University of Ghana http://ugspace.ug.edu.gh GENOTYPING AND TREATMENT OF SECONDARY BACTERIAL INFECTIONS AMONG BURULI ULCER PATIENTS IN THE AMANSIE CENTRAL DISTRICT OF GHANA BY ELIZABETH GYAMFI (10442509) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A MASTER OF PHILOSOPHY DEGREE IN MEDICAL BIOCHEMISTRY JULY, 2015 University of Ghana http://ugspace.ug.edu.gh DECLARATION I ELIZABETH GYAMFI, do hereby declare that with the exception of references to other people’s work, which have been duly acknowledged, this thesis is the outcome of my own research conducted at the Department of Medical Biochemistry, University of Ghana Medical School, College of Health Sciences and the Department of Cell, Molecular Biology and Biochemistry, University of Ghana, College of Basic and Applied Science under the supervision of Dr. Lydia Mosi and Dr. Bartholomew Dzudzor. Neither all nor parts of this project have been presented for another degree elsewhere. ……………………………………………. Date: ………………………. ELIZABETH GYAMFI (Student) ……………………………………………. Date: ………………………… DR. LYDIA MOSI (Supervisor) ………………………………………….. Date: ……………………….. DR. BATHOLOMEW DZUDZOR (Supervisor) i University of Ghana http://ugspace.ug.edu.gh ABSTRACT Background Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans. BU is the third most common mycobacterial disease after tuberculosis and leprosy, but in Ghana and Cote d’ Ivoire, it is the second. M. ulcerans produces mycolactone, an immunosuppressant macrolide toxin which makes the infection painless. However, some patients have complained of painful lesions and delay healing. Painful ulcers and delay healing experienced by some patients may be due to secondary bacterial infections. Main Objective: To identify secondary microbial infections of BU patients, their genetic diversity as well as determine the levels of antibiotics resistance of these microorganisms. Methodology: The study was conducted at Biochemistry, Cell and Molecular Biology, University of Ghana. Subjects were recruited from Amansie Central District, Ashanti Region. Swabs of 51 BU patients were taken and immediately frozen for transport into the laboratory. Microscopy was performed using Ziehl-Neelsen and Gram staining techniques. The samples were also cultured on Luria Bertani, MacConkey, Mannitol, BPA and Sabouraud dextrose agar to identify bacteria and fungi. Antibiotic susceptibility tests were performed on selected bacteria species. DNA was extracted from the samples, after which Polymerase Chain Reaction (PCR) was performed using universal (16S rRNA), MSHA/PA (16S rRNA for mycobacterial) and IS2404 (insertion sequence specific to mycolactone producers) primers to find the different strains of organisms. Finally sequencing was performed on the DNA amplicons that were randomly selected to identify the kinds of microorganisms causing secondary infection. ii University of Ghana http://ugspace.ug.edu.gh Results: All the samples were positive for bacteria. However 49 and 40 positives were obtained from PCR products using the primers MSHA/PA, and IS2404 respectively, thus 40 BU patients were identified out of the total 51 patient samples. Majority of the bacteria identified after sequencing with universal primers for bacteria were Staphylococcus spp (aureus including MRSA, saprophyticus, and lentus), Alcaligene spp (aquatilis and faecalis), Pseudomonas spp (aeruginosa, stutzeri and koreensis) and bacilli cereus group of bacteria. Interestingly, 60% of the sequencing result for mycobacteria detected the presence of Corynebacterium spp (aurimucosum, diphtheria and striatum). Other bacteria identified were Brevibacterium iodinum and Rhodococcus erythropolis. Majority of these bacteria live in muddy areas and dirty water. The selected bacteria were less susceptible to rifampicin, clarithromycin and amikacin. Conclusion: Other bacteria beside M. ulcerans colonize and proliferate on BU lesions. The selected bacteria were less susceptible to clarithromycin and amikacin and rifampicin. The pains and healing delay experienced by some BU patients could be the result of these bacteria colonizing and proliferating on the ulcer or lesions. iii University of Ghana http://ugspace.ug.edu.gh DEDICATION This research work is foremost dedicated to the Almighty God for His undeserved kindness that has been showered upon me throughout my academic life. My profound appreciation goes to my mother for her love, care, support and encouragement over the years. iv University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I am most grateful to the Almighty God for his wisdom, guidance and protection throughout my academic years. I want to thank my supervisor; Dr. Lydia Mosi for her guidance and financial assistance throughout the project. I am very grateful for all the workshops she helped me to attend. I am also grateful to my co-supervisor, Dr. Bartholomew Dzudzor of the Department of Medical Biochemistry (U.G.M.S) for the trouble he took to frequently find out how the work was progressing. I appreciate the corrections and advice. I render an appreciation to Charles Narh and Charles Quaye for the wonderful skills they taught me during the field work. Your criticism and corrections kept me on my toes. My profound gratitude goes to the staffs and students of Molecular Biology Laboratory at the Department of Biochemistry, Cell and Molecular Biology (U.G) especially Mr. Jacob Kwaku Donkor, Adiza Abass, David Abana, Godwin Williams Ametsi, Senyo Gideon Amevinya and Georgia Naa K. Ghartey (Department of Animal Biology) for assisting me whenever help was needed. A big thank you also goes to the staff of the Department of Medical Biochemistry (U.G.M.S) especially, Dr. Nii Ayite Aryee for helping me in the completion of my thesis. Finally, I am grateful to my family and friends especially my mother, Grace Gyamfi, my sister, Esther Gyamfi and Ofori Adomako Ampaw and my colleagues Bismark Bimah, Hayford Narh and Elisha Kwaku Denkyirah for their advice and emotional support. v University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENT DECLARATION ................................................................................................................. i ABSTRACT ........................................................................................................................ ii DEDICATION ................................................................................................................... iv ACKNOWLEDGEMENT .................................................................................................. v TABLE OF CONTENT ..................................................................................................... vi LIST OF FIGURES ........................................................................................................... ix LIST OF TABLES ............................................................................................................. xi LIST OF ABBREVIATIONS ........................................................................................... xii CHAPTER ONE ................................................................................................................. 1 1.0 INTRODUCTION ..................................................................................................... 1 1.1 Background ............................................................................................................... 1 1.2. Justification/Relevance............................................................................................. 3 1.3. Hypothesis ................................................................................................................ 4 1.4. Aim ........................................................................................................................... 4 1.5. Objectives ................................................................................................................. 4 CHAPTER TWO ................................................................................................................ 5 2.0. LITERATURE REVIEW ......................................................................................... 5 2.1. Buruli Ulcer .............................................................................................................. 5 2.1.1. Mycolactone ...................................................................................................... 6 2.1.2. Epidemiology..................................................................................................... 7 2.2. Ecology and Transmission of the Disease.............................................................. 10 2.3. Clinical Presentation of Buruli ulcer ...................................................................... 11 2.4. Diagnosis ................................................................................................................ 14 2.5. Treatment of Buruli Ulcer ...................................................................................... 15 2.6. Suggested treatment other than antibiotics............................................................. 17 2.7. Immunology of the Disease.................................................................................... 18 2.8. Genetic Variation ................................................................................................... 19 2.8.1. Genetic Variation in Mycobacterium ulcerans ...............................................