SUSCEPTIBILITIES OF Salmonella typhi AND OTHER BACTERIAL PATHOGENS TO ANTIBIOTICS AND HOT AQUEOUS EXTRACT OF Hibiscus sabdariffa BY CHUKWUEMEKA CHUKWUMA. K. MB /2008/399 DEPARTMENT OF MICROBIOLOGY AND BIOTECHNOLOGY, FACULTY OF NATURAL SCIENCES CARITAS UNIVERSITY, AMORJI-NIKE ENUGU. AUGUST, 2012. 1 Susceptibilities of Salmonella typhi and other Bacterial Pathogens to Antibiotics and hot aqueous extract of Hibiscus sabdariffa By Chukwuemeka Chukwuma. K MB /2008/399 A Project Research submitted to the Department of Microbiology and Biotechnology Faculty of Natural Sciences Caritas University, Amorji-Nike, Enugu. In Partial Fulfillment of the Requirement for the Award of Bachelor of Science (B.Sc) Degree in Microbiology and Biotechnology Supervisor, Dr Nmema E.E August, 2012 2 CERTIFICATION I certify that this research project was carried out by Chukwuemeka Chukwuma K. with the Registration number MB /2008/399 in the Department of Microbiology and Biotechnology, Faculty of Natural Science, Caritas University, Amorji-Nike, Enugu. The department recognises that Chukwuemeka Chukwuma. K. (MB /2008/399) bears full responsibility of this work. _____________________ ____________ Chukwuemeka Chukwma K. Date Student _______________________ ________________ Dr. Nmema. E.E Date Supervisor ______________________ ________________ Dr. Nmema E.E Date Head of Department _____________________ ________________ External Examiner Date 3 DEDICATION This work is dedicated to Almighty God for his mercies and an unending grace upon my life. 4 ACKNOWLEDGEMENT You won’t win if you don’t begin; for every beginner is a potential winner, God has made me a beginner and a winner. I am grateful. My profound gratitude goes to my ever loving and caring parents Mr. and Mrs. Godwin Chukwuemeka Nwankwo for their support, prayers and provisions throughout this journey. I owe my respect to my very beloved brother Mr. Chukwuemeka Chinomnso N. for his financial support and care towards the achievement of this great goal. Words cannot quantify nor convey in a concise manner what you mean to me. I am greatly indebted to the Head of department as well as my supervisor Dr. Nmema E.E for her constructive criticisms; for bringing out the best in me. I am grateful. I am grateful to my project coordinator Mrs. Amara Ezeme for her support throughout this research and also to all the staffs of microbiology and Biotechnology department of Caritas University, Prof. Ogeneh, Dr. Orji, Miss. Ekwuobi and Mr. Nwobodo for their tireless efforts in making me who I am academically throughout the period of my study. I acknowledge the contribution of my one and only Sister Miss Chioma Chukwuemeka, my brothers Chika Chukwuemeka, Chijioke 5 Chukwuemeka and Iheanyi Chukwuemeka for their assistance towards the actualization of this dream. Special thanks to all my group members, Ezediunor Pepetua, Akujiobi Nkechi, Ezeuko Chinyere, Maduabum Keziah and Nwebeh Chidera for supporting me throughout the course of this work. Permit me to acknowledge in a special way the following people; Pastor David Ibiyeomi; General overseer, Salvation Ministries; you are a role model Father. Evang. (Mrs) Ukeh Pst. (Engr.) John Owolabi. Dr. Karibo George Evang. Wobo Emmanuel To my class of graduating students of Microbiology and Biotechnology department 2012, I say a very big thank you for making my academic programme this past four years (2008-2012) a memorable one. Finally, to everyone that has impacted in my life directly or indirectly, I thank you all in a special way. 6 Table of Content Title ………………………………………………………………i Certification …....................................................................................ii Dedication …………………………………………................iii Acknowledgement ………………………………………….........iv Table of content ………………………………………………….vi List of tables ……………………………………………………….ix List of figures …………………………………………….………...x Appendix …………………………………………………………..xi Abstract ………..………………………………………………….xii Chapter one Introduction …………………………………………………1 1.1 Aims and Objectives ……………………………………….….6 7 Chapter two 2.0 Literature review ……………………………………………....7 2.1 Plants extract review…………….…………………………….7 2.1.1 Brief history of medicinal plant …………………………..….9 2.1.2 Phytochemical components with antimicrobial activity ….…16 2.1.3 Characteristics of phytomedicine …………………………...18 2.1.4 Why the Demand for Phytomedicine ……………………….19 2.1.5 Challenges and Development in the use of phytomedicine…...20 2.1.6 Possible Solutions ……………………………………...…......22 2.2 A review of Hibiscus sabdariffa ………………………..….....24 2.2.1 Scientific classification ……………………………….….…...24 2.2.2 Description ……………………………………….…..……....25 2.2.3 Origin and distribution ……………………………………….26 2.2.4 Various names of H. sabdariffa ……………………………..28 2.2.5 Climate …………….