018; Berinson et al., 2021). University of Ghana http://ugspace.ug.edu.gh 26 Other evolving methods includes the real time PCR, microarray, mass spectrometry, the flow cytometry and the bioluminescent have high sensitivity, reduced time in providing ASTS results and give detailed understanding on their impacts of the antimicrobial agent on the cell of the intended microorganism. The problem associated with this processes are; high cost in purchasing special equipment’s, special probe requirement, technically skilled required in their operation and limited microorganism sputum involvement in their data base (Doerna, 2018; Romney et al., 2018; Berinson et al., 2021). The selection of specific antimicrobial susceptibility testing method is influenced by number of factors including cost, level of flexibility, reproducibility, accuracy, automaton, individual’s preference and familiarity with the method (Romney et al., 2018). In general, AST has been beneficial in epidemiological studies, discovering antimicrobial agents and predicting the outcome for antimicrobial agents in their therapeutic applications. In giving standard interpretations after antimicrobial susceptibility testing, the clinical and laboratory standard institute(CLSI) of USA and the European committee on antimicrobial susceptibility testing EUCAST publish a standard interpretation for all AST (Doerna, 2018; Romney et al., 2018; Berinson et al., 2021; Bertrand et al., 2021) 2.6.1 Principles of Antimicrobial Susceptibility Testing 2.6.1.1 The Diffusion Method. In the diffusion method, a known concentration of antimicrobial agent is used to infuse 6 mm in diameter paper disk. The infused disk is placed on an agar medium seeded with the test organism and incubated within a specified period. During the period of incubation, the agar medium first absorb water from the agar medium through diffusion. After the absorption of water in to the agar University of Ghana http://ugspace.ug.edu.gh 27 medium, the infused antimicrobial compounds (antibiotic) diffuses into the surroundings of the media. The rate of water diffusion is rapid as compared to that of the antimicrobial agent. The difference in diffusion is dependent on factors including; the difference in concentration gradient between the disk and the agar medium, the solubility of the antimicrobial agent and the weight of molecular compounds present in the antimicrobial agent. The rate of diffusion of compounds with higher molecular weight diffuse slower as compared to compounds with lower molecular weight. Each antimicrobial agent has a unique zone of inhibition due to the difference in the rate of diffusion (Berinson et al., 2021; Bertrand et al., 2021). 2.6.1.2 The Dilution Method In this quantitative analysis, different concentration of the antimicrobial agent may be introduced into the broth or agar medium and incubated for about 24 hours. It is expected that the antimicrobial agent (antibiotics) interact directly with the growth of the microorganisms. The minimum inhibitory concentration of an antimicrobial agent is the lowest concentration that can suppress microbial growth after the incubation period. Further comparison of MIC values with a known concentration of those antimicrobial agents obtained from other fluids can be used in checking other responses (Matuschek et al., 2014; Berinson et al., 2021; Bertrand et al., 2021). 2.7 Role of Chromatography in Science The application of chromatographic techniques have been one of the fast-growing areas of science due to its numerous application (Coskun & Öztopuz, 2019). When chromatography is compared to other methods of separation of mixtures such as distillation, sublimation, fractional crystallization, partition, chemical separations, fractionation of mixtures of weakly polar molecules and compounds that may be spread across immiscible solvents, it becomes clear that University of Ghana http://ugspace.ug.edu.gh 28 chromatography has two extremely valuable advantage. It can be used with small amounts of material and the conditions of operation usually cause no change in the components of the mixture being separated ( Kilmer, 2010; Mack et al., 2019). In recent years, chromatography is noted as one of the most essential analytical procedures for the identification and quantification of medication and its metabolites in the medical profession. Several chromatographic approaches have been developed to distinguish medications based on their properties and forms of interactions. Coskun & Öztopuz, 2019; Mack et al., 2019; Pharmacopoeia & Edition, 2019). Chromatography has gained popularity as a potential technique for determining drug-protein binding and examining clinical or pharmaceutical samples (Mack et al., 2019). In pharmacy, its application includes; pharmaceutical analysis, preparative and analytical procedures for a variety of compounds that can be used in their processes. Many antibiotics have been isolated using chromatography on laboratory and industrial scale for characterization and assays as well as their structural research (Mack et al., 2019). After the necessary experimental procedures have been thoroughly studied in the preparation of both herbal and other conventional medicines, chromatographic methods are particularly useful in dealing with three types of analytical problems such as; testing for homogeneity of substances susceptible to contamination with chemically similar substances, identification of pharmaceutical substances and preparations, determination of individual components of complex mixtures