………………………………………..30 2.2.6 Cultivation and harvesting …………………….………….. 30 2.2.7 Pests and diseases ………………………………………… 32 2.2.8 General uses OF H. sabdariffa ……………………………….. 33. 8 2.2.9 Phytomedicine ………………………………………….….38 2.2.3.1 Various research on H. Sabdariffa ……………..….…….38 2.3 Description of Salmonella typhi………………………………..40 2.3.1 Scientific classification OF Salmonella typhi ………….…41 2.3.2 History of typhoid fever ………….……………………….41 2.3.3 Causative Agent ………………………….……………….43 2.3.4 Mode of transmission ………………………………….....44 2.3.5 Signs and symptoms………………………………………46 2.3.6 Epidemiology of typhoid fever ………………………….48 2.3.7 Pathogenesis of Typhoid Fever …………………………..49 2.3.8 Prevention and control ……………………………………50 2.3.9 Diagnosis ………………………..………………………..51 2.34.1Risk factors ……………………………………………….52 2.3.4.2Treatment ……………………………………………...…52 2.3.4.3Other pathogens …………………………………….…….53 9 Chapter three Materials………………………………………………….…..57 3.1.1 Hard Equipments …………………..…………………...57 3.1.2 Soft wares …………………………….………………...57 3.1.3 Media used……………………………….…………......57 3.2. Collection and confirmatory of test organisms……..….58 3.2.1 Gram staining……………………………...…………...58 3.2.2 Biochemical Tests ………………………………..…....59 3.3 Collection and identification of plant material….…..….65 3.4 Extraction of plant material……………………………..65 3.5 Preparation of the Mcfarland’s Standard………….……66 3.6 Preparation of the cell suspension (inocular)……….…....67 3.7 Serial dilutions of the extract ………………………...…67 3.8 Antibiotics Susceptibility testing ………………………..68 3.9 Susceptibility testing using H. sabdariffa…………..……..70. 10 Chapter four 4.1 Results…………………………………………………………..71 Chapter five 5.1 Discussions of results……………………………………....77 5.2 Conclusion……..………………………………………......81 5.3 Recommendation.........................................................................…..82 References…………………………………………….…………….83 11 List of tables Table 1: Some African medicinal plants……………………..…….15 Table 2: Constituents of H. sabdariffa…………………………………37. Table 3: Results of identification test…………………………..….71 Table 4: Inhibitions zone diameters (IZDs) of antibiotics………...72 Table 5: IZDs of H. sabdariffa on the test organisms..………………75 12 List of figures Fig 1: Hibiscus sabdariffa calyces………………………………..26 Fig 2: serial dilution of plant extract………………………………68 Fig 3: Agar plate showing the effect of antibiotics on S. typhi…….73 Fig4: Agar plates showing effect of antibiotics on other test organisms........................................................................................73 Fig 5: Agar plate showing the effect of H. sabdariffa extract on S.typhi……………………………………………………………….75 Fig 6: Agar plate showing effect of H. sabdariffa extract on S. aureus………………………………………………………………………..75 Fig 7: Agar plate showing the effect of H. sabdariffa on E. coli……76 Fig 8: Agar plate showing Effect of H. sabdariffa on klebsiella spp.76 13 APPENDIX Appendix 1: Media preparation…………………………………95 Appendix 2: Reagents used……………………………………..97 Appendix 3: interpretative chart for antibiotic for testing………98 14 ABSTRACT The susceptibilities of Salmonella typhi and other pathogens to antibiotics and hot aqueous extract of Hibiscus sabdariffa were investigated using agar diffusion and agar well diffusion methods respectively. Salmonella typhi was sensitive to ampicillin, cetriaxone, ciprofloxacin, gentamycin of ofloxacin and perfloxacin. Nitrofurantoin, ampicillin, clarithomycin and augumentin are resistant. Escherichia coli, Klebsiella spp, and Staphylococcus aureus were sensitive to 50%, 70% and 60% of the antibiotics respectively. Pseudomonas aeruginosa was resistant to all antibiotics. Hibiscus sabdariffa extract (0.6g in 6ml of sterile distilled water) was active against S. typhi at concentrations of 100mg/ml, 50mg/ml and 25mg/ml (inhibitions zone diameter IZDs = 23mm, 20mm and 16mm respectively). Staphylococcus aureus was susceptible to 100mg/ml, 50mg/ml, 25mg/ml and 12.5mg/ml of the extract with IZDs of 29mm, 18mm, 17mm and 14mm respectively. Klebsiella spp was susceptible to concentrations of 25mg/ml and 12.5mg/ml of the extract with IZDs of 15mm and 10mm respectively. Escherichia coli and Pseudomonas aeruginosa were resistant to all the concentrations of H. sabdariffa extract. It is therefore imperative to note that the use of medicinal plants is recommended to the Government and Industry. 15 CHAPTER ONE INTRODUCTION Long before mankind discovered the existence of microbes, the idea that plants have some healing potentials, i.e. that they contain what we will currently characterize as antimicrobial principle was well accepted (Doughari, mahmood & Tyoyina, 2011). In whatever manner early man gained his knowledge of the curative powers of plants, one must assume that the was able thereafter to recognize