or substances in dilute solution (Mack et al., 2019; Raj, 2020). In plant extraction process, homogeneity tests are especially useful for standardizing substances derived from natural sources, such as alkaloids and glycosides, steroids, and lipids. University of Ghana http://ugspace.ug.edu.gh 29 Chromatography is used in quantitative analysis to isolate the target ingredient in a form that can be determined by a conventional chemical, physical, or biological approach ( Mack et al., 2019; Yang et al., 2020). Alkaloids are progressively being isolated, characterized, and estimated using chromatographic techniques (Yang et al., 2020; Raj, 2020). Several reviews point to the usefulness of chromatography in both qualitative and quantitative analysis of phytochemical compounds and other substances like proteins, peptides, and amino acids (Yakubu et al., 2017; Coskun & Öztopuz, 2019; Mack et al., 2019; Raj, 2020; Yang et al., 2020). 2.8.1 Thin Layer Chromatography This is a separation technique used on a microscale to determine the type and number of compounds or ingredients in a mixture. For this reason, this method is only used to select the appropriate solvent system for chromatographic works that involves the use of liquid solvents at the mobile phase. This method is helpful in monitoring chromatographic works, i.e. for separation and combination of elutes based on visualized spots observed on the TLC plates. In performing of TLC, it has both stationary and mobile phase. The most commonly used stationary phase material is the alumina and the silica. Combination of two solvents are usually used at the mobile phase. Non- polar solvents are used at the initial stage with gradual switch in polarity till the required spots are observed. A small size of the TLC plate is cut with seizers. A thin straight line of about 0.5cm from each end along its length is made and labeled. The plate is then developed in a solvent system at an ambient temperature after spotting the mixture on the TLC plate. After that, the dish is dried in a 90°C oven for about 5 minutes to ensure complete evaporation of the solvent. Plate can be visualized under ultraviolet (UV) light or spraying with University of Ghana http://ugspace.ug.edu.gh 30 10% or 5% ethanol in sulphuric acid, absolute sulphuric acid or vanillin solution followed by heating in an oven at 100°C for 5 minutes. Changing /switching of solvents can be done repeating the process until the required sports are observed during visualization (Matuschek et al., 2014; Raj, 2020) 2.8.2 Column Chromatography This is one of the most commonly used separation technique in organic chemistry. This has been propagated in disciplines like biology, biochemistry, microbiology and medicine due to its usefulness in separating and collecting a single chemical compound from a mixture of compounds dissolved in a solvent. This technique has been helpful over years in separating both small and large scale of mixture of chemical compounds into individual compound. Column chromatography is useful in isolating active ingredients and metabolites from biological fluid, separating chemical compounds in mixtures, estimating fractions in drug preparation and purification of compounds (Raj, 2020) The principle of separation of compounds into individual components is based on differential adsorption of compounds as compounds in fluids moving through the column at different rate are collected in separate fractions. For this reason, column chromatography is sometimes called adsorption chromatography. In performing column experiment, TLC is first performed to determine the solvent to use at the mobile phase of the column. Column have stationary and mobile phase. The common elements used at the stationary phase is the alumina or silica gel. These two solid materials are highly preferred due to their varying properties ranging from less expensive, readily available, good adsorption, uniform shape and size ranging from 60 – 200μ in diameter. They are mechanically stable and chemically inert. Other properties like colorless, polar but do University of Ghana http://ugspace.ug.edu.gh 31 not react with acidic, basic or any other solvents and allow the free flow of mobile phase also increase their chance of use. The mobile phase on the other hand is made up of solvents to use in the column that is determined by the use of TLC. A solvent is selected based on the polarity of the sample. Common examples of solvent used at the mobile phase include ethanol, acetone, water, chloroform, ethyl-acetate, lactic acid, pyridine, etc. The solvent is used in preparing mixture for sample to be introduced in the column. The solvent aid in the separation of individual components in the sample to form separate bands. The individual component is in the solvent mixture separates during the experiment and therefore elute from the column in fractions. Column chromatography have been classified into four based on the method of separation of compounds as; adsorption column chromatography (ACC), Partition column chromatography, Gel column chromatography and ion exchange column chromatography. In ACC, the components that need to be separated from the mixture are adsorbed on the adsorbent's surface whiles in gel column chromatography, separation occurs in column packed with gel making solvents at the stationary phase held at a fixed position. In partition column chromatography, both the stationary and mobile phase are liquid used in partitioning the column but in an ion exchange column chromatography, it’s the stationary phase that is always made of ion exchange resin(Yang et al., 2020). In undertaking column experiments, one must go through processes like packing of the column, adding of samples, monitoring the samples and isolating the separated compounds. Packing this done before samples will be loaded into the column. This is to ensure complete separation of compounds. The two main method of packing are the dry and slurry method packing. The dry packing is used for micro-scale separations whiles the slurry is used in macro-scales separations. Although the dry method gives better results but the slurry method yields best results. Right after packing using either the dry or the slurry method, the sample is loaded on top of the column. Before University of Ghana http://ugspace.ug.edu.gh 32 loading the sample, dissolve the sample with few drops of polar solvent. Add the mixture gently using pipette without disrupting the uniform surface of the column after packing. Samples with thin horizontal bands is best for separation. In other to keep a uniform level of column when adding solvents, add small amount of white sand. After adding sand, continually add the eluting solvent whiles collecting the various fractions in few milliliters. Monitor the column by performing TLC on each fraction collected. Color change can be used for taking fractions of colored samples. After collecting all compounds from samples in fractions, another TLC is performed to combine fractions with similar bands. Fractions with different bands will be left behind. Further purify samples by recrystallization (Yang et al., 2020) 2.8.3 Principles of Column Chromatography The principle of column is based on level of molecules affinity and adsorption. After the introduction of both mobile (solvents to be used) and stationary phase (silica gel or alumina) into the column top, the individual components in the mixture move at a different rate due to the difference in their affinity and adsorption. Components with lower affinity have lower absorption rate to the stationary phase and therefore travels faster than components with higher affinity. Compounds with higher affinity have higher absorption rate at the stationary phase and therefore travels at a slower rate. Chemical components with lower affinity and lower adsorption move faster and elute first in an orderly manner lower to higher affinity compounds. Molecules are adsorbed to the column in an irreversible manner (Yang et al., 2020) 2.9 Gas Chromatography Mass Spectrometer Gas chromatography mass spectrometer (GCMS) is a common analytical technique use in chemistry, microbiology, biomedical science and other disciplines in order to separate and quantify University of Ghana http://ugspace.ug.edu.gh 33 chemicals in a mixture. This method involves the fusion of two methods; the gas chromatography which separates chemical of a mixture and the mass spectroscopy which characterize chemical components of a mixture into individual components. The combination of this two methods makes both qualitative and quantitative analysis of a chemical component in a mixture feasible. This implies that, a single individual component of a mixture can be isolated, quantified and evaluated individually (Friesen & Pauli, 2005; Yakubu et al., 2017) As in other chromatographic techniques, this method has both mobile and stationary phase. The mixture is largely moved toward the stationary phase using an inert gas such as helium in the mobile phase. The stationary phase is located in a tube-like column or stainless steel with varying dimensions. Within the column, the chemical attracts individual components in a selective manner. Compounds in the mixture interact at different rate at both phases. The rate of interaction is specific for each compound as compounds with high interactive power elute the column faster and vice versa. The difference in interaction rate and elution of compounds aid separation of chemical compounds. In other to increase the level of refined components using this process, varying temperature at the stationary phase or varying pressure at the mobile phase can be used (Yakubu et al., 2017) Compounds that elute the column enters the detector designed to create an electronic signal in the presence of every detectable compounds. The signal's size is determined by the concentration of components present. The stronger the signal, the higher the compound concentration, and the smaller the signal, the lower the compound concentration. The detector is directed to a computer to process signals produced (Yakubu et al., 2017). In the electron ionization director, continuous bombardment of compounds with higher energy (70Ev) beam of electrons break compounds into larger and smaller individual fragments. As molecules are bombarded with higher electrons, other University of Ghana http://ugspace.ug.edu.gh 34 molecules attached are removed to get individual molecules. This is why GCMS is able to separate individual molecules with charge ions. This single charged molecule are the molecular ions. Some molecular ions are unstable due to the energy imparted on them by the electrons. These unstable ions further break into smaller units. The charge ions have individual mass but when each mass is divided by a charge, it is termed as the mass charge ratio (M/Z). The mass to charge ratio represent the molecular weight of the fragment since fragments produced by electron ionization have charge of +1. The retention time using this method is the time from introduction of mixture to the time of elution of compounds